PEDIATRIC NEPHROLOGY

F IFT H E DIT ION

PE DIA TRIC N EPHROL OGY

FIFTH EDITION

Edited By

ELLIS D. AVNER, M.D.

Associate Dean for Research
Professor of Pediatrics and Physiology
Medical College of Wisconsin
Director, Children’s Research Institute
Children’s Hospital Health System of Wisconsin
Milwaukee, Wisconsin

WILLIAM E. HARMON, M.D.

Associate Professor of Pediatrics
Harvard Medical School
Director, Division of Nephrology
Children’s Hospital Boston
Boston, Massachusetts

PATRICK NIAUDET, M.D.

Professor of Pediatrics
Department of Pediatric Nephrology
Hôpital Necker Enfants Malades
Paris, France

with 132 contributing authors

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© 2004 by LIPPINCOTT WILLIAMS & WILKINS

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Library of Congress Cataloging-in-Publication Data

Pediatric nephrology / [edited by] Ellis D. Avner, William E. Harmon, Patrick Niaudet.--
5th ed.
p. ; cm.
Includes bibliographical references and index.
ISBN 0-7817-3545-9
1. Pediatric nephrology. 2. Children--Diseases. I. Avner, Ellis D. II. Harmon, William,
1943- III. Niaudet, P.
[DNLM: 1. Kidney Diseases--Child. 2. Kidney Diseases--Infant. 3. Urinary Tract
Infections--Child. 4. Urinary Tract Infections--Infant. WS 320 P371 2003]
RJ476.K5P4344 2003
618.92'61--dc22
2003060615

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tion and dosage set forth in this text are in accordance with current recommendations and
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ernment regulations, and the constant flow of information relating to drug therapy and
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planned for use in their clinical practice.
10 9 8 7 6 5 4 3 2 1
 CON TE N TS

Contributing Authors ix
Preface and Dedication xv SECTION III: RESEARCH METHODS 299

14 Molecular Biology 301

Francesco Emma and Gian Marco Ghiggeri
15 In Vitro Methods in Renal Research 317
SECTION I: DEVELOPMENT 1 Patricia D. Wilson
1 Embryology 3 16 Animal Models 335
Adrian S. Woolf Jordan A. Kreidberg
2 Glomerular Circulation and Function 25 17 Clinical Investigation 347
Valentina Kon and Iekuni Ichikawa Susan L. Furth and Jeffrey Fadrowski
3 Tubular Function 45 18 Genetics 363
Deborah P. Jones and Russell W. Chesney Lisa M. Guay-Woodford
4 Perinatal Urology 73 19 Tissue Engineering 373
David A. Diamond and Craig A. Peters Anthony Atala
5 Renal Dysplasia/Hypoplasia 83
Paul R. Goodyer
6 Syndromes and Malformations of the SECTION IV: CLINICAL METHODS 385
Urinary Tract 93
Chanin Limwongse and Suzanne B. Cassidy 20 Clinical Evaluation 387
T. Martin Barratt and Patrick Niaudet
21 Laboratory Investigations 399
SECTION II: HOMEOSTASIS 123 Jean-Pierre Guignard and Fernando Santos
22 Evaluation of Growth and Development 425
7 Sodium and Water 125
Sandra L. Watkins and Gail E. Richards
Howard Trachtman
8 Potassium 147 23 Diagnostic Imaging 449
George J. Schwartz Fred E. Avni and Michelle Hall
9 Acid-Base Homeostasis 189 24 Renal Pathology 475
James C. M. Chan and Robert H. K. Mak Agnes B. Fogo
10 Calcium and Phosphorus 209
Anthony A. Portale
11 Disorders of Phosphorus, Calcium, SECTION V: GLOMERULAR DISEASE 501
and Vitamin D 237
Craig B. Langman 25 Congenital Nephrotic Syndrome 503
12 Nutrition and Metabolism 255 Christer Holmberg, Karl Tryggvason, Marjo K. Kestilä,
Corina Nailescu, Phyllis J. Kaskel, and Hannu J. Jalanko
and Frederick J. Kaskel 26 Inherited Glomerular Diseases 517
13 Fluid and Electrolyte Therapy in Children 275 Marie Claire Gubler, Laurence Heidet,
Michael J. G. Somers and Corinne Antignac
vi Contents

27 Steroid-Sensitive Idiopathic Nephrotic Syndrome 45 Henoch-Schönlein Purpura 851

in Children 543 Rosanna Coppo and Alessandro Amore
Patrick Niaudet 46 Systemic Lupus Erythematosus 865
28 Steroid-Resistant Idiopathic Nephrotic Syndrome Patrick Niaudet and Rémi Salomon
in Children 557 47 Hemolytic Uremic Syndromes 887
Patrick Niaudet Chantal Loirat and C. Mark Taylor
29 Immune Mechanisms of Glomerular Injury 575 48 Sickle Cell Nephropathy 917
Allison A. Eddy Jon I. Scheinman
30 Acute Proliferative Glomerulonephritis 601 49 Diabetic Nephropathy 931
Endre Sulyok Leonard G. Feld
31 Immunoglobulin A Nephropathy 615 50 Renal Manifestations of Metabolic Disorders 941
Norishige Yoshikawa William G. van’t Hoff
32 Membranoproliferative Glomerulonephritis 629 51 Infectious Diseases and the Kidney 955
C. Frederic Strife, Michael C. Braun, and Clark D. West Amitava Pahari, Sam Walters,
33 Membranous Nephropathy 641 and Michael Levin
Sudesh Paul Makker 52 Nephrotoxins 987
34 Crescentic Glomerulonephritis 655 Russell W. Chesney and Deborah P. Jones
Michael J. Dillon

SECTION VIII: URINARY TRACT

SECTION VI: TUBULAR DISEASE 663 DISORDERS 1005

35 Nephronophthisis-Medullary Cystic 53 Urinary Tract Infection 1007

Kidney Disease 665 Sverker Hansson and Ulf Jodal
Friedhelm Hildebrandt 54 Vesicoureteral Reflux and Scarring 1027
36 Polycystic Kidney Disease 675 H. Gil Rushton, Jr.
Katherine MacRae Dell, Ruth A. McDonald, Sandra L. 55 Obstructive Uropathy 1049
Watkins, and Ellis D. Avner Robert L. Chevalier and Jonathan A. Roth
37 Aminoaciduria and Glycosuria 701 56 Bladder Dysfunction 1077
Israel Zelikovic Dawn L. McLellan and Stuart B. Bauer
38 Tubular Disorders of Electrolyte Regulation 729 57 Urolithiasis 1091
Juan Rodríguez-Soriano Dawn S. Milliner
39 Renal Tubular Acidosis 757 58 Pediatric Renal Tumors 1113
John T. Herrin Christopher S. Cooper and Jack S. Elder
40 Nephrogenic Diabetes Insipidus 777
Nine V. A. M. Knoers and Leo A. H. Monnens
41 Cystinosis and Fanconi Syndrome 789
SECTION IX: HYPERTENSION 1123
John W. Foreman
42 Primary Hyperoxaluria 807 59 Epidemiology of Hypertension 1125
Pierre Cochat and Laure B. D. E. Collard Jean U. Bender, Melvin A. Bonilla-Felix,
43 Tubulointerstitial Nephritis 817 and Ronald J. Portman
Uri S. Alon 60 Pathophysiology of Hypertension 1153
Joseph T. Flynn and Robert P. Woroniecki
61 Evaluation of Hypertension in Childhood
SECTION VII: SYSTEMIC DISEASE 833 Diseases 1179
Eileen D. Brewer
44 Renal Vasculitis 835 62 Treatment of Hypertension 1199
Rudolph P. Valentini and William E. Smoyer Beth A. Vogt and Ira D. Davis
 Contents vii

73 Pediatric Renal Transplantation 1437

SECTION X: ACUTE RENAL FAILURE 1221 William E. Harmon
74 Ethical Considerations 1469
63 Pathogenesis of Acute Renal Failure 1223
Michel J. C. Broyer
Norman J. Siegel, Scott K. Van Why, and Prasad Devarajan
64 Clinical Evaluation and Management 1233
Sharon P. Andreoli
65 Management of Acute Renal Failure 1253 SECTION XII: PEDIATRIC NEPHROLOGY
Mark R. Benfield and Timothy E. Bunchman AROUND THE WORLD 1479

75A Overview 1481

Matthias Brandis and Ira Greifer
SECTION XI: CHRONIC RENAL FAILURE 1267
75B Europe 1485
Jochen H. H. Ehrich and Anita Amina Elgendi
66 Pathophysiology of Progressive Renal Disease 1269
Agnes B. Fogo and Valentina Kon 75C Japan 1491
Koichi Nakanishi, Masataka Honda,
67 Conservative Management of Chronic
and Norishige Yoshikawa
Renal Insufficiency 1291
Richard N. Fine, Dilys A. Whyte, and Ivy I. Boydstun 75D North America 1495
Sharon P. Andreoli
68 Endocrine and Growth Disorders in Chronic
Renal Failure 1313 75E Africa 1499
Franz Schaefer Bahia Hassan Moustafa, Felicia Eke,
and Rajendra Bhimma
69 Renal Osteodystrophy 1347
Beatriz D. Kuizon and Isidro B. Salusky 75F Latin America 1507
Ramon Alfonso Exeni
70 Peritoneal Dialysis 1375
Bradley A. Warady, Bruce Z. Morgenstern, 75G Australia and New Zealand 1513
and Steven R. Alexander John R. Burke and William Wong
71 Hemodialysis 1395 75H Asia 1517
Stuart L. Goldstein and Kathy Jabs Carmelo A. Alfiler, Hui-Kim Yap, and
Man-Chun Chiu
72 Transplantation Immunobiology 1411
Alan M. Krensky and Carol Clayberger Index 1525
 CONTRI B U TING A U THOR S

Steven R. Alexander, M.D. Professor of Pediatrics, Stan- Jean U. Bender, M.D. Fellow in Nephrology and Pediat-
ford University School of Medicine; Chief of Pediatric ric Nephrology, Division of Pediatric Nephrology and
Nephrology, Lucile Packard Children’s Hospital at Stan- Hypertension, University of Texas—Houston Medical
ford, Stanford, California School, Houston, Texas
Carmelo A. Alfiler, M.D. Professor of Pediatrics, Uni- Mark R. Benfield, M.D. Associate Professor of Pediat-
versity of the Philippines Manila College of Medicine, rics, University of Alabama School of Medicine, Birming-
Manila, Philippines ham, Alabama
Uri S. Alon, M.D. Professor of Pediatrics, Department Rajendra Bhimma, M.D., M.B.Ch.B., D.C.H., M.Med.,
of Pediatric Nephrology, University of Missouri—Kansas F.C.P.P. Associate Professor of Pediatrics and Principal
City School of Medicine, Children’s Mercy Hospital, Kan- Specialist, Department of Pediatrics and Child Health,
sas City, Missouri University of Natal, Durban, South Africa
Alessandro Amore, M.D. Professor of Nephrology, Melvin A. Bonilla-Felix, M.D. Associate Professor of
Department of Nephrology, Dialysis, and Transplanta- Pediatrics, University of Puerto Rico School of Medicine,
tion, Regina Margherita Children’s Hospital, Torino, Italy San Juan, Puerto Rico
Sharon P. Andreoli, M.D. Professor of Pediatrics, Indi- Ivy I. Boydstun, M.D. Assistant Professor, Department
ana University School of Medicine, James Whitcomb Riley of Pediatrics, SUNY at Stony Brook School of Medicine
Hospital for Children, Indianapolis, Indiana Health Sciences Center, Stony Brook, New York
Corinne Antignac, M.D., Ph.D. Professor of Medicine,
Matthias Brandis, M.D. Professor of Medicine, Depart-
Inserm U574 and Department of Genetics, Paris 5 Univer-
ment of Pediatrics and Adolescent Medicine, Freiburg Uni-
sity, Hôpital Necker Enfants Malades, Paris, France
versity Hospital, Freiburg, Germany
Anthony Atala, M.D. Surgeon, Associate Professor of
Surgery, Department of Urology, Children’s Hospital, Bos- Michael C. Braun, M.D. Assistant Professor of Pediat-
ton, Massachusetts rics, The Institute of Molecular Medicine, University of
Texas—Houston Medical School, Houston, Texas
Ellis D. Avner, M.D. Associate Dean for Research; Pro-
fessor of Pediatrics and Physiology, Medical College of Eileen D. Brewer, M.D. Professor of Pediatrics, Head,
Wisconsin, Director, Children’s Research Institute, Chil- Renal Section, Department of Pediatrics, Baylor College
dren’s Hospital Health System of Wisconsin, Milwaukee, of Medicine, Texas Children’s Hospital, Houston, Texas
Wisconsin
Michel J. C. Broyer, M.D. Professor Emeritus, Depart-
Fred E. Avni, M.D., Ph.D. Professor of Radiology, ment of Pediatric Nephrology, Hôpital Necker Enfants
Department of Medical Imaging, Erasme Hospital, Malades, Paris, France
Brussels, Belgium
Timothy E. Bunchman, M.D. Professor of Pediatrics,
T. Martin Barratt, M.B., F.R.C.P. Emeritus Professor of University of Alabama School of Medicine, Pediatric
Paediatric Nephrology and Honorary Consulting Nephrol- Nephrology and Transplantation, Children’s Hospital of
ogist, Institute of Child Health, Great Ormond Street Hos- Alabama, Birmingham, Alabama
pital for Children, London, England, United Kingdom
John R. Burke, M.B., F.R.A.C.P. Associate Professor of
Stuart B. Bauer, M.D. Professor of Surgery, Department Medicine, Department of Nephrology, Royal Children’s
of Urology, Harvard Medical School; Senior Associate in Hospital, Mater Children’s Hospital, Princess Alexandra
Urology, Children’s Hospital, Boston, Massachusetts Hospital, Brisbane, Australia
x Contributing Authors

Suzanne B. Cassidy, M.D. Professor of Pediatrics, University Michael J. Dillon, M.B.B.S., F.R.C.P., F.R.C.P.C.H.,
of California, Irvine, College of Medicine, Irvine, California D.C.H. Professor of Pediatric Nephrology, Nephro-Urol-
ogy Unit, Institute of Child Health, Great Ormond Street
James C. M. Chan, M.D. Professor of Pediatrics, Uni- Hospital for Children, London, England, United Kingdom
versity of Vermont College of Medicine, Burlington, Ver-
mont; Director of Research, Barbara Bush Children’s Allison A. Eddy, M.D. Professor of Pediatrics, Univer-
Hospital, Maine Medical Center, Portland, Maine sity of Washington School of Medicine, Children’s Hospital
and Regional Medical Center, Seattle, Washington
Russell W. Chesney, M.D. Le Bonheur Professor and
Chair, Department of Pediatrics, University of Tennessee, Jochen H. H. Ehrich, M.D., D.C.M.T. (London)
Memphis College of Medicine, Memphis, Tennessee Professor of Pediatrics, Hannover Medical School, Chil-
dren’s Hospital, Hannover, Germany
Robert L. Chevalier, M.D. Professor and Chair, Depart-
ment of Pediatrics, University of Virginia Medical Center, Felicia Eke, M.D. Professor of Pediatrics, University of
Charlottesville, Virginia Port Harcourt, Port Harcourt, Rivers, Nigeria

Man-Chun Chiu, M.B.B.S., F.R.C.P., F.R.C.P.C.H., Jack S. Elder, M.D. Carter Kissell Professor of Urology,
F.H.K.A.M. (Paed) Doctor, Department of Pediatrics Case Western Reserve University School of Medicine,
and Adolescent Medicine, Princess Margaret Hospital, Director of Pediatric Nephrology, Rainbow Babies and
Hong Kong, China Children’s Hospital, Cleveland, Ohio
Anita Amina Elgendi, M.D. Department of Pediatric
Carol Clayberger, Ph.D. Professor of Pediatrics and Car-
Nephrology, Children’s Hospital, Hannover, Germany
diothoracic Surgery, Stanford University School of Medicine,
Stanford, California Francesco Emma, M.D. Pediatric Nephrologist and
Co-Director of the Pediatric Nephrology Laboratory,
Pierre Cochat, M.D. Professor of Pediatrics, Hôpital Department of Nephrology and Urology, Bambino Gesù
Edouard-Herriot, Lyon, France Children’s Hospital and Research Institute, Rome, Italy
Laure B. D. E. Collard, M.D. Department of Pediat- Ramon Alfonso Exeni, M.D. Professor of Pediatrics,
rics, Division of Pediatric Nephrology, University of Department of Pediatric Nephrology, University of Bue-
Liège—CHU, CHR, CHC, Liège, Belgium nos Aires, Hospital de Niños de San Justo, Beccar, Buenos
Aires, Argentina
Christopher S. Cooper, M.D. Chief of Pediatric Urol-
ogy, Associate Professor of Urology, University of Iowa Col- Jeffrey Fadrowski, M.D. Fellow, Department of Pediat-
lege of Medicine, Iowa City, Iowa rics, Johns Hopkins University School of Medicine, Balti-
more, Maryland
Rosanna Coppo, M.D. Professor of Pediatric Nephrol-
ogy, Department of Nephrology, Department of Dialysis Leonard G. Feld, M.D., Ph.D., M.M.M. Professor of
and Transplantation, Regina Margherita Hospital, Torino, Pediatrics, UMDNJ—New Jersey Medical School, New-
Italy ark, New Jersey; Chairman of Pediatrics, Atlantic Health
System, Morristown, New Jersey
Ira D. Davis, M.D. Associate Professor of Pediatrics, Case
Western Reserve University School of Medicine; Director of Richard N. Fine, M.D. Professor and Chair, Depart-
Pediatric Nephrology, Rainbow Babies and Children’s Hospi- ment of Pediatrics, SUNY at Stony Brook School of Medi-
tal, Cleveland, Ohio cine Health Sciences Center, Stony Brook University
Hospital, Stony Brook, New York
Katherine MacRae Dell, M.D. Assistant Professor of Pedi-
atrics, Case Western Reserve University School of Medicine; Joseph T. Flynn, M.D. Associate Professor of Clinical
Attending Pediatric Nephrologist, Rainbow Babies and Chil- Pediatrics, Division of Pediatric Nephrology, Albert Ein-
dren’s Hospital, Cleveland, Ohio stein College of Medicine of Yeshiva University, Montefiore
Medical Center, Bronx, New York
Prasad Devarajan, M.D. Louise M. Williams Endowed
Professor of Pediatrics and Developmental Biology, Director Agnes B. Fogo, M.D. Professor of Pathology, Medicine,
of Nephrology and Hypertension, Cincinnati Children’s and Pediatrics, Department of Pathology, Vanderbilt Uni-
Hospital Medical Center, Cincinnati, Ohio versity Medical Center, Nashville, Tennessee
David A. Diamond, M.D. Associate Professor of Surgery, John W. Foreman, M.D. Professor of Pediatrics, Chief
Department of Urology, Harvard Medical School, Children’s of Pediatric Nephrology, Duke University Medical Center,
Hospital, Boston, Massachusetts Durham, North Carolina
 Contributing Authors xi

Susan L. Furth, M.D., Ph.D. Assistant Professor, Depart- Masataka Honda, M.D. Vice President, Department
ment of Pediatrics and Epidemiology, Johns Hopkins Univer- of Pediatrics, Division of Pediatric Nephrology, Tokyo
sity School of Medicine, Baltimore, Maryland Metropolitan Hachioji Children’s Hospital, Hachioji,
Tokyo
Gian Marco Ghiggeri, M.D. Nephrology Unit, Researcher,
Laboratory on Pathophysiology of Uremia, Department of Iekuni Ichikawa, M.D., Ph.D. Professor of Pediatrics
Pediatrics and Basic Science, G. Gaslini Children’s Hospital, and Medicine, Department of Pediatric Nephrology, Vander-
Genoa, Italy bilt University Medical Center, Nashville, Tennessee

Stuart L. Goldstein, M.D. Assistant Professor, Department Kathy Jabs, M.D. Associate Professor of Pediatrics, Direc-
of Pediatrics, Baylor College of Medicine, Houston, Texas tor of Pediatric Nephrology, Vanderbilt University Medical
Center, Nashville, Tennessee
Paul R. Goodyer, M.D. Professor of Pediatrics, McGill
University Faculty of Medicine, Montreal, Quebec, Canada Hannu J. Jalanko, M.D. Department of Pediatric Neph-
rology and Transplantation, University of Helsinki, Hospital
Ira Greifer, M.D. Professor of Pediatrics, Albert Ein- for Children and Adolescents, Helsinki, Finland
stein College of Medicine of Yeshiva University Monte
Fiore Medical Center, Bronx, New York Ulf Jodal, M.D., Ph.D. Professor of Pediatric Nephrol-
ogy, Pediatric Uro-Nephrologic Center, Queen Silvia Chil-
Lisa M. Guay-Woodford, M.D. Professor of Medicine dren’s Hospital, Göteborg, Sweden
and Pediatrics, University of Alabama School of Medicine,
Birmingham, Alabama Deborah P. Jones, M.D. Associate Professor of Pediat-
rics, Memphis College of Medicine, Memphis, Tennessee
Marie Claire Gubler, M.D. Directeur de Recherche,
Hôpital Necker Enfants Malades, Paris, France Frederick J. Kaskel, M.D., Ph.D. Professor of Pediat-
rics, Albert Einstein College of Medicine of Yeshiva Univer-
Jean-Pierre Guignard, M.D. Professor of Pediatrics, Cen- sity; Chief, Section on Nephrology, Vice Chair, Affiliate
tre Hospitalier Universitaire Vaudois, Lausanne, Switzerland and Network Affairs, Children’s Hospital at Montefiore,
Bronx, New York
Michelle Hall, M.D. Professor of Pediatric Nephrology,
Queen Fabiloa Brussels Free University; Pediatric Nephrol- Phyllis J. Kaskel, M.A., R.D., C.D.N. Director, Depart-
ogist, Dialysis and Transplantation, Children’s Hospital, ment of Clinical Nutrition, Mount Sinai Hospital of the
Brussels, Belgium Mount Sinai Medical Center, New York, New York
Sverker Hansson, M.D., Ph.D. Associate Professor of Marjo K. Kestilä, Ph.D. Senior Research Scientist, Depart-
Pediatric Nephrology, Pediatric Uro-Nephrologic Center, ment of Molecular Medicine, National Public Health Insti-
Queen Silvia Children’s Hospital, Göteborg, Sweden tute, Helsinki, Finland
William E. Harmon, M.D. Associate Professor of Pediat- Nine V. A. M. Knoers, M.D., Ph.D. Clinical Geneti-
rics, Harvard Medical School; Director, Division of Nephrol- cist, Professor in Clinical Genetics, Department of Human
ogy, Children’s Hospital Boston, Boston, Massachusetts Genetics, University Medical Centre Nymegen, Nymegen,
The Netherlands
Laurence Heidet, M.D., Ph.D. Hôpital Necker Enfants
Malades, Paris, France Valentina Kon, M.D. Associate Professor of Pediatric
Nephrology, Vanderbilt University Medical Center, Nash-
John T. Herrin, M.B.B.S., F.R.A.C.P. Associate Clini- ville, Tennessee
cal Professor of Pediatrics, Harvard Medical School;
Director of Dialysis, Children’s Hospital Boston, Boston, Jordan A. Kreidberg, M.D., Ph.D. Assistant Professor
Massachusetts of Pediatrics, Department of Medicine, Harvard Medical
School, Children’s Hospital, Boston, Massachusetts
Friedhelm Hildebrandt, M.D. Professor of Pediatrics
and Human Genetics, Department of Pediatrics and Com- Alan M. Krensky, M.D. Shelagh Galligan Professor,
municable Diseases and Human Genetics, University of Department of Pediatrics, Stanford University School of
Michigan Medical School, Ann Arbor, Michigan Medicine, Stanford, California
Christer Holmberg, M.D., Ph.D. Professor of Pediat- Beatriz D. Kuizon, M.D. Department of Pediatrics, Kai-
rics, University of Helsinki, Pediatric Nephrologist, Hospi- ser Permanente Los Angeles Medical Center, Los Angelos,
tal for Children and Adolescents, Helsinki, Finland California
xii Contributing Authors

Craig B. Langman, M.D. Isaac A. Abt, M.D., Professor Amitava Pahari, M.D.D.C.H., D.N.B., M.R.C.P.,
of Kidney Diseases, Department of Pediatrics, Northwestern F.R.C.P.C.H. Senior Consultant, Department of Pediatric
University Medical School; Director of Nephrology, Children’s Nephrology, Apollo Gleneagles Hospital, Kolkatla, India
Memorial Hospital, Chicago, Illinois
Craig A. Peters, M.D. Associate Professor of Surgery,
Michael Levin, Ph.D., M.B.B.C.H., F.R.C.P. Professor Department of Urology, Harvard Medical School, Chil-
of Pediatrics, Imperial College London Faculty of Medi- dren’s Hospital Boston, Boston, Massachusetts
cine, London, England, United Kingdom
Anthony A. Portale, M.D. Professor of Pediatrics, Uni-
Chanin Limwongse, M.D. Assistant Professor of Medi- versity of California, San Francisco, School of Medicine,
cine, Mahidol University Faculty of Medicine, Siriraj Hos- San Francisco, California
pital, Bangkok, Thailand
Ronald J. Portman, M.D. Professor and Director,
Chantal Loirat, M.D. Professor of Pediatrics, Department Division of Pediatric Nephrology and Hypertension, Uni-
of Pediatric Nephrology, Hôpital Robert Debré, Paris, France versity of Texas—Houston Medical School, Houston, Texas
Robert H. K. Mak, M.D., Ph.D. Professor and Chief, Gail E. Richards, M.D. Professor of Pediatrics, Univer-
Division of Pediatric Nephrology, Oregon Health Sciences sity of Washington School of Medicine, Children’s Hospital
University School of Medicine, Portland, Oregon and Regional Medical Center, Seattle, Washington
Sudesh Paul Makker, M.D. Professor and Chief of Pedi-
Juan Rodríguez-Soriano, M.D. Professor of Pediatrics,
atric Nephrology, Department of Pediatrics, University of
Basque University School of Medicine, Hospital de Cruces,
California, Davis, School of Medicine, Davis, California
Baracaldo, Spain
Ruth A. McDonald, M.D. Associate Professor of Pediat-
rics, Department of Nephrology, University of Washington Jonathan A. Roth, M.D. Assistant Professor of Urology,
School of Medicine, Children’s Hospital and Regional University of Virginia School of Medicine, Charlottesville,
Medical Center, Seattle, Washington Virginia

Dawn L. McLellan, M.D., F.R.C.S.C. Fellow in Urol- H. Gil Rushton, Jr., M.D., F.A.A.P. Professor of Urol-
ogy, Children’s Hospital Boston, Boston, Massachusetts ogy and Pediatrics, George Washington University School
of Medicine and Health Sciences, Chairman, Department
Dawn S. Milliner, M.D. Professor of Pediatrics and of Pediatric Urology, Children’s National Medical Center,
Medicine, Division of Pediatric Nephrology, Mayo Clinic, Washington, D.C.
Rochester, Minnesota
Rémi Salomon, M.D., Ph.D. Pediatric Nephrology,
Leo A. H. Monnens, M.D., Ph.D. Professor, Department Hôpital Necker Enfants Malades, Paris, France
of Pediatric Nephrology, Nymegen University, Nymegen, The
Netherlands Isidro B. Salusky, M.D. Professor of Pediatrics, Depart-
ment of Pediatric Nephrology, University of California,
Bruce Z. Morgenstern, M.D. Associate Professor, Depart- Los Angeles, UCLA School of Medicine, Los Angeles,
ment of Pediatric and Adolescent Medicine, Division of Pedi- California
atric Nephrology, Mayo Clinic, Rochester, Minnesota
Fernando Santos, M.D. Professor of Pediatrics, Hospital
Bahia Hassan Moustafa, M.D. Professor of Pediatrics,
Universitario Central de Asturias and School of Medicine,
Pediatric Nephrology Unit, Cairo University, Children’s
Asturias, Spain
Hospital, Cairo, Egypt
Corina Nailescu, M.D. Fellow in Pediatric Nephrology, Franz Schaefer, M.D. Professor of Pediatrics, Pediatric
Department of Pediatrics, Albert Einstein College of Medi- Nephrology Division, University of Heidelberg, Children’s
cine of Yeshiva University, Children’s Hospital at Mon- Hospital, Heidelberg, Germany
tefiore, Bronx, New York
Jon I. Scheinman, M.D. Professor of Pediatrics; Chief,
Koichi Nakanishi, M.D. Instructor of Pediatrics, Pediatric Nephrology, University of Kansas Medical Center,
Wakayama Medical University, Wakayama City, Japan Kansas City, Kansas

Patrick Niaudet, M.D. Professor of Pediatrics, Depart- George J. Schwartz, M.D. Professor of Pediatrics and
ment of Pediatric Nephrology, Hôpital Necker Enfants Medicine, University of Rochester School of Medicine and
Malades, Paris, France Dentistry, Rochester, New York
 Contributing Authors xiii

Norman J. Siegel, M.D. Professor of Pediatrics and Sam Walters, M.B., F.R.C.P. Senior Lecturer in Pediat-
Medicine, Yale University School of Medicine, New ric Infectious Diseases, Department of Pediatrics, Imperial
Haven, Connecticut College London, London, England, United Kingdom

William E. Smoyer, M.D. Robert C. Kelch Professor, Bradley A. Warady, M.D. Professor of Pediatrics; Chief,
Director, Pediatric Nephrology Division, University of Section of Pediatric Nephrology, Director of Dialysis and
Michigan Medical School, Ann Arbor, Michigan Transplantation, Department of Pediatric Nephrology, Uni-
versity of Missouri—Kansas City School of Medicine, Kansas
Michael J. G. Somers, M.D. Assistant Professor of Pedi- City, Missouri
atrics, Division of Nephrology, Harvard Medical School,
Children’s Hospital, Boston, Massachusetts Sandra L. Watkins, M.D. Professor of Pediatrics, Uni-
versity of Washington School of Medicine, Children’s Hos-
C. Frederic Strife, M.D. Professor of Medicine, Depart- pital and Regional Medical Center, Seattle, Washington
ment of Nephrology and Hypertension, University of Cincin-
nati College of Medicine, Cincinnati Children’s Hospital Clark D. West, M.D. Emeritus Professor of Pediatrics,
Medical Center, Cincinnati, Ohio Department of Nephrology and Hypertension, University
of Cincinnati College of Medicine, Cincinnati Children’s
Endre Sulyok, M.D., Ph.D., D.Sci. Director and Profes- Hospital Medical Center, Cincinnati, Ohio
sor of Pediatrics, County Children’s Hospital, Pécs, Hungary
Dilys A. Whyte, M.D. Assistant Professor of Pediatrics,
C. Mark Taylor, F.R.C.P. (London), F.R.C.P.C.H. Stony Brook University Hospital, Stony Brook, New York
Consultant Pediatric Nephrologist, Senior Clinical Lec-
turer, Department of Nephrology, Birmingham Children’s Patricia D. Wilson, Ph.D. Professor, Department of
Hospital, Birmingham, England, United Kingdom Medicine, Mount Sinai School of Medicine of the City
University of New York, New York, New York
Howard Trachtman, M.D. Professor of Pediatrics, Albert
Einstein College of Medicine of Yeshiva University, Schneider William Wong, M.B.Ch.B., F.R.A.C.P. Pediatric Neph-
Children’s Hospital, Bronx, New York rologist, Starship Children’s Hospital, Grafton, Auckland,
New Zealand
Karl Tryggvason, M.D., Ph.D. Professor of Medical
Adrian S. Woolf, M.D., M.A., F.R.C.P.C.H. Professor of
Chemistry, Department of Medical Biochemistry and Bio-
Nephrology, Nephro-Urology Unit, Institute of Child
physics, Karolinska Institutet, Stockholm, Sweden
Health, London, United Kingdom
Rudolph P. Valentini, M.D. Assistant Professor, Direc- Robert P. Woroniecki, M.D. Assistant Professor of Pedi-
tor of Dialysis Services, Department of Pediatrics, Wayne atrics, Division of Pediatric Nephrology, Albert Einstein
State University School of Medicine, Children’s Hospital of College of Medicine of Yeshiva University, Montefiore
Michigan, Detroit, Michigan Medical Center, Bronx, New York
Scott K. Van Why, M.D. Associate Professor, Depart- Hui-Kim Yap, M.D., M.B.B.S., M.Med. (Pediatrics),
ment of Pediatrics, Medical College of Wisconsin, Milwau- F.R.C.P.C.H., F.R.C.P. (Edin) Professor of Pediatrics,
kee, Wisconsin National University of Singapore, National University Hos-
pital, Singapore, China
William G. van’t Hoff, M.D., F.R.C.P., F.R.C.P.C.H.
Consultant Pediatric Nephrologist, Great Ormond Norishige Yoshikawa, M.D. Professor and Chair, Depart-
Street Hospital for Children, London, England, United ment of Pediatrics, Wakayama Medical University, Wakayama
Kingdom City, Japan

Beth A. Vogt, M.D. Associate Professor of Pediatrics, Case Israel Zelikovic, M.D. Associate Professor of Pediatrics,
Western Reserve University School of Medicine, Rainbow Technion—Faculty of Medicine, Director, Department of
Babies and Children’s Hospital, Cleveland, Ohio Pediatric Nephrology, Rambam Medical Center, Haifa, Israel
 PREFACE AND DEDICATION

Through its past four editions, Pediatric Nephrology has mixture of continuity, new ideas, and new perspectives. The
become the standard medical reference for health care pro- current editors are nationally and internationally recognized
fessionals treating children with kidney disease. This new leaders in complementary areas of pediatric nephrology and
edition is focused on providing critical information regard- reflect the international nature of the text and the subspe-
ing the evaluation of children with symptoms of renal dis- cialty it serves. Professor Avner’s focused interest is in devel-
ease, the molecular and cellular pathophysiology of renal opmental renal biology and congenital/genetic diseases of
disorders, and the diagnosis and therapy of specific renal the kidney. He serves as Chairman of the Department of
diseases in children. The text is particularly targeted to Pediatrics at Case Western Reserve University School of Medi-
pediatricians, pediatric nephrologists, pediatric urologists, cine and Chief Medical Officer at Rainbow Babies and
and physicians in training. It is also targeted to the increas- Children’s Hospital in Cleveland, Ohio, and is the current
ing number of allied health professionals involved in the President of the American Society of Pediatric Nephrology.
care of children with renal disease, including geneticists, Professor Harmon is a leading expert in the field of chronic
genetic counselors, nurses, dialysis personnel, nutritionists, renal failure and its therapy in children. He serves as the
social workers, and mental health professionals. It is Director of Nephrology at Harvard Medical School and The
designed to serve the needs of primary care physicians Children’s Hospital, Boston, Massachusetts, and is the cur-
(internists and family practitioners), as well as internist rent President of the American Society of Transplantation
nephrologists who are increasingly involved in the initial and Director of the North American Pediatric Renal Trans-
evaluation and/or longitudinal care of children with renal plant Collaborative Study. Professor Niaudet is an interna-
disease under the managed care systems of health care tionally recognized educator and clinician-scientist in the
delivery evolving throughout the world. evaluation and treatment of kidney disease in children, with
The fifth edition includes a total reorganization of con- a particular focus on childhood nephrosis. Professor Niau-
tent from previous editions and begins with an overview of det directs the Pediatric Nephrology Unit at The University
the basic developmental anatomy, biology, and physiology of Paris and the Hôpital Necker Enfants Malades, is the
of the kidney, which is critical to understanding the devel- former President of the European Society of Pediatric
opmental nature of pediatric renal diseases. This is followed Nephrology, and currently directs the Continuing Medical
by a comprehensive presentation of the evaluation, diagno- Education Programs of the International Pediatric Nephrol-
sis, and therapy of specific kidney diseases of childhood, ogy Association.
including extensive clinical algorithms. Of particular note The editors of Pediatric Nephrology take pleasure in ded-
is the emphasis on how rapidly evolving research advances icating the fifth edition of the text to the senior editor of
in molecular genetics and evidence-based medicine are the previous edition, Professor Martin Barratt, C.B.E.,
being translated into new, effective clinical approaches to F.R.C.P., F.R.C.P.H. Over an extraordinarily productive
many children’s kidney diseases. To keep pace with the dra- career as one of the first pediatricians in the United King-
matic evolution of pediatric renal medicine since the previ- dom to specialize in nephrology, Professor Barratt has held
ous edition, the content of the fifth edition has been leadership positions in the British Association for Pediatric
extensively revised, with approximately 40% of the chap- Nephrology and the International Pediatric Nephrology
ters being entirely new and the remainder being revised by Association and is internationally recognized for his scien-
previous and/or new authors. tific contributions in the fields of childhood nephrotic syn-
As has been the tradition of previous editions, the former drome and hemolytic uremic syndrome. His distinguished
senior editor of the text, Professor Martin Barratt, has career predominantly evolved at the Institute of Child
stepped down, and Professor Ellis D. Avner serves as senior Health and the Great Ormond Street Hospital for Children
editor of the fifth edition. Professor William E. Harmon in London, where he served as Director of Clinical Services
continues as second editor, and Professor Patrick Niaudet from 1989 to 1994. In 1992, he was awarded the coveted
joins the editorial troika as junior editor for this edition. James Spence Medal of the Royal College of Pediatrics and
This rotation of editors continues to provide a dynamic Child Health. Professor Barratt has the reputation of being
xvi Preface and Dedication

the “clinician’s clinician” in recognition of his extraordinary leagues. He has been an inspiration to all of the current edi-
clinical skills and acumen as a pediatrician and pediatric tors of Pediatric Nephrology, as well as to countless trainees,
nephrologist. His work is distinguished for its clarity and colleagues, patients, and their families.
logic—characteristics that he used well in reengineering The editors also wish to acknowledge the outstanding
and guiding Pediatric Nephrology through its second, third, editorial assistance of Shelly Parkhurst (Cleveland),
and fourth editions. On a personal note, he has an extraor- Corinne Fleming (Boston), and Denise Martin, formerly of
dinary sense of humor and the ability to turn the most Lippincott Williams & Wilkins. Their dedicated efforts
tedious of editorial tasks into an exciting adventure. He is a were essential in the publication of the fifth edition.
keen observer of human nature, finds pleasure in the Ellis D. Avner, M.D.
extraordinary aspects of seemingly ordinary events, and William E. Harmon, M.D.
takes great pride in mentoring trainees and junior col- Patrick Niaudet, M.D.
 S E C T I O N

DEVELOPMENT
 1

EMBRYOLOGY
ADRIAN S. WOOLF

In the last two decades, considerable advances have been genesis continues for 1 to 2 postnatal weeks in mice. In
made in understanding the mechanisms that control kidney addition, there are some anatomic differences. The human
development (1,2). In previous years, nephrogenesis was mesonephros contains glomeruli with capillary loops, but
described in purely anatomic terms, but we can now inter- mouse mesonephric tubules have rudimentary glomerular
pret many of the key anatomic events as dynamic processes tufts. Humans have approximately 0.9 × 106 glomeruli in each
driven by the expression of specific genes. Although our kidney (7), with a rather large normal range, from 0.7 to 1.0 ×
long-term aim is to understand human kidney development, 106; mice and rats (8) have proportionately fewer glomeruli
most functional studies have been performed in murine spe- per kidney. Finally, whereas the human renal pelvis has multi-
cies; therefore, these animal experiments are described in ple papillae, the murine kidney has one. An illustration of the
some detail. As is obvious to any pediatric nephrologist, early stages of human metanephric development is depicted in
developmental disorders account for a wide spectrum of kid- Figure 1.1, and a histologic analysis is shown in Figure 1.2.
ney diseases that cause considerable morbidity and mortality Figure 1.3 indicates the major cell lineages derived from the
in the first years of life (3–5). Renal malformations, such as metanephros, and Figure 1.4 addresses the formation of blood
agenesis (absence of the kidney) and dysplasia (failure of nor- vessels in the metanephros.
mal renal differentiation), represent major defects of develop-
ment, whereas enhanced proliferation, a characteristic of
Pronephros and Mesonephros
undifferentiated cells, occurs in Wilms’ tumor and cystic kid-
ney diseases. Although these diseases are described in detail The mesoderm forms during gastrulation, and embryonic
elsewhere in this book, they are alluded to as illustrative kidneys subsequently develop from nephrogenic cords;
examples of “nephrogenesis gone wrong”: In some of these masses of intermediate mesoderm, located behind the
disorders, defined aberrations of cell biology and genetics embryonic coelom between the dorsal somites; and the lat-
shed light on normal human kidney development. eral plate mesoderm. At the height of their development,
the pronephros and the mesonephros extend in series from
the cervical to lumbar levels. They develop in a segmental
manner as tubules that are induced to differentiate from
ANATOMY OF KIDNEY DEVELOPMENT
mesoderm by the adjacent pronephric and mesonephric (or
wolffian) duct. In humans, the pronephros develops from
Overview
the third embryonic week and contains rudimentary
Potter has provided the most complete anatomic description tubules opening into the pronephric duct. The human
of human kidney development (6). The reader is also referred mesonephros begins to develop in the fourth week of gesta-
to more recent reviews (1,2). Three sets of “kidneys” form in tion and contains well-developed nephrons comprising vas-
mammalian embryos: the pronephros, mesonephros, and met- cularized glomeruli connected to proximal and distal-type
anephros. The metanephros is the direct precursor of the adult tubules draining into the mesonephric duct, a continuation
kidney, whereas the others essentially involute before birth. of the pronephric duct. The mesonephric duct extends to
The anatomic events of human and mouse nephrogenesis are fuse with the cloaca, the urinary bladder precursor, at the
similar, but the timetable of development differs. Human ges- end of the fourth week. The pronephros and mesonephros
tation is 40 weeks, but mouse gestation is 20 days. The meta- can be regarded as a single unit, and as the wave of differen-
nephros appears 5 to 6 weeks after fertilization in humans and tiation spreads caudally, the cranial end of this organ com-
on day 11 in mice. The first metanephric glomeruli form by 9 plex begins to regress. By one-third of the way through
weeks in humans and day 14 in mice. The final layer of neph- human gestation, most cells in these organs have involuted.
rons forms by 36 weeks’ gestation in humans, whereas nephro- In the male, mesonephric tubules in the area of the gonad
4 I. Development

FIGURE 1.1. Early development of the metanephros. Cross-sec-

tional diagrams of the human metanephros at approximately 5
weeks’ (A), 6 weeks’ (B), 8 weeks’ (C), and 10 weeks’ gestation
(D). Note that the most primitive structures are located in the
periphery of the maturing organ. c, comma-shaped body; g,
glomerulus; m, mesenchyme; mc, mesenchymal condensate; s, S-
shaped body; u, ureteric bud; w, wolffian duct.

FIGURE 1.2. WT1 immunostaining in human fetal kidneys.

form the efferent ductules, and the mesonephric duct gives A: Normal human fetal kidney shows a gradient of WT1 immu-
rise to the epididymis and ductus deferens. In the female, noreactivity (black) from nephrogenic cortex (right) to maturing
some mesonephric tubules persist as the epoophoron and nephrons (left). Cells in condensates and vesicles (larger arrow-
heads) are weakly positive for WT1. Expression increases in the
para-oophoron. mesenchymal (m) to epithelial transition, with high WT1 levels in
the proximal limb of S-shaped bodies (s; smaller arrowheads) and
the podocytes of fetal glomeruli (g). A ureteric bud branch tip (u) is
Metanephros negative. B: Intense WT1 expression is maintained in the podocytes
of maturing glomeruli. Bars are 10 μm. (Courtesy of Dr. P. J. D. Win-
The metanephros is the last embryonic kidney to develop yard, Institute of Child Health, London.)
and is identified in humans and consists of two compo-
nents (2). These are the ureteric bud epithelium, which
branches from the caudal part of the mesonephric duct at tions of ureteric bud branches, and the minor calyces arise
approximately 4 weeks of gestation, and the metanephric from the next generation of branches. Each minor calyx is
mesenchyme, which condenses from the intermediate associated with 20 ampullae, which will form the papillary
mesoderm around the enlarging tip, or ampulla, of the bud. collecting ducts: These indent the calyx to form the familiar
The human metanephric kidney can be first identified at cup shape seen in intravenous urograms (2).
approximately weeks 5 to 6 of gestation. The ureteric bud Up to 14 weeks’ gestation, the formation of each new
and its branches form epithelia of the collecting ducts, renal collecting duct is associated with the induction of a neph-
pelvis, ureter, and bladder trigone, whereas the metanephric ron from adjacent metanephric mesenchyme. The differen-
mesenchyme differentiates into nephron tubules (glomeru- tiation of each nephron starts with mesenchymal cell
lar, proximal tubule, loop of Henle, and distal tubular epi- aggregation around ureteric bud branch tips. Each conden-
thelia) and interstitial fibroblasts. These lineages may be sate subsequently forms a lumen (the vesicle stage) and
more plastic than previously considered, as discussed later elongates to form a tubule (the comma-shaped body),
in this chapter. In humans, the renal pelvis and major caly- which then shows regional specialization into primitive glo-
ces are apparent by the tenth to twelfth weeks of gestation. merular and proximal tubular epithelia (the S-shaped
The pelvis forms from remodeling of the first six genera- body). The proximal end of each S-shape becomes the glo-
 1. Embryology 5

FIGURE 1.3. Main cell lineages arising in the metanephros.

(Modified from Hardman P, Kolatsi M, Winyard PJD, et al. Branch-
ing out with the ureteric bud. Exper Nephrol 1994;2:211–219.)

merular epithelium, whereas the distal end of each tubule

fuses with the adjacent branch of the ureteric bud. With
each division of the ureteric bud, a new layer of nephrons is
induced from stem cells in the periphery of the organ.
Between 14 and 20 weeks, each ampulla induces three to
six nephrons without dividing. During this process, the
connecting tubule of the older, innermost nephron shifts
the position of its point of attachment away from the
ampulla to the connecting tubule of the next-formed neph-
ron so they are joined together in arcades of four to seven
nephrons. Up to 34 to 36 weeks’ gestation, the ureteric bud
branch tips advance further outward, and another four to
FIGURE 1.4. Capillary formation in the mouse metanephros.
seven nephrons form and attach separately just behind the A: The mouse metanephros at the ureteric bud (u) stage contains
ampullary tips. Thereafter, no new nephrons form, no formed capillaries. Center panel shows diagram of the ureteric
although each tubule continues to mature into the postna- bud stage above a photograph of the intact organ. Flanking pan-
els are histology sections of the organ in different planes: The
tal period. These changes include the elongation of the right panel shows an area of loose connective tissue and capillar-
loops of Henle toward the medulla as well as convolution ies (arrows; between the metanephros itself and the wolffian
of the proximal tubule. duct (w). Upper dashed line is lower limit of metanephros and
lower dashed line marks upper limit of wolffian duct. B: Electron
The adult kidney is highly vascular and receives approxi- microscope image of a glomerulus that has formed in the meta-
mately 20% of the cardiac output. However, at the incep- nephros. m, mesenchyme.
tion of metanephric development, no mature vessels are
present in the renal mesenchyme. The first patent capillar-
ies are evident around the stalk of the ureteric bud, when it metanephros receives its blood supply from the lateral sac-
has branched once or twice, and capillaries later appear in ral branches of the aorta. As development proceeds, the
the glomerular crevices of the S-shaped bodies (9). The organ is located at progressively higher levels and is sup-
primitive, multilayered, visceral glomerular epithelium sub- plied by higher branches of the aorta. By 8 weeks’ gestation,
sequently forms a monolayer of podocytes, which abut glo- the metanephros is located in the lumbar position, and ulti-
merular capillary loops. At 9 to 10 weeks’ human gestation, mately, the definitive renal arteries arise from the aorta at
the most mature nephrons, located toward the center of the the level of the second lumbar vertebra (2).
metanephros, are the first to acquire capillary loops and a
patent Bowman’s space. The forming glomerular basement
Ureter and Urinary Bladder
membrane is believed to be synthesized by both the endo-
thelium and epithelium (10). The fusion of the two embry- The lower urinary tract forms in synchrony with the meta-
onic membranes and its subsequent biochemical nephric kidney (1). The urogenital sinus is the urinary blad-
maturation correlate with the progressive restriction of fil- der rudiment, and it separates from the rectum by 4 weeks of
tration of macromolecules. At its inception, the human human gestation. At this time, its epithelium fuses with that
6 I. Development

of the mesonephric duct, and the ureteric bud arises as a found that the mouse metanephros forms a small kidney in
diverticulum from the posteromedial aspect of the meso- organ culture over days. Of note, other branching organs,
nephric duct near where it enters the forming bladder. such as the lung and the salivary gland, can also be grown
Between 4 and 5 weeks, the human ureter is patent, and it in the same manner (Fig. 1.6). If either the renal mesen-
has been assumed because the cloaca is imperforate at that chyme or the ureteric bud were cultured in isolation, how-
time, mesonephric urine maintains ureteral patency by ever, Grobstein noted that they failed to differentiate. This
increasing intraluminal pressure. The mesonephric duct clearly demonstrated that embryonic tissue interactions are
above the ureteric bud becomes the vas deferens in males but critical for kidney development, and we now know that
involutes in females. At 5 weeks of gestation, the meso- growth factors are important signaling molecules involved
nephric duct below the ureteric bud, the “common excretory in these inductive processes. Growth may be modulated in
duct,” is absorbed into the urogenital sinus to form the tri- organ culture with antisense oligonucleotides that impair
gone; thereafter, ureteric orifices migrate cranially and later- the transcription of metanephric mRNAs or with antibod-
ally. Over the next few weeks, the ureter apparently becomes ies that block the bioactivity of secreted or cell surface pro-
occluded and then recanalizes, the latter event perhaps coin- teins (15–17). Other technologic advances have made it
ciding with urine production from the first metanephric glo- feasible to transfer new genes into the metanephros in vitro
meruli. The embryonic “ascent” of the metanephros from the (18–20). Qiao and Herzlinger used retroviral transduction
level of the sacral segments to the lumbar vertebrae is partly to introduce a reporter gene into renal mesenchymal cells;
associated with ureteric elongation. The urinary bladder they subsequently demonstrated that these precursors not
becomes a recognizable entity by approximately 6 weeks of only formed nephron tubules, as expected, but also differ-
gestation, and the urogenital membrane ruptures at approxi- entiated into a minor proportion of cells within collecting
mately week 7, providing a connection between the bladder duct epithelia (20). Finally, the generation of metanephric
and outside of the body. The allantois, another potential out- cell lines has made it feasible to study the expression of
flow tract on the anterior of the developing bladder, involutes multiple genes in homogeneous populations of precursors
by 12 weeks. Toward the end of the first trimester, the ure- (16) as well as in normal and abnormal differentiation of
teric urothelium assumes a pseudo-multilayered arrange- these cells in response to defined stimuli (21).
ment, and its walls become muscularized; by this time, the
bladder wall has differentiated into circular and longitudinal
smooth muscle fibers which continue to mature in the sec- Functional Studies In Vivo
ond trimester. The murine urinary bladder develops in a sim- In vivo experiments on developing kidneys have used phys-
ilar fashion, in synchrony with the metanephros (11). ical, teratogenic (e.g., chemical), and genetic strategies. For
example, surgical interruption of the avian mesonephric
duct prevents the conversion of intermediate mesoderm
METHODS USED TO STUDY THE BIOLOGY into mesonephric tubules and also prevents the formation
OF NEPHROGENESIS of the metanephros (22). Complete obstruction of the
sheep fetal ureter in midgestation generates hydronephrotic
Descriptive Studies of Gene Expression kidneys, with generation of cysts and disruption of nephro-
genesis resembling human renal dysplasia (23,24). With
Patterns of cell division, death, differentiation, and morpho- regard to teratogenic studies, an example is the generation
genesis can be correlated with changes in spatial and temporal of urinary tract malformations after exposure to ethanol
patterns of gene expression in terms of messenger RNA (25). Welham et al. (8) reported that the imposition of
(mRNA) and protein by using in situ hybridization and mild (9%) dietary protein restriction during rat pregnancy
immunohistochemistry (Figs. 1.2 and 1.5). Recently, “gene reduced numbers of glomeruli per kidney of offspring
chip” technology has been used to study the spectrum of genes when measured postnatally. This was associated with
expressed during murine and human kidney development enhanced apoptotic deletion of renal mesenchymal precur-
(12,13). These observations provide the essential data for the sors at the start of metanephrogenesis (8). In humans, the
generation of hypotheses regarding the molecular control of equivalent developmental time frame is 5 to 7 weeks’ gesta-
nephrogenesis; the hypotheses can be tested by studying the tion, and this might represent a critical window when fetal
effects of diverse interventions during normal development. kidney morphogenesis is susceptible to maternal dietary
These functional experiments can be performed in the intact influences.
animal, in vivo, or “in the test tube,” in vitro. The most powerful in vivo experiments, however, alter
the expression of metanephric molecules by genetic engi-
neering using transgenic animal technology. First, levels of
Functional Studies In Vitro
a specific protein can be increased by inserting a coding
Experiments performed by Grobstein several decades ago DNA sequence, linked to a strong promoter, into the
are classic examples of in vitro studies (14). Grobstein genome of early embryos. This is usually done by microin-
 1. Embryology 7

FIGURE 1.5. Early human metanephros (mt) and the mesonephros (ms). Sections are stained
with antibody to PAX2 transcription factor: Positive nuclei appear black, whereas others are
counterstained with methyl green and appear gray. A: Transverse section of a 5- to 6-week gesta-
tion of human embryo showing, on each side of the embryo, a mesonephros, a metanephros,
and a gonadal ridge (g) (×5). Also shown is the central notochord (n) in a mass of cartilage that
will form the vertebral body, and the coelom (c). B: Enlarged view of A. Note the mesonephros
duct (arrowhead) stains for PAX2, as does the flanking paramesonephric duct (×10). C: High
power of metanephros containing the first branches of the ureteric duct with adjacent mesen-
chymal condensates. The mesonephros duct (arrowhead) is nearby (×20). D: Medulla of an 11-
week human kidney shows a major branch of the fetal ureter (arrowhead) branching to form
collecting ducts: Most nuclei in these structures stain for PAX2 (×20). E: Cortex of an 11-week
human fetal kidney shows presence of a nephrogenic outer cortex with increasingly mature
nephrons and glomeruli (arrowhead) toward the center of the organ. Note that PAX2 is down-
regulated in more mature elements (×20). F: High-powered image of E. Intense staining for PAX2
in the branch tip of a ureteric bud and the flanking renal mesenchymal condensates (×63). (Cour-
tesy of Dr. P. J. D. Winyard, Institute of Child Health, London.)

jection into the male pronucleus of fertilized ova. The phe- genetically engineered in vitro and then incorporated into
notype of such mice illustrates the effects of an excess of a early embryos that develop into chimeric mice. If the
molecule (26). Even more informative is the technique of altered cells contribute to the germline, animals with
homozygous recombination in which the function of a homozygous and heterozygous gene deletions can be gener-
gene can be ablated. Here, mouse embryonic stem cells are ated by further breeding (27). The phenotypes of these null
8 I. Development

TABLE 1.1. NULL MUTANT MICE WITH

RENAL MALFORMATIONS

Transcription factor genes

BF2 (small, fused, and undifferentiated kidneys)
EYA1 (absent kidneys)
EMX2 (absent kidneys)
FOXC1/FOXC2a (duplex and hypoplastic kidneys)
HOXA11/HOXD11a (small or absent kidneys)
LIM1 (absent kidneys)
LMX1B (poorly formed glomeruli)
N-MYC (malformed mesonephric kidneys)
PAX2b (small or absent kidneys)
SALL1 (failure of ureteric bud outgrowth)
WT1b (absent kidneys)
Growth factors and receptor genes
Angiotensin II receptor 1 (poor papillary growth)
AT2 (diverse kidney and lower urinary tract malformations)
EGFR (cystic collecting ducts)
BMP4 (kidney and ureter malformations)
FIGURE 1.6. Growth of mouse embryonic tissues in organ cul- BMP7 (undifferentiated kidneys)
ture. Note branching morphogenesis occurs over 1 week in FGF7 (small kidneys with fewer glomeruli)
organ culture. A–D: The metanephros. E–H: The salivary gland. GDNFb and its receptor, RET (small or absent kidneys)
I–L: The lung. (Modified from Hardman P, Kolatsi M, Winyard NOTCH2 (malformed glomeruli)
PJD, et al. Branching out with the ureteric bud. Exp Nephrol PDGFB chain and its receptor, PDGFRβ (absent mesangial cells)
1994;2:211–219.)
WNT4 (undifferentiated kidneys)
Adhesion molecules and receptor genes
α3 integrin (decreased collecting duct branching)
mutant or “knock-out” mice, which include the complete α8 integrin (impaired ureteric bud branching and nephron
absence of metanephric development, have so far suggested formation)
that approximately 30 genes are essential for normal neph- GPC3 (dysplastic kidneys)
s-laminin/laminin β2 (nephrotic syndrome)
rogenesis in vivo (Table 1.1). Many of these animal models
Other genes
also have defects in nonrenal systems because the same BCL2 (small kidneys)
genes are expressed in, and critical for, the normal develop- COX2 (small kidneys)
ment of organs other than the kidney. Formin (absent kidneys)
Hundreds of molecules are known to be expressed dur- MPV17 (nephrotic syndrome)
Neuronal NOS (bladder outflow impairment)
ing nephrogenesis (28), and tens of these have been consid-
RARαγ/αβ2a (small or absent kidneys)
ered to be functionally important based on organ culture Uroplakin III (hydronephrosis and vesicoureteric reflux)
studies. However, mice with null mutations of the same
genes sometimes have normal kidney development in vivo. Note: Many mutants have aberrations of other organ systems: Please
Hence, we can speculate that numerous metanephric mole- see references for details. Unless otherwise stated, the renal abnor-
cules are of little functional significance or are redundant in malities are detected only in homozygous null mutant animals (i.e.,
both alleles of one gene are nonfunctional).
the intact fetus. It also follows that organ culture must con- aNull mutations of two homologous but separate genes are required

stitute a relatively stressful milieu in which it is compara- to produce the malformation.

bIn these cases, heterozygous null mutations produce kidney malfor-
tively easy to disrupt development by altering levels of a mations that are less severe than the homozygotes. In all other cases,
single molecule. Of note, the genetic background, or strain, heterozygotes appear to have normal nephrogenesis.
of mice with defined mutations can affect the kidney phe-
notype, suggesting the presence of modifying genes (29).
chymal stem cells are believed to reside within this area,
When two structurally similar molecules are expressed at
and such cells are believed to divide to generate a copy of
identical locations in the metanephros, both loci may have
themselves and also another cell. These cells subsequently
to be ablated to generate a renal malformation in vivo
differentiate into nephron epithelia or interstitial cells (21).
(30,31).
More recent evidence, discussed later in this chapter, sug-
gests that cells in the renal mesenchymal compartment can
also differentiate into glomerular capillaries and juxtaglom-
CELL BIOLOGY OF NEPHROGENESIS
erular cells. Stem cells are generally considered to be absent
from the mature kidney; although if they did exist, they
Cell Proliferation and Cell Death
might provide a source of cells for the regeneration of
Proliferation is prominent in the tips of the ureteric bud nephron epithelia after injury.
branches and in the adjacent mesenchymal cells in the Not all cells born in the developing kidney are destined
nephrogenic cortex of the metanephros (32). Renal mesen- to survive the fetal period. In 1926, Kampmeier reported
 1. Embryology 9

that the first layers of metanephric nephrons “disappeared” Therefore, normal nephrogenesis involves a fine balance
before birth (33), a process likely to be associated with the between cell proliferation and death. Excessive proliferation
remodeling of the first divisions of the ureteric bud during is associated with the generation of neoplasms (e.g., Wilms’
formation of the pelvis. More recently, others have tumor) and cysts (e.g., polycystic kidney diseases) (36). Con-
reported the normal occurrence of cell death in the mesen- versely, excessive apoptosis would cause a reduction of kidney
chyme adjacent to primitive nephrons, where it may regu- growth resulting in an organ with fewer nephrons than nor-
late the number of cells in each tubule or the number of mal (e.g., a hypoplastic kidney) or even involution of a meta-
nephrons formed (8). These cells die by apoptosis, a pro- nephric kidney (e.g., some dysplastic kidneys) (37,38).
cess accompanied by nuclear condensation and fragmenta-
tion. These deaths are sometimes called programmed
because they are part of the normal program of develop- Differentiation
ment, and each cell “commits suicide” by an active pro- As individual renal precursor cells become specialized, they
gram of biochemical events, including digestion of undergo differentiation. For example, some renal mesen-
genomic DNA into fragments of approximately 200 nucle- chymal cells differentiate into primitive nephron epithelia,
otides by calcium-dependent endonucleases. Apoptosis also whereas others differentiate into stromal cells or interstitial
occurs in the developing medulla (Fig. 1.7) and it has been fibroblasts (39). Precursor cells later become “terminally
suggested that this process is implicated in the morphogen- differentiated” to enable them to perform specific functions
esis of the thin ascending limb of the loop of Henle (34) of the adult organ. For example, cells within a nephron pre-
and also in deleting excess beta intercalated cells in the col- cursor form the glomerular parietal and visceral epithelia as
lecting duct (35). well as the cells that comprise the proximal tubule and loop
of Henle. The term lineage describes the series of pheno-
types as a precursor differentiates into a mature cell.

Morphogenesis
Morphogenesis describes the developmental process by
which groups of cells acquire complex three-dimensional
shapes. Examples include the formation of nephron tubules
from renal mesenchymal cells and the serial branching of
the ureteric bud to form the collecting duct system. The
process of morphogenesis also occurs during angiogenesis
and vasculogenesis; modes of renal capillary formation is
discussed later in this chapter (9). Angiogenesis also
involves the fundamental cellular process of directional
movement or migration.

MOLECULAR CONTROL OF NEPHROGENESIS

Overview
Three main classes of molecules are expressed during neph-
rogenesis: transcription factors, growth/survival factors, and
adhesion molecules. Note that italics are used when refer-
ring to genomic sequences, whereas regular typescript is
used for gene products (e.g., PAX2 gene and PAX2 mRNA
or protein). The following criteria should be satisfied for a
FIGURE 1.7. Cell death in normal nephrogenesis. A: Apoptotic molecule to be definitively involved in normal nephrogene-
cell death is detected in the outer medulla of the human meta- sis: It must be expressed by the metanephros in an appro-
nephros, as assessed by bright, condensed, propidium-iodide– priate spatial or temporal manner; functional experiments
stained nuclei (between the white bars) in primitive loops of
Henle and other tubules, probably collecting ducts (×63). should demonstrate that its absence perturbs kidney devel-
(Courtesy of Dr. P. J. D. Winyard, Institute of Child Health, Lon- opment in organ culture and in vivo; and the molecule
don.) B,C: Electron microscope images of the medulla of a should have appropriate bioactivity on isolated populations
mouse metanephros to show apoptotic nuclei (curved arrows)
being engulfed by epithelial cells (B) and cells within the inter-
of precursor cells. At present, few molecules have been
stitium (C). shown to fulfill all three criteria.
10 I. Development

Transcription Factors AT, leukemia inhibitory factor, and TNF-α, signal through
different classes of receptors.
Transcription factor proteins bind to DNA and regulate
expression of other genes. Because they can enhance or
switch off the transcription of mRNAs, transcription fac- Adhesion Molecules
tors have been likened to conductors of an orchestra during
The third major class of molecules comprises the adhesion
normal development. These molecules can be classified into
molecules (43). Some mediate the attachment of cells to
families that share similar DNA-binding protein motifs
one another, whereas a second group mediates attachment
and domains. One such motif is called the zinc-finger,
of cells to the surrounding matrix. Examples of the former
which describes a projection of the molecule that interca-
include neural cell adhesion molecule, whose adhesive
lates with DNA. An example of a transcription factor with
properties are independent of calcium, and E-cadherin,
multiple zinc-fingers is the WT1 protein, which is
whose adhesive properties depend on calcium. Molecules in
expressed at the inception of the metanephros (32). Other
the second group include collagen, fibronectin, laminin,
examples of transcription factors expressed during nephro-
nidogen, and tenascin. Many bind to cell surfaces via inte-
genesis include members of the HOX family, which con-
grin receptors to provide a physical framework for epithelial
tain DNA-binding homeodomains, as well as the PAX
tubules and endothelia. Some of these interactions also
family, which contain DNA-binding paired domains (40).
modulate growth and differentiation in an analogous fash-
At present, little is known about the specific targets of
ion to the binding of growth factors to their receptors. Pro-
many of the transcription factors expressed in the develop-
teoglycans, including syndecan and heparan sulfate,
ing kidney.
constitute another type of adhesion molecule. They also
bind growth factors, such as FGFs and VEGF, hence
Growth Factors sequestering and storing these molecules as well as modu-
lating their binding to receptor tyrosine kinases.
The metanephros is rich in growth factors that modulate
cell survival, proliferation, differentiation, and morphogen-
esis (41). Factors that have a positive effect on growth are
CONVERSION OF METANEPHRIC
epidermal growth factor (EGF), transforming growth fac-
MESENCHYME INTO NEPHRON EPITHELIA
tor (TGF) α, fibroblast growth factor (FGF)-2, glial cell
line–derived neurotrophic factor (GDNF), hepatocyte
Uninduced Metanephric Mesenchyme
growth factor (HGF), insulin-like growth factor (IGF)-1
Is Preprogrammed to Form Nephrons
and -2, keratinocyte growth factor (KGF; also called FGF-
7), leukemia inhibitory factor, neurotrophin 3, pleiotro- Isolated metanephric mesenchyme can be induced to form
phin, and TGFβ. Factors that have been reported to have a nephrons in vitro by recombination with the ureteric bud
negative effect on growth include activin, TGFβ, and or by apposition to embryonic spinal cord. However, mes-
tumor necrosis factor-α (TNF-α). In some cases, a single enchyme from other embryonic organs cannot be stimu-
factor can have multiple effects by virtue of binding to lated to produce nephrons by either the ureteric bud or
more than one different receptor; for example, angiotensin heterologous inducers (14). Hence, by the time the meta-
(AT) II generally acts to promote growth through its type 1 nephros can first be detected, the renal mesenchyme has
receptor but also stimulates apoptosis through the AT 2 already been programmed to form nephrons, but it requires
receptor (42). additional, inductive signals from the ureteric bud to per-
When acting on the neighboring cell, growth factors mit its differentiation. The molecules responsible for this
are paracrine factors, but when acting on the producing preprogramming of the renal mesenchyme are currently
cell, they are autocrine factors. Growth factors bind to undefined. The transcription factor LIM1 is expressed in
cell-surface receptors, many of which are receptor tyrosine the intermediate mesoderm before it forms the renal mes-
kinases that, after ligand binding, dimerize and become enchyme, and the metanephros fails to form in mice, which
phosphorylated, thereafter transducing signals into the lack LIM1 (44). However, the embryonic expression of this
cell. Factors acting via receptor tyrosine kinases include gene is widespread, and diverse nonrenal organs are mal-
angiopoietin, EGF, TGFα, FGFs, HGF, IGF-1 and -2, formed in null mutants.
KGF, neurotrophin 3, nerve growth factor, platelet-
derived growth factor A and B chains, and vascular endo-
Uninduced Metanephric Mesenchyme
thelial growth factor (VEGF). Others, including bone
Is Preprogrammed to Die
morphogenetic proteins (BMP) types 4 and 7 and TGFβ,
bind receptors with threonine and serine kinase activity. When cultured in isolation, murine renal mesenchyme fails
GDNF is a distant relative of TGFα but signals through a to survive. In contrast, the recombination with either ureteric
receptor tyrosine kinase after binding to an accessory bud epithelium or embryonic spinal cord rescues mesenchy-
receptor. Yet, other metanephric growth factors, including mal cells from death and induces them to form nephrons.
 1. Embryology 11

Koseki et al. demonstrated that death of the isolated renal

mesenchyme was mediated by apoptosis and that it was an
active process, as assessed by a requirement for mRNA and
protein synthesis (45). They also reported that isolated renal
mesenchyme could be rescued from death by the addition of
EGF (the adult homolog of TGFα) or by phorbol ester (a
chemical that enhances the activity of protein kinase C).
Perantoni et al. reported that FGF-2 could also facilitate sur-
vival of isolated renal mesenchyme (46)—an important
observation considering that the ureteric bud secretes this
factor (47). Barasch et al. (48) have reported that metallopro-
teinase-2 stimulates mesenchymal growth by preventing this
cell population from dying.
The WT1 gene produces multiple transcripts, some of
which act as transcription factors, whereas others are likely
to affect splicing of mRNA before export from the nucleus
(49). WT1 is expressed at low levels in metanephric mesen-
chyme, and levels are upregulated during differentiation
into nephron precursors (32) (Fig. 1.2). In vivo, absence of
WT1 protein causes fulminant death of the intermediate
mesoderm, which would normally form the metanephric
mesenchyme, producing renal agenesis (50). A similar final
phenotype can be generated in mice that are homozygous
null mutants for PAX2, formin, or GDNF/RET. In these
cases, the primary defect is a failure of outgrowth of the
ureteric bud from the mesonephric duct: The defect in the
renal mesenchyme is secondary to the loss of its normal
inducing tissue in vivo. Nishinakamura et al. (51) have
recently reported that mesenchymal expression of the
SALL1 transcription factor is necessary for early inductive
events in the murine kidney; the human homologue is
mutated in the Townes-Brocks syndrome, a disorder associ-
ated with urinary tract malformations.

FIGURE 1.8. Hepatocyte growth factor and its receptor, MET, in

Condensation of Renal Mesenchyme the normal metanephros. A–D: Nine-week gestation of human
metanephros. A: Positive MET staining in ureteric bud branches
The first morphologic step in nephron formation is the (u) and primitive nephrons, such as vesicles (v) and S-shaped bod-
ies (s). Note marked staining on basal surface of primitive epithe-
aggregation of renal mesenchymal cells to form a condensate. lia (arrows). Condensing renal mesenchyme (m) expresses a much
At the same time, these nephrogenic precursor cells undergo lower level of MET in the cytoplasm. B: Similar field as A, after pri-
a burst of proliferation and upregulate the expression of the mary antibody was preabsorbed with MET peptide: no specific
staining. C: MET staining in basal surface of epithelia of Bow-
transcription factors WT1 (32), PAX2 (Fig. 1.5) (32), and man’s capsule (open arrows) of fetal glomeruli (g). Mesangial and
N-MYC. Other genes that are switched on or upregulated endothelial cells in the fetal glomeruli are MET-positive. D: The
include MET (the HGF receptor) (Fig. 1.8) (52), α8β1 inte- stalk of the ureteric bud shows positive MET immunostaining on
the basal epithelial surface (immunostaining appears black). E: By
grin (53), and BCL2 (32). Inhibition of PAX2 by antisense Western blot, HGF protein is detected at 62 kDa in conditioned
oligonucleotides prevents condensation in metanephric media from D4-ras NIH 3T3 cells (positive control) and from a
organ culture (54), and mice lacking one copy of the PAX2 mouse renal mesenchyme cell line (M5) but was not present in
media from a more mature metanephric clone (A1) or in uncondi-
gene are born with small kidneys (27,55). Mice with tioned medium. [Panels A–D from Kolatsi-Joannou M, Moore R,
homozygous mutations of N-MYC have malformed meso- Winyard PJD, et al. Expression of hepatocyte growth factor/scatter
nephric tubules but die before development of the meta- factor and its receptor, MET, suggests roles in human embryonic
organogenesis. Pediatr Res 1997;41:657–665; panel E from Woolf
nephros (56). HGF induces epithelioid characteristics in AS, Kolatsi-Joannou M, Hardman P, et al. Roles of hepatocyte
MET-transfected 3T3 embryonic fibroblasts (57), a model growth factor/scatter factor and the MET receptor in early devel-
for the conversion of mesenchymal into epithelial cells. This opment of the metanephros. J Cell Biol 1995;128:171–184, with
permission.]
growth factor also induces the expression of epithelial mark-
ers in a murine renal mesenchymal cell line (58). Mice defi-
cient in α8 integrin have small, severely malformed kidneys,
12 I. Development

with defective nephron formation and impaired ureteric bud gene encoding this protein develop an infantile nephrotic
branching (53). A ligand for α8β1 integrin dimers is syndrome, despite the presence of grossly normal-appearing
expressed on the surface of ureteric bud branches; therefore, glomeruli at birth (67).
these molecules most likely coordinate morphogenetic inter- Other types of molecules have been implicated in the
actions between the mesenchymal condensate and the ure- growth of primitive nephrons. WNTs are glycoproteins,
teric bud epithelium. BCL2 is located in the nuclear and which are believed to be secreted signaling factors. WNT4,
mitochondrial membranes and prevents apoptosis, perhaps a member of this family, is upregulated in renal mesenchy-
by interfering with lipid peroxidation. Homozygous null mal cells as they differentiate into nephrons and may have
mutant mice have fulminant renal apoptosis during develop- an autocrine role in this process. Mice with WNT4 homo-
ment and are born with small kidneys containing fewer zygous null mutations have metanephroi in which the renal
nephrons than normal (renal hypoplasia) (37,59). mesenchyme is induced but “frozen” at the condensate
Putting the above experiments together, the following sce- stage (68). BMPs are members of the TGFβ superfamily
nario can be envisaged: WT1 appears necessary for the and transduce growth signals through types 1 and 2 recep-
induction and survival of renal mesenchymal cells; PAX2 tor serine/threonine kinases. BMP-7 is expressed by the
and HGF/MET may drive the proliferation of nephron pre- branches of the ureteric bud and is also upregulated in
cursors; BCL2 may protect cells in the condensate stage from primitive nephrons (69,70). Nephrogenesis is impaired in
an untimely programmed death; and α8β1 integrin plays a BMP-7 null mutant mice with formation of only a few
role in aggregation of nephron precursor cells around the nephrons and ureteric bud branches. Recent data suggest
ureteric bud branch tips. The application of either FGF-2 or that leukemia inhibitory factor, a member of the interleu-
lithium ions to uninduced renal mesenchyme in vitro kin-6 family, is secreted by the ureteric bud and can trans-
appears to stimulate differentiation up to but not beyond form renal mesenchyme into epithelia, including proximal
this stage of mesenchymal condensation (46,60). tubules and glomeruli, acting together with FGF-2 and
TGFα (71). Indeed, several growth factors may have a per-
missive effect on epithelial growth at this stage, including
Morphogenesis into Nephron Tubules
HGF, FGF-2, IGF-1, IGF-2, and TGFα (16,71–73).
Next, the mesenchymal condensate forms a lumen and dif- At present, little is known about the molecular controls
ferentiates into an increasingly mature nephron via the vesi- of differentiation of primitive nephrons into the specialized
cle, comma shaped, and S-shaped stages. This process is cells in the mature mammalian nephron. However, this
associated with the replacement of the intermediate filament type of detailed analysis has been performed for cells in the
vimentin by cytokeratin in all segments apart from glomeru- Malpighian excretory tubule of embryonic Drosophila fruit
lar podocytes. In addition, there are profound changes of flies (74). In mammals, there is evidence that expression of
expression of adhesion molecules. The neural cell adhesion WT1 is essential for the maintenance of the mature
molecule becomes downregulated (61), whereas E-cadherin podocyte (32), where it downregulates the transcription of
appears at sites of cell-cell contact (adherens junctions) in the PAX2 (75). In humans, mutation of a single WT1 allele
primitive nephron (17). Numerous studies have implicated causes the Denys Drash syndrome in which nephrotic syn-
the latter molecule as playing a part in the genesis of epithelia drome and structural glomerular abnormalities are key fea-
(62,63). At the same time, the expression of the extracellular tures (76), whereas the transgenic overexpression of PAX2
matrix molecules, collagen I and fibronectin, are downregu- causes epithelial overgrowth and a congenital nephrotic
lated, and primitive tubular epithelia begin to synthesize a syndrome in mice (26).
basement membrane containing collagen IV, laminin, hepa-
ran sulfate, and nidogen (17,43). Evidence from organ cul-
Negative Regulators of Nephrogenesis
ture experiments supports the concept that the interaction of
laminin-1, a cruciform trimeric molecule, with a cell-surface The final form of the kidney is determined by not only
receptor, α6β1 integrin, is essential for lumen formation of growth but also negative processes, such as programmed
the primitive nephron (17,64). Other interactions of lami- cell death and as yet poorly defined mechanisms that termi-
nin-1 with the α-dystroglycan complex (located on the cell nate morphogenesis. In organ culture, TNF-α, a classical
surface) and with nidogen (a mesenchymal-derived matrix inflammatory cytokine, inhibits nephron differentiation
protein) are also critical for epithelial morphogenesis (65,66). and morphogenesis at the stage of mesenchymal condensa-
As the nephron epithelia differentiate further into specific tion (77). This protein is localized at mesenchymal/epithe-
segments, other integrins are expressed: α3 subunit is found lial interfaces and is expressed by renal mesenchymal cells
in glomerular podocytes and binds laminin, and the α2 sub- and possibly also by macrophages that populate the meta-
unit is expressed by distal tubules and collecting ducts and nephros from its inception. Rogers et al. reported that these
interacts with collagen IV and laminin (43). As the glomeru- in vitro actions of TGFβ are similar to those of TNF-α (78).
lar epithelium matures, the basement membrane becomes Another factor that can inhibit nephrogenesis in organ cul-
rich in s-laminin, the β2 chain of laminin. Mice without the ture is activin, a TGFβ-like molecule (79).
 1. Embryology 13

STROMAL CELL LINEAGE (85). When the ureteric bud is cultured as a monolayer, it dies
by apoptosis over a few days, but GDNF can partially reverse
Little is known about the mechanisms that control the differ- this process (85). The factor also stimulates survival and mor-
entiation of renal mesenchyme into stromal cells, or intersti- phogenesis of RET-transfected collecting duct cells (93).
tial fibroblasts (80), but there is evidence that stromal cells Hence, GDNF is a survival factor and morphogen for the ure-
are essential for epithelial development. For example, the teric bud epithelium.
GD3 ganglioside is expressed by stromal cells surrounding the Other metanephric growth factors affect ureteric bud
stalk of the ureteric bud, and antibodies to this molecule pre- growth. HGF induces branching morphogenesis of Madin-
vent bud morphogenesis (81). Metanephric stromal cells also Darby canine kidney collecting duct cells grown in a collagen I
express the BF2 winged-helix transcription factor. Mice that matrix. This model system mimics ureteric bud morphogene-
were homozygous null mutants for this gene have impaired sis (94). HGF is secreted by renal mesenchymal cells, whereas
branching of the collecting ducts and also perturbed conver- its receptor, tyrosine kinase MET, is located on the basal sur-
sion of renal mesenchyme to nephrons (82). Other evidence face of ureteric bud epithelia (16,52) (Fig. 1.8). Functional
emphasizes that metanephric stromal and epithelial cells are studies demonstrate that HGF is required for growth and
involved in complex reciprocal signaling loops, with expres- branching in metanephric organ culture (16,95,96) (Fig. 1.9).
sion of retinoic acid receptors in the stroma, playing impor- Transgenic mice overexpressing HGF commonly develop
tant roles (83). A subset of metanephric stromal cells has renal failure, although details of renal histopathology have not
neuronal characteristics, staining positively with neurofila- yet been reported (97). Mice with homozygous HGF or MET
ment markers (84). The role of these cells is unknown, but null mutations die at embryonic day 13 or 14 with placental,
their survival can be modulated by neurotrophin 3 (84). liver, and muscle pathologies, but early nephrogenesis is sur-
Although fibroblasts in the mature kidney are heterogeneous prisingly normal (98,99). ROS is another receptor tyrosine
(e.g., only some make the hormone erythropoietin), it is not kinase expressed in the ureteric bud lineage (100). Its ligand is
understood how this diversity is generated. unknown but is likely to be a mesenchymally derived factor,
using the GDNF/RET and HGF/MET signaling systems as
paradigms. Antisense oligonucleotides to ROS mRNA cause
impaired growth in organ culture (100), but mice with homo-
BRANCHING MORPHOGENESIS
zygous ROS null mutations have normal nephrogenesis in vivo
OF THE URETERIC BUD
(101). Antisense experiments in metanephric organ culture
have also implicated low-affinity nerve growth factor receptor
Mesenchymal Factors and
in branching (15), but again, mice genetically engineered to
Ureteric Bud Growth
lack this molecule have grossly normal nephrogenesis (102).
When the mouse ureteric bud is grown in organ culture with Thus, it is easy to perturb branching morphogenesis in organ
its adjacent renal mesenchyme, it undergoes branching mor- culture, but the process is more robust when the whole animal
phogenesis; however, it fails to differentiate when cultured in is challenged in vivo.
isolation (14,85). It is established that the epithelia of the ure- EGF and its embryonic homolog, TGFα, bind to the
teric bud express various receptors, mostly tyrosine kinases that EGF receptor. EGF induces limited branching of isolated
transduce differentiation signals by binding to mesenchyme- ureteric buds in collagen I gel (103), whereas blocking anti-
derived growth factors (85,86). The most important signaling sera to TGFα inhibit metanephric development in organ cul-
system involves RET, a receptor tyrosine kinase expressed in ture (73). Mice with homozygous null mutations of EGFR
mesonephric duct, ureteric bud, and its branching tips. have collecting duct dilation and kidney failure, a strain-
GDNF causes tyrosine phosphorylation of RET after binding dependent phenotype, implicating the activity of modifying
to a membrane-linked accessory receptor called GDNF recep- genes (29). An isoform of FGF receptor-2 with high affinity
tor α (87). The ligand is expressed by condensing renal mesen- for KGF (FGF-7) is expressed in mesonephric duct and its
chyme, whereas GDNF receptor α is expressed in the same derivatives (104). Furthermore, administration of KGF
cells as RET (85). Lower levels of the accessory receptor are enhances proliferation of adult urothelial cells in vivo (105),
found in renal mesenchyme, where it may concentrate the whereas the factor modulates ureteric bud morphogenesis in
ligand and also prevent its diffusion with initiation of ectopic organ culture (106). Finally, pleiotrophin, a heparin-binding
ureteric bud branches (85). Mice with homozygous null muta- protein, has recently been identified as a renal mesenchymal-
tions of RET or GDNF do not develop kidneys because of secreted factor involved in ureteric bud branching (107).
deficient outgrowth of the ureteric bud (88–92). Experiments
using culture of whole metanephric rudiments with blocking
Cell Adhesion Molecules and
antibodies to GDNF demonstrate that this signaling system is
Ureteric Bud Growth
critical for stimulation branching after initial outgrowth of the
ureteric bud (85). Equally impressive, the addition of recombi- The stalk of the ureteric bud is surrounded by a basement
nant GDNF to cultured metanephroi induces ectopic ureters membrane composed of laminin-1 and collagen IV, as well
14 I. Development

FIGURE 1.9. Blockade of hepatocyte growth factor in metanephric organ culture. A–C: Mouse
rudiments after 3 days of culture in basal media (A), nonimmune immunoglobulin G (B), and
anti-HGF immunoglobulin G (C). Ureteric bud (u) branching and nephron formations are limited
in organs treated with anti-HGF antibody. D,E: Photomicrographs of sections of a metanephros
grown for 3 days in basal medium (D) or in the presence of anti-HGF antibody (E). Note the con-
densations of mesenchyme (m) around the tips of the ureteric bud in D compared with the cystic
dilation of the bud surrounded by loose mesenchyme with pyknotic nuclei in E. Arrowheads indi-
cate the tips of the ureteric bud, and arrows indicate mesenchyme condensates (D). Bars are 100
μm. Arrowheads in A, C, and E indicate ureteric bud branch tips. (Modified from Woolf AS,
Kolatsi-Joannou M, Hardman P, et al. Roles of hepatocyte growth factor/scatter factor and the
MET receptor in early development of the metanephros. J Cell Biol 1995;128:171–184.)

as nidogen/entactin and tenascin. As assessed by electron ses, such as matrix metalloproteinases, and plasminogen
microscopy, the basement membrane is attenuated around activating proteases, such as urokinase) (109). TGFβ inhib-
the tips of the ureteric bud, and branching epithelia may be its branching and decreases the ratio of matrix-degrading
exposed to a renal mesenchymal matrix rich in collagen I molecules versus others that inhibit this process (e.g., tissue
and fibronectin. There is evidence that matrix molecules inhibitor of metalloprotease-1 and plasminogen activator
affect branching morphogenesis in vitro. In monolayer cul- inhibitor-1) (109). Further evidence that the basement
ture of ureteric bud epithelia, proliferation is enhanced by a membrane plays a critical role in collecting duct morpho-
fibronectin versus laminin substrate (85), consistent with genesis is provided by experiments that disrupt the signal-
the observation that cells at the branching tips have a high ing of extracellular matrix molecules to the cell surface.
proliferation rate (32). Collagen I appears permissive for Mice genetically engineered to lack α3 integrin, which
branching of Madin-Darby canine kidney cells, whereas forms functional dimers with β1 subunits, have a reduced
some components of the basement membrane are inhibi- number of medullary collecting ducts (110). Finally, galec-
tory (108). Using the Madin-Darby canine kidney model, tin-3 is a cell adhesion molecule expressed in the ureteric
HGF-induced branching into collagen I is accompanied by bud lineage, which may regulate the growth of collecting
an increase in matrix degrading molecules (e.g., collagena- duct epithelia by interaction with laminin and other extra-
 1. Embryology 15

cellular matrix molecules (111,112), whereas polycystin 1, sels of the yolk sac, endocardium, and dorsal aorta arise by
the product of a gene mutated in autosomal-dominant vasculogenesis, but thereafter, descriptive studies alone can-
polycystic kidney disease, is a putative cell adhesion mole- not ascertain the origin of embryonic vessels. When avascular
cule expressed in the ureteric bud lineage (113). murine renal mesenchyme is induced to differentiate in
organ culture, the glomeruli that develop lack capillaries as
assessed by light and electron microscopy (121), a result used
Transcription Factors and
to argue against the possibility of glomerular vasculogenesis.
Ureteric Bud Growth
When mouse metanephroi were transplanted onto avian
As yet, little is known about how the expression of trans- chorioallantoic membranes, forming glomeruli acquired cap-
cription factors orchestrates the expression of growth fac- illary loops but these were of host origin as assessed by a
tors and matrix molecules that directly affect the quail-specific nuclear marker and antisera to chick collagen
morphogenesis of the ureteric bud lineage. PAX2 appears IV, a component of endothelial basement membrane
to be particularly important because it is expressed in the (10,122). These results were used to argue that glomerular
mesonephric duct and in the ureteric bud branch tips vessels form by angiogenesis in vivo. However, neither organ
(32,112). The expression of PAX2 in the human ureteric culture nor chorioallantoic membrane is likely to provide a
bud lineage correlates with proliferation, and both are normal environment for growth of glomerular capillaries.
downregulated as the ducts mature (32). In mice geneti- During endothelial differentiation, cells in the lineage
cally engineered to lack PAX2, the ureteric bud fails to express a defined sequence of receptor tyrosine kinases (9):
branch from the mesonephric duct, producing renal agene- VEGF receptor (VEGFR)-2, VEGFR-1, TIE2, and TIE1.
sis (27). Finally, mice lacking the expression of formin show VEGF is expressed at sites of embryonic vessel formation
no outgrowth of the ureteric bud from the mesonephric and is a ligand for VEGFR-1 and VEGFR-2, whereas
duct. The function(s) of this gene, which produces multiple angiopoietin 1 is a ligand for TIE2. VEGFR-1/-2 and TIE1
transcripts, is unclear (114). are expressed by uninduced metanephric mesenchyme,
hence suggesting the presence of endothelial precursors
(123). When a TIE1/LACZ transgenic mouse was used, in
Further Differentiation of Collecting Ducts
which a β-galactosidase reporter gene product can be
As the bud branches, the stems mature into the collecting detected histochemically, endothelial precursors were visu-
ducts, which contain three types of cells: the potassium- alized in intermediate mesoderm condensing around the
handling principal cells and the proton-handling α and β caudal end of the mesonephric duct, with a similar pattern
intercalated cells (115). Corticosteroids enhance collecting noted in renal mesenchyme at the ureteric bud stage
duct differentiation, and there is also some plasticity regard- (123,124). Later in nephrogenesis, TIE1/LACZ was
ing the lineage of these cells based on in vitro studies expressed by all endothelia, including glomerular endothe-
(116,117). During this period of maturation, the Na+-K+– lia. Loughna et al. (123) determined whether these precur-
adenosine triphosphatase becomes relocated from the apical sors could differentiate into endothelia using a model in
to basal plasma membrane, and this process is perturbed in which metanephroi form filtering glomeruli after trans-
some polycystic kidney diseases (118). Other cells that arise plantation into the neonatal nephrogenic cortex (125).
from the ureteric bud differentiate into the pseudostratified When transgenic avascular metanephroi were transplanted
urothelium, which lines the renal calyces, pelvis, and ureter: into wild-type hosts, differentiated donor tissue contained
The KGF receptor is expressed by these cells and the admin- transgene-expressing glomerular arterioles and capillary
istration of KGF enhances urothelial proliferation in vivo loops (123) (Fig. 1.10). Other experiments support the
(106). The human fetal ureter is not patent in early gestation, conclusion that glomerular capillaries originate from endo-
but the mechanisms of its canalization are unknown (119): a thelial precursors present at the inception of nephrogenesis.
failure of this process is believed to occur in human multicys- When wild-type mouse metanephroi were grafted into the
tic dysplastic kidneys (3). AT II, acting through its AT 1 anterior eye chamber of adult ROSA26 transgenic mice, a
receptor, has recently been shown to be important for matu- strain in which the β-galactosidase reporter gene is
ration of the ureter, partly by enhancing peristalsis and partly expressed in all cells, glomerular endothelia were found to
by a direct effect on morphogenesis (120). be of donor origin (126). Moreover, exposure of murine
metanephroi to hypoxia increases endothelial growth in
organ culture, whereas other experiments in mice and rats
FORMATION OF GLOMERULAR CAPILLARIES have demonstrated that addition of VEGF or angiopoietin-
1 enhance the formation of metanephric capillaries in
Embryonic blood vessels arise by vasculogenesis or angiogen- organ culture (127,128). One can speculate that capillary
esis (9). In vasculogenesis, mesenchyme differentiates in situ formation within the metanephros is at least in part driven
to form capillaries. In contrast, angiogenesis involves by hypoxia and that low oxygen tension increases expres-
ingrowth from existing capillaries. The first embryonic ves- sion of diverse vascular growth factors and their receptors.
16 I. Development

MESANGIAL CELL LINEAGE

It has been speculated that mesangial cells arise from the

same lineage as glomerular capillaries because neither cell
forms when the metanephros is cultured under standard
organ culture conditions (121). It was assumed that mesan-
gial cells were derived from cells outside the embryonic kid-
ney. However, mesangial cells develop when metanephroi
are grown in oculo (126), and thus, mesangial precursor
cells may be present in renal mesenchyme. Whatever their
origin, platelet-derived growth factor B and its receptor,
tyrosine kinase platelet-derived growth factor receptor β,
are crucial for the differentiation of mesangial cells in vivo.
Mice with null mutations of either of these genes lack
mesangial cells and develop bizarre, malformed glomeruli
(133,134). Platelet-derived growth factor B is expressed by
primitive nephron epithelia, and mesangial precursors
express the receptor, suggesting a paracrine mode of action.
As mesangial cells mature, both ligand and receptor are
coexpressed, suggesting autocrine activity. Other growth
factor–signaling systems may play roles in development of
this lineage. For example, HGF and MET are coexpressed
by immature mesangial cells (135).

NEURONS IN EARLY NEPHROGENESIS

Grobstein found that when an embryonic spinal cord was

placed on the opposite side of a filter from metanephric
FIGURE 1.10. Transplantation of TIE1/LACZ metanephroi into mesenchyme, nephrons were induced to differentiate (14).
wild-type neonates. A: After 1 week, the E11 transplant differ-
entiated (arrows) in the host cortex (c). Transgene expression Further investigations showed that neurons had penetrated
(black) was confined to the transplant. No staining was detected the mesenchyme through the microscopic pores of the fil-
in host cortex or medulla (m). B: Donor glomeruli expressed the ter, and if the neurons in the spinal cord were destroyed,
transgene intensely. C: Positive capillary loops in glomeruli are
visible. Podocytes (open arrow) do not express the transgene. induction did not occur (136). As assessed by antibodies
Bars: 120 μm in A; 30 μm in B; and 10 μm in C. (From Loughna S, against neurofilaments and neural cell surface gangliosides,
Hardman P, Landels E, et al. A molecular and genetic analysis of neuronal cell bodies can be observed around the ureteric
renal glomerular capillary development. Angiogenesis 1997;1:
84–101, with permission.) bud in vivo, and their terminals surround mesenchymal
condensates (137). These observations support the hypoth-
esis that kidney development may partly depend on inner-
vation. It is also interesting that embryonic neural tissues
Other molecules are implicated in vascular growth are capable of inducing the formation of mesonephric
within the metanephros. These include the Eph/ephrin tubules from intermediate mesoderm (138).
family of membrane receptors, which appear to be critical
in cell-cell recognition (129). Renin is widely expressed in
perivascular cells in the arterial system of the metanephros
HUMAN RENAL MALFORMATIONS:
but becomes restricted to juxtaglomerular cells during
NEPHROGENESIS GONE WRONG
maturation. Recent evidence suggests that the meta-
nephric mesenchyme contains renin-expressing precursor
Overview
cells (130). In addition, other molecules required to gen-
erate bioactive AT are expressed in the metanephros, as Renal malformations are the major causes of end-stage renal
are AT 1 and AT 2 receptors. In rats, there is evidence that failure in children (3–5) (see Chapter 6). The term renal
AT II may enhance glomerular endothelial growth in vivo malformation encompasses a heterogeneous group of devel-
(131,132). This axis is also important in fetal glomerular opmental aberrations. In the most extreme example, renal
hemodynamics, as discussed elsewhere in this text (see agenesis, the kidney is absent. Renal dysplasia describes an
Chapter 2). organ comprised of undifferentiated and metaplastic cells
 1. Embryology 17

ney development is a highly dynamic process of prolifera-

tion, death, morphogenesis, and differentiation controlled
by the temporal and spatial expression of specific genes (dis-
cussed above). It is interesting to reassess multicystic dys-
plastic kidneys with these perspectives in mind. Serial
ultrasound scans performed both prenatally and postnatally
have shown that some of these organs can increase to a mas-
sive size only to subsequently regress and even involute
completely (139). Epithelia lining dysplastic cysts have a
high rate of proliferation and overexpress both PAX2, a
potentially oncogenic transcription factor, and BCL2, a sur-
vival molecule (32). Other evidence also suggests that mes-
enchymal-derived growth factors, such as TGFβ, HGF (Fig.
1.12), and IGFs, are involved in dysplastic epithelial growth
(52,140,141). Conversely, undifferentiated tissues around
cysts have a high rate of apoptosis associated with a lack of
BCL2 expression, and these cells fail to form nephrons (38).
Hence, phases of growth and involution correlate with cel-
lular events and aberrant expression of nephrogenesis genes.
An understanding of the dynamics that underlie these mal-
formations now allows potential therapies, especially the
administration of growth factors that prevent apoptosis and
enhance normal differentiation into nephrons, to be envis-
aged (19). The deregulated expression of genes such as
PAX2, BCL2, and TGFβ, although intriguing, is likely to
be a secondary event. A major question remains: What are
the primary causes of human malformation? To date, there
are at least three answers: teratogens, physical obstruction of
the urinary tract, and sporadic or inherited mutations of
genes expressed in the developing kidney (2,142).
FIGURE 1.11. Histology of a human dysplastic kidney. A: Typical
histology of a human dysplastic kidney. Primitive ducts, believed Teratogens
to be branches of the ureteric bud (u), are surrounded by undif-
ferentiated tissue that superficially resembles normal renal mes- As discussed, animal experiments have implicated a wide
enchyme (m). Note that no nephrons have differentiated from
the mesenchyme compartment. B: For comparison, this plate variety of agents, including vitamin A and ethanol, as renal
shows a normal human metanephros or embryonic kidney at 10 teratogens. In humans, both glucose (i.e., mothers with
weeks after fertilization. Note that there is a superficial similarity diabetes) (143) and AT-converting enzyme inhibitors (used
to the dysplastic kidney depicted in A because both structures
contain primitive ureteric bud ducts and mesenchyme. In the nor- to treat maternal hypertension) are associated with renal
mal embryonic kidney, however, there is evidence of conversion malformations. It is unusual, however, to elicit a history of
of mesenchyme to epithelial nephron precursors (n). (Courtesy of exposure to known teratogens from the parents of children
Dr. P. J. D. Winyard, Institute of Child Health, London.)
with renal malformations, but it remains possible that
occult exposure is important. For example, a recent study
surrounding poorly branched ureteric bud derivatives (Fig. suggested that the incidence of various malformations,
1.11). These organs may be tiny (renal aplasia) or large including those of the genitourinary tract, was increased
enough to distend the abdomen (multicystic dysplastic kid- with a daily intake of vitamin A over 10,000 IU (144).
neys). A hypoplastic kidney is small and has fewer nephrons
than normal. These nephrons may be grossly enlarged in oli-
Physical Obstruction of the
gomeganephronia. Associated malformations of the urinary
Developing Urinary Tract
tract include agenesis, duplications, obstruction, and vesi-
coureteric reflux. It has long been recognized that a significant minority of
kidney malformations in girls and perhaps half of all mal-
formations in boys are associated with obstructed lower uri-
Renal Malformations as Dynamic Processes
nary tracts (3–5). Obstruction during the last third of
The above classification of renal malformations is based on gestation is associated with hydronephrosis, poor renal
pathologic end points. Yet it is now known that normal kid- parenchymal growth, and subcortical cysts, whereas kid-
18 I. Development

lies with dominant inheritance. Polymorphic markers were

used, spaced at 10 centiMorgan (cM) through the genome,
with affecteds-only parametric and nonparametric linkage
analyses. The most positive locus spans 20 cM on chromo-
some 1p13, with a nonparametric lod score of 5.76 (p =
.0002) and a parametric lod score of 3.16. Hence, VUR maps
to chromosome 1, although the gene has yet to be defined.
There was also evidence of genetic heterogeneity, and 12 addi-
tional loci were identified with p <.05. The results support the
hypotheses that VUR is a genetic disorder and offers the future
prospect of genetic screening to replace invasive cystograms for
diagnosis. Other loci, yet to be defined, might be implicated in
the rare families reported with inherited nonsyndromic kidney
aplasia and dysplasia (149,150).

Genetic Causes: Multiorgan

Malformation Syndromes
In some cases, congenital kidney or lower urinary tract disor-
ders occur with a multiorgan malformation syndrome com-
FIGURE 1.12. Hepatocyte growth factor (HGF) and MET immuno-
histochemistry in a dysplastic kidney. A: A dysplastic tubule within a monly affecting central nervous, cardiovascular, and skeletal
postnatal human kidney malformation. HGF immunostaining is systems. For a listing of such syndromes, see Chapter 6 and
localized to the epithelium and also to scattered cells in the sur- reference 151. Although individually rare, malformation syn-
rounding undifferentiated stromal/mesenchymal tissues. B: In a sec-
tion adjacent to that depicted in A, MET immunostaining was dromes collectively account for significant morbidity. Some are
confined to the epithelium. C: Other parts of the same organ con- associated with gross chromosomal anomalies, such as mono-
tained large cysts. Faint HGF/SF immunoreactivity was noted in somies, trisomies, 4p- syndrome, and dup (10p)/del (10q);
three locations: stromal; around the cyst, apparently coating the
lining of the cyst; and in amorphous material inside the cyst. D: In a however, gross cytogenetic defects are absent in most cases of
section adjacent to that depicted in (C), MET immunostaining was children born with syndromic renal tract malformation. Many
prominent in epithelium, lining the cyst (cy). Bar is 50 μm. SF, scat- renal malformation syndromes are inherited in Mendelian pat-
ter factor; dt, dysplastic tubule. (From Kolatsi-Joannou M, Moore R,
Winyard PJD, et al. Expression of hepatocyte growth factor/scatter terns, and in some, specific mutations have been defined
factor and its receptor, MET, suggests roles in human embryonic (Table 1.2) (151). Here, four such syndromes are highlighted.
organogenesis. Pediatr Res 1997;41:657–665, with permission.) The ablation of a single PAX2 allele in mice causes
impaired metanephric growth as well as megaureter, a find-
ing consistent with gross VUR (27,55). These animals are
neys associated with obstruction during early gestation are blind because of maldevelopment of the retina, another site
often dysplastic. Data suggest that experimental obstruc- of embryonic PAX2 expression. There is a human syndrome
tion of murine kidneys in the neonatal period causes that is strikingly similar to these mouse models, namely the
enhanced cell death by apoptosis, as well as aberrant expres- renal-coloboma syndrome. This comprises blindness caused
sion of BCL2, TGF-β1, AT II, and EGF (145). Similar by optic nerve colobomas and renal failure and hypertension
molecules are implicated in the pathogenesis of renal dys- associated with vesicoureteric reflux and small, malformed
plasia, a phenotype that can be generated in animals, such kidneys. This resemblance was noted by a New Zealand–
as sheep, by ureteric obstruction much earlier during devel- based laboratory that described heterozygous mutations of
opment (23,24). All of these molecules have been function- PAX2 in individuals with this syndrome (152). These muta-
ally implicated in metanephric growth in various other tions most likely result in haploinsufficiency (a partial lack of
experiments using organ culture or transgenic mice. functional protein), and they can arise de novo or be inherited
in an autosomal-dominant manner. PAX2 mutations have
also been implicated in human inherited renal hypoplasia
Genetic Causes: Malformations
with a very mild ocular phenotype, emphasizing the utility of
Confined to the Urinary Tract
careful examination of the optic fundus in such cases (153).
Primary vesicoureteric reflux (VUR) affects approximately 1% X-linked Kallmann syndrome is caused by mutations
of white infants, and associated reflux nephropathy causes up of KAL-1 (154,155). Anosmia and hypogonadotrophic
to 15% of end-stage renal failure in children and adults hypogonadism occur in affected male patients because of
(4,146). There is a 20- to 50-fold increased risk of VUR in defective prenatal elongation of axons of olfactory neu-
immediate relatives of probands (147). Feather et al. (148) rons and migration of gonadotrophin-releasing hormone–
reported the first genome-wide search of VUR in seven fami- synthesizing neurons from the nasal placode into the forebrain.
 1. Embryology 19

TABLE 1.2. EXAMPLES OF HUMAN RENAL

MALFORMATION SYNDROMES WITH A
GENETIC BASIS

Associated with syndromes

Apert syndrome (FGFR2 mutation)
Bardet Biedl syndrome (several loci and BBS genes defined)
Beckwith-Wiedemann syndrome (p57KIP2 mutations in a
minority of patients)
Branchio-oto-renal syndrome (EYA1 mutation)
Camptomelic dysplasia (SOX9 mutation)
Denys Drash syndrome (WT1 mutation)
Di George syndrome (locus on 22q11, possibly several genes
mutated)
Glutaric aciduria type II (glutaryl–coenzyme A dehydroge-
nase mutation)
Fanconi anemia (FAA mutation)
Kallmann syndrome (KAL-1 mutation)a
Meckel syndrome (locus on 17q21–q24)
Nail-patella syndrome (LMX1B mutation)
Oral-facial-digital syndrome type 1 (OFD1 mutation)
Renal-coloboma syndrome (PAX2 mutation)a
Renal cysts and diabetes syndrome (HNF1β mutation)a
Simpson-Golabi-Behmel syndrome (GPC3 mutation)a
Townes-Brocks syndrome (SALL1 mutation)
Smith-Lemli-Opitz syndrome [ δ(7)-dehydrocholesterol
reductase mutation]
WAGR syndrome (WT1 and PAX6 contiguous gene defects)
Zellweger syndrome (peroxisomal protein mutation)
Nonsyndromic
Congenital anomalies of the kidney and urinary tract (AT2
polymorphism)
Renal adysplasia (locus not defined)
Primary vesicoureteric reflux (locus on 1p13 but condition
is genetically heterogeneous)a

aConditions discussed in more detail in the current text. For updated

information on the genetics of these and other renal and urinary
tract malformation syndromes, see reference 151.

Moreover, the olfactory bulb fails to grow and is hypoplas-

tic. Approximately 30 to 40% of patients have a solitary
functioning kidney, with presumed renal agenesis (156).
Patients with urinary tract agenesis also lack the vas defe-
rens, a structure derived from the mesonephric duct which
also gives rise to the ureteric bud and its derivatives. Two
children with X-linked Kallmann syndrome and unilateral
multicystic dysplastic kidney have been reported (157):
Perhaps the apparent “agenesis” phenotype seen in older
individuals with the syndrome might be the result of spon-
taneous involution of multicystic organs, as is known to
occur in non-Kallmann patients. In vitro studies demon- FIGURE 1.13. KAL expression in the developing human meta-
strate an adhesive role for the protein coded by KAL-1. In nephros. A: Bright-field photomicrograph of section of the
vivo KAL-1 is expressed in the embryonic human central organ at 11 weeks’ gestation counterstained with hematoxylin
and eosin, showing the cortex (c) and medulla (m). B: In situ
nervous and excretory systems (Fig. 1.13) (155,157). The hybridization with dark field illumination: KAL antisense probe
protein, called anosmin-1, immunolocalizes to the epithelial shows moderate signal over the nephrogenic cortex (arrows)
interstitial matrix and basement membranes of the meso- with a weak signal from deeper tissues. C: Background signal
from KAL sense probe. Bar is 40 μm. (From Duke V, Winyard PJD,
nephric collecting tubules and mesonephric duct and to the Thorogood PV, et al. KAL, a gene mutated in Kallmann’s syn-
first generations of metanephric collecting duct branches of drome, is expressed in the first trimester of human develop-
the ureteric bud (158). It is possible that failure of growth ment. Mol Cell Endocrinol 1995;110:73–79, with permission.)
of either the ureteric bud or its first branches, perhaps due
20 I. Development

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 2

GLOMERULAR CIRCULATION
AND FUNCTION
VALENTINA KON
IEKUNI ICHIKAWA

The essential function of the kidney is to preserve con- Like most organs, the kidneys possess intrinsic autoregu-
stancy of body fluid and electrolytes by removing water and latory mechanisms that adjust local RVR when RPP
potentially harmful metabolic end-products (e.g., uric changes. This autoregulation maintains RBF relatively con-
acids, sulfates, phosphates) while preserving blood pressure stant in the face of changing BP and RPP under physio-
(BP) and essential solutes (e.g., sodium, chloride, bicarbo- logic conditions (2). Many hormonal systems regulate RVR
nate, sugars, amino acids). The process begins in the renal and, hence, RBF. The nature and magnitude of their effects
glomerulus, where plasma is ultrafiltered under pressure are often age specific because anatomic factors (innerva-
through a semipermeable glomerular capillary wall. The tion), presence and distribution of receptor subtypes
ultrafiltration separates plasma water and crystalloids from [angiotensin (AT) II receptors], and post–receptor signaling
blood cells and protein macromolecules, which remain in events change with development (3–6). In addition, auto-
the glomerular circulation. The magnitude of this filtration regulatory efficiency is impaired in several conditions,
process is enormous and requires a high rate of renal blood including extracellular fluid depletion, diuretic exposure,
flow (RBF). Indeed, the entire plasma volume is cycled congestive heart failure, and renal parenchymal damage (7–
through the glomerular system 20 times per hour. The RBF 10). Each of these conditions makes the organism more
and glomerular filtration rate (GFR) are interrelated such susceptible to acute renal failure in the face of relatively
that maintenance of an adequate RBF is crucially impor- minor changes in BP that cause substantial changes in RBF
tant for optimal GFR while the glomerulus is an active par- (see later).
ticipant in determining the RBF.
Development of Renal Blood Flow
Prenatal Renal Blood Flow
RENAL BLOOD FLOW
Fetal RBF is low but increases with gestational age. Dop-
Blood flow to the kidneys comprises 20 to 30% of cardiac pler ultrasound at 25 weeks of gestation shows the RBF to
output (CO) and is determined by two factors: renal perfu- be 20 mL/min, whereas at 40 weeks, the RBF is 60 mL/
sion pressure (RPP), which is approximately equal to the min (11). Within the kidney, the relative perfusion varies
systemic arterial BP, and renal vascular resistance (RVR), with cortical depth, with deeper nephrons of the cortex
which is determined primarily by the afferent and efferent receiving more blood flow than nephrons in the superficial
arterioles. The relationship can be expressed as RBF = BP/ layers (12). This distribution of blood flow parallels mor-
RVR. Although RBF is the parameter usually discussed, it phologic maturation because deeper nephrons are the first
is the renal plasma flow (RPF) that is clinically relevant. to form and mature; superficial nephrons are not com-
Thus, at a given level of RBF, RPF may vary with the vol- pleted until near term (13).
ume of packed red cells. For example, RPF increases with Although the kidneys in the human embryo produce
anemia. Lambs bled to decrease their hematocrit from 33 urine by 12 weeks’ gestation, the role of kidneys in fetal
to 14% double their RPF (1). Because RBF is partly deter- homeostasis is minor compared to the placenta. The per-
mined by the need for oxygen delivery, RPF may be high centage of CO perfusing the kidneys is low during intra-
with severe chronic anemia such as occurs with sickle cell uterine life. During late gestation, the kidneys of fetal
disease. In such circumstances, both the RPF and the GFR lambs receive only approximately 2.5% of the CO whereas
are elevated because of decreased volume of red blood cells. the placenta receives 40%. The kidneys of 10- to 20-week
26 I. Development

human fetuses receive only 3 to 7% of CO (14). Thus, the increases exponentially during fetal life, structural immatu-
clinical relevance of intrauterine RBF and also glomerular rity of resistance vessels (narrower vascular lumina), as well
filtration is less for the clearance of fetal plasma than for the as unique modulation by vasoactive compounds (e.g., AT
formation of urine and, hence, amniotic fluid. II, prostaglandins, kinins, and NO).
Fetal hemodynamics and urine formation are affected by
maternal factors such as the maternal volume status, drugs,
Postnatal Renal Blood Flow
and vasoactive substances that cross the placenta. For exam-
ple, acute oral hydration, which is sufficient to decrease RBF, measured as a clearance of paraamino hippurate
plasma osmolality of healthy pregnant women, increases (CPAH) and corrected for body size, is low in human neo-
fetal urine production in near-term fetuses (15). Furo- nates and correlates with gestational age. For example,
semide given to pregnant women induces diuresis in the CPAH is 10 mL/min/m2 in babies born at 28 weeks and 35
fetus; likewise, maternally administered indomethacin less- mL/min/m2 in those born at 35 weeks of gestation (31).
ens urine production and may lead to glomerular hypofil- After birth, RBF increases steadily, doubling by 2 weeks
tration even after the baby is born (16,17). AT II has and reaching mature levels by 2 years of age (32). The post-
distinct effects on the maternal, ureteroplacental, and fetal natal change in RBF primarily reflects the considerable
hemodynamics. Maternal circulation appears to be most increase in the relative RBF to the outer cortex (33–35).
sensitive to the vasoconstrictive effects of AT II, which RBF is governed by two factors: CO and the ratio of
would tend to preserve ureteroplacental and fetal circula- renal to systemic vascular resistance. After birth, both an
tions (18,19). However, this fetoprotective effect disappears increase in CO and a decrease in RVR favor an increase in
with long-term exposure to elevated levels of AT II. Thus, RBF. Furthermore, RVR decreases much more than sys-
although the first 4 hours of AT II infusion into pregnant temic vascular resistance (12) allowing for a progressive
ewes did not compromise ureteroplacental/fetal perfusion, increase in the renal fraction of CO. For example, RBF
more than 20 hours of heightened AT II caused a dramatic increases 18-fold in newborn pigs during the first 5 weeks
decrease in the placental perfusion and compromised fetal of life, whereas CO (corrected for body surface area)
gas exchange (20). These observations illustrate the impor- increases only 7.2 times during the same period (Fig. 2.1).
tant effect of AT II on the ureteroplacental circulation and, RVR is a function of the arteriolar resistance offered not
by extension, on fetal well-being. AT’s actions to maintain only by the sum of the glomerular vessels, but also by the
BP in utero and at the same time maintain RVR at a high number of existing vascular channels. New nephron forma-
level can be compromised by inhibitors of its actions. AT- tion increases the number of channels and, hence, decreases
converting enzyme inhibitors (ACEIs) (e.g., captopril) lead RVR. New nephron formation contributes to the postnatal
to a decrease in fetal BP, RVR, and GFR in ewes, resulting decrease in RVR and increase in RBF only in premature
in oligoanuria (21) and have been observed to cause anuria infants born before 36 weeks of gestation (13).
in the human fetus and newborn (22,23). These hemody-
namic changes may result from decreased AT II synthesis or
accumulation of bradykinin. AT II receptor antagonism did
not affect BP or RVR in fetal piglets or puppies (24,25),
whereas bradykinin receptor antagonism attenuated renal
vasodilation after AT II receptor antagonism of neonatal
rats (26). These findings indicate a role for vasodilatory role
of kinins offsetting AT II–modulated vasoconstriction in
the maturing kidney and complement the observations that
the developing kidney expresses high levels of immunoreac-
tive bradykinin and receptors (27,28). It is interesting that
the kinin components localize to the deeper parts of the
kidney (29), promoting preferential perfusion of the deeper
rather than superficial nephrons characteristic of the fetal
kidney.
Another vasodilator that likely plays an important role
in fetal renal hemodynamics is nitric oxide (NO) (30).
Basal production of NO in third-trimester fetal sheep
maintains baseline RBF; its inhibition increased RVR by
50% and blocked the increase in GFR and the natriuresis FIGURE 2.1. Renal blood flow as a percentage of cardiac out-
that accompany volume loading. The NO effects may be put, plotted versus age, in growing rats between 17 and 60 days
of age. (From Aperia A, Herin P. Development of glomerular per-
direct or mediated by modulation of AT II actions. Overall, fusion rate and nephron filtration rate in rats 17 to 20 days old.
the low RBF in utero reflects incomplete renal mass that Am J Physiol 1975;228:1319, with permission.)
 2. Glomerular Circulation and Function 27

Other factors that control the postnatal decrease in RVR tubular secretion. Most solutes are freely permeable across
largely affect the resistance of glomerular arterioles. In rats, the glomerular capillary and undergo filtration, followed by
both afferent and efferent arteriolar resistances decrease by a tubular reabsorption or secretion along the various nephron
factor of three between 40 days of life and maturity (36). segments. Renal clearance of a substance X (Cx) is the vol-
This decrease in RVR may be linked to a decrease in vaso- ume of plasma from which X is removed (or “cleared”) by
constrictors or activation of potent vasodilators. Catechola- the kidney within a period of time. Glomerular filtration
mines, but especially components of the renin-angiotensin and tubular secretion facilitate clearance of a solute,
system, are high in the early postnatal period of premature whereas tubular reabsorption impedes it. Clearance is cal-
and term infants (37–41). The role of the renin-angiotensin culated as follows:
system has been extensively studied. Angiotensinogen under-
Cx = (Ux × V)/Px
goes a dramatic postnatal increase in liver expression before
decreasing and settling to adult levels (42). Renin production where Ux and Px are the concentrations of X in urine and
in neonates is robust and expands beyond the juxtaglomeru- plasma, respectively, and V is the urine flow. The units of
lar apparatus to include more proximal segments of the renal clearance are volume per unit time, usually in milliliters per
arterial tree (43). ACE is also abundant. Renal ACE increases minute. For example, if Px = 40 mg/mL, Ux = 80 mg/mL,
postnatally such that within 2 weeks of birth the renal level and V = 100 mL/min, then Cx = 200 mL/min. Clearance is
surpasses the adult level as does the level of circulating ACE a more appropriate concept in describing the renal han-
(44). Both the AT 1 and AT 2 receptor subtypes are expressed dling of a certain substance than is urinary excretion rate
in the neonatal kidney (3,39–46). The AT 2 receptor is (i.e., Ux × V) because clearance takes into account the
believed to play an important role in the apoptotic processes plasma level of X.
during organogenesis, which wanes postnatally (45,47). If a plasma substance is totally excreted on a single pas-
Expression of AT 1 receptors peaks postnatally at twice that sage through the kidneys, it can be used as a marker of RPF.
of adult levels (46). Overall, the substrate, receptors, and the However, in reality, only approximately 92% of the total
enzymes required for production and actions of AT II are RPF passes through the functioning excretory tissue, a frac-
abundantly expressed in the neonatal kidney and contribute tion termed effective RPF. Effective RPF is commonly mea-
to the vasoconstriction of the neonatal kidney. In addition, AT sured as a CPAH, a weak acid that is almost completely
II has been shown to have an important role in the develop- extracted by the renal tubule cells and eliminated in the
ment and function of the renal outflow tract by inducing the urine (55). Measurement of effective RPF as CPAH
development of the renal pelvis and by stimulating the prolif- requires a constant infusion of paraamino hippurate and
eration and differentiation of smooth muscle cells around the multiple plasma and urine specimens. A simplified modifi-
ureters, thereby promoting ureteral peristalsis (48). Absence of cation using a single injection technique, although less
these AT II–mediated effects promotes hydronephrosis. accurate, can be used. The use of CPAH as an estimate of
Counteracting these vasoconstrictive effects are postna- RBF has a major limitation in young infants because the
tal increases in the activities of prostaglandins, NO, and renal tubular extraction of paraamino hippurate is incom-
kinins, which contribute to the maturational increase in plete; it is 65% in infants younger than 3 months of age
RBF. Indomethacin (which inhibits prostaglandin) lowers and reaches adult levels only by 5 months of age (56).
RBF in newborn rabbits (49) and decreases renal function Thus, CPAH underestimates RBF in infants younger than
in infants indicating an important role for vasodilator pros- 5 months of age.
taglandins (49–52). Endothelium-derived NO release by
the renal artery, as well as constitutive NO synthase activity Indirect Assessment of Renal Blood Flow
in the renal microvasculature, increases with fetal and post- Radionuclide markers and radiographic techniques can be
natal maturation of guinea pigs (53). This increase in NO used to assess RBF. Radiopharmaceuticals used in imaging
production is paralleled by increased sensitivity of vascular of the kidneys can provide estimates of RBF or GFR. They
smooth muscle to NO after birth, which contributes to have gained wide use in clinical studies of both children
NO’s modulation of postnatal RBF. As in utero, vasodilators and adults because they do not require biochemical assays.
contribute to the maturational increase in RBF. However, The markers are usually labeled with radioactive iodine or
as in the fetus, it may be linked to the renin-angiotensin technetium. Because of concern that radioactive iodine
system through the AT 2 receptor, a potent stimulator of may accumulate in the thyroid, noniodine radioactive tags
prostaglandin, NO, and kinins (54). are preferred in children. A major value of nuclear methods
is the ability to obtain “split” renal function (i.e., separate
measurements for each kidney).
Measurement of Renal Blood Flow
Radioactive hippuran is another agent used for assessing
Concept of Clearance renal function. It is excreted by glomerular filtration (20%)
Substances reaching the kidney through circulation may and tubular secretion (80%), both governed by RPF. After
undergo glomerular filtration, tubular reabsorption, or intravenous injection, timed images are obtained and a
28 I. Development

computer-generated time activity curve is obtained for the The number of glomeruli in healthy humans has been
region of interest drawn around each kidney. Split renal taken to be, on the average, approximately 1 million in
function is also calculated from the renogram by computer each kidney. However, new observations indicate that
analysis. Other markers include iothalamate (57), ortho- there is considerable interindividual variability in the total
iodohippurate (handled by the kidney in a manner similar to number of nephron units. The final complement of neph-
paraamino hippurate and, hence, a marker of RBF), pen- rons has been shown to be affected by an assortment of
taacetic acid, and dimercaptosuccinic acid (58,59). Although prenatal factors. For example, low birth weight, especially
the risk of radiation injury from most radionuclide agents in fetal growth retardation; protein malnutrition; vitamin A
use is minimal, they are usually reserved for selected cases deficiency; drugs, such as gentamicin, amino-penicillins,
when accurate estimation of renal function is necessary, such cyclosporine A, and glucocorticoids; as well as metabolic
as in the evaluation of a child before urologic surgery (e.g., disorders, such as maternal hyperglycemia, have all been
nephrectomy) or in the follow-up of renal transplantation. shown to cause a significant nephron deficit in the fetus
Doppler ultrasonography is a radiologic method that (74–81). Due to the very large number of individual
assesses blood velocity in the renal vessels. Although limited nephron units contributing to the filtration process and
in its sensitivity, this method can provide screening informa- the capacity to increase filtration within a single glomeru-
tion regarding the patency and flow through the renal vessels lus, it is unlikely that this interindividual variability in
and detect significant arterial stenosis (60). The resistive nephron number impacts whole kidney GFR, although
index is a crude index of the resistance of the kidneys to no studies exist on this possibility. Instead, the relevance
blood flow. It takes into account systolic and diastolic blood of these findings stems from observations that individuals
flow in the renal vessels, as measured by Doppler, and may be who have even a modest decrease in the complement of
helpful in the diagnosis and follow-up of some forms of number of nephrons are at an increased risk of developing
acute renal failure, such as transplant rejection. hypertension and progressive chronic renal dysfunction
(74,82,83).

GLOMERULAR FILTRATION
Theoretic Considerations of
Glomerular Filtration
Whole kidney GFR represents filtration occurring in both
kidneys and is the product of single-nephron glomerular As a filtering structure, the glomerulus is essentially a tuft
filtration rate (SNGFR) and the number of filtering neph- of capillaries, and filtration is transudation of fluid across
rons. Formation of new nephrons (nephrogenesis) occurs the capillary wall into Bowman’s space (Fig. 2.2). Two char-
mainly during intrauterine life and proceeds at different acteristics distinguish glomerular ultrafiltration from trans-
rates in different species. In humans, it is complete by 36 capillary exchange in other organs: (a) The glomerular
weeks’ gestation and proceeds at the same pace when capillary wall exhibits an extraordinarily high net perme-
infants are born prematurely (13). Nephronogenesis con- ability to water and small solutes, with up to 33% of intra-
tinues postnatally in rats until 1 week (33), in dogs until 3 glomerular plasma being filtered; and (b) the glomerulus
weeks (61), and in guinea pigs until 6 weeks of age (62). almost completely excludes plasma proteins the size of
However, regardless of the species, once nephrogenesis is albumin and larger from its filtrate. The filtration rate is
complete, it is not reactivated even in the face of reduction determined by the same Starling forces governing move-
in the functional renal mass (i.e., disease or surgical resec- ment of fluid across other capillary walls (i.e., imbalance
tion). Any increase in GFR after nephrogenesis reflects between transcapillary hydraulic and oncotic pressure dif-
increased filtration of residual nephrons. The degree of this ferences). These can be summarized as follows:
compensatory increase correlates with the magnitude of the
initial loss and is more pronounced in the young (63–72). 1. Mean glomerular transcapillary hydraulic pressure dif-
Apart from lack of new nephron formation after nephron ference [ΔP = (PGC – PBS)]
loss, compensatory renal growth reflecting increased tubule 2. Systemic plasma colloid osmotic pressure (πA)
length and interstitial expansion can start in utero. For 3. Glomerular plasma flow rate (QA)
example, in the model of unilateral obstruction in fetal 4. Glomerular capillary ultrafiltration coefficient (Kf =
lambs at 60 days and contralateral kidney, weight increased k × S)
by 50%, together with an increase in indices of cell prolifer-
ation (hyperplasia); however, there is no increase in glomer- When the individual pressures are expressed as average val-
ular number (66,67). The increase in single nephron ues over the entire length of the capillary, SNGFR is given
function that follows a loss of other nephrons early in life is by the equation:
greater in glomeruli in the outer cortex; however, when loss SNGFR = (k × S) × (ΔP – Δπ)
occurs later in life, the increase is more evenly distributed
among all residual nephrons (73). = Kf × PUF
 2. Glomerular Circulation and Function 29

FIGURE 2.2. Schematic representation of a glomerulus. Blood

enters the glomerular capillaries through the afferent arteriole,
courses through the capillary tuft, and exits through the effer-
ent arteriole. Filtration takes place across the capillary wall into
Bowman’s space. Mesangial cells are strategically located to con-
trol the filtration surface area. The juxtaglomerular apparatus,
one of the sites of glomerulotubular feedback regulation, is
depicted. Terminals from the renal nerve are also shown. (From
the artist, Dr. W. Kriz, with permission.)

where PGC is the glomerular capillary hydraulic pressure and

PBS is the hydraulic pressure in the tubule; ΔP – Δπ are the
mean glomerular transcapillary hydraulic and colloid
osmotic pressure differences, respectively; k is the permeabil-
ity of the glomerular capillary, and S is the total surface area FIGURE 2.3. Schematic portrayals of the process of ultrafiltration
as it proceeds from the afferent (A) to efferent (E) arteriole. A:
available for filtration; and Kf, the glomerular ultrafiltration Glomerulus with reduced mean transcapillary hydraulic pressure
coefficient, is the product of k and S. In this equation, aver- difference (ΔP). B: Increased systematic colloid osmotic pressure
age values are used for ΔP – Δπ because ΔP decreases and Δπ (πA). C: Reduced glomerular plasma flow rate (QA). D: Reduced
ultrafiltration coefficient (Kf). The shaded areas represent normal
increases as the plasma flows from the beginning to the end mean net ultrafiltration pressure (PUF), determined by the normal
of the capillary within a given glomerulus. profiles of hydraulic (ΔP) and oncotic (Δπ) pressure differences. The
altered ΔP profile as a consequence of each of the above changes is
given by an interrupted curve in each panel. Curve 1 in C and curve
3 in D represent conditions of filtration pressure disequilibrium,
Effect of Perturbation in Determinants of whereas curve 3 in C and curve 1 in D represent equilibrium. The
Single-Nephron Glomerular Filtration Rate Starling equation is also given and describes the determinants for
single-nephron glomerular filtration rate (SNGFR).
Mean Glomerular Transcapillary Hydraulic
Pressure Difference
Changes in ΔP seldom play a significant role in altering tration diminishes to zero. Conversely, when an increase in
SNGFR because the autoregulatory mechanism in the affer- BP is extreme, PGC and filtration increase despite a marked
ent arteriole sustains glomerular capillary pressure despite concurrent increase in preglomerular vascular resistance.
large changes in systemic BP (Fig. 2.3). For example, PGC During a decrease (or increase, respectively) in RPP, autoreg-
remained unchanged in Munich-Wistar rats despite a drop ulation of PGC is largely determined by the ability of the
in RPP from 115 to 80 mm Hg induced by aortic constric- afferent arteriole to constrict/dilate and of the efferent arteri-
tion (8). Similarly, GFR in patients with mild to moderate ole to respond conversely. It is important to note that the
hypertension is usually normal (84). As BP rises, spontane- autoregulatory range is not fixed. For example, renal autoreg-
ously hypertensive rats maintain normal PGC by increased ulation is impaired by volume contraction. In volume-
afferent arteriolar resistance (85). However, when BP depleted animals, lowering BP leads to a reduction in PGC
changes outside the autoregulatory range, both PGC and and GFR at a relatively high RPP, whereas little or no change
GFR change accordingly. In circulatory collapse, GFR is occurs in euvolemic animals (8). This effect and low circulat-
severely reduced. Significant reductions in PGC and SNGFR ing volume play significant roles in the reduction in GFR
are noted in rats when BP falls below 80 mm Hg (8). As ΔP that accompanies hypovolemic shock. Renal autoregulation
becomes equal to systemic plasma osmotic pressure (πA), fil- is also readjusted in severe chronic hypertension in which
30 I. Development

acute hypotensive treatment may lead to a decrease in GFR Glomerular Plasma Flow Rate
and a rise in serum creatinine (SCr). Such a phenomenon in
The impact of a change in QA on SNGFR depends on
the setting of chronic hypertension might reflect a structural
whether the other determinants of SNGFR have been concur-
(i.e., not readily reversible) rather than functional narrowing
rently modified (Fig. 2.3). For example, infusion of isooncotic
of arteriolar lumen as a consequence of long-standing eleva-
plasma selectively increases QA, whereas constriction of the
tion in RPP. Although this change may be beneficial in main-
aorta or the renal artery decreases both QA and ΔP (8). Under
taining PGC and GFR in the steady state, it leads to a loss of
certain circumstances, filtration does not occur along the
responsiveness of the renal vasculature and to an acute reduc-
entire length of the glomerular capillary but ceases at some
tion in RPP.
point before its end. This is because plasma oncotic pressure
Changes in ΔP can also occur because of changes in PBS.
increases progressively from the beginning to the end of the
These occur with acute urinary tract obstruction and some
glomerular capillary. This progressive rise in oncotic pressure is
forms of acute renal failure. Early in acute ureteral obstruc-
accelerated when Kf is high or QA is low. Thus, a decrease in
tion, whether partial or complete, SNGFR is well main-
QA results in cessation of glomerular filtration at an earlier por-
tained despite marked elevation in PBS due to compensatory
tion of the glomerular capillary tuft and, hence, a reduction in
increases in both PGC and QA. Twenty-four hours after com-
GFR. Experimental administration of renal vasodilators, such
plete ligation of a ureter, PBS returns to near normal, yet GFR
as prostaglandin E1, acetylcholine, bradykinin, or histamine
remains low as a result of a low QA secondary to vasocon-
causes substantial increase in RBF and RPF in humans or in
striction. In chronic, mild unilateral ureteral obstruction,
animals, but GFR is unaffected. SNGFR remains constant
total kidney GFR, SNGFR, PBS, and QA are nearly the same
because of the opposing influences of an increase in QA and a
as values in unobstructed control kidneys. This preservation
decrease in Kf that occur in response (90). The unexpected
of GFR is a result of the development of two opposing
decrease in Kf could represent a direct action of these sub-
changes in SNGFR determinants: an increase in PGC and a
stances on the glomerular capillary, distinct from their known
reduction in Kf.
dilatory effects. Conversely, vasoconstrictors, such as AT II and
norepinephrine, are capable of producing substantial reduc-
Systemic Plasma Colloid Osmotic Pressure tions in RPF but little resultant change in GFR. Again, this is a
result of a significant compensatory increase in PGC as a conse-
Derived primarily from serum proteins, πA is a function of
quence of the pressor-induced increase in efferent arteriolar
the number of molecules of protein present per unit volume
resistance (91).
of solution. At the same concentration, a small protein (e.g.,
albumin) contributes more to oncotic pressure than does a
large protein (e.g., globulin). An isolated change in systemic
Glomerular Capillary Ultrafiltration Coefficient
plasma protein concentration (CA), and hence in πA, would
theoretically be expected to change SNGFR in an opposite Glomerular capillary ultrafiltration coefficient is the product
direction (Fig. 2.3). However, this does not occur. Acute of the glomerular capillary permeability to water (k) and the
reductions in CA from 5.5 g/dL to 3.5 g/dL, induced by infu- surface area available for filtration (s). Because changes in Kf
sion of colloid-free solutions into rats, did not increase inevitably lead to directionally similar changes in Dp (Fig.
SNGFR because the fall in πA and, hence, the rise in PUF elic- 2.3), changes in Kf, unless extreme, are not expected to cause
ited a decrease in Kf; the opposing influences of an increase in major changes in SNGFR. Nevertheless, a profound fall in Kf
PUF and a decrease in Kf maintained SNGFR nearly constant can affect GFR as demonstrated in rats with various experi-
(34). Whether changes in πA occurring chronically are also mental conditions. Many of these are disease models, such as
accompanied by changes in Kf is unknown. In the experimen- minimal change nephrotic syndrome, acute renal failure,
tal rat model of nephrotic syndrome, Kf decreases signifi- acute and chronic extracellular fluid depletion, and congestive
cantly, accounting for a large decrease in SNFGR (86). This heart failure in which a reduction in Kf is the main factor in
reduction of Kf observed in the nephrotic syndrome may decreasing SNGFR (10,89,91). A variety of hormones and
account for the low or normal GFR rather than higher values vasoactive substances, including antidiuretic hormone, aden-
that would be predicted when CA is low and circulation vol- osine, AT II, endothelin, catecholamines, prostaglandins, ace-
ume is not appreciably affected (87). tylcholine, and histamine, modulate SNGFR by reducing Kf
Hypoproteinemia, in association with severe malnutri- (10,89,92). The mesangial cells are believed to be the main
tion, is often accompanied by a decrease in GFR (88,89). locus of their actions because they appear to have a capillary
This decrease in GFR may be overlooked because SCr may surface area–regulating function. They possess intracellular
not be elevated, consequent to a low muscle mass and creat- contractile myofilaments, bear receptors to vasoactive agents,
inine production. GFR is reduced in protein malnutrition and visibly contract in response to these agents (Fig. 2.4) (93).
even when CA is normal. The reduction in SNGFR is a It is speculated that hormones and vasoactive substances regu-
result of reduced filtering surface area because glomerular late glomerular capillary filtering surface areas, and hence
size is smaller in protein-malnourished animals. GFR, by affecting mesangial contractility.
 2. Glomerular Circulation and Function 31

duced. The mechanisms that maintain GFR stable depend

on adjustments at the glomerular loci, namely the afferent
and efferent arterioles, and likely also in the glomerular
capillary bed itself (Fig. 2.5). Two mechanisms, namely the
myogenic reflex and tubuloglomerular feedback, are impor-
tant for the autoregulation of GFR during changes in BP.
In the young, autoregulation of RBF is maintained through
the same mechanisms but over a lower range of RPP that
reflects the lower prevailing BP in the young (2,24).

Myogenic Reflex
The myogenic reflex describes the theory that an increase in
transmural pressure increases vascular tone. In the renal
circulation, this is particularly important in the afferent
arteriole, which dilates in response to a decrease in RPP.
This dilation also serves to preserve PGC. At the same time,
PGC (and GFR) is also maintained in the adult animal
through stimulation of renin release and the selective vaso-
constrictor effect of AT II on the efferent renal arteriole
(10). This reflex is independent of renal nerves or macula
densa mechanisms and reflects the inherent characteristics
of the vessel (24,95–97). This response has been demon-
strated in isolated perfused renal vessels in which a change
in vasomotor tone occurs in response to changes in the
perfusion pressure in mature animals. However, a defini-
tive role of the myogenic reflex during gestation and early
FIGURE 2.4. Structural expression of a reduction in the glomer- life has not been defined.
ular capillary ultrafiltration coefficient (K f). The top panel repre-
sents the cast of a normal glomerulus; the bottom panel
represents a glomerulus after a stimulus (renal nerve stimulation Tubuloglomerular Feedback
known to induce contraction of mesangial cells and a decrease
in Kf) is applied. Mesangial cell contraction leads to obliteration Constancy of GFR is also determined by the tubuloglomer-
of some of the glomerular capillaries (i.e., anatomic reduction in
the surface area available for filtration), reflected as a decrease ular feedback system, which describes the coupling of the
in the functional parameter Kf. (From Ichikawa I, Kon V. Fed Proc distal nephron flow and SNGFR. In each nephron, the dis-
1983;42:3078, with permission.) tal tubule returns to the parent glomerulus and contributes
to the formation of the macula densa, which consists of
specialized cells of the ascending loop of Henle located
Defense of Glomerular Filtration Rate
between the afferent and efferent arterioles and the glomer-
Nonmammalian vertebrates have effective homeostatic ulus. In this system, the stimulus to adjust SNGFR is
mechanisms to drastically alter GFR, which is critically related to the rate of distal flow and also to the composition
important to maintain hydration in these species (94). of the tubular fluid, particularly the chloride concentration
They can afford to markedly alter GFR because toxic and the tubule fluid osmolality (98–102). The signal is per-
nitrogenous wastes are excreted through nonrenal organs ceived in the macula densa and transmitted to the vascular
such as gills, skin, and cloacae. In contrast, mammals, with structures of the nephron, particularly the afferent arteriole,
their highly and variable fluid intake, have developed a but also to the efferent arteriole and the glomerular capil-
greater capability to conserve and eliminate water from the laries, which in concert adjust the rate of filtration. This
body, largely through an expanded and highly regulated feedback system is well suited to adjust the rate of filtration
reabsorptive capacity of the renal tubules. However, and maintain constancy of salt and water delivery to the
because glomeruli are the only route for elimination of distal nephron where tubular reabsorption is precisely regu-
metabolic wastes and toxins, the GFR in mammals is lated. Thus, an inverse relationship between filtration and
remarkably constant and high relative to other species. tubular flow is established such that a decrease in tubular
Mammals have developed specific mechanisms that main- flow is anticipated to increase the rate of SNGFR and vice
tain GFR stable over a wide range of BP and extracellular versa. The vascular response has been linked to several vaso-
fluid volume, which ensures an effective removal of large active substances, including adenosine, thromboxane A2,
amounts of nitrogenous waste that are constantly pro- endothelin, NO, and particularly AT II (103–106).
32 I. Development

FIGURE 2.5. Mechanisms contributing to the

autoregulatory maintenance of renal blood
flow and glomerular filtration rates (GFR) in the
face of a reduction in renal perfusion pressure.
In the young animal, high baseline levels of
angiotensin II, the inability to maximally acti-
vate the renin-angiotensin system on stimula-
tion under certain circumstances, and low
responsiveness of the vasculature to the con-
strictor action of angiotensin II may limit the
ability to autoregulate GFR. PGC, glomerular cap-
illary hydraulic pressure; QA, glomerular plasma
flow rate; RA, afferent arteriolar resistance; RE,
efferent arteriolar resistance. (From Badr KF,
Ichikawa I. Prerenal failure: a deleterious shift
from renal compensation to decompensation. N
Engl J Med 1988;319:623, with permission.)

The existence of a tubuloglomerular feedback mecha- AT II including efferent arteriolar constriction (3,109).
nism has been established in the superficial nephrons of Glomerular AT II hyporesponsiveness in the neonatal kidney
young (30-day-old) rats (107). Its sensitivity (i.e., the does not appear to reflect inadequate AT 1 receptor density, as
change of SNGFR induced by a given change in tubule kidney AT 1 expression peaks postnatally at twice the adult
flow rate) is maximal around the values of SNGFR and level (46). Further, as noted above, AT II availability is also
tubule flow rate prevailing under normal undisturbed con- maximized, reflecting an abundance of renal angiotensinogen,
ditions. As SNGFR and tubule flow rates increase with renin, and ACE (42–44). The observed hyporesponsiveness
growth, adjustments in the tubuloglomerular feedback of the neonatal kidney, therefore, appears to reflect inadequate
mechanism take place to maintain this relationship, and the postnatal maturation of post-receptor processes. It is possible,
relative sensitivity of the system remains unaltered. however, that the blunted vasoconstriction of the neonatal
As noted earlier, afferent arteriolar dilation and RBF adjust efferent arteriole in response to AT II reflects the vasodilatory
to decreasing RPP in both adults and immature animals. contribution of the AT 2 receptor, which is abundant during
However, one study found that although decreasing the RPP development but wanes with maturation. This may occur
by approximately 30% from baseline was accompanied by a through a direct effect of the AT 2 receptor or through AT 2–
minimal fall in the GFR in adult rats; in young rats, GFR mediated stimulation of NO and bradykinin (54).
plummeted by more than 80% (108). Micropuncture experi-
ments revealed that the profound hypofiltration in the young
rats reflected decreased glomerular capillary pressure. Because Development of Glomerular Filtration Rate
glomerular capillary pressure, in large part, reflects efferent
Prenatal Glomerular Filtration Rate
arteriolar vasoconstriction maintained by AT II, the autoregu-
latory decompensation observed in the young animals likely GFR in the fetus correlates with gestational age and body
reflects incompetence in the AT II–mediated vasoconstriction weight and parallels the increase in renal mass (110,111).
of the efferent vessels. In this connection, a similar degree of There remains controversy as to whether this increase contin-
water deprivation causes a greater increase in the plasma renin ues throughout gestation (112) or plateaus between 28 and 35
activity in adult animals than in immature animals, and a weeks of gestation (113). However, even corrected for body
higher dose of AT II is required in immature animals than in weight, prenatal GFR at every stage of development is much
adult animals to effect a similar increase in glomerular capillary lower than that in adults. For example, creatinine clearance
pressure (108). Taken together, it appears that the young have (CCr) measured within 24 to 40 hours of birth in 30-week pre-
limited ability to activate AT II and that the immature efferent mature infants is less than 10 ml/min/1.73 m2 body surface
arteriole has a limited responsiveness to AT II. Thus, even in area (11); at 34 weeks, it is less than 15 mL/min/1.73 m2,
the face of afferent vasodilation after decreasing RPP, young whereas at 40 weeks, it ranges between 10 and 40 mL/min/
animals develop hypofiltration. These observations provide a 1.73 m2 (Fig. 2.6) (114). Direct measurement of intrauterine
mechanism for dissociation between RBF and the GFR in that glomerular function is obviously limited, and creatinine is not
dilation in the afferent arteriole without sufficient vasocon- an ideal indicator of fetal renal function because it freely
striction in the efferent arteriole is insufficient to maintain a crosses the placenta such that the fetal level actually reflects
transcapillary pressure that promotes glomerular hypofiltration maternal levels. Recently, endogenous low-molecular-weight
in the young (Fig. 2.5). proteins such as β2-microglobulin and cystatin C have been
As noted above, of the two currently recognized receptors shown to be useful in assessing renal function of adults, chil-
for AT II, AT 1 and AT 2, the AT 1 is most abundantly dren, and infants and have been used to assess prenatal renal
expressed and transduces the bulk of the recognized actions of function (115–121). Cordocentesis measurements of β2-
 2. Glomerular Circulation and Function 33

FIGURE 2.7. Glomerular filtration rate (GFR) during the first

year of life. (From Aperia A, Broberger O, Thodenius K, et al.
FIGURE 2.6. Creatinine clearance measured within 24 to 40 Development of renal control of salt and fluid homeostasis dur-
hours of birth in 30-week premature to 40-week full-term ing the first year of life. Acta Paediatr Scand 1975;64:393.)
infants. [From Chevalier RL. Developmental renal physiology of
the low birth weight preterm newborn. J Urol 1996;156(2 Pt
2):714–719, with permission.]
been shown in immature animals (36). Indeed, the BP in
babies born at 28 to 43 weeks of gestation predicted their CCr
microglobulin and cystatin C have generated reference values (33,38). Plasma protein concentration, and therefore the resul-
in fetuses with normal amniotic fluid volume, normal chro- tant πA, is lower in newborns than in older children (5 to 6 g/
mosomes, and absence of sonographic evidence of renal/extra- dL vs. 6 to 8 g/dL) and is a factor that increases ultrafiltration.
renal abnormalities as well as fetuses with abnormalities in However, the maturational increase in πA that hinders ultrafil-
these parameters or postnatal evidence of renal dysfunction tration is offset by a more profound increase in PGC having the
(115). The study found that β2-microglobulin has higher sen- net effect on PUF to promote ultrafiltration. Experimental
sitivity, and cystatin C has higher specificity of predicting studies during later postnatal maturation indicate that πA and
impaired postnatal renal function, suggesting that these mea- PGC are at adult levels and remain constant (36). The further
surements may be a useful adjunct to the analysis of prenatal increase in SNGFR is attributable to an increasing plasma flow
renal/urinary tract abnormalities. rate, QA, that reflects an increasing caliber of afferent and effer-
ent arterioles and decreasing resistances in these arterioles.
Experimental and human observations support the parallel
Postnatal Glomerular Filtration Rate
increase in plasma flow and GFR. Thus, increasing circulating
At birth, the placental function of regulating fetal homeosta- blood volume by delayed clamping of the umbilical cord or
sis becomes shifted to the kidneys. Compared to the adult, intravenous fluid infusion increases inulin clearance
the GFR of a term newborn baby is less than 10% of the (128,129). Finally, rising hydraulic conductivity as well as sur-
adult level whether expressed per gram of kidney weight, face area of the glomerular capillaries likely contributes to mat-
body weight, or surface area and correlates closely with the urational increase in the capillary ultrafiltration coefficient, Kf.
gestational age (Figs. 2.6 and 2.7) (122,123). However, dur- Glomerular size, glomerular capillary basement membrane
ing the first 2 weeks of life, the GFR doubles and continues surface area, and capillary permeability to macromolecules all
to increase, reaching adult levels by 2 years of age (112,123). increase from neonatal period to adulthood (130–132). How-
This increase lags in premature babies (124). As with RBF, ever, neither human nor animal data can provide the precise
development of GFR proceeds centrifugally, and the matura- contribution of such changes to the increasing GFR.
tional increase in whole kidney GFR reflects primarily an
increase in the SNGFR within superficial nephrons and less
CLINICAL ASSESSMENT OF GLOMERULAR
in the juxtamedullary nephrons (33,125,126).
FILTRATION RATE
All four determinants of SNGFR—ΔP, πA, QA, and Kf —
contribute to the maturational increase in GFR to varying
Inulin Clearance
degrees. In early stages, the systemic BP in humans averages 40
to 70 mm Hg, which is below the autoregulatory range Assessment of GFR is the single most important measure-
(126,127) and likely contributes to a low PGC and DP that has ment of renal function. Substances reaching the kidney
34 I. Development

may undergo one of several processes, including glomerular Creatinine as a Marker of Glomerular
filtration, tubule reabsorption, tubule secretion, and intra- Filtration Rate
renal metabolism. These considerations necessitated the
In clinical practice, GFR is most often estimated from mea-
search for an “ideal GFR marker.” Among various sub-
surements of SCr concentrations and the clearance of endoge-
stances considered, inulin emerged and has remained the
nously produced creatinine. These require only collections of
standard against which all other techniques of measuring
urine or blood samples. Most commonly, renal function is esti-
GFR are compared to validate their accuracy (133). Inulin
mated simply by obtaining an SCr measurement as the initial
is a polymer of fructose, containing, on average, 32 fructose
screening assessment and in monitoring the increase as an esti-
residues and has a molecular weight of approximately 5700
mate of the rate of deterioration of renal function. In the steady
daltons. Natural inulin is derived from plant tubers such as
state, UCr × V (creatinine excretion rate) equals creatinine pro-
dahlias, chicory, and Jerusalem artichokes. Although the
duction rate, which is constant. GFR is inversely proportional
molecular configuration of inulin varies depending on the
to plasma creatinine concentration because GFR = (UCr × V)/
source, the Stokes-Einstein radius that affects filtration is
PCr. Thus, an increase in plasma creatinine from 1 to 2 mg/dL
constant at approximately 5.0 nm. Inulin fulfills the follow-
or from 4 to 8 mg/dL represents a functional loss of 50% of
ing criteria:
GFR between measurements, although the absolute decline in
1. It is freely and completely filterable at the glomerulus. function is less in the latter as is the fractional residual function.
2. It is neither secreted nor reabsorbed by tubules. The utility of SCr measurement stems from the relatively con-
3. It is neither metabolized nor synthesized by the kidney. stant production of daily creatinine and is independent of diet,
4. It is not bound to plasma proteins, or if it is, the free protein catabolism, and physical activity. Creatinine produc-
unbound as well as the bound components can be tion is a function of muscle mass. Approximately 1 g creatinine
measured separately. is derived from 20 kg muscle mass in 1 day (i.e., 50 mg/kg
5. It is physiologically inert. muscle) (134). In individuals of average proportions, creatinine
production is 15 to 20 mg/kg/day in boys and 10 to 15 mg/kg/
Because of these characteristics of inulin, the rate of inulin day in girls and infants. This is the amount that is expected to
filtered into Bowman’s space equals the urinary excretion of be found in a complete 24-hour urine collection. Thus, if GFR
inulin. Moreover, inulin concentration in Bowman’s space completely ceases (e.g., acute renal failure), the plasma creati-
equals that of plasma. Thus, the flow rate of the fluid fil- nine concentration is expected to increase by approximately
tered into Bowman’s space is 1.5 mg/mL/24 hr.
GFR = CIn = UIn × V/PIn CCr is a well-accepted estimate of GFR (as measured by CIn)
in patients who are older than 1 month of age (135) who have
where CIn is the clearance of inulin; UIn and PIn are the normal or have only moderately decreased renal function.
inulin concentrations in the urine and plasma, respec- However, in some settings, CCr is an unreliable estimate of
tively; and V is the urinary flow rate. For example, if PIn GFR. CCr overestimates GFR with severely decreased renal
= 0.5 mg/mL, UIn = 50 mg/mL, and V = 1.1 mL/min, function; when GFR falls below 20 mL/min/1.73 m2, CCr
then GFR = CIn = 110 mL/min. Although this is a overestimates CIn by approximately 20%. CCr has been shown
straightforward relationship, there are several points to overestimate CIn in adults with nephrotic syndrome (136)
worth emphasizing. It is plasma, not urine, that is being and in kidney transplant donors and recipients (137,138).
cleared of inulin. In the above example, all inulin is Both CCr and CIn underestimate GFR in some forms of acute
removed from 110 mL of plasma each minute. The inu- renal failure in which the functional integrity of the renal
lin clearance is independent of the rate of urinary flow tubule is disrupted. Under these conditions, filtered creatinine
rate. Thus, the concentration of inulin in the urine and inulin can be reabsorbed by passive back diffusion. In
increases as the volume decreases and vice versa at a acute tubular necrosis after cardiac surgery, 50% of filtered inu-
given GFR. The inulin clearance is also independent of lin undergoes tubular back leak (139). Thus, measurements of
the concentration of inulin in the plasma; thus, as GFR in the setting of acute renal failure with tubule injury are
plasma inulin concentration increases, its appearance in invalid. Notably, although the value of GFR is relatively pre-
the urine increases as more is filtered. Although inulin served in this setting, the clearance of creatinine, inulin, and
clearance remains the most accurate method of assessing the toxic metabolic wastes represented by these markers is com-
GFR, it is cumbersome in routine clinical settings. The promised. In practice, therefore, changes in GFR are reflected
drawbacks include difficulty in obtaining the inulin, the by changes in SCr with sufficient accuracy to follow the course
preparation of the inulin, and requirement for continu- of patients with acute renal failure.
ous intravenous infusion to maintain constancy in its Values for SCr in human newborns are somewhat higher
plasma concentration. Moreover, measurements of inu- than the maternal SCr level, reflecting a gradient that allows
lin levels are not routinely available in hospital clinical fetal creatinine to be cleared largely by the maternal kidneys.
laboratories. These drawbacks have led to the develop- Over the first few days of life, SCr decreases and represents
ment of other methods to estimate GFR. GFR maturation, which eliminates both the maternal creati-
 2. Glomerular Circulation and Function 35

FIGURE 2.8. Plasma creatinine values during

the first 3 months of life in low-birth-weight
infants (less than 2000 g). (From Stonestreet BS,
Oh W. Plasma creatinine levels in low-birth-
weight infants during the first three months of
life. Pediatrics 1978;61:788, with permission.)

nine and the newborn’s endogenous creatinine production ligrams per deciliter; and δ, a constant of proportionality, is
(Fig. 2.8). In normal premature infants, SCr values in the first age and sex dependent. Although this formula is useful in
10 days of life range between 0.1 and 1.8 mg/dL with a mean day-to-day management, a more precise measurement of
of 1.3. During the first month of life, SCr declines gradually GFR (using CIn or iothalamate clearance) should be obtained
and becomes less than 1 mg/dL after 1 month of age (140). whenever a high accuracy is desirable. New equations have
Low-birth-weight infants have daily urinary creatinine excre- been developed from the Modification of Diet in Renal Dis-
tion rates during the first 2 weeks of life that correlate with ease study that more accurately estimate GFR in adults than
birth weight, gestational age, and body length (141). SCr con- GFR measurements obtained by CCr (144). The equations
centration in normal infants and children increases with age take into account the individual’s age, gender, race, and body
and is slightly higher at any age in male patients than in female size; however, the utility of these equations have not been
patients (Fig. 2.9) (142). Estimation of GFR by CCr entails tested in children.
obtaining an accurately timed urine collection over a long
period, ideally two 24-hour periods (see above). Investigators
Modifications of the Standard
have attempted to derive formulas to estimate CCr from SCr
Clearance Method
level, in conjunction with anthropometric measurements such
as height, weight, or body surface area. The following formula, Because of the difficulty in maintaining intravenous access
derived by Schwartz and others, yields values of GFR that cor- and obtaining urine samples in newborn infants and young
relate with those obtained from CCr and CIn (143): children, various modifications of the standard clearance
tests have been used to yield indirect assessments of GFR.
GFR = δL/SCr
One modification uses an intravenous infusion of a GFR
where GFR is expressed in milliliters per minute per 1.73 marker but does not require urine collection. Because at
m2; L represents body length in centimeters; SCr is SCr in mil- steady state the amount of marker infused per unit time (I)

FIGURE 2.9. Mean plasma creatinine concen-

tration (mg/dL) plotted against age for both
sexes. The regression equations are for males: y
= 0.35 + 0.025 × age, and for females: y = 0.37 +
0.018 × age. (From Schwartz GJ, et al. Plasma
creatinine and urea concentration in children:
normal values for age and sex. J Pediatr
1976;88:828, with permission.)
36 I. Development

equals the amount excreted in the urine, as well as the infants with gestational age ranging from 24 to 36 weeks
amount filtered, (154). The data are shown in Figure 2.10. The data obtained
in this study reiterate the high creatinine values at birth and
Ux × V = I
during the first week of life (see above) and the fall that
= GFR × Px
occurs over the first month. Creatinine levels then remain
hence, GFR = I/Px
constant until 2 years of age when they rise to adolescent val-
Thus, only blood sampling is required, together with accu- ues. The authors emphasize that, by contrast, cystatin C val-
rate knowledge of infusion parameters (infusion rate and ues more closely parallel functional clearance studies. Thus,
marker concentration) and certainty that a steady state has premature babies have the highest levels of cystatin C (0.43
been attained. The method uses unlabeled iothalamate to 2.77 mg/L; mean, 1.56), followed by infants less than 1
infused subcutaneously via a portable minipump (145). year of age reflecting kidney immaturity (0.59 to 1.97 mg/L;
After steady state (8 to 24 hours), the marker clearance can mean, 1.20). By 1 year of age, the range 0.50 to 1.27 mg/L
be calculated, assisted by a computer program to correct for (mean, 0.82) approximates levels found in adults of 0.51 to
the child’s age, gender, and body size. Some advantages of 0.98 mg/L and indicates essentially constant cystatin C con-
this method include its suitability for use in infants, in centration beyond 1 year of age. Recent studies also find that
terms of its accuracy, simplicity, safety, and low cost. cystatin C may be a more accurate serum marker than creati-
Clearance of a marker has also been measured by a single nine in individuals with impaired renal function (155–157).
injection of that marker, followed by analysis of its disap- Finally, even fetal serum concentrations of cystatin C appear
pearance rate as assessed from repeated plasma samples. to be useful predictors of postnatal renal function suggesting
This method bypasses the necessity of continuous intrave- that this may well become a more universal marker of glo-
nous infusion and urine collection. A dose of inulin or merular function (115).
polyfructosan (0.5 mL/kg of a 25% solution) is given intra-
venously, and seven or eight capillary blood samples are col-
lected at 5, 10, 15, and 20 minutes, and then after 10- to GLOMERULAR SIEVING OF
15-minute intervals; clearance of the marker is calculated MACROMOLECULES
using a mathematic model (146,147). Radiolabeled mark-
ers such as chromium-51–ethylene diameinetetraacetic acid The enormity in the quantity of filtration generated by the
(147) or iodine-125–iothalamate (57) have been used glomerulus underscores specific features of the glomerular
because they do not require biochemical assays. However, capillary bed that allows high permeability to water and
they remain costly and are still reserved for selected cases small molecules while at the same time providing efficient
when accurate estimation of renal function is necessary. selectivity that bars cells, proteins larger than albumin, and
charged molecules (158,159). This barrier function of the
glomerular capillaries is influenced by the size, shape, and
Cystatin C as a Marker of Glomerular
charge of the macromolecules. Micropuncture studies and
Filtration Rate
urinary clearance analyses that compare concentration of a
Although inulin clearance remains the gold standard and SCr given macromolecule in Bowman’s space/urine to plasma
concentration and CCr are currently most widely used to esti- have been used to obtain the glomerular sieving coefficient
mate GFR, a new endogenous marker has emerged that may for a variety of macromolecules. Sieving coefficients are
obviate some of the limitations of the traditional methodolo- inversely correlated with the effective radius of the macro-
gies. Cystatin C is a 13.6 kDa protease inhibitor constitu- molecules. Thus, clearance of the larger proteins, such as
tively synthesized by all nucleated cells that is freely filtered albumin and globulin, is markedly less than that of smaller
through the glomerulus and essentially completely reab- proteins such as monomeric immunoglobulin light chains
sorbed and catabolized by tubular cells. Its measurement can (160,161). Molecules without charge, including dextran
now be performed by readily available immunoassay and polyvinylpyrrolidone, that are neither reabsorbed nor
(118,148–151). Studies in adults and children, including secreted (unlike proteins), have been used extensively to
premature babies, show that serum concentrations are not study glomerular capillary size selectivity both in experi-
affected by gender, height, or muscle mass. It is important to mental settings and in human diseases (160–170). Greater
note that the assay is also free from analytical interference restriction of anionic than of neutral or cationic molecules
from bilirubin and hemoglobin, which represent consider- suggests an electrostatic barrier that is charge selective. The
able confounders of SCr in newborns with jaundice or hemol- observation that for a given chromatographic radius and
ysis from difficult blood draws. charge density, the protein sieving coefficient is smaller
A reference range has been established for adults (152– than that of neutral dextrans suggests that the glomerular
154). Recently, a pediatric reference range for cystatin C capillary barrier is also shape selective. Thus, proteins are
together with levels of SCr was published based on measure- believed to behave as rigid spheres, whereas dextrans are
ments in 291 children aged 1 day to 17 years, including more compliant so as to have a smaller effective radii (161).
 2. Glomerular Circulation and Function 37

FIGURE 2.10. Range for cystatin C (A) and creati-

nine (B) measured in 291 children aged 1 day to 17
years. [From Finney H, Newman DJ, Thakkar H, et al.
Reference ranges for plasma cystatin C and creati-
nine measurements in premature infants, neonates,
and older children. Arch Dis Child 2000;82(1):71–75,
with permission.]

It appears that each of the three major components of the passage. The composition of the GBM includes type IV col-
glomerular capillary wall (endothelial cells, basement mem- lagen, laminin, and proteoglycans that provide size and charge-
brane, and epithelial cells with their podocytes and slit dia- selective restriction to glomerular filtration. Type IV collagen is
phragms) provide impedance to macromolecular filtration a trimeric basement membrane specific protein that represents
(Fig. 2.11). Both endothelial cells and epithelial cells are six genetically distinct forms that assemble in specific combi-
important in this barrier function as evidenced by the observa- nations in utero and in different combinations postnatally
tion that permeability of the isolated glomerular basement (172). Defects in adult collagen IV lead to distortion of the
membrane (GBM) is much higher than in the intact glomeruli GBM that characterizes Alport’s syndrome (172). Laminin
(171). Indeed, native anionic ferritin particles accumulate in plays a role in basement membrane structure, cellular differen-
the endothelial fenestrae and in the lamina rara externa of the tiation, and adhesion. Laminin also provides sieving imped-
basement membrane. The GBM is increasingly being recog- ance, as mice deficient in laminin-2 chain develop nephrotic
nized as providing an important barrier to macromolecular syndrome (173). Perlecan and agrin are the major proteogly-
38 I. Development

FIGURE 2.11. Molecular model of the glomerular filtration bar-

rier. The three layers of the barrier are the fenestrated endothe-
lial cells lining the inside of the glomerular capillary, the
glomerular basement membrane (GBM), and the podocyte foot
processes with their intervening slit diaphragm. CD2AP, CD2-
associated protein; ZO-1, zonula occludens-1. (From Tryggvason
K, Wartiovaara J. Molecular basis of glomerular permselectivity.
Curr Opin Nephrol Hypertens 2001;10:543, with permission.)

cans in the GBM that contain high levels of negatively charged FIGURE 2.12. Schematic representation of the relationship
heparan sulfate moieties (174–176). Overall, it is currently between glomerular capillary pressure (P GC), functional pores
in the glomerular capillary wall, and glomerular macromolecular
believed that the endothelial cells together with the basement sieving. An increase in PGC, as induced by angiotensin II, increases
membrane components provide electrostatic impedance for nonselective pores on the capillary wall allowing the bulk of
negatively charged components of glomerular filtrate. macromolecules to escape into Bowman’s space and then the
urine. When the high PGC is attenuated by saralasin, an angioten-
The epithelial cell layer of the glomerular capillary network sin II antagonist, the abnormal presence of large nonselective
is the site of restrictive size selection of macromolecular filtra- pores and the sieving defect are largely corrected. (From Ichikawa
tion. Indeed, the epithelial cells appear to provide the most sig- I, Harris RC. Angiotensin actions in the kidney: renewed insight
into the old hormone. Kidney Int 1991;40:586, with permission.)
nificant element in glomerular permselectivity, and loss of
epithelial podocytes has been shown to parallel the degree of
proteinuria in patients with immunoglobulin A and diabetic
nephropathies (177,178). Significant proteinuria is typically vidual determinants of SNGFR also impact filtration of
accompanied by dissolution of the normal interdigitation of macromolecules (Fig. 2.12). Glomerular capillary flow rate,
epithelial podocytes. Notably, infusion of exogenous polyca- but particularly the glomerular capillary pressure, modu-
tions, which cause proteinuria, also produce this podocyte lates membrane pore structure such that an increase in glo-
lesion (179). Recent studies have clarified the molecular struc- merular pressure augments proteinuria. The mechanism for
ture and functional implications of the epithelial podocyte slit this effect has been attributed to AT II–induced pro-
diaphragms. Positional cloning has identified the gene teinuria. Thus, infusion of AT II, or endogenous stimula-
mutated in congenital nephrotic syndrome of the Finnish tion of AT II activity, increases the fractional excretion of
type. The NPHS1 gene product, nephrin, is a transmembrane protein, whereas decreasing the pressure has the opposite
glycoprotein similar to immunoglobulin-like cell adhesion effect (186–188). These studies underscore that glomerular
molecules, and nephrin is located in the podocyte slit dia- hemodynamic changes, through increasing the large nonse-
phragms such that two adjacent molecules form the porous lective pores in the glomerular membrane, can allow mac-
filter (180–184). Intracellularly, nephrin interacts with CD2- romolecules to escape into the urinary space (Fig. 2.12). It
associated protein as well as other proteins such as podocin is of interest that acute exercise-induced proteinuria, in
and zonula occludens-1 that connect with actin (185). Mice which the sieving defect is believed to be linked to
deficient in nephrin or CD2-associated protein have massive increased intraglomerular pressure, is lessened by pretreat-
proteinuria and early death (173,181). These studies under- ment with ACEI (189). Conversely, antagonism of AT II
score the recent appreciation of the pivotal role of the actions by ACEI or AT II receptor antagonist acutely less-
podocyte and slit diaphragm in the glomerular filtration bar- ens proteinuria and is well documented to decrease protein
rier function. excretion in many different chronic settings (190–196).
In addition to the structural characteristics of the capil- The mechanism for this antiproteinuric effect is in part
lary wall components that determine its permeability, indi- related to decreased efferent arteriolar resistance and there-
 2. Glomerular Circulation and Function 39

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 3

TUBULAR FUNCTION
DEBORAH P. JONES
RUSSELL W. CHESNEY

ANATOMIC ORGANIZATION ularly electrolytes, and water. This fine-tuning preserves

ECF and whole-body composition within the narrow mar-
The renal tubule forms an epithelial surface that, through gins we describe as normal and buffers change in ECF com-
the processes of reabsorption and secretion, adjusts glomer- position through disease.
ular filtrate so that excretion balances input and body com-
position is kept close to normal. In particular, this function
Developmental Pattern
maintains extracellular fluid (ECF) volume and electrolyte
and acid-base homeostasis, maintains substrate levels, and At birth, the infant is transformed from an organism
excretes a wide variety of organic solutes, including drugs. dependent on the maternal placenta to an independent
The renal tubule is divided into 12 segments; each has organism that relies largely on renal function to maintain
distinct morphologic and functional characteristics. The homeostasis. From midgestation, the kidney develops rap-
differences that set apart each segment result from the idly so as to be able to fulfill its function at birth. Nephro-
changes in distribution of transport systems on the apical genesis is complete by 36 weeks’ gestation; tubular
and basolateral surface of the epithelial cells of each of these maturation begins early in the second trimester, is very
segments and to the anatomic arrangement in which cells active during weeks 32 to 36 of gestation, and continues
from one segment influence the environment and function well into postnatal life (2,3). Factors that affect this matura-
of another segment. The membrane properties of segments tion and transport capacity include the density, structure,
change as the tubule progresses from its proximal origin to and turnover of specific transporter proteins, in particular,
its distal terminus. A series of at least 16 different tight the activity of Na+-K+-ATPase; the electrochemical gradient
junction proteins, termed claudins, are found in segment- across the cell membrane; hormone responsiveness; mem-
specific expression patterns along the nephron in each of brane surface area, phospholipid content, and fluidity; and
these 12 segments (1). Changes in the volume and compo- cell metabolic activity (3).
sition of peritubular capillary fluid and of tubular luminal This chapter reviews the transport functions of the indi-
fluid also influence these transport processes. vidual classes of solutes that are transferred, beginning with
Reabsorption is the net transfer of solutes from luminal those specific to the proximal segments and proceeding to
fluid to pericapillary fluid; secretion is the reverse. The those transferred along the 12 segments of the nephron
transfer may be transcellular across apical and basolateral from origin to terminus. In each case, we consider overall
membranes or paracellular across epithelial tight junctions tubular handling and the cellular mechanisms and regula-
into intercellular spaces. In many cases, it is bidirectional, tory systems involved in solute transfers. We also describe
and the net transfer may change from reabsorptive to secre- developmental features affecting each class of solutes.
tory as conditions dictate. Before describing the transport mechanisms for each
Some solutes, particularly organic substrates and phos- class of solute, we note that three levels of active transport
phates, are reabsorbed almost exclusively in the proximal operate along the nephron. The primary active transport
convolution. Electrolytes, including hydrogen ions, are systems are those in which energy derived from adenosine
transferred by segments along the entire nephron, some- triphosphate (ATP) hydrolysis is used. The major compo-
times being reabsorbed and then secreted, or the reverse. nent of this primary system is Na+-K+-ATPase. Others
Often, there are different transport systems for the same include H+-ATPase and Ca2+-ATPase. Secondary active
solute, such as glucose or organic acids. The proximal seg- transporters are those in which sodium transport is coupled
ments reabsorb the bulk of glomerular filtrate. The distal to the antiport or cotransport of another solute. These
segments effect more precise adjustments of solutes, partic- include Na+-H+ exchange; the Na+-K+-2Cl– cotransporter;
46 I. Development

FIGURE 3.1. A: Model of Na+-K+–adenosine triphosphatase (ATPase) and biochemical reactions

involved in the sodium-driven conformational changes of the α subunit. The 3 Na+ bind to E1-ATP
to form phosphorylated E1-P, which releases the 3 Na+ at the outer surface and undergoes a con-
formational change to the E1 form, then binds to 2 K+ and is dephosphorylated at the inner sur-
face, where it binds to ATP and releases the 2 K +. The intermediate pathway represents reactions
in the absence of extracellular K+. B: Model of the cellular orientation of the Na +-ATPase. ADP,
adenosine diphosphate. (From Guder WG, Morel F. Biochemical characterization of individual
nephron segments. In: Windhager EE, ed. Handbook of physiology: renal physiology. New York:
Oxford University Press, 1992:2120–2164, with permission.)

and the amino acid, glucose, and phosphate cotransporters. is located on the basolateral cell membrane and is the energy-
These symport and antiport systems couple the downhill dependent step that regulates sodium chloride reabsorption.
influx of sodium with the uphill flux of the other solute. Sodium reabsorption begins with its passive transport from
Tertiary active transport systems are those that are coupled luminal fluid across the apical membrane into the intracellu-
to secondary active transport processes. Examples are the lar space by secondary transporters (see earlier). It is actively
Cl–-formate exchanger, which is coupled to Na+-H+ transported out of the cell into peritubular fluid by Na+-K+-
exchange, and the organic anion exchanger, which is cou- ATPase (Fig. 3.1). This step creates the favorable electro-
pled to ketoglutarate exchange. Selective ion channels chemical gradient necessary for continued transport of solutes
enable ions to diffuse down their concentration gradients. from luminal fluid into the cell and then into pericapillary
The use of human cell lines with extended in vitro growth fluid, which completes the reabsorptive process. The high
potential, especially if they are fully differentiated, have permit- level of renal Na+-K+-ATPase activity accounts for the high
ted new insights into tubular transport function (4). Likewise, level of renal oxygen consumption and energy expenditure
the use of tissue-engineered bioartificial renal tubules, in which (6). With the hydrolysis of ATP, this pump exchanges three
porcine proximal tubule cells are seeded into the intraluminal intracellular sodium ions for two extracellular potassium
space of a high-flux hollow-fiber hemofiltration cartridge, has ions, thus maintaining a low intracellular sodium concentra-
permitted study of differentiated transport as well as the meta- tion and creating a sodium concentration gradient from
bolic and endocrine functions of the kidney (5). lumen to cell. In addition, the high intracellular potassium
concentration establishes a cell membrane potential differ-
ence (PD) of –60 mV inside the cell.
Na+-K+-ATPase
Na+-K+-ATPase is composed of two subunits, α and β,
Na+-K+-ATPase transports sodium from cells in exchange for which link the hydrolysis of ATP to the translocation of
potassium uptake into cells. In the renal tubule, this activity Na+ and K+ (Fig. 3.1). The phosphorus from ATP phos-
 3. Tubular Function 47

phorylates an amino acid residue of the α subunit, which and manipulations that increase the Na+-H+ exchanger
binds to potassium on the extracellular face. This binding activity stimulate Na+-K+-ATPase (14). The rate of sodium
site is the same as that for ouabain. Na+ binds to the inter- entry into cells and intracellular sodium concentration reg-
nal cell surface of the α subunit and is translocated to the ulate transcription of both subunits of Na+-K+-ATPase
extracellular face of the membrane along with adenosine (15). Inhibition of Na+-K+-ATPase leads to an increased cell
diphosphate. The stoichiometry of the process is 3Na+ sodium concentration, which, within hours, stimulates syn-
exchanged for 2K+ per ATP hydrolyzed. Three isoforms of thesis of Na+-K+-ATPase (15). Glucocorticoid hormones
the α subunit (α1, α2, and α3) and two isoforms of the β increase transcription of both subunits of Na+-K+-ATPase
subunit (β1 and β2) have been identified (7). The α1 and β1 in the newborn rat kidney (16). Na+-K+-ATPase activity is
forms are found in the mature kidney. acutely affected by other hormones and intracellular second
messenger systems. The immature kidney has a blunted
response to dopamine and protein kinase C activation (17).
Regulation of Na+-K+-ATPase Activity
Agents with a natriuretic action, such as dopamine, atrial
natriuretic factor (ANF), parathyroid hormone (PTH), PROXIMAL TRANSPORT SYSTEMS
endothelin, and prostaglandin E2, inhibit Na+-K+-ATPase,
whereas agents such as norepinephrine, α-adrenergic recep- The proximal tubule has three nephron segments (S1, S2,
tor agonists, insulin, and angiotensin (low dosages) stimu- and S3), which differ from each other morphologically. Cells
late Na+-K+-ATPase. In the dopaminergic system, CA1 of the early, or S1, segment have a greater density of
receptors are coupled to adenylate cyclase such that dopa- microvilli and deeper basolateral membrane infoldings, as
minergic agonists simultaneously increase cyclic adenosine well as an abundance of mitochondria. Transport capacity for
monophosphate (cAMP) and phospholipase C2 with acti- most solutes (NaCl, bicarbonate, glucose, amino acids, and
vation of protein kinases, which phosphorylate a regulatory phosphate) is greatest in the S1 and less in the S2 and S3 seg-
protein and potentially specific phosphorylation sites on ments. This difference may result from the high permeability
the α subunit of Na+-K+-ATPase. Phosphorylation of the α of the epithelium to solutes and water or to intrinsic differ-
subunit reduces ATP hydrolysis and changes the equilib- ences in the density or kinetic properties of specific transport
rium between sodium binding E1 and potassium binding processes. Changes in the transport rate do not automatically
E2 forms (8). imply a greater intrinsic transport capacity; for example, a
decrease in transport rate may follow decreasing substrate
concentration as luminal fluid moves distally along the prox-
Developmental Changes in
imal tubule (5,18). Later segments may be described as hav-
Na+-K+-ATPase Activity
ing less surface area and decreased capacity for transport,
The activity of the Na+-K+-ATPase increases during devel- both transcellular and paracellular. However, these later seg-
opment, as does the expression of the α and β subunits. ments are also less permeable to solutes and fluid because
Striking increases occur during weanling (9). The activity they have tighter junctional complexes; backleak is minimal
of the Na+-K+-ATPase in homogenized whole rabbit kidney in the region of these segments, and a higher concentration
and isolated tubules from rabbit is lower in neonatal kidney gradient is thereby achieved. This illustrates a common
than in the adult kidney; however, the distribution and the theme: The arrangement of transcellular transporters and
affinity constants for Na+ and K+ are identical (10,11). The intercellular permeability characteristics in series in the prox-
increase in Na+-K+-ATPase that occurs during development imal tubule favors a higher flux in the proximal segment and
parallels the increase in sodium reabsorption. In developing creation of steeper concentration gradients in the later seg-
rats, an increase in sodium reabsorption precedes an ments. This arrangement achieves almost complete absorp-
increase of Na+-K+-ATPase (12). Studies performed in tion of nutrients and organic solutes as luminal fluid passes
guinea pig renal cortex demonstrate that pump activity through the proximal tubule (5,18).
increases fourfold to fivefold during transition from fetus to Functionally, the proximal tubule performs two major
newborn. Most of the postnatal increase in basolateral functions: the reabsorption of almost all of the filtered glu-
pump activity was related to increased pump synthesis cose, amino acids, phosphate, and bicarbonate and reabsorp-
rather than redistribution of preformed pumps from inter- tion of more than one-half of the sodium, chloride, and
nal stores. The increase in pump protein abundance was water. Figure 3.2 describes the relative reabsorption of vari-
greater than the increase in mRNA for both α and β sub- ous solutes along the length of the proximal tubule. The
units, leading to the conclusion that posttranscriptional tubular fluid (TF):plasma (P) ratio of each solute relative to
upregulation might account for the marked increase in that of inulin is represented; a TF:P ratio less than 1 denotes
pump abundance (13). Inhibition of the Na+-H+ pump, the a drop in TF concentration and indicates net reabsorption.
major mechanism for active sodium entry into the cell, The PD across the proximal tubular epithelium is lumen
reduces the activity of the Na+-K+-ATPase in weanling rats, negative and that of the later (S3) is lumen positive (18).
48 I. Development

erular filtration rate (GFR), intracellular sodium concentra-

tion, and the electrical potential across the tubular epithelium
(19). A number of membrane-associated polypeptides are also
found at the inner side of the plasma membrane. These influ-
ence incorporation and/or retrieval of transporters into the
membrane, a process which alters membrane capacitance (20).
The membrane protein, termed RS1, also influences the
organic cation transporter (OCT2).

Heterogeneity of Glucose Transport

Along the Tubule
The bulk of filtered glucose is reabsorbed by the first 3 mm
of the proximal tubule (S1), with negligible reabsorption
by S2 and S3 segments (Fig. 3.2). There are two compo-
nents of glucose flux: nonsaturable, passive paracellular dif-
fusion and saturable, active transport, which is transcellular.
The rate-limiting step in glucose reabsorption is the activ-
ity of the glucose transporter, which is located on the
brush-border membrane surface. The kinetic properties of
the glucose transporter vary among the segments of the
proximal tubule: in the S1 segment, there is a high-capacity
(Jmax), low-affinity (Km) system; in the S2 segment, a high-
affinity, lower-capacity system; and in the S3 segment, an
even higher-affinity, lower-capacity system (Table 3.1)
(21). The permeability of the diffusion pathway decreases
with increasing distance along the proximal tubule. Hence,
the rate of glucose reabsorption decreases along the length
of the proximal tubule as the character of the transport
proteins change, the permeability of the diffusive pathway
FIGURE 3.2. Reabsorption of solutes along the proximal
tubule in relation to the TF:P ratios and potential difference (PD) changes, and the concentration of glucose in luminal fluid
across the tubular epithelium. TF/P represents the ratio of tubule declines. This diversity allows high glucose flux in the early
fluid to plasma concentration. The area above the line denotes proximal tubule and then a steep uphill reabsorption as the
that the substance is secreted, and below the line that the sol-
ute is reabsorbed. OSM, osmolality; PAH, p-aminohippurate. affinity for glucose increases along the late proximal tubule,
(Adapted from Rector FC Jr. Sodium, bicarbonate, and chloride where backleak is minimal. A question still remains: Do
absorption by proximal tubule. Am J Physiol 1983;244:F461.) these heterogenous functions of the uphill glucose trans-
porter (SGLT1) indicate two independent transporters or
different transport modes through an oligomeric protein?
Glucose
The most likely explanation is that heteroassociation with
Glucose is almost completely reabsorbed by the proximal regulatory subunit(s) contribute to this heterogeneity of
tubule; less than 0.05% of the filtered load appears in urine glucose handling (22).
when the serum level is within the normal range. As the filtered
load of glucose increases, the quantity of glucose reabsorbed
also increases until the maximum rate of glucose reabsorption TABLE 3.1. COMPARISON OF KINETIC PARAMETERS
(TmG) is achieved. The plasma level of glucose at which glu- FOR GLUCOSE TRANSPORT IN PROXIMAL
cose reabsorption is maximal is known as the threshold for glu- TUBULE SEGMENTS
cose. The transport capacity (milligrams per minute) when the Segment Vmax (pmol/min/mm) Km (mM)
threshold is reached is called the TmG. The limited capacity for
glucose transport is probably related to the number of carrier Convoluted (S1) 83.2 ± 5.0 1.64 ± 0.19
Early straight (S2) 12.9 ± 1.1 0.70 ± 0.12
or transport molecules on the brush-border membrane surface Late straight (S3) 7.9 ± 0.5 0.35 ± 0.05
and the basolateral membrane. These carrier systems work in
series to transport glucose from the luminal fluid to the intra- Vmax, maximum transport.
cellular space and then into the pericapillary fluid. The trans- From Silverman M, Turner RJ. Glucose transport in the renal proxi-
mal tubule. In: Windhager EE, ed. Handbook of physiology: renal
port maximum for glucose may be modified by factors other physiology. New York: Oxford University Press, 1992:2017–2038,
than the number of glucose transporters: ECF volume, glom- with permission.
 3. Tubular Function 49

tioned earlier. There are two distinct sodium-dependent lumi-

nal sugar transporters in the proximal tubule. The SGLT2,
which is localized to the early proximal segment, is specific for
glucose, has a 1:1 stoichiometry with sodium, and accounts for
approximately two-thirds of glucose absorption. The SGLT1
transporter accepts galactose as well as glucose, has a 2:1 stoi-
chiometry (Na:sugar), and is found along the brush border of
all proximal segments (22,26). The gene for the sodium-
dependent glucose transporter has been localized to chromo-
some 16. The human transporter protein is 664 amino acids
FIGURE 3.3. Model of glucose transport by the proximal tubu- in length, with ten transmembrane helical domains and a
lar cell. (1) Glucose enters the proximal tubular cells by an apical molecular weight of approximately 73 kDa (26). The renal
brush-border cotransporter along with either one or two sodium
ions, depending on the location of the transporter along the cortical, sodium-dependent glucose transporter is similar to
proximal tubule. (2) A transporter for other hexose sugars in addi- that found in small intestine (27).
tion to glucose is present on the apical surface. (3) Exit across the The basolateral glucose transport systems (GLUT) do
basolateral membrane of the cell occurs through a sodium-inde-
pendent transporter. The Na+-K+-ATPase is also illustrated. not require Na+ and are not inhibited by phlorizin. At least
five distinct GLUT transporters are distributed among
numerous tissues, with two predominantly expressed in
Cellular Processes Involved in
renal tubule and intestine (28). One has higher affinity and
Glucose Reabsorption
lower capacity (GLUT 1), and the other has lower affinity
Three steps are involved in the reabsorption of glucose: and higher capacity (GLUT 2). These two systems are dis-
uptake of glucose into the cell by a sodium-dependent glu- tributed differently along the renal tubule: GLUT 1 is
cose transporter in the apical membrane, diffusion of free present in the glomerulus and along the entire proximal
glucose in the intracellular fluid, and exit from the cell via a tubule, whereas GLUT 2 is localized exclusively to the
specific sodium-independent transport process in the baso- basolateral membrane of early proximal convoluted tubule
lateral membrane (Fig. 3.3). The transport of glucose across (S1) (29). The specific locations of the two basolateral glu-
the apical membrane is by secondary active transport cose transport systems parallel those of the apical, sodium-
dependent on the electrochemical sodium gradient that is dependent systems. The basolateral glucose transporter
produced by Na+-K+-ATPase. The transport is carried out system of renal cells is structurally similar to that of erythro-
by two structurally and kinetically distinct apical glucose cytes and hepatocytes (23,29).
transporter proteins (19,23): One has a high capacity and a
low affinity (SGLT2) and the other a low capacity and high
Developmental Changes in Glucose Transport
affinity (SGLT1) for glucose. The transporter-coupling
ratios of sodium to glucose are 1:1 for the S1 segment and The increase in glucose reabsorption during late fetal life
2:1 for the S2 and S3 segments (24). The sensitivity of the through infancy and childhood parallels the increase in
two systems to phlorizin, a competitive inhibitor of the glu- GFR. However, glucosuria is more common among neo-
cose transporter, also differs. Phlorizin binding appears to nates, and the levels of glucose in urine are highest in pre-
influence both the glucose- and Na+-binding sites (25). The term infants (65 ± 78 mg/dL), lower in term infants (15
transitional steps involved in glucose uptake at the brush- mg/dL), and even lower in adults (6 ± 2 mg/dL) (24). TmG,
border membrane have been elucidated. The initial binding when adjusted to standard surface area and to GFR, tends
of sodium at the luminal outside surface induces a confor- to be lower in preterm and term infants (Table 3.2) than it
mational change that increases affinity of glucose binding. is in adults (30).
This is followed by presentation of sodium and glucose to Factors that increase the glucose absorptive capacity dur-
the cytoplasmic side, in which the low sodium concentra- ing growth from fetal life to maturity include development
tion favors disassociation of sodium, which reduces the of new nephrons, increases in cell membrane surface area
affinity of the transporter for glucose that is then released and in basolateral Na+-K+-ATPase, and changes in expres-
into the cytosol. Then, the transporter orients sodium- and sion and density of transporter proteins (31–34). The max-
glucose-binding sites back toward the extracellular domain imal rate of glucose transport into brush-border vesicles is
for repeated transport (26). lower in preparations from fetal kidneys than in those from
adult kidneys (Fig. 3.4); the density of transporter proteins
is higher in adults (31). Age-related differences in glucose
Molecular Studies of Sodium-Dependent
transport capacity correlate with differences in sodium con-
and Sodium-Independent Systems
ductance. Changes in membrane permeability to sodium
The distribution of the two apical sodium-dependent glucose affect membrane potential, a factor that modifies glucose
transport systems may account for the kinetic differences men- reabsorption.
50 I. Development

TABLE 3.2. NORMAL VALUES FOR TUBULAR

MAXIMUM FOR GLUCOSE
TmGa (mg/min/1.73 m2) TmG/GFRb (mg/mL)

Adults 290–375 2.3-2.7

Children 250–400 1.8–2.9
Infants
Term 35–290 0.9–2.9
Premature 25–190 2.3

aMaximum tubular reabsorption of glucose.

bMaximum tubular reabsorption of glucose per mL glomerular filtra-
tion rate (GFR).
Adapted from Brodehl J, Franken A, Gelissen K. Maximal tubular
reabsorption of glucose in infants and children. Acta Paediatr Scand
1972;61:413–420.

Maturation of the glucose transport system parallels

maturation of bicarbonate flux, fluid absorption, and baso-
lateral surface area of the proximal tubule (Fig. 3.5). The
reduced capacity for glucose absorption that characterizes
the immature kidney is related to a less-developed basolat-
eral membrane, in addition to the previously mentioned
differences in membrane transporter density (35). In sum-
mary, the maturational increase in the capacity for tubular
reabsorption of glucose is related to an increased number of
apical symporter proteins, a more favorable electrochemical
FIGURE 3.5. Absorption of fluid (Jv), bicarbonate (JHCO3), and
gradient, decreased turnover rate of transporter proteins, glucose (JGlu) and surface area of basolateral membrane (B-L sur-
and decreased membrane fluidity (31). In addition, the face) per millimeter of proximal convoluted tubule during devel-
membrane of the immature animal is leakier; nearly one- opment in the rat. (From Schwartz GJ, Evans AP. Development of
solute transport in rabbit proximal tubule: I. HCO3– and glucose
absorption. Am J Physiol 1983;245:F382–F390, with permission.)

third of the glucose reabsorbed by the neonatal animal leaks

back across the tubular membrane, compared with one-
sixth of that in the adult (3,36).

AMINO ACIDS

The bulk of amino acid reabsorption occurs within the first 2

mm or the proximal one-third of the proximal tubule (S1
segment) (Fig. 3.2) (37). The luminal fluid concentration of
amino acids drops as it passes this portion of the tubule and
then remains fairly constant. This is indirect evidence for
reabsorption of amino acids along the entire length of the
proximal tubule. Under normal conditions, amino acids
other than glycine, histidine, and taurine are completely
reabsorbed. The capacity for amino acid reabsorption along
the entire proximal tubule is high. The renal tubule has a
mechanism to increase reabsorption in the event that the fil-
tered load of any amino acid increases (38,39).
FIGURE 3.4. Maturation of glucose transport. Time course of Although the loop of Henle is difficult to study, bidirec-
glucose uptake into brush-border membrane vesicles from fetal tional amino acid transport for neutral amino acids and acidic
and adult rabbits. (From Beck JC, Lipkowitz MS, Abramson RG. amino acids has been reported (40,41). The physiologic role
Characterization of the fetal glucose transporter in rabbit kidney.
Comparison with the adult brush border electrogenic Na +-glucose of loop amino acid transport has not been elucidated, and it
symporter. J Clin Invest 1988;82:379–387, with permission.) seems unlikely that it would make a significant contribution
 3. Tubular Function 51

to total tubular reabsorption because delivery of amino acids transporters achieve the net reabsorption of amino acids
to the loop is minimal under normal conditions. from luminal to peritubular fluid. There is also bidirec-
tional movement of amino acids. At the apical membrane,
backleak occurs via a paracellular route, through the leaky
Cellular Mechanisms of
“tight junctions” of the proximal tubule or via a transepi-
Amino Acid Transport
thelial route, using the apical amino acid transporters.
The transepithelial transport of amino acids is an active, Studies in brush-border membrane vesicles (BBMVs) indi-
concentrative, sodium-requiring process that involves sev- cate that the transporters located on the brush border are
eral specific steps (Fig. 3.6) (37). Luminal cell-surface trans- stimulated by intravesicular, or cytoplasmic, amino acids
port is performed by specific amino acid transport systems (transstimulation). Therefore, the transport system on the
that operate by secondary active transport, cotransporting apical membrane is probably bidirectional.
amino acids uphill against their concentration gradient Amino acids may be coupled to sodium in a 1:1 or a 2:1
along with sodium. The electrochemical sodium gradient ratio, which allows the transport process to rely on the luminal
created by Na+-K+-ATPase enables amino acids to be accu- to cellular transmembrane potential (Em) and the chemical
mulated. Once inside the cell, the amino acids may be used potential for sodium. In addition to cellular accumulation of
by the cell or may exit the cell by passive, downhill trans- amino acids from the luminal membrane, amino acids may be
port. Together, the apical and the basolateral amino acid taken up by cells from the basolateral surface to provide sub-
strate, especially in the portions of the nephron that do not
have high flux of amino acids via a luminal system (42).
The kinetics of glycine transport (43) in the convoluted
tubule differ from those in the straight tubule. The trans-
port maximum and the Km are lower in the straight por-
tion. This allows the tubule to drastically reduce the
intraluminal concentration of an amino acid by increasing
the affinity of the transporter for substrate. In addition, the
forces that favor backleak of amino acids are progressively
reduced from convoluted to straight tubule.

Amino Acid Transport Systems

Seven specific transport systems for amino acids have been
identified (Table 3.3). Most of the amino acid transport pro-
cesses are sodium dependent and have some degree of sub-
strate specificity. The electrogenic properties of the sodium–
amino acid transporter complex depend on the charge of the
amino acid, the coupling ratio for sodium, the charge on the
protein carrier complex, and the ionic dependence (Cl– or K+)
(38). Acidic amino acids require potassium for transport (44).
The anionic or acidic amino acids L-glutamate and L-aspartate
are reabsorbed by a transporter that is stereospecific, requiring
a primary two-amino group along with a positively charged
two- to four-carbon chain for transport to occur (45,46). The
glutamate transport system is unique in that it requires 2 Na+
and a proton, which are exchanged for a potassium ion after
the transporter undergoes a conformational change. The β-
amino acid transport system requires chloride in addition to
sodium (47). Protein kinase C activation may also modify tau-
rine transporter activity by phosphorylation of a critical serine
residue (48). Dibasic amino acids L-arginine and L-lysine and
L-ornithine share at least one transport system (49,50); the
FIGURE 3.6. Proposed factors responsible for the physiologic
aminoaciduria of the immature animal. Factors include decreased luminal uptake step is sodium independent and passive. A
activity of the amino acid-sodium cotransporter, increased Na+-H+ sodium-dependent, electrogenic system on the basolateral
exchange at the luminal membrane, and decreased activity of the membrane allows amino acid exit.
Na+-K+-ATPase at the basolateral membrane. (From Zelikovic I,
Chesney RW. Development of renal amino acid transport systems. A large number of amino acid transport systems have
Semin Nephrol 1989;9:49–55, with permission.) now been cloned and characterized by structural homolo-
52 I. Development

TABLE 3.3. AMINO ACID TRANSPORT SYSTEMS but a few amino acids is greater in immature animals. The
increased excretion of amino acids results from reduced
Acidic L-Glutamate
L-Aspartate
reabsorption. Factors that might account for the immatu-
Basic L-Arginine rity of the reabsorptive process include differences in the
L-Lysine luminal transport system, decreased efflux of the amino
L-Ornithine acid from the contraluminal membrane, increased backleak
Cystine L-Cystine
through paracellular or transepithelial pathways, or nonspe-
L-Cysteine
Glycine Glycine cific maturational differences in the cell membrane or cell
Imino acids L-Proline metabolism (Fig. 3.6) (55). Altered renal amino acid han-
L-Hydroxyproline dling is a highly sensitive marker for nephrotoxicity in
β-Amino acids Taurine immature rats (56).
β-Alanine
The aminoaciduria that occurs in preterm and term
γ-Aminobutyric acid
Neutral L-Phenylalanine
neonates probably is not caused by a generalized abnormal-
L-Serine ity, because not all of the filtered amino acids are wasted to
L-Alanine the same degree (55). The excretion of glycine, imino acids
L-Leucine (proline, hydroxyproline), dibasic amino acids, and taurine
L-Phenylalanine
is preferentially higher than that of other amino acids (57–
L-Valine
L-Asparagine
61). Acquisition of mature reabsorptive capacity of the
Glycine L-Tryptophan amino acid system and the glycine transporter occurs at dif-
L-Citrulline ferent developmental stages (57). When compared, the
L-Methionine renal cortical accumulations of glycine, leucine, and
L-Tyrosine
glutamic acid are lower than those of lysine and alanine
L-Glutamine
(55,62–64). Lysine uptake is similar in adult and neonatal
animals, whereas cystine accumulation into cortical slices
from neonatal kidney is lower than it is in adult kidneys
gies to be members of distinct gene families: the glycine until the age of 2 to 3 weeks (65). Urinary levels of proline
transport system, the neutral amino acid transport system are decreased by the postnatal age of 7 days, whereas those
A, the basic amino acid transport system y+, and the acidic of glycine remain elevated until 3 weeks of age (66,67).
amino acid transport system X–. Most studies thus far have failed to demonstrate a
A sodium-independent amino acid transport system change from one transporter protein isoform to another
(rBAT) for the dibasic amino acids, neutral amino acids, and with maturation. Early studies of glycine and proline trans-
cystine has been identified (51). As a member of the type II port led investigators to conclude that the specific, high-
membrane glycoproteins, this transporter is found in the affinity transporter was not present at birth but appeared at
brush-border membrane of both the intestine and the kidney the time that amino acid reabsorption capacity reached
proximal tubule, in which it enables electrogenic exchange of mature levels. Later studies found that both a common
neutral and dibasic amino acids. It shares sequence homology glycine-proline transport system and individual transport
with a variety of amino acid transporters and α glucosidases as systems were present from birth and that no new systems
part of a family of transport regulators. The gene is located on were acquired with maturation (55,66,67).
human chromosome 2. Mutations of this protein have been Intrinsic differences in the activity and transport capac-
identified in patients with cystinuria, a transport defect of ity for certain individual transport systems exist. There is
ornithine, lysine, cystine, and lysine. This transport system is evidence of a decreased rate of efflux (66). Analysis of
inhibited by heavy metals such as lead and mercury (52). kinetic studies involving the uptake of proline by rat renal
These heteromeric amino acid transporters (HATs) have inter- BBMVs reveals that the magnitude of proline uptake is
esting biologic properties (53). If mutated, they form the reduced in neonates and that the characteristic uptake pat-
molecular basis of the primary aminoacidurias cystinuria and tern is less pronounced in vesicles from young rats. This
lysinuric protein intolerance (54). They are composed of two phenomenon is explained by differences in membrane per-
polypeptides, a heavy and light subunit, which also may play a meability to sodium. The sodium gradient that is necessary
role in integrin activation. The genes encoding these transport- for cellular or vesicular uptake of an amino acid is dissi-
ers are SLC 3A1, SLC 7AG, and SLC 7A7 (54). pated more rapidly in the vesicles from 7-day-old rats than
in those from older rats (66).
Studies using isolated renal tubule preparations reveal
Developmental Changes in
that the initial transport rate of proline into neonatal rat
Amino Acid Transport
tubules equals that of adults, yet the cellular amino acid
In general, urinary amino acid levels are higher in neonates levels are higher in the neonatal rats (60). This phenome-
than in mature animals, and the fractional excretion of all non is partially explained by metabolic studies. Metabolism
 3. Tubular Function 53

of proline by tubules from adult rats is twice that of tubules is attained at 1 to 2 weeks of age in the rat. The matura-
from neonates. The maturational changes in the excretion tional factors responsible for this capacity are unknown.
of proline are related to metabolic changes within the cell as The urinary levels of taurine in low-birth-weight neo-
well as to changes in the transport system. nates are significantly elevated, with fractional excretion
Two cystine transport systems have been detected in iso- ranging from 38 to 68% (70). These infants were receiving
lated proximal renal tubules from neonatal dogs. Both sys- parenteral nutrition devoid of taurine and had low plasma
tems are present from birth, yet the activity of both systems, taurine levels. Therefore, immature animals cannot reab-
as judged by the transport maximum (Jmax), is less in the sorb as much filtered amino acids and are less able to adapt
tubules from puppies; these increase to adult levels by 3 to deficiency, predisposing them to significantly reduced
weeks of age. The authors speculate that the postnatal plasma taurine values. Because taurine is needed for normal
increase in transport capacity is secondary to an increase in retinal and CNS development, this may have deleterious
the number of transport sites for the sulfur amino acids (65). consequences in the developing preterm infant.
Changes in the phospholipid composition of the mem-
brane during maturation also affect the ionic and amino
acid permeability characteristics. The change in composi- PEPTIDE AND PROTEIN REABSORPTION
tion of rat tubular brush-border membranes parallels
change in membrane permeability to sodium (55). This Peptides are reabsorbed in the proximal tubule by a variety
may impair the ability of the immature renal tubular cell to of processes, including reabsorption by endocytosis, lumi-
maintain the electrochemical gradient necessary for reab- nal hydrolysis with reabsorption as free amino acids, car-
sorption of amino acids. rier-mediated reabsorption of small intact peptides, and
The steady-state renal cell accumulation of the amino peritubular uptake. Peptides reabsorbed by the proximal
acids glycine, proline, and taurine is greater in young ani- tubule include angiotensin II, bradykinin, and glucagon.
mals than it is in adults, probably because of slower egress These peptides are rapidly hydrolyzed by peptidases found
of amino acids from immature animals (55). The intracell- along the brush-border membrane and are then reabsorbed
ular concentration of amino acids is higher in younger ani- as single amino acids. Certain properties confer resistance
mals. Although the luminal transport process is similar, the to peptidases, because peptides with disulfide bonds or β-
efflux step at the basolateral membrane is reduced. The net amino acids may not undergo hydrolysis (37).
effect is decreased net transepithelial amino acid transport. Dipeptides and tripeptides that are resistant to hydrolysis in
The kinetic characteristics of taurine accumulation by the gastrointestinal tract are presented to the brush-border
BBMVs have been extensively studied and are similar in membrane, in which they are encountered by enzymes that
adult and 28-day-old rats (68). The young rats responded may be reabsorbed by a Na+-independent, electrogenic peptide
to alterations in the supply of the substrate amino acids transporter. The peptide transporter is located in cortex and
with changes in accumulation that were identical to medulla and shares similarities with the intestinal peptide
responses in adults. Despite this, the urinary excretion of transport system (71). Peptide transport is sodium indepen-
taurine is much greater in the immature animals. This dent and energized by an inwardly directed transmembrane
probably is related to differences in the membrane perme- proton gradient that depends on the inward negative mem-
ability to taurine or reduced basolateral efflux in the imma- brane potential. This type of system is unique to the renal
ture rats. An age-related increase in the accumulation of the tubule, is more commonly found among prokaryotic cells, and
amino acid into BBMVs was found in younger rats (59). remains the only example of uphill solute transport energized
Taurine transport into vesicles isolated from nursing rats of primarily by proton movement. Two distinct dipeptide trans-
three different ages (7, 14, and 21 days) was measured to port systems have been characterized: a high-affinity system
examine the influence of dietary supply on uptake. Taurine that is highly dependent on proton gradient and a lower-affin-
uptake by the vesicles from 7-day-old rats was significantly ity system that may operate in the absence or presence of sub-
lower than that of the older rats; there was little response to strate concentration or inward proton gradient (71). A diverse
changes in dietary taurine intake (59). The efflux of taurine group of dipeptides or tripeptides and xenobiotics with a pep-
from cortical slices is decreased in the 7-day-old pups (69). tide backbone are accepted by these systems, but free amino
Taurine accumulation increased as a function of age, being acids, or peptides with more than three amino acids, do not
lower in the 14- and 21-day-old animals and increasing to interact. Hydrophobicity seems to favor binding to trans-
adult levels by 28 days of age. These results suggest that porter. Specific basolateral systems for peptide transport have
age-related differences in taurine reabsorption and urinary not been demonstrated. However, many of these substances
excretion are related to reduced intrinsic apical transporter interact with basolateral systems for organic cations or anions.
activity early in development and to differences in mem- The p-aminohippurate (PAH) transporter reveals significant
brane permeability to taurine and Na+ later in develop- affinity for dipeptides and tripeptides and also seems to favor
ment, when the transporter capacity is at adult levels. The hydrophobic compounds. Peritubular uptake of peptides
ability of the renal tubular cell to respond to dietary taurine appears to be negligible.
54 I. Development

Although not filtered freely like smaller peptides, pro- TABLE 3.4. RENAL PHOSPHATE HANDLING IN
teins may reach the urinary space through glomerular filtra- NEWBORN AND ADULT GUINEA PIGS
tion, in which they are reabsorbed by nonspecific Newborn Adult p Value
adsorptive endocytosis or receptor-mediated endocytosis,
GFR (mL/g kidney) 0.28 ± 0.06 0.55 ± 0.05 <.01
or undergo hydrolysis by brush-border membrane enzymes
Plasma phosphate 2.18 ± 0.15 2.16 ± 0.14 >.90
such as peptidases (72). Handling of proteins by the brush- (mM)
border membrane depends on size. The low-molecular- Tubular reabsorption of Pa
weight proteins are 10 to 44 kDa. Examples include growth % Filtered load 89.93 ± 2.55 78.25 ± 2.89 <.01
hormone, PTH, many enzymes, immunoprotein fragments μmol/mL GFR 1.87 ± 0.14 1.53 ± 0.12 <.05
Fractional reabsorption of Pb (% filtered load)
such as Bence Jones, and tissue-specific antigens such as β2-
Proximal 76.66 ± 2.75 67.21 ± 2.74 <.001
microglobulin. These proteins are absorbed and then Distal 15.62 ± 2.11 10.51 ± 1.83 <.005
undergo hydrolysis.
Intermediate-molecular-weight proteins of 44 to 90 kDa GFR, glomerular filtration rate.
aWhole kidney tubular reabsorption.
(e.g., albumin) are normally filtered to a very small degree bSingle nephron tubular reabsorption.
and are subsequently endocytosed and then hydrolyzed Adapted from Kaskel FJ, Kumar AM, Feld LG, et al. Renal reabsorp-
within lysosomes (72). Larger proteins, such as fibrinogen tion of phosphates during development: tubular events. Pediatr
Nephrol 1988;2:129–134.
and immunoglobulins, normally do not reach the urinary
space. If the normal barrier to filtration of high-molecular-
weight proteins is disrupted, they may undergo endocytosis.
Examples of low-molecular-weight proteins that undergo large substrate flux proximally and more complete absorp-
hydrolysis by a nonspecific system are angiotensin II and tion distally, has been alluded to in the discussion of glu-
bradykinin. Peptides such as insulin, PTH, and ANF are cose transport. Higher affinity of the transporter for
absorbed by specific receptor-mediated endocytosis hydrol- substrate allows more complete absorption by increasing
ysis. The nonspecific system is a high-capacity, low-affinity the transporter efficiency in the face of lower tubular sub-
system, whereas the specific systems are usually low-capacity, strate concentrations.
high-affinity systems (72).
Cellular Mechanisms
Phosphate
Phosphate reabsorption is a unidirectional, transcellular
The fraction of plasma phosphate that is filtered depends movement of phosphate across the proximal tubule from
on the plasma phosphate concentration and the distribu- luminal to peritubular fluid (Fig. 3.7A). Phosphate is reab-
tion of ionized phosphate as determined by the Donnan sorbed by a sodium-dependent, cotransport system located
equilibrium. In the rat, 92 to 98% of phosphate is present on the apical, or brush-border, membrane. Uptake occurs
in the aqueous phase of plasma, and 90% of total plasma against a small chemical gradient and a large electrical gra-
phosphate is filterable. The ultrafilterability of phosphate dient (76). Phosphate uptake is driven by the electrochemi-
declines with increases in plasma calcium (73). cal gradient for sodium, with a sodium-coupling ratio of
Phosphate reabsorption occurs primarily in the proximal 3:1 (77). The rate-limiting process for transepithelial phos-
tubule, in which approximately 76% of the filtered phos- phate reabsorption is the apical transport step (76,78). The
phate is reabsorbed (Table 3.4). Phosphate reabsorption transporter prefers the divalent form (HPO4–2) of inorganic
occurs in both the convoluted and the straight portions of phosphate to the monovalent form (H2PO4–), although
the proximal tubule (73). A small fraction of phosphate is both are candidates for transport. Luminal fluid pH influ-
reabsorbed in the distal tubule under conditions of sodium ences the activity of the Na+-phosphate cotransporter;
loading and in the absence of PTH. Phosphate reabsorp- kinetically distinct transporter proteins also cause the rate
tion rates are three to four times greater in the early proxi- of phosphate transport to vary.
mal tubule than in the S2 portion of the convoluted tubule Sodium is required for cotransport with phosphate and
and are lower still in the straight tubule (74). This axial het- is postulated to activate the binding of phosphate to its
erogeneity in phosphate transport is postulated to be the transport site (78). The Na+-phosphate cotransporter prob-
result of TF acidification, which decreases the ratio of the ably functions in a “glide symmetry” model, as illustrated
divalent form (preferred substrate) to monovalent form in Figure 3.7C: Na+ binds to the transporter protein first
(75) (see Peptide and Protein Reabsorption, Cellular Mech- and then induces a conformational change to increase affin-
anisms). There are differences in the affinity and capacity ity of the protein for phosphate. The substrate-transporter
for phosphate transport between the straight and convo- complex is translocated to the interior of the cell, in which
luted portions of the proximal tubule (74,75). Affinity of sodium and then phosphate dissociates (76).
the transporter for phosphate is low in the S1 and increases Phosphate transport is stimulated by decreased intracell-
as one travels distally. This characteristic, which enables a ular pH (an outwardly directed proton gradient) (75). Two
 3. Tubular Function 55

FIGURE 3.7. A: Model for sodium-phosphate cotransport by the proximal tubular cell (1). Both
monovalent and divalent phosphates are cotransported with sodium at the brush-border mem-
brane. Phosphate exits the basolateral membrane by a sodium cotransport process (2), or by
anion exchange (3). B: Model for conformational changes and ionic interactions of the sodium-
phosphate cotransporter. At high intratubular sodium concentrations, sodium binds to the trans-
porter first and favors binding of phosphate to the transport site, followed by movement of the
transporter-substrate complex to the cytoplasmic side of the cell where sodium dissociates first,
decreasing protein affinity for phosphate, which then dissociates (glide symmetry model). C: Sec-
ondary structure prediction for the type II Na-P 1 cotransporter. Eight transmembrane-spanning
regions are proposed. The transporter is N-glycosylated at the two positions indicated. (From
Murer H. Cellular mechanisms in proximal tubular Pi reabsorption: some answers and more ques-
tions. J Am Soc Nephrol 1992;2:1649–1665, with permission.)

factors are responsible: Increasing the luminal fluid pH demonstration of sodium-coupled phosphate uptake into
increases divalent phosphate, which is more readily trans- basolateral BBMVs; however, these findings may have
ported; and there appears to be a direct effect of pH on the resulted from contamination of the basolateral vesicles with
transporter protein, which alters its binding properties vesicles from apical membranes (76).
(79). Protons and sodium compete for binding sites of the
transporter both inside and outside the cell. A decrease in
Molecular Characterization
the external pH reduces the affinity of the transporter and
the rate of transport, whereas a decrease in intracellular pH Structural characterization of at least two distinct Na-phos-
increases the rate of transport (75,76,79). phate cotransport systems (NPT1 and NPT2) in the kid-
Intracellular phosphate has many important functions ney indicates that the two classes are different, sharing only
in oxidative metabolism, protein synthesis, and enzyme 20% homology. The NPT1 transporters are 465 amino
action. The mechanism of phosphate exit has not been as acids in length, with a predicted seven to nine transmem-
clearly characterized; there is probably a passive component brane domains. The NPT2 transporters have 635 amino
leading to phosphate transfer down its electrochemical gra- acids and eight transmembrane domains (Fig. 3.7). Both
dient. Phosphate also exits the cell by the sodium-indepen- transporters have been localized to the brush-border mem-
dent system, using a phosphate-OH– antiport system brane of the proximal tubular cell, although the NPT1 is
(anion exchanger) (76,80). A basolateral, Na+-phosphate homogenously found throughout, whereas the NPT2 is
cotransport system has been proposed that is based on the most concentrated in the S1 segment (81). NPT1 trans-
56 I. Development

porter is less specific for phosphate and also accepts anions

such as probenecid, chloride, and penicillin. The NPT2 is
the PTH- and hormone-sensitive transporter that also
responds to changes in dietary phosphate. The type II
(NPT2) transporter forms the rate-limiting step in brush-
border membrane transport (82). In addition to these two
systems, Na-independent phosphate transporters have been
identified that are also cell surface viral receptors Glvr-1
and Ram-1 and are widely expressed in mammalian tissues,
in which they are proposed to serve a housekeeping func-
tion (81).

Hormonal Regulation
PTH is the principal hormone regulator of Na+-phosphate
transport. PTH reduces the transport maximum without
changing the affinity of the transporter for substrate (76).
PTH inhibits Na-dependent phosphate transport by mech-
anisms dependent on AMP protein kinase A and protein
kinase C-phosphoinositide pathways. Other factors that
stimulate cAMP production also inhibit phosphate trans-
port (83). Apical and basolateral PTH receptors mediate
FIGURE 3.8. Relationship between the filtered load of phos-
the effect of PTH to reduce phosphate transport through phate and phosphate reabsorption in isolated perfused kidneys
an acute mechanism dependent on endocytosis or phos- from mature and newborn guinea pigs. At a comparable filtered
phorylation (81). Growth hormone, insulin-like growth load, the reabsorption of phosphate is significantly greater in
newborn animals than in adult animals. (From Johnson V,
factor-I, insulin, thyroid hormone, and 1,25-dihydroxyvi- Spitzer A. Renal reabsorption of phosphate during develop-
tamin D3 stimulate phosphate absorption, whereas PTH- ment: whole-kidney events. Am J Physiol 1986;251:F251–F256,
related peptide, calcitonin, ANF, epidermal growth factor, with permission.)
transforming growth factor α, and glucocorticoids inhibit
this process. Hormonal effects may be mediated through
ligand-activated receptors that bind to response elements in formed in the rat, guinea pig, and dog indicate that
the promotor region of the transporter gene. enhanced tubular reabsorption of phosphate is the primary
Two major circulating factors also greatly influence contributing factor to the greater reabsorption and positive
phosphate reabsorption. The first, a membrane-bound phosphate balance that characterize early extrauterine life
endopeptidase encoded by the PHEX (protein with homol- (87). The tubular maximum for the reabsorption of phos-
ogy to endopeptidases on the X chromosome) gene, serves phate is high in infants at a time when the accretion of
as an enzyme to degrade a protein, fibroblast growth factor phosphate is highest; it declines when the accretion of
23, which in the absence of PHEX acts to massively reduce phosphate declines (89). The rate of phosphate reabsorp-
phosphate reabsorption (84). PHEX also appears to tion at any given filtered load of phosphate is greater in
degrade PTH related peptide (85). Mutations of PHEX newborn guinea pigs than in adults (Fig. 3.8).
and fibroblast growth factor 23 have been associated with a The mechanisms responsible for higher phosphate reab-
number of hereditary or tumor-induced phosphaturic syn- sorption by the neonatal kidney are intrinsic ones controlling
dromes (84,86). the renal handling of phosphate and differences in hormonal
regulation of phosphate transport. Investigators have used
Developmental Changes in microperfusion techniques, the isolated perfused kidney, and
Phosphate Reabsorption BBMVs to assess whether the increased tubular reabsorption
of phosphate is related to differences in the transport of
In contrast to most other transport processes, fractional phosphate at the brush-border surface of the proximal
reabsorption of phosphate is greater in the immature or tubule. The fractional reabsorption of phosphate is higher in
neonatal animal than it is in the adult (Table 3.4) (87). the newborn than it is in the adult guinea pig (Table 3.4)
Clearance studies performed in infants and children ini- (90,91). The proximal tubular reabsorption is 77% of the fil-
tially suggested that the increased plasma phosphate levels tered load in the newborn and 67% in the adult guinea pigs.
and reduced fractional excretion of phosphate during The distal reabsorption of phosphate in newborns is 16%
infancy are related, in part, to the lower GFR during the and in mature guinea pigs is 11%. Both proximal and distal
first year of life (88). However, subsequent studies per- tubular phosphate reabsorption are greater in newborns. This
 3. Tubular Function 57

is remarkable in light of the relative structural immaturity of

the newborn proximal tubule. Compared with adults, the
maximum rate of phosphate reabsorption is greater in new-
born guinea pigs (92). There is no significant difference in
affinity (Km) of the transporter for phosphate. BBMVs from
2-month-old and 8- to 9-month-old rats demonstrate no dif-
ference in the activity of sodium phosphate cotransport (89).
However, newborn rats have enhanced phosphate uptake
due to changes in the affinity of the transporter (Km) for
phosphate (90). Therefore, greater apical membrane density
rather than conformational changes or structural isomers of
the sodium phosphate cotransporter probably accounts for
the greater phosphate absorption characteristic of the devel-
oping animal.
Increased phosphate reabsorption by the developing ani- FIGURE 3.9. Age-related changes in the effect of dietary
mal or child might be explained by relative insensitivity to phosphate on maximal capacity for phosphate reabsorption in
thyroparathyroidectomized rats given low-, normal-, and high-
the phosphaturic effect of PTH. The fractional excretion of phosphate diets. GFR, glomerular filtration rate; Pi, inorganic
phosphate after administration of PTH was lower in 6- phosphate; RPI, resting pressure index. (From Mulroney SE,
week-old rats than in 20-week-old rats. However, studies Lumpkin MD, Haramati A. Antagonist to GH-releasing factor
inhibits growth and renal Pi reabsorption in immature rats. Am
indicate that the greater phosphate reabsorption character- J Physiol 1989;257:F29–F34, with permission.)
istic of the newborn is intrinsic to the immature tubule and
is independent of PTH. In parathyroidectomized rats, the
capacity for phosphate reabsorption was greatest in the either low- or high-phosphate diets, depending on whether
immature, or 3-week-old, animals and decreased with the whole organ or the proximal tubule is studied. Studies
growth and development (91). Parathyroidectomy is of whole-kidney phosphate reabsorption in the rat indicate
accompanied by a greater increase in the maximal tubular that the immature kidney has enhanced capacity to respond
reabsorption of phosphate in immature rats than in the to both decreases and increases in dietary phosphate and
mature rats (89). In the presence of parathyroid glands, the the filtered load of phosphate (Fig. 3.9) (89,91,95). Imma-
phosphate reabsorptive capacity was lower in all groups, ture rats receiving low-phosphate diets had greater phos-
but a decline in capacity was still noted as rats matured. phate reabsorption than either immature animals receiving
Therefore, it appears that there is an enhanced intrinsic normal dietary phosphate or mature animals on low-phos-
ability of the immature kidney to reabsorb phosphate, phate diets. In contrast, a low-phosphate diet did not
which is not simply related to noted maturational differ- induce any changes in phosphate uptake by BBMVs from
ences in PTH action on the renal tubule. PTH receptor– the newborn guinea pig, nor did a high-phosphate diet
associated adenylate cyclase is less responsive to PTH stim- decrease phosphate uptake. The newborn animals experi-
ulation in the immature renal tubule than in mature con- enced greater fluctuations in plasma phosphate (92). Proxi-
trols; however, hormone responsiveness in the rat increases mal phosphate transport by immature rats, as assessed by
threefold during the first several weeks of life (93). BBMV studies, is greater than that of adults, yet there is
The rate of phosphate transport is also regulated by the diminished ability of the developing rat to sustain plasma
availability of phosphate. Dietary phosphate deprivation phosphate as dietary phosphate is changed. These develop-
results in enhanced reabsorption of phosphate (92). A par- mental changes in phosphate handling are analogous to
allel has been drawn between the phosphate-replete state of those described for taurine (see earlier). Although imma-
the immature kidney and the adult kidney during phos- ture animals have similar intrinsic transporter function
phate depletion, because in both conditions phosphate with greater relative capacity, the ability of the renal proxi-
reabsorptive capacity is enhanced independent of PTH mal tubule to adapt to its environment is diminished.
(91,94). Factors observed to contribute to the enhanced The ability to respond to alterations in the filtered load
reabsorption of phosphate during dietary deprivation of phosphate does not require PTH (91,95,96). The stimu-
include increased activity of the brush-border transport sys- lation of phosphate reabsorption in the immature animal
tem, significant phosphate reabsorption in both distal and may be related to reduced intracellular levels of phosphate
proximal tubules, and decreased responsiveness to PTH (97). Intracellular phosphate as measured by nuclear mag-
(90). As mentioned, these same factors contribute to the netic resonance is lower in 3-week-old rats than in 12-
greater increase in phosphate reabsorption observed in the week-old rats (97). The investigators postulate that a low
young kidney. intracellular phosphate concentration in developing rats
Conflicting results have been obtained from the study of may stimulate synthesis and expression of phosphate trans-
the response of the newborn rat and guinea pig kidney to porter proteins.
58 I. Development

Growth hormone also may regulate renal phosphate TABLE 3.5. ORGANIC ANIONS
reabsorption during the neonatal period (96). Long-term
p-Aminohippuric acid Phenol red
administration of the growth hormone antagonist (GRF- Ascorbic acid Phenylbutazone
AN) to immature and mature rats resulted in growth failure Benzyl penicillin Probenecid
and increased urinary phosphate excretion in the immature Carbenicillin Riboflavin
rats only. Immature rats (4 weeks old) receiving GRF-AN Folic acid Salicylic acid
Methotrexate Sulfisoxazole
exhibited decreased tubular capacity for phosphate reab-
Nicotinic acid Taurocholic acid
sorption, whereas adult animals did not. These data indicate Pantothenic acid Uric acid
that the release of growth hormone during development Phenobarbital
may contribute to the elevated phosphate reabsorption char-
acteristic of this period (95,98). Insulin-like growth factor-1
induces the increase in activity of the sodium phosphate
transporter and prevents the decrease observed after GRF- A wide variety of organic anions are secreted by this trans-
AN administration (98,99). Preliminary studies suggest a port system (Table 3.5). Although the secretion of all
role for tyrosine kinases in transduction of hormone-trans- organic anions occurs in the proximal tubule, the rate of
porter events (99). secretion by different proximal segments varies, depending
In summary, the renal retention of phosphate in the on the compound and on the species studied (103). In the
newborn is not an expression of an immature transport sys- rat and the rabbit, PAH secretion is greatest in the straight
tem; rather, it is an appropriate physiologic adaptation to tubule, specifically the S2 segment, whereas in the pig, it is
the demands for phosphate during a period of rapid greater in the convoluted portion of the proximal tubule
growth. The postnatal changes in phosphate reabsorption (103). These differences in the secretory patterns probably
do not necessarily depend on GFR, PTH, or dietary phos- are related to differences in transporter density on the baso-
phate. They may be regulated by growth hormone and lateral membrane of the tubule (104).
insulin-like growth factor-1, as well as by intracellular Other factors that influence the excretion of organic
phosphate concentration. anions include the degree of protein binding, the competi-
tion by other similar compounds for the same basolateral
transport system, and the metabolic state of the animal,
ORGANIC ANIONS because many of these compounds are metabolic interme-
diates (103).
The proximal renal tubule is the primary site for the major
organic anion transporters hOAT1 and hOAT3 (100).
Cellular Mechanisms
PAH is the prototypical substrate for the organic anion
transport pathway. Organic anions may be filtered, reab- Secretion of organic anions is accomplished by the coopera-
sorbed, and secreted by the nephron. Excretion of a given tive interaction of several cotransport and countertransport
anion is the sum of filtration and secretion less reabsorp- systems located on the basolateral and apical membrane sur-
tion. There is a great deal of variability in the renal han- faces (Fig. 3.10A) (101,103). As the interior of the cell has a
dling of organic anions, depending on the species and substantial net negative charge, the uptake of anions occurs
organic anion studied (101). In humans, the major trans- against a steep electrochemical gradient. The basolateral trans-
porters are in the proximal tubule (100), whereas in mice, port of PAH is sodium- and energy-dependent but, as a ter-
they are present in proximal tubule, Henle’s loop, and corti- tiary active transport system, is not directly coupled to either
cal collecting duct (102). These anions may undergo bidi- sodium or energy expenditure. PAH enters the basolateral cell
rectional transport, with secretion being the predominant surface via an anion exchanger; ketoglutarate is thought to be
direction of flux. Many of the organic anions are highly the principal anion exchanged for PAH. This anion exchanger
protein bound and are not filtered to any extent at the is coupled to a basolateral sodium-anion (ketoglutarate)
glomerulus. Rather, these compounds may reach luminal cotransporter that allows the energy from ATP hydrolysis to
fluid via transcellular transport movement across basolat- be coupled to that of the anion exchanger through the recy-
eral and apical membranes. PAH is secreted at a very high cling of ketoglutarate at the basolateral membrane. This pro-
rate; as much as 90% is extracted from plasma by the prox- cess is similar to the apical C1-formate exchanger (see Sodium
imal tubule in a single pass through the kidney. Because of and Chloride: Whole Nephron). Once inside the cell, the
this characteristic, PAH is used as a marker of renal blood organic anion diffuses through the cytoplasm and exits the
flow. The urinary levels of PAH increase along the length of apical side of the cell through another anion exchanger. The
the proximal tubule (Fig. 3.2). substrate specificity of the apical anion exchanger differs from
Tubular capacity for the secretion of organic anions that of the basolateral anion exchanger. Ketoglutarate is the
increases as the plasma concentration increases, up to a preferred substrate for exchange with PAH on the basolateral
maximum at which the transport system is saturated (101). surface, but many anions (lactate, pyruvate, hydroxybutyrate,
 3. Tubular Function 59

on the basolateral membrane (100,102). The rat kidney

transporter (OAT1) has 551 amino acid residues with 12
proposed transmembrane domains (105). OAT1 has wide
substrate specificity, including cyclic nucleotides, prosta-
glandins, uric acid, and a variety of drugs. Expression is
limited to an isolated basolateral segment of the proximal
tubule (105). OAT1 shows approximately 38% structural
homology to the organic cation transporter (OCT1). The
human kidney transporter (hOAT3) has 543 amino acids
and is localized to the basolateral membrane (106), with
comparable structural homology.

Developmental Changes in
Organic Anion Transport
The secretion of organic acids is low during the neonatal
period; it increases gradually over the first few years of life
(107,108). Limitations on the excretion of organic acids
include low GFR, anatomic immaturity, reduced number
of transporter sites, and incomplete metabolic capacity. In
addition, the limited distribution of blood flow to the deep
cortical structure reduces the concentration of organic acids
available for secretion.
FIGURE 3.10. A: Organic anions [p-amino hippurate (PAH)]
enter the proximal tubular cell on the basolateral surface by an The kidney’s ability to produce a concentration gradient
anion exchanger (3) in association with a sodium anion cotrans- of organic anions is present before birth and increases with
porter (4), which allows recycling of the anion across the baso- maturation. Studies in newborn humans, dogs, and rats
lateral membrane. PAH exits the cell by another anion
exchanger (2) with different substrate specificity than the baso- demonstrate a gradual increase in renal extraction of PAH
lateral system. The apical anion exchanger is also associated with age (109–111). When PAH transport is examined
with an anion sodium cotransport system (1). B: Organic cations using tissue slices, the slice:medium ratios (in rats) peak at
[tetraethylammonium (TEA)] enter the proximal tubular cell on
the basolateral surface by one of two mechanisms: cation approximately 1 month of age and then decline to adult
exchange (2) or passive, facilitated diffusion (1). TEA exits the values. The PAH extraction (GFR) of infants 3 months or
luminal membrane via a proton exchanger (4) that operates in younger is approximately 30% that of older children.
association with a sodium-proton exchanger to allow recycling
of a proton across the luminal membrane. (Adapted from Roch- The increased secretory ability that occurs with matura-
Ramel F, Besseghir K, Murer H. Renal excretion and tubular tion is the result of the intrinsic ability of the proximal tubule
transport of organic anions and cations. In: Windhager EE, ed. to transport organic acids without appreciable changes in the
Handbook of physiology: renal physiology. New York: Oxford
University Press, 1992:2179–2262.) membrane permeability to PAH. When organic acid trans-
porter activity is standardized for tubule length, an almost
fivefold increase in transport capacity occurs with maturation
acetoacetate, succinate, ketoglutarate, urate, hydroxyl ions, (108). Both increased transporter density and tubular length
bicarbonate, and chloride) may be exchanged for PAH by the contribute to the increased capacity for organic acid secretion
apical exchanger. In addition to the anion exchanger on the that occurs with development.
apical surface, there is a sodium-coupled anion transporter In addition to age-related changes in transporter capac-
that reabsorbs organic substances such as ketoglutarate for the ity, organic anion transport is regulated by substrate avail-
reabsorption of metabolic substrates, as well as one specific for ability and hormones (112,113). The administration of
ketoglutarate that may be exchanged for PAH by the basolat- organic acids induces the transport system (112,114).
eral anion exchanger. Therefore, the net movement of PAH is Administration of penicillin to 2- and 4-week-old rabbits
from peritubular fluid to luminal fluid, and the net move- resulted in enhanced basolateral uptake and excretion of
ment of the anion ketoglutarate is the reverse. Lithium inhib- PAH in the 2-week-old, but not in the 4-week-old, rabbits
its the sodium-organic anion cotransporters, and probenecid (112). Administration of the drugs to pregnant rabbits dur-
inhibits the anion exchangers. ing the later part of pregnancy results in enhanced newborn
PAH transport. The ability to induce transport activity
depends on the species and the anion studied.
Molecular Characterization
Premature infants have a reduced renal ability to excrete
The basolateral organic anion transporter system accom- organic anion drugs because of the immaturity of the
plishes organic anion-dicarboxylate exchange and is found organic anion transport system. Thyroxin administration to
60 I. Development

TABLE 3.6. ORGANIC CATIONS The fetal kidney is able to secrete organic cations; the
ability to do so is probably less than that in adult animals
Amiloride Norepinephrine
Amphetamine Procainamide (108,109,117,118). In general, the capacity to secrete
Atropine Pseudoephedrine organic cations precedes that of organic anions (101,103).
Chloroquine Quinine
Cimetidine Tetraethylammonium
Histamine Thiamine
URIC ACID
Levamisole Tolazoline
Methadone Triamterene
Morphine Uric acid, a weak organic acid, is the biologic end-product of
dietary and endogenous purine metabolism. Approximately
75% is excreted by the kidney. Urate is freely filtered at the
glomerulus and is transported exclusively by the proximal
young rats increases PAH uptake (115). This increased tubule (119). In a number of species (human, monkey, rat,
uptake is blocked by inhibitors of protein synthesis, sug- mongrel dog), the fractional excretion of urate is less than
gesting that the thyroxin effect depends on synthesis of reg- 100%, indicating net reabsorption. In other species (pig and
ulatory or transporter proteins. rabbit), the fractional excretion of urate exceeds 100%, indi-
cating net secretion. The degree to which there is secretion or
reabsorption varies and depends on the species studied (119).
ORGANIC CATIONS Urate reabsorption is inhibited by probenecid, high luminal
fluid flow rate, hexose, and anion exchange inhibitors. Urate
Organic cations include endogenous primary, secondary, ter- secretion is inhibited by pyrazinoic acid, the active metabo-
tiary, and quaternary amines in addition to many drugs that lite of the antituberculous drug pyrazinamide (120). The
are secreted by the proximal tubule (103). A partial list of decrease in urinary excretion of urate after the administration
organic cations is found in Table 3.6. Tetraethylammonium of pyrazinamide or its metabolite pyrazinoate was initially
(TEA) is the model substrate for describing the organic cat- thought to be related to a selective effect of the drug on the
ion system. The clearance of TEA is similar to that of PAH; secretory component of urate excretion. However, recent
there is nearly complete extraction with a single pass through work suggests that pyrazinoate may inhibit the reabsorptive
the kidney. Secretory fluxes are greatest in the proximal con- component in addition to the secretory pathway. Thus, the
voluted (S1) segment and decrease in S2 and S3 (116). pyrazinoate-suppressible excretion of urate is not totally
Changes in the activity of the organic cation transport sys- caused by inhibition of the secretory component (120,121).
tem appear to be related to regional differences in luminal Urate handling along the proximal tubule is theorized to pro-
permeability to TEA along the proximal tubule and are not ceed as follows: Initial reabsorption of urate occurs in the
caused by changes in basolateral cation uptake (101). early proximal convoluted tubule (S1), and secretion occurs
in the late convoluted, early straight tubule (S2) via a secre-
tory pathway similar to that for PAH (121). Postsecretory
Cellular Mechanisms
reabsorption may occur but has not been well characterized.
Organic cations move into the negatively charged cell inte- Clearance studies performed in human subjects demon-
rior by a saturable, electrogenic process that is located on strated that blocking reabsorption prevented an increase in
the basolateral cell surface. Some organic cation uptake is excretion that would be expected to follow probenecid
through facilitated diffusion (Fig. 3.10B) (101,103). A administration (103).
luminal organic cation-proton antiporter that uses poten-
tial energy stored in a pH gradient to drive uphill move-
Cellular Mechanisms
ment of cations has been demonstrated in human, snake,
dog, rat, and rabbit BBMVs. Cations diffuse through the Because of the bidirectional nature, axial heterogeneity, and
cell cytosol and are either taken up into cell organelles or species-related variability of urate transport, any model
vesicles or exit through a luminal proton exchanger. At the describing urate transport is complex. Most likely, passive,
luminal membrane, TEA is exchanged for a proton. This is paracellular, and active transcellular transport mechanisms
another example of a tertiary transport process: The energy- affect urate secretion and reabsorption. A urate anion
requiring process is the Na+-K+-ATPase, which creates the exchanger has been demonstrated in rat and dog BBMV
major driving force for the apical Na+-H+ exchanger. In (121–123). This system may use the hydroxyl, Cl–, HCO3–,
turn, the Na+-H+ exchanger provides protons for apical lactate, or succinate anion in exchange for urate. This system
exchange with TEA. The luminal proton-TEA exchange is is specific for urate, but PAH is also accepted. Probenecid
electroneutral. Therefore, the net movement of TEA is inhibits urate transport by this system (121–123). A sodium-
from the capillary fluid to the lumen; protons recycle across urate cotransport system has been proposed, because the
the apical membrane. reabsorption of urate is closely coupled to sodium in studies
 3. Tubular Function 61

involving intact animals and isolated perfused tubules. Such

a system has not been demonstrated in isolated BBMVs.
Most likely, urate transport is sodium dependent through the
sodium-dependent cotransport of organic anions or dicar-
boxylates and is linked as a tertiary transport process to
sodium flux.
An anion exchange system for urate has been described
in basolateral membrane vesicles from the rat; this trans-
port system is postulated to be distinct from the apical
transport system because PAH and other organic anions do
not interact with it (124).
The initial step for urate reabsorption is the anion
exchanger at the apical membrane, with exit on the basolat-
eral membrane by another anion exchanger or by a passive
mechanism. Secretion of urate occurs by basolateral
exchange of urate for chloride, with secretion by the apical
FIGURE 3.11. Fractional excretion of uric acid (clearance of
anion exchanger. The differential distribution of these two uric acid/creatinine clearance ×100) during the first day of life. A
systems along the proximal tubule probably determines the significant inverse relationship was observed between the frac-
net flux of urate in a given segment. In the S1 segment, tional excretion of urate and gestational age. (From Stapleton
FB. Renal uric acid clearances in human neonates. J Pediatr
reabsorption predominates; in the S2 segment, secretion 1983;103:290–294, with permission.)
(pyrazinamide-sensitive) predominates. Both fluxes could
be present in the S3 segment, with the net flux being in the
direction of reabsorption in the human (119). tion is approximately 10% (131). Sulfate is transported into
The human urate transporter (uHAT) has been cloned the proximal tubular cell by a luminal, sodium-dependent
and it has two membrane-spanning domains. It acts as a process with a stoichiometry of 2 Na+ to 1 SO4– (Fig. 3.12).
highly selective urate ion channel. A second urate trans- The system is electroneutral and requires a proton. The pro-
porter (uHAT2) has also been cloned (125). ton acts as an allosteric activator of the transport system and
modifies the stoichiometry of the transporter with respect to
Developmental sodium. With an acidic internal pH, stoichiometry is 2:1; in
an alkaline environment, it is 1:1 (132,133). On the basolat-
The fractional excretion of urate is much higher in infants eral cell surface, sulfate is transported by an anion exchanger
than in adults or older children. Plasma levels tend to be that is sodium independent. Sulfate is exchanged for another
higher in premature and term newborns than in older chil- anion (hydroxyl radical, bicarbonate, or thiosulfate). This
dren and adults (126). Urate fractional excretion is 70% at a anion exchanger does not accept Cl–, phosphate, or other
gestational age of 29 to 31 weeks and 38% at term (127– organic anions such as PAH or lactate, but it is inhibited by
129). The fractional excretion of urate decreases further dur-
ing the early postnatal period, and this effect appears to be
independent of gestational age (Fig. 3.11). Uptake of urate
by neonatal kidney slices and by fetal and neonatal mouse
kidney cortex slices is lower than uptake by slices from adult
animals (128). This probably indicates decreased ability for
urate secretion in the immature rodent (127,129). Therefore,
the increased fractional excretion probably is not related to
increased secretion. Instead, increased excretion of urate
could be related to the relative volume-expanded state of the
infant, as well as to the potential immaturity of reabsorptive
pathways (130). Maturation of the superficial renal cortex,
with its greater reabsorptive capacity, could also contribute to FIGURE 3.12. Model of sulfate transport by the proximal
the postnatal decline in fractional urate excretion (127). tubule. Sulfate is cotransported with two sodium ions at the
brush-border membrane (1) and exits the cell by way of passive,
facilitated diffusion on the apical membrane (backleak, 2) or by
a sulfate-anion exchanger (3) on the basolateral surface of the
SULFATE cell. (From Murer H, Manganel M, Roch-Ramel F. Tubular trans-
port of monocarboxylates, Krebs cycle intermediates and inor-
ganic sulfate. In: Windhager EE, ed. Handbook of physiology:
Sulfate and thiosulfate are freely filtered at the glomerulus. renal physiology. New York: Oxford University Press, 1992:2164–
Both are reabsorbed by the proximal tubule; fractional excre- 2188, with permission.)
62 I. Development

typical anion transporter inhibitors, SITS and DIDS. Lumi-

nal sodium sulfate cotransport enables significant luminal
chloride transport in the proximal tubule, through the coor-
dinated luminal oxalate-sulfate exchanger and oxalate-chlo-
ride exchange systems.

CARBOXYLATES

The renal tubule has several specific transport systems for

the reabsorption and absorption of monocarboxylates,
dicarboxylates, and tricarboxylates (131). These substrates
are divided into the monocarboxylates (lactate, pyruvate,
acetoacetate, hydroxybutyrate, and fatty acids) and dicar-
boxylates/tricarboxylates (citrate, succinate, ketoglutarate,
oxaloacetate, and fumarate) (131). Most of these com-
pounds are metabolic intermediates or substrates that are
important energy sources, hence the need for absorption
and reabsorption, respectively.
Reabsorption of these compounds is predominantly in
the proximal tubule. In general, tubular reabsorption is
incomplete, which may be the combined result of incom- FIGURE 3.13. A: Model for monocarboxylate transport. Mono-
plete absorption by the apical membrane surface or the carboxylates enter the proximal tubular cell by one of two mecha-
nisms: either a sodium cotransporter (1), where X = lactate,
efflux of these substrates from the cell back into the lumen pyruvate, acetoacetate, or β hydroxybutyrate, or by an anion
when the intracellular concentrations are high (131). exchanger (2) with different substrate specificity (Y = OH –, Cl–,
urate, bicarbonate, p-aminohippurate, ketoglutarate, lactate,
pyruvate, acetoacetate, or β hydroxybutyrate). On the basolat-
Cellular Mechanisms eral membrane, an anion exchanger enables the exit of these sub-
stances (3) (Z = lactate, pyruvate, OH–, formate, Cl–, sulfate,
Monocarboxylates phosphate, or oxalate). B: Model for dicarboxylate/tricarboxylate
transport. The substances enter the luminal membrane of the cell
There are both apical and basolateral transport processes by a sodium cotransport system (1) (Z = pyruvate, succinate, cit-
rate, malate, fumarate, or ketoglutarate) or an anion exchanger
for the monocarboxylates (Fig. 3.13A). Reabsorption (2) (bicarbonate, lactate, pyruvate, acetoacetate, β hydroxybu-
from luminal fluid is by an apical-specific transporter that tyrate, malate, or succinate). In addition, a sodium cotransport
is sodium dependent and is not inhibited by other organic system (3) similar to the apical system enables the basolateral
transport of these substrates. (From Murer H, Manganel M, Roch-
compounds such as PAH, urate, or dicarboxylates. The Ramel F. Tubular transport of monocarboxylates, Kreb’s cycle
transporter requires a single carboxyl group, but it accepts intermediates and inorganic sulfate. In: Windhager EE, ed. Hand-
both the D- and the L-isomer of the carboxylate. Studies book of physiology: renal physiology. New York: Oxford Univer-
sity Press, 1992:2164–2188, with permission.)
using BBMVs indicate that there may be both a high-
affinity/low-capacity system and a low-affinity/high-
capacity system (131,134).
In addition to the sodium-monocarboxylate cotransport tional and allows the entry as well as the exit of monocar-
system, there is an apical anion exchange system. This sys- boxylates. This system can affect the net reabsorption of
tem accepts a variety of anions (Cl–, hydroxyl, bicarbonate, monocarboxylates from luminal fluid and release into peri-
urate, PAH, and dicarboxylates). It is sensitive to SITS and capillary fluid, as well as effect basolateral uptake of these
DIDS, as well as furosemide and probenecid, indicating compounds as substrate for cell metabolism. The basolat-
that this organic anion exchanger is nonspecific. This sys- eral transport system is located in the S2 and S3 segments,
tem probably functions to facilitate exit of metabolic inter- which do not have an apical transport system for the mono-
mediates and the shuttling of protons (formate-Cl carboxylates. In these segments, the transport system is cru-
exchanger), which are necessary for the electroneutral cial to the uptake of monocarboxylates as substrates needed
absorption of NaCl. for cell energy metabolism.
The basolateral system is sodium independent and spe-
cific for the D-isomer of lactate. The basolateral system is
Dicarboxylates/Tricarboxylates
thought to be an anion exchanger that exchanges lactate for
the hydroxyl ion or bicarbonate in a fashion similar to that The model substrate for the transport of dicarboxylates/
described for sulfate (135–137). This system is bidirec- tricarboxylates is α-ketoglutarate. Approximately 75% of
 3. Tubular Function 63

the filtered load is reabsorbed by the proximal tubule, 20% in the S1, and sodium channels are more important in the
of which is by the S3 segment. Secretion also has been dem- S2 and S3 segments (144). Active Na+ transport occurs
onstrated in the rat (138). Ketoglutarate reabsorption is across the apical membrane by the coupling of Na+-H+
effected by cotransport with sodium (stoichiometry 3:1) exchange and the Cl–-formate exchanger (147).
(Fig 3.13B) (139). This system accepts all the Krebs cycle A significant amount of chloride reabsorption occurs in
intermediates as well as pyruvate, which is actually a mono- the proximal tubule by passive diffusion of Cl; the rate of
carboxylate (140). Sodium must bind to the transporter reabsorption is lower in the early proximal tubule, in which
protein to induce a conformational change that facilitates sodium and bicarbonate reabsorption are favored. As TF
binding and transport of substrates. Lithium inhibits this moves distally, it becomes acidified, and the chloride con-
process by binding in place of sodium (141,142). The same centration rises to a level greater than that of plasma (Fig.
system is located on the basolateral surface of the cell to 3.2). The transtubular PD is negative in the early proximal
effect uptake of key substrates from peritubular fluid. The tubule, but it becomes lumen positive later in the proximal
presence of arginine-349 and aspartate-373 of the Na+/ tubule. In the early proximal tubule, the major driving
dicarboxylate cotransporter is important in the conforma- force for passive Cl reabsorption is the lumen-negative PD
tional states of the transport cycle (143). There is an addi- and, in the later proximal tubule, an outwardly directed Cl–
tional anion exchanger that has affinity for many substrates concentration gradient. Active Cl reabsorption also occurs
and is probably the same transport system as that previ- in the proximal tubule by tertiary active transport, because
ously mentioned for the monocarboxylates. the driving force is generated by one of two secondary
active transport systems: the Na+-H+ exchanger in parallel
with Cl– formate or the Na-sulfate exchanger in parallel
Sodium and Chloride: Whole Nephron
with Cl– oxalate. Cl– transport is coupled indirectly to H+
Approximately 60% of the filtered sodium and 50% of fil- transport because the cell concentration of formate is
tered chloride is reabsorbed by the proximal tubule (144). affected by pH, and hydrogen ions are pumped out of the
The preferential reabsorption of amino acids, glucose, cell in exchange for sodium. In the case of Cl-oxalate
phosphate, and bicarbonate in the early proximal tubule exchange, oxalate is accumulated in exchange for sulfate,
creates a TF NaCl concentration greater than the plasma which is cotransported with sodium. Renal potassium-
concentration. This augments the reabsorption of NaCl chloride cotransporters, which are electroneutral, are found
through both active and passive processes (144,145). The in the proximal tubule and thick ascending limb (148).
passive reabsorption of NaCl through a paracellular path- Because Na-H exchange is vital in both NaHCO3 and
way accounts for two-thirds of the total NaCl flux in the S2 NaCl reabsorption, regulation of Na-H activity could be
and S3 segments (144). In the early proximal tubule, a expected to affect both processes. In the setting of meta-
lumen-negative potential resulting from sodium-coupled bolic acidosis, a need for enhanced NaHCO3 absorption
absorption of substrates such as glucose and amino acids results in upregulation of Na-H exchange via increased
favors passive paracellular Cl transport. Sodium bicarbo- expression of NHE3 protein. NaCl transport would be
nate reabsorption via the luminal Na-H exchange is accom- expected to increase as well, but decreased formate-induced
panied by fluid absorption so that the luminal Cl NaCl transport, decreased oxalate-induced NaCl transport,
concentration increases in the late proximal tubule favoring and Na-sulfate cotransport are observed during acidosis,
Cl absorption, which creates a positive luminal charge that thereby counteracting the increase in Na-H exchange activ-
favors passive transcellular sodium absorption in the late ity on net Cl reabsorption (149). Phosphorylation of
proximal tubule (145). Additional Cl absorption is serine-982 in the Na+ bicarbonate cotransporter shifts the
achieved via apical Cl exchange with formate or oxalate. HCO3–:Na+ stoichiometry from 3:1 to 2:1 (150).
This chloride-formate exchanger, expressed in the brush-
border membrane, can also be found in the pancreas and
Na+-H+ Exchanger
heart (146).
The Na+-H+ exchanger is responsible for the bulk of
NaHCO3 and NaCl reabsorption in the proximal tubule
Cellular Mechanisms: Proximal Tubule
(147). The Na+-H+ exchanger requires Na+ and H+ and is
Three apical transport systems operate in conjunction with inhibited by amiloride in concentrations lower than those
the basolateral Na+-K+-ATPase transport system to effect required for inhibition of the Na+-K+-ATPase but higher
sodium reabsorption (Fig. 3.14) (147): the Na+-H+ than those required for inhibition of sodium channels
exchanger; the sodium-coupled transport with amino acids, (149). The antiporter accepts NH4+, Li+, Na+, and H+ but
phosphate, glucose, sulfate, and organic acids; and the does not accept K+, Rb+, Cs+, or choline (23). Na+, Li+,
sodium channels. The Na+-H+ exchanger accounts for the NH4+, and H+ compete for the external binding site. There
greatest fraction of reabsorbed sodium in the S1-3 segment. are two internal cation-binding sites; one is for transport
Sodium-coupled transport mechanisms are more important and the other binds hydrogen ions as a means of regulating
64 I. Development

FIGURE 3.14. The Na-(K)-Cl cotransport-

ers. A: Localization of cloned Na-(K)-Cl
cotransporters along the mammalian neph-
ron. B: Schematic model of the topology of
the Na-(K)-Cl cotransporters. The proposed
membrane-spanning helices are numbered
1 to 12. Putative N-glycosylation sites on the
loop between membrane spans 7 and 8, and
the amino-(NH2) and carboxy-(COOH) ter-
mini are indicated. DCT, distal convoluted
tubule; IMCD, inner medullary collecting
duct; TAL, thick ascending limb. [From
Hebert SC, Gamba G, Kaplan M. The electro-
neutral Na+-(K+)-Cl-cotransport family. Kid-
ney Int 1996:1638–1641, with permission.]

the activity of the transporter (151). Increases in the hydro- Several isoforms of the Na+-H+ exchanger have been
gen ion concentration induce protonation of the regulatory characterized. The eukaryotic housekeeping isoform gene,
site and activation of the antiporter. Increasing intracellular NHE1, encodes a protein that is composed of 10- to 12-
pH inactivates the exchanger (23). Therefore, acidification membrane–spanning α helical segments and a large hydro-
of the cell cytosol activates the Na+-H+ exchanger by provi- philic C-terminal domain with potential phosphorylation
sion of protons and by allosteric activation. sites (145). The housekeeping form is expressed only on the
The activity of the exchanger is regulated by acidosis, basolateral membrane of proximal tubule segments. A total
renal ablation, thyroxine, glucocorticoids, angiotensin II, of five isoforms, NHE1-5, have been identified, with pre-
catecholamines, and PTH (145,147). Activation of the Na+- dicted proteins that have highly conserved sequences except
H+ exchanger occurs either through the action of tyrosine- for the C-terminal domains. All isoforms except NHE5 are
specific kinases or through activation of protein kinase C. found in the kidney and intestine. NHE3 is specifically
Epidermal growth factor, platelet-derived growth factor, and expressed on the brush-border membrane of the proximal
insulin activate the Na+-H+ exchanger through the tyrosine- tubule, and expression is increased after acid loading (145).
specific kinase. Thrombin, angiotensin II, vasopressin, and This isoform appears to be unique in that it is resistant to
α-adrenergic agonists activate the Na+-H+ exchanger via pro- amiloride. In addition, apical membrane expression of
tein kinase C (152–154). The Na+-H+ exchanger also main- NHE3 increases during maturation and after administra-
tains intracellular pH and the regulation of cell growth. The tion of glucocorticoids (156).
Na+-H+ exchanger is regulated in parallel with regulation of
the basolateral system that is involved with bicarbonate reab-
Chloride Formate Exchanger
sorption and the Na-3HCO3 cotransporter. The latter also
increases its transport capacity in response to chronic acidosis The Cl–-formate exchanger is a tertiary transport system
(155). The turnover of both transporters probably is regu- that recycles a base across the membrane and couples pro-
lated by the need for transepithelial bicarbonate transport or ton secretion and sodium reabsorption with chloride reab-
proton secretion. sorption. The base, which is usually formate, reenters the
 3. Tubular Function 65

cell, dissociates, and is again available for exchange with Cl–. Cs+, and NH4+ may be transported instead of K+. The
The Cl–-formate exchanger has been studied in both high-affinity site is specific for Cl– or Br–. Once the high-
BBMVs and intact proximal tubules (157–159). Addition affinity site is occupied, the remaining site is a low-affinity
of formate to luminal fluid increases NaCl reabsorption site that may accept Br–, Cl–, I–, nitrate, or thiocyanate
when the lumen pH is lower than the intracellular pH. The (166). This transport system provides the energy for coun-
effect of pH is related to the H+ gradient between luminal tercurrent multiplication, which concentrates solutes in the
fluid and cell fluid and the recycling of the necessary base. renal medullary interstitium, allowing generation of a con-
Other bases have been considered as substrates for this centrated urine. Regulation of urinary-concentrating ability
exchanger, such as OH–, bicarbonate, HCO3–, and oxalate is, in part, a function of this system. The Na+-K+-Cl– trans-
(160). Oxalate has recently been noted to contribute to net porter is inhibited by furosemide and bumetanide. A sepa-
Na reabsorption in a manner similar to formate, with anion rate secretory isoform of the Na-+K+-Cl– cotransporter
exchange at the apical membrane of the proximal tubule. (NKCCl) (BSG-2) localizes to the basolateral membrane of
Oxalate is recycled via oxalate-sulfate exchange in parallel the ∝ intercalated cell of the collecting duct. This form of
with oxalate-Cl exchange. the transporter serves to regulate cell volume, as well as to
facilitate electrolyte and water transport (167).
The transporter gene (NKCC2) has recently been
Basolateral
cloned (168). Homozygous mutations have been found to
Several different pathways for Cl exit at the basolateral account for the Bartter’s syndrome in numerous kindreds
membrane include KCl cotransport, Na-dependent (168). As a member of the electroneutral cation-chloride
Cl-HCO3 exchange, and Cl channels. Basolateral SITS— cotransporter family, this transport system serves both an
an anion exchange inhibitor—reduces NaCl reabsorption absorptive and a secretory function (cell volume regulation)
in rat proximal tubule by 50%; this suggests a role for a Cl– (169). Sequence analyses show that these cotransporters are
base exchange in the basolateral Cl– exit (147,161). The structurally similar, with 12 transmembrane-spanning heli-
process is sodium dependent with a stoichiometry of Na:2 ces and variable-length amino and carboxyterminal seg-
HCO3–:Cl– and probably is responsible for most basolateral ments, which are cytosolic (Fig. 3.14). A long extracellular
Cl– exit (147). Recent evidence indicates that the largest loop is proposed to be located between the seventh and
component of Cl movement across the basolateral mem- eighth transmembrane domains with potential glycosyla-
brane is via Cl channels (sensitive to diphenylamine-2-car- tion sites. There is approximately 50% homology between
boxylate) (145). the two Na+-K+-Cl– cotransporters and the thiazide-sensi-
tive Na-Cl cotransporter found in the distal tubule. Two
isoforms of the Na+-K+-Cl– cotransporter are found within
Cellular Mechanisms: Thick Ascending
the kidney. One is the absorptive form (BSC1), which is
Limb of the Loop of Henle
localized to the apical membrane of the cortical thick
Sodium reabsorption in this segment proceeds via a lumi- ascending limb of the loop of Henle. The more ubiquitous
nal Na+-K+-Cl– transporter that couples the reabsorption of basolateral form, BSC2, is found in chloride secretory epi-
1 Na+ and 1 K+ with 2 Cl– (Fig. 3.14) (128). Chloride thelia (169). In the kidney, BSC2 is localized to the basolat-
leaves the cell via a basolateral Cl channel or KCl cotrans- eral membrane of the inner medullary collecting duct and
porter (162,163). Potassium is recycled via K channels to the extraglomerular mesangium (juxtaglomerular) and to
located on the apical membrane. These K channels are a subpopulation of smooth muscle cells in the afferent arte-
inhibited by choline, ammonium, ATP, and verapamil and riole (167,169). The function of the BSC2 cotransporter in
are not regulated by Ca2+ (162). In addition, there is a the inner medullary collecting duct may be NaCl secretion
mechanism for potassium exit from the basolateral mem- through an ANF-inducible response. In addition, this
brane either by K+ channels or by the previously mentioned transporter may enable H+ and/or NH3+ secretion. The role
KCl cotransport system (164). Polarization of most K+ con- of the BSC2 transporter in the afferent arteriole and juxta-
ductance on the apical membrane and most Cl channels on glomerular region could be that of tubuloglomerular feed-
the basolateral membrane generates a lumen-positive charge back. Increased Cl delivery to the cortical thick ascending
that drives as much as 50% of total sodium reabsorption limb has been postulated to be detected by a furosemide-
through passive paracellular pathways (162). inhibitable Na+-K+-Cl cotransporter, suggesting a direct
The Na+-K+-Cl– cotransporter is an electroneutral trans- role of chloride as the signal between cortical thick ascend-
port system and is located on the apical surface of the thick ing loop chloride delivery and response from the macula
ascending limb of the loop of Henle and the inner medul- densa to alter afferent arteriolar tone (170). Recent studies
lary collecting duct. In the presence of sodium and potas- have indicated the precise distribution pattern of transport
sium, two Cl– ions are transported with 1 Na+ and 1 K+. proteins in the human distal tubule (171).
Lithium may be transported instead of sodium; however, The main regulating factors for the Na+-K+-Cl cotrans-
other cations may not substitute for sodium (165). Rb+, porter are the cAMP/cyclic guanosine monophosphate system,
66 I. Development

the state of phosphorylation (phosphorylation stimulates), and erally located Na+-K+-ATPase. Sodium enters the principal
cell shrinkage. Antidiuretic hormone stimulates the adenylate cell by way of highly selective, low-conductance Na+ chan-
cyclase system, which increases the rate of NaCl reabsorption nels located on the apical membrane (147). Sodium reab-
in the medullary thick ascending limb of the mouse. sorption is regulated acutely by antidiuretic hormone (175)
Enhanced luminal transport of NaCl occurs after insertion of and chronically by aldosterone and dietary sodium intake
additional transporter units into the plasma membrane (23). (176). The reabsorption of sodium in this segment creates a
In addition, urea at high concentration inhibits the Na+-K+- lumen-negative charge. There is no chloride reabsorption by
Cl– cotransporter independent of changes in cell volume the principal cell; rather, chloride is reabsorbed by a paracel-
(172). This is prevented by prior phosphorylation, which sug- lular route or via a transcellular pathway through intercalated
gests that exposure to urea results in dephosphorylation of the cells. In addition, both arginine and lysine enhance cortical
cotransporter or a closely linked regulatory protein. Urea collecting duct Na+ transport. Because lysine is not a nitrous
appears to alter cotransporter activity by decreasing the num- oxide (NO) precursor, this is a cationic amino acid effect
ber of functional transporters. Long-term saline administra- rather than a vasodilatory effect (177).
tion to rats increases the expression of the gene (BSC1) for the Comparison of the mechanisms of sodium reabsorption
Na+-K+-Cl– cotransporter. Long-term furosemide use also in the proximal tubule (S1-S3), loop of Henle, and the col-
increases gene expression, as well as possibly inducing glycosy- lecting duct reveals major differences. In the proximal
lation of the cotransporter (173). tubule, a large amount of sodium is reabsorbed at low
energy cost to the tubule because there is a substantial com-
ponent of passive paracellular sodium flux; however, the
Cellular Mechanisms:
ability of this segment to generate a concentration gradient
Distal Convoluted Tubule
is limited by the high sodium conductance (147). There-
The thiazide-sensitive NaCl cotransporter of the distal col- fore, a large fraction of sodium and chloride reabsorption
lecting tubule is the primary source of active Na reabsorp- takes place; however, because the luminal concentrations of
tion distal to the loop of Henle. This transporter has Na+ and Cl– are similar to their plasma concentrations, a
structural homology with the Na+-K+-Cl– cotransporter, as significant concentration gradient is not generated. In con-
previously mentioned. trast, sodium reabsorption in the collecting duct occurs by
Also contributing to luminal sodium entry are the sodium a transcellular route that is expensive with respect to cellu-
channels. A ubiquitous class of transporters sensitive to lar energy. However, in the absence of a significant paracel-
amiloride, Na channels are regulated acutely by cAMP- lular conductive pathway, the tubule can generate a large
dependent hormones such as vasopressin; G-proteins; and sodium concentration gradient. The thick ascending limb
intracellular Na+, Ca+, and H+, and chronically by aldoster- is a compromise between the proximal and collecting
one. There is a great deal of heterogeneity among amiloride- tubules; up to 50% of sodium reabsorption is passive.
sensitive sodium channels. They may be characterized by their
conductance, amiloride-binding affinity, and epithelial origin
Developmental Changes in Sodium
(174). Na channels are multimeric, with various combina-
and Fluid Transport
tions of subunits. Channel activity has been shown to be
modified by phosphorylation (cAMP dependent). The guan- The reabsorptive capacity for fluid in the proximal tubule
osine triphosphate-binding protein, GαI-3, is associated with increases approximately twofold from 3 to 6 weeks of age in
the channel protein complex and directly participates in the rat (178) in parallel with increased luminal and basolat-
channel activity. The G protein pathway is activated eral membrane surface area. The postnatal increase in reab-
through phospholipase- and lipoxygenase-generated phos- sorptive ability can be attributed to the increased activity of
pholipids. Nonphosphorylated channels are primarily closed. the Na+-K+-ATPase. The sodium-dependent proton flux in
Exposure to protein kinase A in the presence of ATP shifts isolated, perfused tubules from rabbits is approximately
channel activity to a more open position (174). Other cova- one-third of adult levels at 2 weeks of age and increases to
lent modifications of the Na channel that affect its activity adult levels by 6 weeks of age (179). Studies in both proxi-
include pertussis toxin-induced ribosylation and aldosterone- mal tubular cells in culture and BBMVs confirm that the
mediated carboxyl methylation. All covalent modifications Na+-H+ exchange system is less active in immature animals
are induced by exposure of the channel complex from the than it is in adults (180,181). This is related to differences
internal surface and the opposite site of amiloride binding in the abundance of the transport protein, because the Na+-
and depend on the state of phosphorylation (174). H+ exchanger from newborn rats has characteristics identi-
cal with those of the adult rats except that the transport
maximum (Vmax) is one-fourth that obtained from adult
Cellular Mechanisms: Collecting Tubule
rats (Fig. 3.15). Increases in Vmax occur after the adminis-
In this segment, sodium is reabsorbed by transcellular path- tration of glucocorticoids, indicating an increase in abun-
ways only (Fig. 3.14). The energy is provided by the basolat- dance of the transport protein (181).
 3. Tubular Function 67

proteins, in mouse nephron segments. J Am Soc Nephrol

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3. Schwartz GJ, Evan AP. Development of solute transport in
rabbit proximal tubule: I. HCO3– and glucose absorption.
Am J Physiol 1983;245:F382–F390.
4. Racusen LC, Monteil C, Sgrignoli A, et al. Cell lines with
extended in vitro growth potential from human renal proxi-
mal tubule: characterization, response to inducers, and
comparison with established cell lines. J Lab Clin Med
1997;129:318–329.
5. Humes HD, MacKay SM, Funke AJ, et al. Tissue engineer-
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6. Guder WG, Morel F. Biochemical characterization of indi-
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FIGURE 3.15. Developmental changes in Na+-H+ exchanger
of physiology: renal physiology. New York: Oxford University
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8. Aperia A, Fryckstedt J, Holtback U, et al. Cellular mecha-
This chapter has outlined various renal tubular transport nisms for bi-directional regulation of tubular sodium reab-
sorption. Kidney Int 1996;49:1743–1747.
processes that are vital to homeostasis. Subsequent chapters
9. Emanual JR, Garetz S, Stone L, et al. Differential expression
on water, acid-base, and potassium homeostasis detail those of Na+, K+-ATPase alpha- and beta-subunit mRNAs in rat tis-
tubular transport systems. Transepithelial movement of sol- sues and cell lines. Proc Natl Acad Sci U S A 1987;84:9030–
ute and fluid tends to be massive in the early parts of the 9034.
nephron with most of the renal tubular fine-tuning occur- 10. Beyth Y, Gutman Y. Ontogenesis of microsomal ATPase in
ring in the more distal segments. Recent advances in the the rabbit kidney. Biochim Biophys Acta 1969;191:195–197.
ability to characterize transporter proteins using molecular 11. Schmidt U, Horster M. Na-K-activated ATPase: activity
techniques has provided invaluable insights into the unique maturation in rabbit nephron segments dissected in vitro.
proteins that are arranged along the nephron in a polarized Am J Physiol 1977;233:F55–F60.
and highly segment-specific distribution. Distinct differ- 12. Larsson SH, Rane S, Fukuda Y, et al. Changes in Na influx
ences in protein structure allow kinetic variation along the precede post-natal increase in Na-K-ATPase activity in rat
renal proximal tubule cells. A study of cells in short primary
proximal tubule so that an impressive concentration gradi-
culture. Acta Physiol Scand 1990;138:243–244.
ent from cell to lumen is maintained, allowing nearly com- 13. Guillery EN, Huss DJ, McDonough AA, et al. Posttran-
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 4

PERINATAL UROLOGY
DAVID A. DIAMOND
CRAIG A. PETERS

The routine use of maternal-fetal ultrasound has intro- tion of some degree of hydronephrosis permits appropriate
duced the field of perinatal urology. Fetal hydronephrosis, postnatal evaluation, even if the responsible mechanism
with an incidence of 1:100 to 1:500 maternal studies, is the and precise level of obstruction are not discernible. Diag-
most common abnormality detected. Other urologic nostic accuracy of hydronephrosis in the fetus remains
abnormalities have been diagnosed prenatally as well, imperfect. An early report by Hobbins et al. suggested that
including renal cystic disease, renal agenesis, and even the correct prenatal identification of the site of obstruction
stones and tumors. For the pediatric urologist, these prena- could be confirmed postnatally in 88% of cases (6). Subse-
tal findings have opened a Pandora’s box of questions that quent studies reported fairly high false-positive rates rang-
challenge our understanding of normal and abnormal renal ing from 9 to 22% (3). The majority of false positives in
embryology and physiology. In this chapter, we discuss the these studies was nonobstructive causes of hydronephrosis,
diagnosis of prenatal urologic abnormalities, the rationale such as high-grade reflux or large, nonobstructed, extrare-
behind prenatal intervention, and our clinical experience in nal pelves or transient hydronephrosis.
managing children with prenatal urologic abnormalities. Accurate diagnosis of posterior urethral valves (PUV),
in which intervention might be considered, has proven
difficult. In one series, the false-positive rate was as high
DIAGNOSIS as 58% (7), but the criteria for diagnosing valves were
quite liberal and perhaps inappropriate. In another popu-
In a large prospective Swedish study, the incidence of prena- lation-based series, the sensitivity in detecting valves was
tally detected renal anomalies was 0.28% (1). Of these, two- as low as 23% (3). Diagnosis of obstructive conditions
thirds (0.18%) were hydronephrosis. A British study (2), in based on the criteria of increased renal echogenicity and
which 99% of the pregnant population in Stoke-on-Trent decreased amniotic fluid is very accurate (8). The distinc-
were scanned at 28 weeks’ gestation, demonstrated hydrone- tion between mild and moderate degrees of obstruction
phrosis prenatally in 1.40% of cases, which was confirmed and conditions such as reflux may be more challenging.
postnatally in 0.65% (2). These authors defined prenatal The clinical impact of these distinctions depends on
hydronephrosis as an anteroposterior (A-P) diameter of the whether intervention should be considered for anything
renal pelvis greater than 5 mm but noted the lack of consen- less than severe.
sus on the definition of antenatal hydronephrosis (3–5).
When an abnormality of the urinary tract is determined
Ureteropelvic Junction Obstruction
by maternal-fetal ultrasound, several questions should be
raised by the ultrasonographer and consulting urologist. The basic features of ureteropelvic junction obstruction
Combinations of specific findings direct the differential (UPJO) in the fetus include dilation of the renal pelvis and
diagnosis and permit more accurate prognosis and tailoring collecting system with no evidence of ureteral dilation (Fig.
of postnatal evaluation. The principal findings and their 4.1). The best way to detect ureteral dilation is at the level of
implications are listed in Table 4.1. the bladder, preferably in transverse view. The degree of dila-
tion becomes the critical issue determining postnatal recom-
mendations. The threshold for recommending postnatal
Diagnostic Accuracy
follow-up is largely arbitrary, yet most would agree that a
The importance of accurate diagnosis is dependent on the unilateral A-P diameter over 7 or 8 mm in the third trimester
context of the case. If intervention is to be considered, diag- (5 to 6 mm with bilateral dilation) mandates postnatal fol-
nostic accuracy needs to be high. In most cases, the detec- low-up (9). In the case of unilateral UPJO, there is little
74 I. Development

TABLE 4.1. MAJOR DIAGNOSTIC FINDINGS IN PRENATAL IMAGING

Finding Comment

Kidney Hydronephrosis Assess degree

Unilateral vs. bilateral May be different degrees
Parenchymal echogenicity Should be less than spleen or liver; if increased and organ enlarged, sug-
gests autosomal recessive polycystic kidney disease
Duplication Often with dilation of upper pole; may be lower pole dilation
Cysts Small cysts associated with dysplasia; simple cyst of upper pole suggests
duplication with ureterocele or ectopic ureter; genetic cystic disease
Urinoma Perinephric or subcapsular
Ureter Dilation/tortuosity Obstruction or reflux
Bladder Distended Variation with time
Wall thickness In relation to filling status
Intravesical cystic structure Ureterocele
“Keyhole” pattern Dilated posterior urethra; PUV
Not visible Exstrophy
Amniotic fluid Absence; oligohydramnios Impaired urine output
Polyhydramnios May be seen with mild-moderate hydronephrosis
Gender Penis/scrotum/testes Sex-associated conditions (e.g., PUV)
Spine Meningocele Neural tube defect

PUV, posterior urethral valves.

rationale for in utero intervention. In a few cases with mas- unclear. The findings of multiple noncommunicating cysts,
sive dilation, therapeutic aspiration has been recommended minimal or absent renal parenchyma, and the absence of a
for dystocia. In the case of bilateral UPJO, the efficacy of in central large cyst are diagnostic of a multicystic dysplastic
utero intervention is difficult to assess. kidney. Bilaterally enlarged echogenic kidneys, particularly if
Attempts to correlate prenatal ultrasound appearance associated with hepatobiliary dilatation or oligohydramnios,
with postnatal outcomes have been complicated by the suggests autosomal recessive polycystic kidney disease (Fig.
long-standing controversy regarding postnatal evalua- 4.3). A more challenging finding is normal-sized, diffusely
tion and management of UPJO. Grignon et al. devel- echogenic kidneys that are not associated with other urologic
oped a system of grading hydronephrosis secondary to lesions. Estroff et al. described 19 cases (14 bilateral), includ-
UPJO based on the diameter of the renal pelvis (RPD) ing 10 with normal function who survived and 4 with auto-
and degree of calyceal dilatation (10). Mandell et al. somal recessive polycystic kidneys who died (14).
attempted to correlate the degree of A-P RPD pelvic
diameter relative to gestational age with subsequent need
for postnatal surgical intervention (Fig. 4.2) (11). They
found the “at risk” diameter to be greater than or equal
to 5 mm at 15 to 20 weeks’ gestation, greater than or
equal to 8 mm at 20 to 30 weeks’ gestation, and greater
than 1 cm at over 30 weeks’ gestation. An alternative
system proposed by Kleiner et al. defined hydronephrosis
as the ratio of A-P RPD to A-P diameter of kidney as
being greater than 0.5 cm (12); caliectasis was later
added as an additional indicator of significant hydrone-
phrosis. Mild degrees of renal pelvic dilatation may
resolve in utero. Mandell et al. noted this to occur in
23% of cases, with 66% remaining stable and 9% wors-
ening over the course of the pregnancy (11). Severe
forms of UPJO may be associated with urinary ascites or
perinephric urinomas, which often precede nonfunction
of the kidney (13).

Cystic Kidneys
FIGURE 4.1. Bilateral ureteropelvic junction obstruction mani-
The distinction between severe unilateral hydronephrosis fested as symmetric hydronephrosis with caliectasis and no ure-
and a multicystic dysplastic kidney may occasionally be teral dilation in a fetus.
 4. Perinatal Urology 75

FIGURE 4.4. Fetal image of duplex kidney with marked upper

pole hydronephrosis (open arrow) in contrast to a normal lower
pole (closed arrows). This image is consistent with either ectopic
ureter or ectopic ureterocele with duplication.
FIGURE 4.2. Prenatal renal pelvic diameter in operative and non-
operative patients with hydronephrosis. [From Mandell J, Blyth BR,
Peters CA, et al. Structural genitourinary defects detected in utero.
Radiology 1991;178(1):193–196, with permission.] pole hydroureteronephrosis, associated with an obstructing
ureterocele within the bladder or ectopic ureter, inserting
outside of the bladder (15) (Fig. 4.4). Lower pole hydrone-
Ureterovesical Junction Obstruction phrosis may be present as a result of reflux or UPJO. Occa-
Less common than UPJO, ureterovesical obstruction is sionally, lower pole dilation is due to upper and lower
characterized by ureteral dilation along with varying ureteral obstruction by a large ureterocele.
degrees of renal pelvic and calyceal dilation. More extreme
cases may be confused with single system ectopic ureters, Vesicoureteral Reflux
particularly in males. In general, the differentiation can be
made postnatally. One cannot make a firm diagnosis of vesicoureteral reflux
based on prenatal ultrasound, although intermittent hydro-
nephrosis or hydroureter is highly suggestive. Vesicoureteral
Duplication Anomalies reflux may be present in as many as 38% of children with
Duplication anomalies, among the most interesting of pre- prenatal hydronephrosis (16). Reflux occurred in 42% of
natal urologic findings, are recognized on the basis of upper children in whom postnatal imaging revealed persistent
upper tract abnormalities and in 25% of those with normal
findings on postnatal ultrasound but having a history of pre-
natal dilation. Tibballs and Debrun reported that in patients
with prenatal dilation and postnatally normal renal units by
ultrasound, 25% had grade III–V reflux (17). The incidence
of high-grade reflux was greater in males than in females as
noted in previous studies.

Posterior Urethral Valves

Perhaps the most important diagnosis to be made prena-
tally is that of PUV in the male fetus—a disorder that, at
the very least, mandates prompt postnatal surgical correc-
tion. In some cases, prenatal intervention may be war-
ranted. Fetal sonographic findings of PUV include bilateral
hydroureteronephrosis, a thick-walled bladder with dilated
posterior urethra, and, in more severe cases, dysplastic renal
FIGURE 4.3. Markedly enlarged echogenic (“bright”) kidneys
(arrows) in a fetus with oligohydramnios, consistent with auto- parenchymal changes with perinephric urinomas and uri-
somal-recessive polycystic kidney disease. nary ascites (Figs. 4.5 and 4.6) (18). When characteristic
76 I. Development

FIGURE 4.5. Image of fetal bladder associated with posterior FIGURE 4.6. Echogenic hydronephrotic kidney (arrow) associ-
urethral valves showing bladder wall thickening and a dilated ated with posterior urethral valves in a 20-week fetus.
posterior urethra (arrow), “keyhole sign.”

sonographic findings are present, the differential diagnosis globulin as an indicator of tubular damage. Using this
includes prune belly syndrome (with or without urethral parameter, poor renal outcome has been predicted with a
atresia), massive vesicoureteral reflux, and certain cloacal specificity of 83% and sensitivity of 80% (26).
anomalies (in genetic females) (19,20). Prenatal diagnostic The time of onset of oligohydramnios has been shown
accuracy for PUV is far from perfect but is probably better to be an important determinant of outcome by Mandell et
than the 40% reported by Abbott et al. (7). al. (27). In fetuses in which adequate amniotic fluid was
documented at up to 30 weeks’ gestation in association
with a urologic abnormality, pulmonary outcomes were sat-
isfactory, and postnatal clinical problems were related to
RATIONALE FOR PRENATAL INTERVENTION
renal disease. It seems inappropriate to recommend late uri-
nary tract decompression from a pulmonary or renal basis.
The scientific rationale for prenatal treatment of hydrone-
It is unclear whether early delivery, to permit earlier postna-
phrosis is to maximize normal development of renal and pul-
tal urinary decompression, is beneficial.
monary function. These two aspects of fetal development are
closely linked because urine comprises 90% of amniotic fluid
volume, and oligohydramnios during the third trimester has
been causally related to pulmonary hypoplasia. CLINICAL EXPERIENCE WITH INTERVENTION
Before prenatal surgical intervention for obstructive urop- FOR PRENATAL HYDRONEPHROSIS
athy, it is critical to assess the risk-benefit ratio. The most
widely accepted indicator of salvageable renal function is The ability to diagnose severe prenatal hydronephrosis and
analysis of fetal urine. When the urinary sodium is less than knowledge of its outcomes has led to our desire to treat it. In
100 mg/dL and urine osmolarity less than 200 mOsm/dL, 1982, Harrison et al. described the initial report of fetal sur-
renal function appears to be salvageable with in utero inter- gery in a 21-week-old fetus with bilateral hydroureterone-
vention (Table 4.2) (21). The accuracy of these predictors has phrosis due to PUV (28). More interventions followed. After
been challenged (22,23). More recently, serial aspirations of the 1986 report of the International Fetal Surgery Registry
fetal urine have been reported to yield more valuable results (29) in which outcomes did not seem to justify risk, a de
(24). Guez et al. (25) reported ten fetuses who underwent facto moratorium on in utero urinary tract shunting evolved.
multiple urine samplings and in whom severe obstruction More recently, with improved technology and renewed inter-
reduced sodium and calcium reabsorption. They concluded est in fetal shunting, most cases have been referred to a small
that fetal urinary chemistries were reasonably predictive of number of highly specialized centers actively engaged in pre-
severe but not moderate postnatal renal impairment. Other natal surgery. The initial method of decompression with
investigators have suggested the use of fetal urinary β2-micro- open surgery has largely been replaced by in utero shunt
 4. Perinatal Urology 77

TABLE 4.2. PRENATAL ASSESSMENT OF RENAL FUNCTIONAL PROGNOSIS

Good Poor

Amniotic fluid Normal to moderately decreased Moderate to severely decreased

Sonographic appearance of kidneys Normal to echogenic Echogenic to cystic

Fetal urine Glick et al. (21) Johnson et al. (24) Glick et al. (21) Johnson et al. (24)

Sodium (mEq/L) <100 <100 >100 >100

Chloride (mEq/L) <90 — >90 —
Osmolarity (mOsm/L) <210 <200 >210 >200
Calcium (mg/dL) — <8 — >8
β2-Microglobulin (mg/L) — <4 — >4
Total protein (mg/dL) — <20 — >20
Output (mL/h) >2 — <2 —
Sequential improvement in urinary values — X — —

X, only in this series was the criterion used.

placement, although this has been complicated by technical been suggested as a possible indication for in utero inter-
problems of shunt dislodgement and, in the case of the dou- vention. The principal goal of intervention is not to prevent
ble-J shunt, bowel herniation (30). Some investigators have pulmonary hypoplasia and deaths but to prevent or delay
explored the use of fetoscopic methods for direct interven- end-stage renal failure. Although some reports have shown
tion to provide prolonged bladder drainage (31–33), whereas the ability to distinguish those fetuses with likely early renal
others have attempted direct endoscopic valve ablation (34). failure from those with later-onset failure (40,41), the spec-
Harrison et al. have clearly outlined the indications and ificity and accuracy of methods using a combination of
contraindications of intervention for prenatal obstructive ultrasound and urinary chemistries (sodium, β2-microglob-
uropathy (Table 4.3). (35) The principal reason for consid- ulin, and calcium) has not been well defined. In summary,
ering vesicoamniotic shunting is to prevent early neonatal precise identification of those situations in which interven-
pulmonary insufficiency and death. The risks that one tion may benefit the fetus with obstructive uropathy
accepts with intervention include induction of premature remains unclear.
labor, perforation of fetal bowel and bladder, as well as
hemorrhage and infection in the fetus and mother.
The need to consider in utero intervention for obstruction POSTNATAL MANAGEMENT OF
is rare. In a recent study, only 9 of 177 fetuses with a diagno- INFANT WITH PRENATALLY
sis of hydronephrosis were considered to have PUV, and only DIAGNOSED HYDRONEPHROSIS
3 warranted serious consideration of intervention (11).
The results of prenatal intervention for fetal hydrone- A child with a prenatal diagnosis of hydronephrosis
phrosis between 1982 and 1985 were reported by the Inter- should be carefully evaluated and followed by a pediatric
national Fetal Surgery Registry (29). The largest group of
fetuses that were treated had an indeterminate cause of their
TABLE 4.3. PRENATAL INTERVENTION
prenatal hydronephrosis (45%). Among those fetuses with a FOR HYDRONEPHROSIS
clear etiology of their hydronephrosis, PUV was the leading
cause (29%). Surgical intervention in these cases led to a sur- Indications (prerequisites) Contraindications
vival rate of 76%. Among the several reports of outcomes Presumed obstructive hydro- Unilateral hydronephrosis
with fetal intervention for obstructive uropathy, true benefit nephrosis, persistent or pro- with an adequately func-
is difficult to assess. This is due to variability in diagnosis, gressive, bilateral or in tioning contralateral kidney
solitary unit
length of follow-up, and documentation of postnatal care. A
Otherwise healthy fetus Chromosomal abnormalities
compilation of these reports is shown in Figure 4.7. These or presence of associated
data demonstrate improvement in survival and reduction in severe anomalies
renal failure in patients with both good and poor prognostic Oligohydramnios Bilateral hydronephrosis with-
features (36–38). Longer follow-up periods have demon- out oligohydramnios
No overt renal dysplasia Severely dysplastic kidneys
strated a high incidence of later renal failure and growth
Adequate renal functional Evidence of urethral atresia
retardation (39). No direct, prospective trial of prenatal potential based on urinary
intervention for fetal hydronephrosis has been performed. indices (see text)
More recently, the ability to influence renal outcome in Informed consent Presence of a normal twin
male patients with PUV but without oligohydramnios has
78 I. Development

For the child with bilateral hydroureteronephrosis sug-

gestive of bladder outlet obstruction, an ultrasound and
voiding cystourethrogram should be performed promptly.
In boys, PUV is the most important diagnosis to be ruled
out. In girls, an obstructing ectopic ureterocele would be
the most likely cause for bladder outlet obstruction. In
the event that an obstructive lesion is discovered, it should
be corrected promptly. If high-grade reflux is noted, anti-
biotic prophylaxis with appropriate radiologic follow-up
is indicated.
The timing of postnatal cystography in the setting of
prenatally detected unilateral hydronephrosis remains
controversial. Although some groups advocate postnatal
cystography in any child with a history of prenatal hydro-
nephrosis, others have questioned the value of this
approach (44). A practical threshold for performing cys-
tography used in many programs has been the presence of
caliectasis, a dilated ureter, or variable hydronephrosis.
In a neonate with prenatally detected hydronephrosis,
FIGURE 4.7. Survival and renal function in fetal interventions
for bladder outlet obstruction comparing treated and untreated the importance of diagnosing vesicoureteral reflux
fetuses with good and poor prognostic indices. C.I., confidence remains controversial. Several studies have demonstrated
interval; Rx, treatment. [Data from Crombleholme TM, Harri- that a high incidence of reflux is a major factor contrib-
son MR, Golbus MS, et al. Fetal intervention in obstructive
uropathy: prognostic indicators and efficacy of intervention. uting to prenatally detected hydronephrosis, although its
Am J Obstet Gynecol 1990;162:1239–1244; Johnson MP, clinical significance is unclear. In several studies, the
Bukowski TP, Reitleman C, et al. In utero surgical treatment of incidence of reflux is approximately 20 to 30% in all
fetal obstructive uropathy: a new comprehensive approach to
identify appropriate candidates for vesicoamniotic shunt ther- grades of hydronephrosis (45–50). The grade of hydro-
apy. Am J Obstet Gynecol 1994;170(6):1770–1776; discussion nephrosis does not predict the likelihood or grade of
1776–1779; and Coplen DE, Hare JY, Zderic SA, et al. 10-year reflux, and the absence of hydronephrosis does not rule
experience with prenatal intervention for hydronephrosis. J
Urol 1996;156(3):1142–1145.] out high-grade reflux. As with hydronephrosis in the
neonatal period, reflux (and particularly high-grade
reflux) occurs more commonly in boys. The natural his-
urologist from birth. The vast majority of these children tory is less well defined, with some series demonstrating
(with unilateral hydronephrosis and a contralateral nor- rapid resolution of higher grades of reflux (48,51).
mal kidney) appear entirely healthy and, in the absence of When neonates with grades 4 and 5 and higher reflux
prenatal ultrasound findings, would not have any indica- are studied with dimercaptosuccinic acid (scintigraphy)
tions for regular urologic follow-up. Parental anxiety is renal scanning, renal abnormalities are detected in up to
common and should be addressed directly with prenatal 50% of children with no history of infection (49,52–
counseling and education. The presence of unilateral dila- 54). This challenges current dogma that renal injury
tion of the kidney warrants postnatal evaluation in a associated with reflux is only due to infection. It may be
timely but nonurgent fashion (3 to 8 weeks of life) with argued that the observed renal abnormalities are dysplas-
an ultrasound (42). Early ultrasound is unlikely to miss a tic and will not progress postnatally, yet this remains
significant abnormality, but a normal result adds confu- unproven. The relationship of bladder function to neo-
sion as to the need for further evaluation (43). The precise natal reflux is currently being defined and may be of crit-
indications for antibiotic prophylaxis (amoxicillin, 10 ical importance, particularly in boys. Significant bladder
mg/kg/day or 50 mg/day) are unclear. A reasonable rule of instability has been documented in infants with high-
thumb is that if a voiding cystourethrogram is requested, grade reflux (55–57), and for this reason, managing
prophylactic antibiotics should be used. There are no out- bladder dysfunction is appropriate.
comes-based assessments of follow-up protocols for chil-
dren with mild to moderate dilation. An approach that
seems reasonable and safe is to recommend functional URETEROPELVIC JUNCTION OBSTRUCTION
imaging (diuretic renography or intravenous pyelography
if there is calyceal dilation followed by serial ultrasonogra- Perhaps the most challenging aspect of managing prenatal
phy. Serial ultrasonography with some plan for extending hydronephrosis is determining when postnatal surgical cor-
the interval between imaging studies seems reasonable but rection for obstruction is appropriate. Despite the improved
unproven. anatomic detail afforded by real-time ultrasound and the
 4. Perinatal Urology 79

from 30 to 40% are controversial: The London group rec-

ommends surgical repair based on a high incidence of later
functional loss (58), whereas Koff and colleagues observe
these patients with frequent diuretic renograms (59,60).
Indeed, Koff has recommended scans as frequently as every
2 weeks for those with significantly reduced uptake. In kid-
neys with less than 10 to 15% uptake on renogram, it is
questionable whether any attempt to salvage renal function
is appropriate. The greatest difficulty lies in those patients
with a unilateral kidney having severe dilation and normal
uptake (i.e., greater than 45%). Some are found to have
uptakes greater than 50%, which is as yet unexplained (61).
The most predictive clinical parameter identified to date is
FIGURE 4.8. Renal pelvic dilation and occurrence of surgical the degree of renal pelvic dilation (A-P diameter). Dhillon et
intervention (based on declining function, increasing hydro-
nephrosis, or symptoms). [Adapted from Dhillon HK. Prenatally
al. have shown that with increasing A-P diameter, there is a
diagnosed hydronephrosis: the Great Ormond Street experi- progressive risk of decrease in relative renal function or
ence. Br J Urol 1998;81(Suppl 2):39–44.] development of symptoms (Fig. 4.8) (62). These are mini-
mal risks if the A-P diameter is less than 20 mm, whereas in
those with dilation greater than 50 mm, it is almost 100%.
increasing experience with functional nuclear medicine stud- It remains unknown if this relationship is time dependent
ies (mercaptotriglycylglycine) (see Chapter 25), no gold stan- and whether the risk of deterioration will increase at lesser
dard for physiologically significant obstruction exists. Over degrees of dilation with increasing time. The few reports of
time, some kidneys have been seen to improve, whereas oth- prospective assessments of the risk of renal injury (func-
ers appear to lose function. The natural history of prenatal tional deterioration, infection, or symptoms) are listed in
hydronephrosis remains to be clearly defined. Table 4.4 and indicate a 20 to 25% likelihood of patients
The debate over the appropriate management of infants with unilateral, significant UPJO warranting pyeloplasty by
with prenatally detected unilateral hydronephrosis remains objective criteria. In most cases, functional loss was cor-
unsettled and will ultimately be determined by three key fac- rected by surgical intervention. A retrospective study by
tors. It will be necessary to define the degree of hydronephro- Chertin et al., however, observed that the functional status
sis in a reproducible way to correlate initial presentation with of comparable kidneys was better if the children had under-
outcome. Next, the natural history of various degrees of gone earlier rather than delayed pyeloplasty (63).
hydronephrosis must be defined in current clinical terms, as Other indicators of potential deterioration have been
well as using novel means of marking the progression of renal proposed. Koff attempted using contralateral renal growth
function or response to the hydronephrotic condition. as an indirect indicator of ipsilateral obstructive injury, but
Finally, clinically useful indicators of the state of the develop- this has not proved to be clinically useful (64). Urinary
ing kidney in the hydronephrotic state need to be defined to markers, including N-acetyl-β-D-glucosaminidase (65) and
permit identification of “at risk” patients. transforming growth factor-β1 (66), have been explored in
At present, it is commonly considered that a unilaterally limited series with suggestive but as yet clinically unproven
hydronephrotic kidney with significantly reduced relative predictive value. All of these markers have suggested that
uptake on a renogram (i.e., less than 30%) should be con- affected kidneys are not normal, but few data have been
sidered for surgical repair. The group of kidneys with uptake rigorously correlated with functional outcomes.

TABLE 4.4. REPORTS OF NONOPERATIVE MANAGEMENT OF

UNILATERAL URETEROPELVIC JUNCTION OBSTRUCTION
Crossover to
Author (reference) No. of patients Study type pyeloplasty (%)

Ransley et al. (58) 100 Prospective, single 23

arm
Cartwright et al. (68) 41 Prospective, single 15
arm
Palmer et al. (69) 16 Prospective, double 25
arm, randomized
Ulman et al. (60) 104 Prospective, single 22
arm
80 I. Development

From a practical standpoint, the clinician must balance 12. Kleiner B, Callen PW, Filly RA. Sonographic analysis of the
possible spontaneous resolution of hydronephrosis without fetus with ureteropelvic junction obstruction. AJR Am J
impairment of renal development with losing the opportunity Roentgenol 1987;148(2):359–363.
to maximize potential renal function through surgical inter- 13. Mandell J, Paltiel HJ, Peters CA, et al. Prenatal findings
vention. As in all of pediatric practice, the emphasis should associated with a unilateral nonfunctioning or absent kid-
ney. J Urol 1994;152:176–178.
remain on permitting maximal childhood growth and devel-
14. Estroff JA, Mandell J, Benacerraf BR. Increased renal paren-
opment while minimizing medical intrusion (67). Our current chymal echogenicity in the fetus: importance and clinical
approach is to consider surgical repair in the setting of outcome. Radiology 1991;181(1):135–139.
impaired relative renal function (i.e., less than 40%), marked 15. Vergani P, Ceruti P, Locatelli A, et al. Accuracy of prenatal
or increasing dilation, decreasing function on serial radionu- ultrasonographic diagnosis of duplex renal system. J Ultra-
clide imaging, or the development of symptoms. Also critical is sound Med 1999;18(7):463–467.
parental preference, with many parents remaining uncomfort- 16. Zerin JM, Ritchey ML, Chang AC. Incidental vesicoureteral
able with a “watchful waiting” approach. Early counseling by reflux in neonates with antenatally detected hydronephrosis
all members of the pediatric healthcare delivery team can help and other renal abnormalities [see comments]. Radiology
avoid confusion and allay anxiety. 1993;187(1):157–160.
17. Tibballs JM, DeBruyn R. Primary vesicoureteric reflux-how
useful is postnatal ultrasound? Arch Dis Child 1996;75:444–
447.
ACKNOWLEDGMENT 18. Peters CA. Lower urinary tract obstruction: clinical and
experimental aspects. Br J Urol 1998;81(Suppl 2):22–32.
The authors acknowledge the assistance of Beryl Benacerraf, 19. Kaefer M, Barnewolt C, Retik AB, et al. The sonographic
M.D., for providing many of the fetal ultrasound images. diagnosis of infravesical obstruction in children: evaluation
of bladder wall thickness indexed to bladder filling [see
comments]. J Urol 1997;157(3):989–991.
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39. Holmes N, Harrison MR, Baskin LS. Fetal surgery for pos- infants with pre-natal hydronephrosis. Br J Urol 1997;80
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Med 1991;1:283–287. 59. Koff SA, Campbell KD. The nonoperative management
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1993;82(5):813–820. 60. Ulman I, Jayanthi VR, Koff SA. The long-term followup
42. Clautice-Engle T, Anderson NG, Allan RB, et al. Diagnosis of newborns with severe unilateral hydronephrosis ini-
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Roentgenol 1995;164:963–967. 61. Oh SJ, Moon DH, Kang W, et al. Supranormal differential
43. Docimo SG, Silver RI. Renal ultrasonography in newborns renal function is real but may be pathological: assessment by
with prenatally detected hydronephrosis: why wait? J Urol 99m technetium mercaptoacetyltriglycine renal scan of con-
1997;157(4):1387–1389. genital unilateral hydronephrosis. J Urol 2001;165(6 Pt
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46. Anderson PA, Rickwood AM. Features of primary vesi- 63. Chertin B, Fridmans A, Knizhnik M, et al. Does early
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1991;67(3):267–271. gical outcome in terms of renal function? J Urol 1999;162(3
47. Paltiel HJ, Lebowitz RL. Neonatal hydronephrosis due to Pt 2):1037–1040.
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82 I. Development

by measuring the size of the opposite kidney. J Urol 1994; 67. Peters CA. Urinary obstruction in children. J Urol 1995;
152(2 Pt 2):596–599. 154:1874–1884.
65. Carr MC, Peters CA, Retik AB, et al. Urinary levels of the 68. Cartwright PC, Duckett JW, Keating MA, et al. Managing
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thy. J Urol 1993;151:442–445. born. J Urol 1992;148(4):1224–1228.
66. Furness PD 3rd, Maizels M, Han SW, et al. Elevated blad- 69. Palmer LS, Maizels M, Cartwright PC, et al. Surgery versus
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beta1 correlates with upper urinary tract obstruction in hydronephrosis: a report from the Society for Fetal Urology.
children. J Urol 1999;162(3 Pt 2):1033–1036. J Urol 1998;159(1):222–228.
 5

RENAL DYSPLASIA /HYPOPLASIA

PAUL R. GOODYER

During normal kidney development, interactions between described. Although syndromes involving marked renal dys-
the branching ureteric bud and the metanephric mesen- plasia are categorized in Chapter 6, examples of renal dyspla-
chyme generate the nephrons of each kidney. Nephrons sia and the evidence that obstruction of the fetal urinary tract
eventually hang like apples, each on its own branch of the can disturb mesenchymal cell fate during kidney develop-
arborized collecting system. At birth, this crop of nephrons ment are considered in this chapter. Abnormalities of kidney
constitutes the individual’s nephron endowment for life position and macroscopic patterning, particularly multicys-
and ranges widely among normal humans from 0.3 to 1.1 tic/dysplastic kidneys (MCKDs), are reviewed in regard to
million nephrons per kidney (1). “Normal” children at the failure of mechanisms normally involved in the terminal dif-
low end of this nephron endowment spectrum are thought ferentiation of renal epithelia.
to be at increased risk for hypertension and renal insuffi-
ciency after an acquired renal insult later in life; suboptimal
nephron number may also influence longevity of renal
RENAL AGENESIS
allografts (2–4). On occasion, renal hypoplasia may be
more extreme. In one antenatal ultrasound screening study,
Bilateral Renal Agenesis
it was estimated that approximately 1 in 400 neonates are
born with at least one hypoplastic kidney (5). When renal Bilateral failure of primary nephrogenesis during fetal life
hypoplasia is bilateral, nephron number may be insufficient causes a characteristic pattern of facial compression and pul-
for normal extrauterine life. As somatic growth outstrips monary hypoplasia (Potter syndrome) due to the absence of
nephron endowment, these children develop progressive amniotic fluid. When severe, oligohydramnios is evident clini-
renal insufficiency, generating approximately 20% of the cally or by ultrasonography in the second trimester (21 to 23
pediatric end-stage renal disease population (6). weeks), and should raise major concern about the postnatal
In this chapter, those clinical settings in which primary viability of the infant (8). High-resolution color Doppler
nephron number is suboptimal from birth are considered. In ultrasonography is helpful in detecting the fetal renal arteries
some, nephron number may be reduced even though the and distinguishing renal hypoplasia from renal agenesis (9).
structure and function of nephrons is normal (pure renal Complete absence of renal parenchyma (renal agenesis) and
hypoplasia). More often, renal hypoplasia is associated with amniotic fluid predicts severe pulmonary hypoplasia, often
histopathologic evidence of aberrant developmental fates of resulting in pneumothorax and/or inability to oxygenate in
the metanephric mesenchyme and profound disturbances of the newborn period. In those who initially survive, the deci-
the normal patterning of renal tissue (renal dysplasia). As sion about whether to embark on chronic peritoneal dialysis is
suggested by Pohl et al., current approaches to classification usually based on whether: (a) lung development is sufficient to
of human kidney hypoplasia/dysplasia must integrate recent allow oxygenation without respiratory support beyond the
advances in the understanding of nephrogenesis with clinico- perinatal period; (b) there is any functional renal parenchyma
pathologic observations (7). Because the former field is so [identifiable by mercaptoacetyltriglycerine (MAG-3) imaging
rapidly evolving, any classification system should be consid- and/or ultrasonography]; (c) urine volume is sufficient to per-
ered a work in progress. In this chapter, primary renal agene- mit minimal long-term oral nutrition (100 kcal/kg/day); and
sis and pure renal hypoplasia are considered in the context of (d) family and institutional resources can sustain dialytic ther-
key molecules that are either essential for initial steps in renal apy long enough to achieve growth and development suffi-
development or which optimize the rate of branching neph- cient to permit successful renal transplantation.
rogenesis. Then, renal dysplasia, which is often linked to The causes of renal agenesis in humans are not well under-
aberrant outgrowth of the primary ureteric bud and/or dis- stood, but reports of absent renal arteries during the second
turbed interactions of bud with metanephric mesenchyme, is trimester suggest arrest of kidney development at an early
84 I. Development

stage. Study of homozygous knock-out mice has identified a URA has been associated with developmental abnormal-
number of critical developmental genes causing experimental ities of other tissues, particularly the ear, genital tract, and
BRA. For example, inactivation of either glial cell line– axial skeleton. Among 40 girls with URA, 4 out of 40
derived neurotrophic factor (a growth factor expressed in the (10%) had an ipsilateral mild-moderate sensorineural hear-
undifferentiated mesenchyme) or c-ret (the glial cell line– ing deficit and 14 (35%) had mullerian duct abnormalities
derived neurotrophic factor receptor, which is expressed at the (16). In a retrospective study of patients with mullerian
surface of ureteric bud cells) results in failure of primary out- duct abnormalities, 30% had URA (17); this association
growth of the ureteric bud (10,11). Knock-out mice lacking was particularly strong for girls with uterus didelphys (13
key transcription factor genes, such as PAX2 or WT1, are also of 16 cases), uterine agenesis (two of five cases), and unicor-
anephric (12,13). Presumably, homozygous mutations for any nuate uterus (two of seven cases). URA was seen in all 11
one of these genes could account for occasional sporadic cases cases of obstructed uterus didelphys, ipsilateral to the side
of complete renal agenesis in humans, but this has not yet of the obstructing transverse hemivaginal septum. The inci-
been demonstrated. Most cases of Potter syndrome are associ- dence of genital tract anomalies in boys with URA is not
ated with obstruction of the urinary tract or severe bilateral precisely known, but ipsilateral cystic dysplasia or seminal
renal hypoplasia rather than complete BRA. Antenatal ultra- vesicle cysts and bilateral absence of the vas deferens (with
sonography of 4000 normal Japanese pregnancies uncovered ipsilateral syndactyly) have all been described (18–20). In a
no examples of BRA, indicating its rarity (5). prospective study of 202 patients with congenital vertebral
abnormalities, 54 (26.7%) had at least one genitourinary
abnormality detected by intravenous pyelography or ultra-
Unilateral Renal Agenesis
sonography; the most frequent being URA (16). Although
In a recent study of American infants, the incidence of uni- the pathogenetic mechanisms are not well understood, it is
lateral renal agenesis (URA) was 1 per 2900 births and was evident that defective developmental pathways or genes
more common in infants of diabetic mothers and among may disturb morphogenesis of mesenchyme in the ear, gen-
black mothers than in others (adjusted odds ratio of 4.98 and ital tract, and skeleton, and it is not unreasonable to screen
2.19, respectively) (14). In a screening study of normal Japa- children with URA for these associated anomalies.
nese newborns, 52 of 4000 newborns (1.3%) had evidence
of unilateral hypoplastic/dysplastic kidneys (5). During fol-
low-up of three of these cases, the abnormal renal tissue invo- PRIMARY RENAL HYPOPLASIA
luted in early postnatal life so that eventually no detectable
kidney was evident (5). Thus, adults with congenital absence Normal mature kidneys contain approximately 617,000
of one kidney may be born with at least some partial, though glomeruli with mean glomerular size of approximately 6
abortive, renal tissue that regresses at an early stage. In μm3 (21). In the 1970s, pediatric nephrologists recognized
murine models, knock-out of both copies of the c-ret gene a familial form of oligomeganephronia in which renal
usually results in anephric fetuses. However, a small percent- hypoplasia and progressive proteinuric renal failure in
age manage to achieve outgrowth of the ureteric bud and childhood were associated with unusually large but other-
generate a small, though suboptimal, kidney on one side wise normal-appearing glomeruli (22). During the same
(10). This suggests that some cases of URA may be caused by period, an autosomal dominant syndrome of renal hypo-
inherited mutant genes. Occasional cases of autosomal dom- plasia associated with colobomas of the optic nerve [renal-
inant URA have been reported (15). coloboma syndrome (RCS)] was also characterized (23). In
At birth, nephron number is suboptimal in children 1995, Eccles showed that RCS was caused by heterozygous
with URA, and the contralateral kidney is stimulated to mutations of the PAX2 gene and drew attention to the high
undergo postnatal compensatory hypertrophy. In most frequency of vesicoureteral reflux (VUR) in affected fami-
cases, plots of renal length or volume versus body length lies (24–26). When patients with oligomeganephronia were
demonstrate gradual compensatory hypertrophy of the subsequently reinvestigated, these patients also proved to
unaffected contralateral kidney over the first 3 to 4 months have heterozygous PAX2 mutations, though the optic nerve
postnatally, crossing percentiles established for normal pop- abnormality was often barely detectable (27–29). Approxi-
ulations. Failure to undergo compensatory hypertrophy mately 15 different inactivating mutations of the PAX2
usually indicates contralateral renal dysplasia and may pre- gene have been described, and the most common (a single
dict progressive renal insufficiency. In addition to monitor- base-pair insertion in the second exon of PAX2) also causes
ing contralateral growth, it is important to screen for RCS in the inbred 1Neu mouse strain (30). Studies of fetal
residual functional renal tissue by nuclear dimercaptosuc- kidney development in 1Neu mice have led to the hypothe-
cinic acid (DMSA) or MAG-3 scanning. The presence of sis that a critical function of the PAX2 gene is to prevent
severely hypoplastic/dysplastic tissue is sometimes associ- apoptosis of cells in the branching ureteric bud during kid-
ated with hypertension, which might require excision of the ney development (31,32). Absence of one PAX2 gene is
dysplastic kidney. sufficient to permit increased ureteric bud cell apoptosis,
 5. Renal Dysplasia/Hypoplasia 85

The otic defects in BOR syndrome can usually be delin-

eated by computed axial tomography, but nuclear magnetic
resonance imaging may be useful in diagnosis (36). It has
been argued that renal defects are not common enough in
children with external ear anomalies alone to warrant rou-
tine renal ultrasonography (37). On the other hand, several
other syndromes involve renal anomalies and external ear
deformities (38). Although renal hypoplasia can be quite
variable in BOR, branchial arch defects with normal kid-
neys (BO syndrome) probably result from mutations of a
different gene on chromosome 1q31 (39).
In the 1940s and 1950s, Wilson reported that severe
vitamin A (retinol) deficiency caused renal agenesis (40,41).
More recent observations indicate that even modest mater-
nal retinol deficiency (50% reduction in circulating levels
of retinol) can cause significant renal hypoplasia in rodents.
In such studies, postnatal kidney weight is decreased by
24%, and nephron number is reduced by 20% (42). In
these rodent studies, nephron number correlated with kid-
ney size and varied directly with circulating retinol concen-
FIGURE 5.1. Hypoplastic kidney bisected to show presence of tration. Normally, the fetus acquires retinol from the
ureter and symmetric collecting system derived from initial
branches of the ureteric bud. The thin outer rim of renal cortex
maternal circulation and converts it to an active metabolite,
has normal architecture, but nephron number is dramatically all-trans-retinoic acid, in the kidney and other peripheral
reduced. tissues (43). In fetal rat kidneys cultured ex vivo, all-trans-
retinoic acid (0.1 to 1.0 μmol) accelerates new nephron for-
mation by two- to threefold (44,45). Vitamin A deficiency
compromising nephron number by slowing the rate of is widespread (15 to 25% of pregnant women) in many
branching nephrogenesis. Presumably, PAX2 function also third world regions, and in North America a small but sig-
influences structure of the ureterovesical junction. In pure nificant fraction (1%) of pregnant women have retinol
primary renal hypoplasia, there is little evidence of dyspla- levels in the low-normal range that could effect nephron
sia, and glomerular hypertrophy is presumably the normal number. As yet, however, it is unknown whether human
compensatory response to the deficit in nephron number maternal vitamin A deficiency limits nephrogenesis as it
(Fig. 5.1). does in rats.
At least one other form of hereditary renal hypoplasia,
branchiootorenal (BOR) syndrome (OMIM 113650), can
be attributed to mutation of a single gene. The autosomal RENAL DYSPLASIA/HYPOPLASIA AND
dominant BOR syndrome, involving variable degrees of VESICOURETERAL REFLUX
renal hypoplasia, congenital lateral cervical fistulas or cysts,
and ear abnormalities (preauricular tags or pits, a mal- During early fetal life, the two nephric (wolffian) ducts arise in
formed auricle, atresia of the canal, anomalies of the middle the pronephric region of the intermediate mesoderm and
ear, and hypoplasia of the cochlea or semicircular canals) is migrate caudally. Just before arrival at the cloaca (approxi-
caused by mutations of the EYA1 gene (32,33). More than mately somite 25 in the mouse), ureteric buds emerge from
20 different EYA1 mutations have been reported; end-stage the nephric ducts at a site corresponding to the future vesico-
renal failure may develop anywhere between 12 and 36 ureteral junction and induce the metanephric kidneys from
years of age. EYA1 is the human homolog (chromosome laterally adjacent metanephric mesenchyme. In 1975, Mackie
18q13.3) of a small family of transcription factors origi- and Stephens observed (by ureteroscopy) that many children
nally identified in Drosophila because of their importance born with duplex collecting systems and/or VUR also had
to eye development, but in humans the eye is unaffected. malpositioned ureteric orifices (46). In Japanese perinatal
Within the same family, heterozygous mutations of EYA1 screening studies, renal hypoplasia (approximately 1 in 400
may cause renal defects ranging from BRA to mild unilat- normal births) was strongly correlated with VUR (5). Among
eral hypoplasia (34). During normal renal development neonates investigated for VUR, renal hypoplasia (reduced
EYA1 is expressed in the metanephric mesenchyme, and renal length by ultrasonography and reduced DMSA uptake
homozygous EYA1 knock-out mice lack primary out- by scintigraphy) was frequent, being symmetric in approxi-
growth of the ureteric bud with subsequent failure of neph- mately one-third of cases and involving patchy scars of the
ron induction (35). renal parenchyma in others (47,48). Thus, disturbance of the
86 I. Development

FIGURE 5.2. Renal hypoplasia associated with ipsilateral vesico-

ureteral reflux in a 2-year-old boy who presented with his first
lower urinary tract infection. Dimercaptosuccinic acid scintigra-
phy demonstrates normal reniform appearance of the hypoplas-
tic parenchyma, which contributes only approximately 10% of
total renal function.

factors that regulate the proper timing or positioning of ure-

teric bud outgrowth appear to lead to ectopic ureteric orifices,
abnormalities of ureter structure, and faulty induction of meta-
nephric mesenchyme by the ectopic ureteric bud (Fig. 5.2).
This hypothesis is supported by knock-out mouse experiments
in which homozygous inactivation of a single transcription
factor gene (Foxc2) may cause various anatomic abnormalities
of the ureters associated with renal hypoplasia in approxi-
mately 60% of offspring (49). Foxc2 is normally expressed in FIGURE 5.3. A: Renal dysplasia: peritubular fibrous cuff. B: Renal
the metanephric mesenchyme and is thought to be involved in dysplasia: island of cartilage amid dysplastic tissue represents aber-
guiding outgrowth of the ureteric bud (49). The gross abnor- rant cell fate of metanephric mesenchyme.
malities were often unilateral (70 to 85% of offspring) and
were highly dependent on the genetic background of the mice
(49). To date, single gene mutations causing VUR and renal well understood, fetal UTO appears to modify survival of
hypoplasia/dysplasia have not been identified in humans, but selected cell lineages and affect signals regulating develop-
putative VUR loci have been reported (50). mental cell fates. Timing of the fetal obstruction may be
important; very early (50 days’ gestation) obstruction of the
urinary tract in lambs causes severe hypoplasia and interstitial
RENAL HYPOPLASIA/DYSPLASIA AND FETAL fibrosis, whereas obstruction at 60 days results in a larger,
URINARY TRACT OBSTRUCTION cystic kidney with less fibrosis (55). In humans, striking
obstruction has sometimes been reported in association with
Numerous observations indicate that antenatal obstruction normal renal histology, prompting the speculation that UTO
of the urinary tract (UTO) may be associated with distur- late in fetal life may have less effect on determinants of devel-
bances of normal nephrogenesis. Most commonly, renal opmental cell fate (56).
hypoplasia/dysplasia is reported in males with posterior ure- As in other forms of renal hypoplasia/dysplasia, the risk
thral valves but is also evident in other UTO settings such as of progressive renal insufficiency due to congenital UTO is
prune-belly syndrome and urethral atresia (51–53) (Fig. 5.3). highly influenced by the number of functioning nephrons
Dysplastic areas containing cysts, fibrotic interstitial zones, at birth. The likelihood of developing end-stage renal dis-
and even islands of cartilage may be scattered amid fairly ease ranges from 22 to 70% in various studies of boys with
normal-appearing renal tubules. In fetal monkeys, obstruc- posterior urethral valves. A key predictor of long-term out-
tion produced by injection of agaral beads into the collecting come is serum creatinine level 1 year after relief of obstruc-
system at mid-gestation causes progressive diminution in tion (53,57). In 29 patients with prune-belly syndrome
renal size associated with evidence of apoptosis (particularly (deficient abdominal musculature, evidence of UTO, and
of collecting duct and glomerular cells), cysts with pericystic cryptorchidism), approximately one-third die in the perina-
fibrotic collars, and a defect in branching of the ureteric bud tal period from Potter syndrome, and most survivors
(54). Thus, although the molecular mechanisms are not yet develop end-stage renal disease (58,59).
 5. Renal Dysplasia/Hypoplasia 87

SYNDROMIC RENAL
HYPOPLASIA/DYSPLASIA

Renal dysplasia is associated with many different recogniz-

able patterns of malformation, presumably involving genes
or developmental pathways shared by multiple organs.
Although a more complete list appears in Chapter 6, several
syndromes of interest are mentioned briefly here. Muta-
tions in the hepatocyte nuclear factor-1β gene (TCF2) are
responsible for an autosomal dominant syndrome charac-
terized by maturity-onset diabetes of the young, nondia-
betic progressive nephropathy, genital malformations, and
liver dysfunction (60). The hepatocyte nuclear factor-1β
gene is normally expressed in the wolffian duct, meta- FIGURE 5.4. Nonfunctional multicystic/dysplastic right kidney
nephric tubules, and mullerian duct during fetal life; its and grossly normal-appearing left kidney from an infant who
absence results in a form of cystic renal dysplasia in which died in the perinatal period of nonrenal causes.
glomerular number is significantly reduced (61,62). Early-
onset progressive nephropathy may be seen without evi-
dence of diabetes mellitus in some cases, and renal dysplasia ing little or no urine formation from early fetal life. The
is quite variable, producing unilateral renal hypoplasia with cystic mass usually lacks a reniform shape; scanty tissue
cystic dysplasia in the contralateral kidney (62). between cysts is hyperechoic, and there is usually no detect-
Renal dysplasia is also part of the autosomal dominant able renal artery by Doppler ultrasonography. Microscopic
HDR syndrome (hypoparathyroidism, deafness, and renal analysis reveals disorganized renal architecture with islands
anomalies) caused by mutations of another transcription fac- of undifferentiated mesenchymal cells, occasional bizarre
tor gene, GATA3 (63) and is part of the Bardet-Biedl syn- differentiation (e.g., cartilage), and few if any normal-
drome caused by mutations at six different loci, including the appearing nephrons. Cysts are often rimmed by collars of
MKKS gene (64). Other monogenic syndromes with renal fibromuscular cells. The incidence of unilateral MCKD is
dysplasia include Townes-Brock syndrome (SALL1 gene) approximately 1 in 4000 live births (75). Rarely, bilateral
(65) and nail-patella syndrome (LMX1B gene) (66). MCKD has been reported and is fatal in the newborn
Variable degrees of renal dysplasia have been described in period (76).
numerous syndromes for which the etiology is unknown. More recently, antenatal detection and long-term follow-
The causes of the VATER syndrome (vertebral defects, anal up studies of unresected MCKDs have suggested that the
atresia, trachio-esophageal fistula, and renal dysplasia) are pathogenesis and prognosis for this entity are more complex
thought to be heterogeneous but presumably identify a com- than was initially appreciated. In approximately 15% of uni-
mon developmental pathway in these tissues (67). In the lateral cases, postnatal nuclear scans show some minimal
VACTERL syndrome (with additional limb and cardiac functional renal tissue amid the dysplastic areas, so complete
defects), the majority of patients have renal dysplasia, which absence of renal function is no longer the sine qua non (77).
may be associated with vesicoureteral reflux (39%), unilateral Numerous cases of localized (restricted to one pole) cystic
aplasia (23%), or unilateral multicystic dysplasia (7%) (68). dysplasia have been reported (78,79). More important, the
Similarly, renal dysplasia may be seen in association with contralateral kidney often (20 to 30% of cases) exhibits some
developmental defects of the pancreas and liver (Ivemark form of limited dysplasia (80–83). Approximately one-
syndrome) (69), jejunal atresia (70), and encephalocele fourth of contralateral kidneys exhibit VUR, and this may be
(Meckel syndrome) (71), and with pancreatic fibrosis, liver associated with recurrent urinary tract infections and pro-
dysgenesis, and situs inversus (72). In Perlman syndrome, gressive renal insufficiency (84). Careful evaluation of con-
renal dysplasia and Wilms’ tumor are associated with fetal tralateral renal growth by serial postnatal ultrasonography,
gigantism and multiple congenital anomalies (73,74). DMSA nuclear scans to detect foci of dysfunctional paren-
chyma, and voiding cystourethrography to identify contra-
lateral VUR may be considered to identify cases with
MULTICYSTIC/DYSPLASTIC KIDNEY significant contralateral dysplasia (85).
Because experimental obstruction may produce cystic
Classically, MCKDs have been identified as unilateral mul- dysplasia, it has been proposed that first-trimester UTO
tiloculated abdominal masses with thin-walled cysts that do might account for MCKD (75,86). However, the putative
not appear to connect (Fig. 5.4), distinguishing them from obstruction would have to be intrarenal as MCKD ureters
hydronephrotic kidneys. Nuclear scans often show no func- are atretic, and lower tract obstruction could not explain
tional parenchyma, and ureters are usually atretic, indicat- cases of localized dysplasia. Furthermore, there are reports
88 I. Development

of autosomal dominant MCKD and chromosomal anoma- and uretero-pelvic junction obstruction in 23% (108). The
lies (87,88), suggesting that failure of key genes can lead to risk of Wilms’ tumor is slightly increased in children with
MCKD by perturbing the normal pattern of nephrogene- horseshoe kidney, and there are more than 40 reports of
sis. A high incidence of subtle genital and other nonrenal this association in the literature (109). However, regular
abnormalities suggests aberrations in shared developmental screening for this complication is probably unwarranted.
programs rather than UTO as the primary etiology (81,82). Although 0.5% of Wilms’ tumors arise within a horseshoe
On occasion, unilateral MCKD has been associated with kidney, this is only approximately three times the expected
hypertension; in some cases, hypertension resolves when the incidence. The arterial supply to the horseshoe kidney is
dysplastic tissue is resected (89). Ectopic renin gene expres- highly variable, presenting a special technical challenge for
sion has been documented in macrophage-like interstitial transplantation. Nevertheless, horseshoe kidneys can be
cells (90). Although hypertension is not commonly identi- transplanted into recipients en bloc or after division of the
fied in children with unilateral MCKD (less than 5%), its isthmus, and the success rate is equivalent to normal cadav-
prevalence may be underestimated. Subtle abnormalities of eric transplantation with either approach (106).
blood pressure were found in 5 of 25 such children when
studied by ambulatory blood pressure monitoring (91).
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 6

SYNDROMES AND MALFORMATIONS

OF THE URINARY TRACT
CHANIN LIMWONGSE
SUZANNE B. CASSIDY

Birth defects involving the kidney and urinary system are abnormal, and sometimes a malformation (e.g., renal agen-
common and often occur in association with other struc- esis) can predispose patients to a deformation (e.g., Potter’s
tural abnormalities. A congenital urinary tract anomaly sequence from oligohydramnios).
may provide the first clue to the recognition of multiorgan Disruption refers to a single structural anomaly that
developmental abnormalities. Nevertheless, many renal results from a destructive event after normal morphogene-
anomalies remain asymptomatic and undiagnosed. sis. Such events can be caused by lack of vascular supply,
Although the number of single malformations involving infectious process, or mechanical factors. Examples include
the kidney and urinary system is limited, combinations of limb amputation from amniotic bands and abdominal wall
such malformations with anomalies involving other organ defects from vascular insufficiency related to maternal
systems are found in more than 500 syndromes. In addi- cocaine use.
tion, many well-known sequences and associations involve Sequence refers to a cascade of abnormalities that results
the kidney and urinary tract. This chapter discusses com- from a single initiating anomaly. Sequences can be malfor-
mon malformations, sequences, and associations involving mational, deformational, or disruptive, and they sometimes
the kidney and urinary tract and provides a summary of represent more than one of these categories. Obstruction of
conditions that have these anomalies as one of their fea- urine flow at the level of the ureter during early gestation,
tures. In addition, Tables 6.1 through 6.3 summarize more for example, can cause malformation of the kidneys, intes-
detailed information about a large number of disorders tines, and abdominal wall—a malformation sequence. At
associated with urinary tract anomalies. These tables can be the same time, decreased urine flow produces oligohydram-
used to (a) provide readily available information about nios, fetal compression, and multiple deformities of the
potential urinary tract anomalies in patients with a diag- face, limbs, and chest wall—a deformation sequence.
nosed genetic syndrome and (b) develop a differential Syndrome refers to a consistently observed pattern of
diagnosis when specific anomalies are identified. Readers multiorgan anomalies consisting of malformations, defor-
interested in additional details about a specific syndrome mations, or disruptions. Examples include Turner syn-
are referred to standard reference textbooks and databases drome and fetal alcohol syndrome.
for further reading (4–7). Association refers to a constellation of anomalies that
To understand the pathophysiologic basis of structural occur together more often than expected by chance alone
abnormalities, it is important to define certain terms used but cannot be explained by a single cause or sequence of
in describing malformations and syndromes. events. Vertebral, anus, tracheoesophageal, radial, and renal
Malformation refers to a single structural anomaly that (VATER) association, which is discussed later in this chap-
arises from an error in organogenesis. Such an error may be ter, is a common example.
due to the failure of cells or tissues to form, to regress (pro-
grammed cell death), or to induce others’ anlagen. Exam-
ples include renal agenesis, horseshoe kidney, and bladder PREVALENCE OF URINARY
exstrophy. TRACT ANOMALIES
Deformation refers to a single structural anomaly that
arises from mechanical forces (e.g., intrauterine constraint). The true incidence of urinary tract anomalies is difficult to
Examples include metatarsus adductus, torticollis, and con- ascertain because they are asymptomatic and, therefore,
genital scoliosis. The underlying tissue may be normal or remain undetected. The reported incidence of many anoma-
 TABLE 6.1. SYNDROMES AND DISORDERS THAT HAVE URINARY TRACT ANOMALIES AS A FREQUENT FEATURE

94
Urinary tract abnormalities

Inheritance

Renal agenesis
Ectopia/horseshoe
Cystic/dysplasia
Duplication
Hypoplasia
Hydronephrosis/ureter
Diverticulae
Atresia/stenosis
Reflux
Nephritis/sclerosis
Tumor/nephromegaly
Syndromes Other associated anomalies pattern Reference

Abruzzo-Erickson H Coloboma, cleft palate, hypospa- Uncertain 44

dias, deafness, short stature
Acrocephalopolydactylous + + + Acrodactyly, hand hexadactyly, AR 45
dysplasia (Elejalde syn- overgrowth, visceromegaly, glob-
drome) ular body, redundant neck skin
Acrorenal (Dieker) 1 E + + + Ua Ectrodactyly, oligodactyly, hypo- Sporadic 46
plastic carpal/tarsal bones
Acrorenal (Johnson-Mun- 1, 2 U, Ua Aphalangy, hemivertebrae, genital/ AR 46
son) intestinal/anal dysgenesis
Acrorenal (Siegler) E + U + Short stature, hypoplastic radii/ Uncertain 46
ulnae/humeri, oligodactyly
Acro-renal-mandibular 1 + Ectrodactyly, hypoplastic mandible AR 47
Acro-renal-ocular 1 E + B Hypoplastic thumb, optic AD 48
coloboma, cleft lip/palate
Adrenoleukodystrophy, See Pseudo-Zellweger
neonatal
Aglossia-adactylia 1 Micrognathia, cranial nerve palsy Sporadic 49
Agnathia-holoprosenceph- H + Arrhinencephaly, situs inversus, Sporadic 50
aly midline defects
Alagille 1 + + + Cholestasis, peripheral pulmonic AD 51
stenosis, characteristic face
Alport + Nephritis, proteinuria, deafness AD, X-linked 52
Alsing + Nephritis, nephronophthisis, optic AR 53
coloboma, hip dislocation
Alstrom + Diabetes mellitus, retinopathy, AR 54
short stature, deafness
Amelogenesis imperfecta + Enamel hypoplasia, nephrolithia- AR 55
sis, enuresis
Amyloidosis type 5 + Nephropathy, proteinuria, cranial AD 56
nerve palsy, cutis laxa
Angiotensin converting + IUGR, oligohydramnios, patent Sporadic 57
enzyme inhibitor, mater- ductus arteriosus, limb anoma-
nal use lies, renal artery stenosis, pro-
gressive renal failure
Aniridia-Wilms’ tumor + Aniridia, ambiguous genitalia, AD 58
hypospadia, short stature
 Axial mesoderma dysplasia 1 Bladder exstrophy, vertebral anom- Uncertain 59
alies, Goldenhar-like
Baldellou 1 Hypoparathyroidism, ocular Uncertain 60
coloboma, MR, seizures
Baller-Gerold + E + + Craniosynostosis, radial aplasia, AR 61
malformed ear, anal atresia
Barakat + Mesangial sclerosis, MR, optic atro- AR 62
phy, nystagmus
Bardet-Biedl + + + + + + Obesity, polysyndactyly, MR, reti- AR 63,64
nopathy, hypogonadism
Beckwith-Wiedemann E + + + + Overgrowth, macroglossia, ompha- Sporadic, AD 65–68
locele, embryonal tumors
Berardinelli + + Insulin resistance, lipodystrophy, AR 69
acanthosis nigricans, MR
Brachymesomelia-renal + Micrognathia, corneal opacity, Uncertain 70
craniofacial dysmorphism
Braddock-Carey + Robin sequence, thrombocytope- Uncertain 71
nia, ACC
Branchio-oculo-facial 1 Philtrum hypertrophy, cleft lip/pal- AD 72
ate, branchial remnant
Branchio-oto-renal 1, 2 E + + + + + Branchial remnant, preauricular AD 73
pit/tag, microtia, deafness
Branchio-oto-ureteral + Branchial remnant, preauricular AD 74
pit/tag, microtia, deafness
Braun-Bayer + U + Deafness, bifid uvula, digital anom- Uncertain 75
alies
Cat eye See Table 6.3 Chromosomal
Caudal duplication 1, 2 E + + + Duplication of colon, sacrum, geni- Sporadic 76
talia, vertebral defects
Caudal regression 1, 2 E, H + + Ua + Atresia of colon, anus, genitalia, Sporadic, AR 77,78
vertebral defects, transesoph-
ageal fistula
Cerebro-hepato-renal (Pas- + Hypotonia, abnormal ear, AR 79
sarge) hepatomegaly, hypospadias
Cerebro-oculo-hepato- + Cerebellar hypoplasia, hepatic AR 80
renal fibrosis, Leber amaurosis
Cerebro-osteo-nephro- + + Rhizomelic limb shortening, cere- AR 81
dysplasia bral atrophy, MR, seizures
CHARGE association + E + + + + U + See text for details 30–33
Chondroectodermal dyspla- 1 + + + Acromelic dwarfism, polydactyly, AR 82,83
sia (Ellis van Creveld) nail dystrophy, tooth hypoplasia
narrow thorax, CHD
Cocaine, maternal use 1, 2 + + Ua + Vascular disruption anomalies Sporadic 84
affecting multiple organs
Cornelia de Lange 1 + + + + SS, microcephaly, limb defect, hir- Uncertain 87
sutism, synophrys
Crossed renal ectopia-pel- E + U Clubbing of fingers, gynecomastia Uncertain 85
vic lipomatosis
Czeizel + U Ectrodactyly, spina bifida, megacystis AD 86

95
continued
 TABLE 6.1. CONTINUED.

96
Urinary tract abnormalities

Inheritance

Renal agenesis
Ectopia/horseshoe
Cystic/dysplasia
Duplication
Hypoplasia
Hydronephrosis/ureter
Diverticulae
Atresia/stenosis
Reflux
Nephritis/sclerosis
Tumor/nephromegaly
Syndromes Other associated anomalies pattern Reference

Diabetic mother, infant of 1, 2 E, H + + + + Ua + Neural tube defect, cardiac/limb Sporadic 88,89

anomalies, sacral agenesis
DiGeorge/velocardiofacial 1, 3 E + + + + + See Table 6.3 Chromosomal
Denys-Drash E + + + Pseudohermaphroditism, Wilms’ Sporadic 90
tumor, proteinuria
Down See Table 6.3 Chromosomal
Ectrodactyly-ectodermal 1 + + + + Ectrodactyly, hypohidrosis, sparse AD 91
dysplasia-clefting (EEC) hair, cleft lip/palate
Elejalde See Acrocephalopolydactylous dys-
plasia
Epstein + Thrombocytopenia, nerve deaf- AD 92
ness, cataract
Facio-cardio-renal H + U Cardiomyopathy, conduction AR 93
defect, MR, typical face
Fanconi anemia 1 E, H + + + + Pancytopenia, limb defects, leuke- AR 94,95
mia, lymphoma
Fetal alcohol 1 E, H + + + + IUGR, DD, microcephaly, short Sporadic 96
palpebral fissure
Fibromatosis, infantile + + Multiple myofibromatosis, myosi- AR 97
tis ossificans
Fraser cryptophthalmos 1, 2 + U Fused eyelids, ear/genital anoma- AR 98
lies, syndactyly
Frasier + + Male pseudohermaphrodite, Uncertain 99
amenorrhea, ovarian cysts
Goeminne + + Congenital torticollis, keloids, cryp- X-linked 100
torchidism
Goldenhar (oculo-auriculo- 1 E + + + + + Hemifacial microsomia, ear anoma- Sporadic, AD 101
vertebral) lies, vertebral defects
Goldston + Dandy-Walker malformation, cere- Uncertain 102
bellar malformation
Graham + + Cystic hamartoma of lung and kid- Sporadic 103
ney
Hemifacial microsomia See Goldenhar (oculo-auriculo-ver-
(oculo-auriculo- tebral)
vertebral)
Hemihyperplasia + + Asymmetry, vascular malformation, Sporadic 104
embryonal tumors
 Hepatic fibrosis + Congenital hepatic fibrosis Sporadic 105
Holzgreve 2 Potter sequence, cardiac defect, Uncertain 106
polydactyly, cleft palate
Hypertelorism-microtia- E + Microcephaly, cleft lip/palate, MR AR 107
clefting
Ivemark 1 + + + Poly/asplenia, complex CHD, later- Sporadic, AR 108
ality defects
Jeune + Narrow chest, short limbs, polydac- AR 82,83
tyly, glomerulosclerosis
Joubert + Vermis aplasia, apnea, jerky eyes, AR 109
retinopathy, ataxia
Juberg-Hayward H + Microcephaly, cleft lip/palate, AR 110
abnormal thumbs/toes
Kabuki H + + U + MR, characteristic Kabuki-like face, Uncertain 111
large ears, cleft palate
Kallmann 1 + Anosmia, cleft lip/palate, hypogo- AD, AR, +R 112
nadotrophic hypogonadism
Kaufman-McKusick E + + + Ua Hydrometrocolpos, polydactyly, AR 113
anal/urogenital sinus defect
Kivlin E + Short stature, Peters’ anomaly, MR, AR 114
genital/cardiac defects
Klippel-Feil 1 E + Short neck, cervical vertebral Sporadic, AD 115
fusion, low posterior hairline
Kousseff 1 Sacral meningocele, hydroceph- AR 116
alus, cardiac defects
Leprechaunism (Donohue) + + Insulin resistance, lipodystrophy, AR 117
hirsutism
Limb–body wall complex 1 E + U, Ua Lateral body wall defect, limb Sporadic 118
reduction, CHD
Mammo-renal + Ipsilateral supernumerary breasts/ Sporadic 119
nipples
Marden-Walker + + Microcephaly, blepharophimosis, AR 120
micrognathia, contractures
Meckel-Gruber + + U, Ua Encephalocele, cardiac defects, AR 121
cleft lip/palate, polydactyly
Megacystis-microcolon + U Large bladder, intestinal hypoperi- AR 122
stalsis, oligohydramnios
Melnick-Needles osteo- + U Bowing long bones, short upper X-linked 123
dysplasty limbs, micrognathia
Mendelhall + Insulin resistance, acanthosis nigri- AR 124
cans
Microgastria–upper limb 1 E + Hypoplastic spleen, limb reduction Uncertain 125
anomaly defects
Miranda + + Brain malformation, liver dysplasia Uncertain 126
Moerman 1 + + + + Short limbs, brain malformation, Uncertain 127
cleft palate
MURCS association 1 E + U See text for details Sporadic

continued

97
 TABLE 6.1. CONTINUED.

98
Urinary tract abnormalities

Inheritance

Renal agenesis
Ectopia/horseshoe
Cystic/dysplasia
Duplication
Hypoplasia
Hydronephrosis/ureter
Diverticulae
Atresia/stenosis
Reflux
Nephritis/sclerosis
Tumor/nephromegaly
Syndromes Other associated anomalies pattern Reference

Nager acrofacial dysostosis + + + + Facial bone hypoplasia, cleft eyelid, AD 128

radial ray defect
Nail-patella + Absent/hypoplastic nails and patel- AD 129
lae
Neu-Laxova 1 IUGR, lissencephaly, CHD, pterygia, AR 130
ichthyosis
Neuro-facio-digito-renal 1 Prominent forehead with vertical Uncertain 131
groove, MR, ear anomaly
Neurofibromatosis type I 1 Ua + Renal artery stenosis, hyperneph- AD 132
roma, café-au-lait spots
Nezelof + + Arthrogryposis, hepatic impair- AR 133
ment, hypotonia, club feet
Noonan + + + Webbed neck, short stature, MR, AD 134
pulmonic stenosis
Occipital horn + B Ua Cranial exostosis, hyperextensibil- X-linked 135
ity, cutis laxa
Ochoa + + B, U, Facial grimacing with lateral dis- Uncertain 136
Ua + placement of mouth
Oculo-auriculo-vertebral See Goldenhar syndrome
Oculo-hepato-encephalo- + + Encephalocele, hepatic fibrosis, AR 137
renal coloboma
Oculorenal See Pierson syndrome 139
Oculorenal (Karcher) 1 + + Optic nerve coloboma AD 139
Oculo-renal-cerebellar + MR, spastic diplegia, choreoatheto- AR 138
sis, retinopathy
OEIS complex + + Ua + Malrotation of colon, sacral defect, Sporadic
tethered spinal cord, pelvic bone
abnormalities
Otorenal + + + U Renal pelvis diverticulae, nerve AD 140
deafness
Pallister-Hall 1, 2 E, H + + Hypothalamic hamartoblastoma, AD 141,142
polydactyly
Pierson + Hypoplastic retina, cataract, ante- AR 143
rior chamber anomalies
Penoscrotal transposition E + + + B U Abnormal placement of external Sporadic 144
genitalia
 Perlman + + + + + Early overgrowth, typical face, AR 145
nephroblastomatosis
Polydactyly-obstructive + Ua + Postaxial polydactyly of hands and Uncertain 146
uropathy feet
Potter (oligohydramnios) 1, 2 + + Ua See text for details Uncertain
Prune belly + + + Ua + See text for details Sporadic
Pseudo-Zellweger + + Hypotonia, seizures, MR, typical AR 147–149
face, FTT, hepatomegaly
Pyloric stenosis H + + + + Cystic kidney Sporadic 150
Rapadilino + Radial/patella hypoplasia, diarrhea, AR 152
short stature, long nose
Rass-Rothschild + + Klippel-Feil anomaly, sacral agene- Uncertain 151
sis, cryptorchidism
Renal dysplasia or adyspla- 1 E + + + Ua + Abnormal uterus in some patients AD 154
sia
Renal-hepatic-pancreatic + Pancreatic cysts, extrahepatic bil- AR 82,83
dysplasia iary atresia, Caroli disease
Renal/müllerian hypoplasia H + + Absent uterus, broad forehead, AR 153
DD, large fontanel
Retinoic acid, maternal use + + U Ear anomalies, CHD, cleft palate, Sporadic 155,156
neural tube defect
Roberts 1 H + + Limb reduction, oligo/syndactyly, AR 157
CHD, dysmorphic face
Robson + MR, macrocephaly, deafness, pro- X-linked 158
teinuria, Alport-like
Rokitansky-Mayer-Kuster- 1 E + U + Absence of vagina, uterine anoma- Sporadic 159
Hauser lies, amenorrhea
Rubella, congenital 1 + + CHD, MR, deafness, cataract, Sporadic 160
growth retardation
Rubinstein-Taybi 1 E + + + Ua SS, MR, broad thumbs and great Sporadic 161,162
toes, typical face
Russell-Silver + Ua SS, triangular face, asymmetry, clin- Sporadic, AD 163
odactyly, hypoglycemia
Santos 1 Hirschsprung’s disease, hearing AR 164
loss, postaxial polydactyly
Say + SS, microcephaly, micrognathia, AD 165
large ear, cleft palate
Schimke + SS, spondyloepiphyseal dysplasia, AR 166
immunodeficiency
Schinzel-Giedion E U + U CHD, distinctive face, figure 8 head AR 167
shape, eyelid groove
Senior-Loken + + Nephronophthisis, tapeto retinal AR 168
degeneration
Setleis + U Cutis aplasia with temporal scar- AD, AR 169
ring, abnormal eyelashes
Short rib, Beemer Langer + + + U Hydrops, cleft lip, bowed long AR 170
bones, atretic ear canal
Short rib–polydactyly, types 1 + Ua Urethral fistula, CHD, cloacal/uro- AR 170,171
1–3 genital sinus anomalies

99
continued
 TABLE 6.1. CONTINUED.

100
Urinary tract abnormalities

Inheritance

Renal agenesis
Ectopia/horseshoe
Cystic/dysplasia
Duplication
Hypoplasia
Hydronephrosis/ureter
Diverticulae
Atresia/stenosis
Reflux
Nephritis/sclerosis
Tumor/nephromegaly
Syndromes Other associated anomalies pattern Reference

Silverman (dyssegmental + U SS, flat face, cleft palate, general- Uncertain 172
dwarfism) ized skeletal dysplasia
Simopoulos + Hydrocephalus, polydactyly AR 173
Simpson-Golabi-Behmel + + + Overgrowth, polydactyly, typical X-linked 174
face, arrhythmia
Sirenomelia sequence 1, 2 E + + + + + + + See text for details Sporadic
Smith-Lemli-Opitz 1 + + SS, ambiguous genitalia, two to AR 175,176
three toe syndactyly, brain
anomalies
Sommer 1 Iris aplasia, corneal opacity, glau- AD 177
coma, prominent forehead
Sorsby (coloboma-brachy- 1 Ocular coloboma, brachydactyly AD 178
dactyly) type B, bifid thumbs
Sotos Ua + Overgrowth, MR, embryonal Sporadic 179
tumors, advanced bone age
Supernumerary nipples, E + + + + Familial polythelia AD 180
renal anomalies
Thalidomide, maternal use 1, 2 E, H + + + + + Limb reduction, phocomelia, neu- Sporadic 181,182
ral tube defect
Thymic-renal-anal-lung + + U SS, absent thymus, parathyroid AR 183
agenesis, urethral fistula
Tolmie + Lethal multiple pterygia, long bone X-linked 184
abnormalities
Townes-Brocks 1 + + Ua + Triphalangeal thumb, imperforate AD 185
anus, skin tag, deafness
Trimethadione, maternal 1 U SS, CHD, omphalocele, distinctive Sporadic 186
use face
Tuberous sclerosis + + MR, seizures, cortical tuber, facial AD 187
angiofibroma
Turner See Table 6.3 Chromosomal
Ulnar-mammary 1 Oligodactyly, ulnar ray defect, nip- AD 188
ple aplasia, genital defects
Urogenital adysplasia 1, 2 + Absent uterus, vaginal atresia, AD 189
hydrometrocolpos
VATER (VACTERL) associa- 1 E, H + + + + + + + See text for details
tion
Velocardiofacial 1, 2 E, H + + + + + See Table 6.3
 von Hippel–Lindau + + Cerebello-retinal angiomatosis, AD 190,191
pheochromocytoma
Weinberg-Zumwalt + + Multiple lung cysts, ascites, acces- Uncertain 192
sory spleen
Wenstrup 1 + + + U Female pseudohermaphrodite, Uncertain 193
imperforate anus
Weyers H + + + U Oligodactyly, pterygia, sternal AR 194
defect, cleft palate
Wiedemann-Beckwith See Beckwith-Wiedemann syn-
drome
Williams E + + B, U U See Table 6.3 Chromosomal
Wilms’ tumor–horseshoe H + Possible association Sporadic 195
kidney
Wilms’ tumor–hemi- + Ipsilateral vascular malformation, Sporadic 104
hypertrophy café-au-lait spots
Wilms’ tumor–radial apla- + Hypoplastic fibula/tibia, abnormal Sporadic 196
sia thumbs
Winter (oto-renal-genital) 1, 2 + Middle ear anomalies, deafness, AR 197
vaginal atresia
Wolfram + + Diabetes mellitus/insipidus, optic Mitochon- 198
atrophy, nerve deafness drial
Zellweger 1 + + Hypotonia, seizures, hepatospleno- AR 147–149
megaly, growth delay

+, Feature present or reported; 1, unilateral renal agenesis; 2, bilateral renal agenesis; ACC, agenesis of corpus callosum; AD, autosomal-dominant; AR, autosomal-recessive; B, bladder; CHARGE,
coloboma, heart disease, atresia choanae, retarded growth and development, genital hypoplasia, and ear anomalies; CHD, congenital heart disease; DD, developmental delay; E, ectopia; FTT,
failure to thrive; H, horseshoe; IUGR, intrauterine growth retardation; MR, mental retardation; MURCS, müllerian, renal, cervicothoracic, somite abnormalities association; OEIS, omphalocele-
cloacal exstrophy-imperforate anus-spinal dysraphism; SS, short stature; U, ureter; Ua, urethra; VACTERL, vertebral, anal, cardiac, tracheal, esophageal, renal, limb; VATER, vertebral, anus, tra-
cheoesophageal, radial, and renal.

101
 TABLE 6.2. WELL-KNOWN SYNDROMES ASSOCIATED WITH OCCASIONAL URINARY TRACT ANOMALIES

102
Urinary tract abnormalities

Inheritance

Renal agenesis
Ectopia/horseshoe
Cystic/dysplasia
Duplication
Hypoplasia
Hydronephrosis/ureter
Diverticulae
Atresia/stenosis
Reflux
Nephritis/sclerosis
Tumor/nephromegaly
Syndromes Other associated anomalies pattern Reference

Aase Ua Triphalangeal thumb, hypoplastic anemia AD, AR 199

Achondrogenesis + + Micromelic dwarfism, short trunk, fetal hydrops AR 200
Acrocallosal 1 + ACC, macrocephaly, polymicrogyria, polydactyly, CHD AR 201
Acro-facial dysostosis H Abnormal thumb/toe, facial bone defect, ear AR 202
anomalies
Acromelic frontonasal dys- E + Polydactyly, ACC, encephalocele, Dandy-Walker Sporadic 203
plasia anomaly
Adrenal hypoplasia-MR + U + Aminoaciduria, MR, muscular dystrophy, visual X-linked 205
abnormality
Adrenogenital 1 + + Ambiguous genitalia, vomiting, salt losing, ure- AR 204
teropelvic junction obstruction
Antley-Bixler H + Craniosynostosis, radio-humeral synostosis, cardiac AR 206
defects
Apert (acrocephalosyndac- + + Acrocephaly, craniosynostosis, syndactyly AD 207
tyly)
Bloom + Short stature, telangiectasias, leukemia, lymphoma AR 208
Bowen-Conradi H + Micrognathia, arthrogryposis, cloudy cornea, brain AR 209
anomaly
Brachydactyly type E 1 + Vertebral anomalies, narrow auditory canal Uncertain 210
3C (Ritscher-Schinzel) + SS, Dandy-Walker anomaly, typical face, CHD AR 211
C-trigonocephaly 1 Polysyndactyly, abnormal ear, hypospadias, dislo- AR 212
cated joints
Campomelic dysplasia 1 + + Tibial bowing, pretibial dimples, ambiguous genitalia AD 213
Carbohydrate deficient gly- + FTT, abnormal fat pad, hepatosplenomegaly, neu- AR 214
coprotein rodegeneration
Carpenter + Aminoaciduria, polysyndactyly, craniosynostosis AD 215
CHILD 1, 2 + Unilateral erythroderma, ipsilateral limb defect X-linked 216
Chondrodysplasia punctata, + Flat face, microcephaly, cataract, short femora/ AR 217
nonrhizomelic humeri, stippled epiphyses
Coffin-Siris 1 + MR, sparse scalp hair, hirsutism, coarse face, thick lips AR 218
Cutis laxa type I B GI tract diverticulae, emphysema, diaphragmatic AR 219
defect
Disorganization-like + Polydactyly, duplication of lower limbs, skin Sporadic 220
appendages
Duane anomaly–radial 1 Limited ocular abduction, radial defects, blepharo- AD 221
defects phimosis
 Ehlers-Danlos + Joint hypermobility, skin hyperextensibility, easy AD, AR 222
bruising
Epidermolysis bullosa + U Skin blistering, pyloric stenosis, dystrophic nails, AR 223,224
sparse hair
Femoral hypoplasia-unusual 1 Various leg deformities, abnormal genitalia, typical Sporadic, 225
facies face AD
Floating-Harbor E SS, typical face, DD, delayed bone age Uncertain 226
Focal dermal hypoplasia H Atrophy/linear skin pigmentation, hand/vertebral X-linked 227
anomalies
Freeman-Sheldon + + Whistling face, ulnar deviation of hands, talipes AD 228
equinovarus
Fronto-metaphyseal dyspla- + + Prominent supraorbital ridges, contractures, deafness X-linked 229
sia
Frontonasal dysplasia + E Hypertelorism, broad nasal tip, median cleft nose Sporadic 230
Fryns + Digital hypoplasia, diaphragmatic defect, cleft palate AR 231
G (Opitz/BBB) + + + See Opitz (G/BBB)
Glutaric aciduria, type II + Cerebral anomalies, pancreatic dysplasia, biliary AR 232
dysgenesis
Grieg cephalopolysyndactyly + Macrocephaly, polydactyly, hypertelorism AD 233
Hajdu-Cheney + SS, Wormian bones, acro-osteolysis, osteoporosis AD 234
Hydrolethalus + Ua Hydrocephalus, polydactyly, polyhydramnios, cleft lip AR 235
Jarcho-Levin + Spondylothoracic dysplasia, fused ribs, hemivertebrae AR 236
Johanson-Blizzard + Pancreatic insufficiency, spiky hair, small alae nasi AR 237
Killian-Pallister See Table 6.3 Chromo-
somal
Lacrimo-auriculo-dento-digi- 1 Nasolacrimal duct stenosis, malformed ears/ AD 238
tal enamel/digits
Larsen 1 + Multiple joint dislocations, flat face AD, ?AR 239,240
Lenz microphthalmia 1 + + Ocular coloboma, ear/facial anomalies, syndactyly/ X-linked 241
camptodactyly
LEOPARD (multiple lentigi- 1 Hypertelorism, deafness, abnormal electrocardio- AD 242
nes) gram, genital anomalies
Marfan + + Tall thin habitus, aortic root dilatation, lens sublux- AD 243,244
ation, arachnodactyly
Marshall-Smith + MR, FTT, accelerated bone maturation, broad pha- Sporadic 245
langes
Miller-Dieker (lissencephaly) 1 + See Table 6.3 Microdele-
tion
Moebius-peripheral neurop- + Peripheral neuropathy, anosmia, hypogonadism Sporadic 246
athy
Mohr-Majewski See Orofaciodigital, type IV
Multiple pterygium + Multiple soft tissue contractures, camptodactyly AR 247
Myotonic dystrophy + Myotonia, muscle weakness, cataract, arrhythmia, AD 248,249
diabetes
Nijmegen breakage + MR, SS, microcephaly, immunodeficiency AR 250
Opitz (G/BBB) Hypertelorism, hypospadias, cleft lip/palate, dys- AD, X-linked 251,252
phagia
Orofaciodigital, type I + + Midline cleft lip, multiple frenulae, polydactyly, X-linked 253
tongue nodules

103
continued
 TABLE 6.2. CONTINUED.

104
Urinary tract abnormalities

Inheritance

Renal agenesis
Ectopia/horseshoe
Cystic/dysplasia
Duplication
Hypoplasia
Hydronephrosis/ureter
Diverticulae
Atresia/stenosis
Reflux
Nephritis/sclerosis
Tumor/nephromegaly
Syndromes Other associated anomalies pattern Reference

Orofaciodigital, type IV 1 + Cleft palate, multiple frenulae, polysyndactyly, AR 253,254

lobed tongue
Orofaciodigital, type VI 1 + Midline cleft lip, multiple frenulae, polydactyly, AR 253
tongue nodules
Pallister-Killian See Table 6.3 Chromo-
somal
Peutz-Jeghers + Hamartomatous intestinal polyposis, lip hyperpig- AD 255,256
mentation
Poland anomaly 1 + + Hypoplastic pectoralis, ipsilateral upper limb reduc- Sporadic 257,258
tion
Restrictive dermopathy U Aplasia cutis, rigid skin, contractures, typical face AR 259
Robinow + + Mesomelic dwarfism, typical face, abnormal genitalia AD, ?AR 260
Rothmund-Thomson + Poikiloderma, alopecia, dysplastic nails, photosensi- AR 261
tivity
Serpentine fibula + Elongated curved fibulae, hirsutism, hypertelorism Uncertain 262
Spondylocostal dysostosis 1 + Sacral agenesis, anal atresia, bifid thumb, skin tags Uncertain 263
Spondyloepimetaphyseal + U Joint laxity, kyphoscoliosis, talipes equinovarus, CHD AR 264
dysplasia
Spondylometaphyseal dys- + SS, platyspondyly, coxa vara, vertebral/long bone AD 265
plasia anomalies
Syndactyly, type V E + Bladder exstrophy, fusion of 4th and 5th metacar- AD 266
pal bones

+, Feature present or reported; 1, unilateral renal agenesis; 2, bilateral renal agenesis; ACC, agenesis of corpus callosum; AD, autosomal-dominant; AR, autosomal-recessive; B, bladder; CHD,
congenital heart disease; CHILD, hemidysplasia with ichthyosiform erythroderma and limb defects; DD, developmental delay; E, ectopia; FTT, failure to thrive; H, horseshoe; LEOPARD, lentigi-
nes, electrocardiographic abnormality, ocular hypertelorism, pulmonic stenosis, abnormal genitalia, retardation of growth, and deafness; MR, mental retardation; SS, short stature; U, ureter;
Ua; urethra.
 TABLE 6.3. CHROMOSOMAL DISORDERS AND THEIR CONSISTENT ASSOCIATED URINARY TRACT ANOMALIES

Urinary tract abnormalities

Reported
familial

Renal agenesis
Ectopia/horseshoe
Cystic/dysplasia
Duplication
Hypoplasia
Hydronephrosis/ureter
Diverticulae
Atresia/stenosis
Reflux
Nephritis/sclerosis
Tumor/nephromegaly
Chromosomal disorders Other associated anomalies cases References

3p deletion E, H + MR, growth delay, ptosis, postaxial polydac- No 269

tyly, micrognathia
3q duplication H + + + MR, SS, seizures, hirsutism, typical face, cardiac No 270
defects
Williams syndrome (7q dele- E + + B, U U SS, typical face, supravalvar aortic stenosis, Yes 267
tion) hypercalcemia
Trisomy 9 mosaicism + + B MR, joint contractures, cardiac defects, brain No 268
anomalies
10q duplication H + MR, ptosis, short palpebral fissures, campto- Yes 269
dactyly
Aniridia-Wilms’ tumor (11p13 + Ambiguous genitalia, hypospadias, short stature AD 270
deletion)
Pallister-Killian syndrome (tetra- + SS, MR, hypogonadism, seizures, diaphrag- No 270
somy 12p) matic defect
Patau syndrome (trisomy 13) 1, 2 H + + + Holoprosencephaly, midline anomalies, cleft No 268
lip/palate
Miller-Dieker syndrome (17p13 1 + MR, lissencephaly, microgyria, agyria, typical No 271
deletion) face, seizures
Edward syndrome (trisomy 18) + E, H + + + IUGR, CHD, clenched hands, rocker bottom feet Yes 268
18q deletion H SS, MR, microcephaly, narrow external ear Yes 272
canals, long hands
Down syndrome (trisomy 21) H + + MR, hypotonia, CHD, typical face, clinodactyly Yes 273
Cat eye syndrome (tetrasomy 1 H + + U MR, CHD, colobomas, anal/digital anomalies Yes 274
22p)
Velocardiofacial syndrome 1, 2 E, H + + + + + + Conotruncal CHD, thymic aplasia, typical face, Yes 275,276
(22q11 deletion) cleft palate
Turner syndrome (45,X or 1 E, H + + + + + SS, amenorrhea, webbed neck, cubitus valgus, No 277
46,X,i(Xq)) hypogonadism
Triploidy H + + Large molar placenta, IUGR, syndactyly of No 270
third and fourth digit, others

+, Feature present or reported; 1, Unilateral renal agenesis; 2, bilateral renal agenesis; AD, autosomal-dominant; B, bladder; CHD, congenital heart disease; E, ectopia; H, horseshoe; IUGR, intra-
uterine growth retardation; MR, mental retardation; SS, short stature; U, ureter.

105
106 I. Development

TABLE 6.4. PREVALENCE OF URINARY TRACT ANOMALIES PER 1000 BIRTHS FROM METROPOLITAN ATLANTA
CONGENITAL DEFECTS PROGRAM (MACDP), CALIFORNIA BIRTH DEFECTS MONITORING PROGRAM (CBDMP),
AND WASHINGTON STATE BIRTH DEFECTS REGISTRY (WSBDR)
Rates per 1000 liveborn infants

Anomalies MACDP 1983–1988 CBDMP 1983–1994 WSBDR 1987–1989

Renal agenesis/dysplasia 0.47 0.480 0.58

Horseshoe kidney No data 0.040 0.16
Cystic kidney No data 0.030 0.05
Obstruction of kidney/ureter 0.80 1.270 1.27
Double ureter No data 0.004 0.05
Exstrophy of bladder 0.03 0.030 0.02
Obstruction of bladder/urethra 0.23 0.160 0.20
VATER, CHARGE, and MURCS associ- No data 0.210a No data
ations
Sirenomelia No data 0.090b No data

CHARGE, coloboma, heart disease, atresia choanae, retarded growth and development, genital hypoplasia, and ear anomalies; MURCS, mülle-
rian, renal, cervicothoracic somite abnormalities; VATER, vertebral, anus, tracheoesophageal, radial, and renal.
aRates include 39 other syndromes with related anomalies.
bRates include 34 other syndromes with related anomalies.

lies reflects ascertainment bias because data are often and for any other type of congenital or developmental
derived from symptomatic individuals. Reliable data have anomalies must be obtained. Many genetic disorders have
been reported from several birth defects registries. In the variable expression even within the same family. A careful
United States, these include the Metropolitan Atlanta Con- physical examination looking specifically for major and
genital Defects Program, California Birth Defects Monitor- minor anomalies should be performed. Sometimes, a pat-
ing Program, and the Washington State Birth Defects tern of multiple anomalies can be recognized immediately
Registry (1,2). Table 6.4 summarizes prevalence data for a as a well-described syndrome. Patterns of anomalies that
number of distinct urinary anomalies reported from these cannot be recognized may require a literature or database
registries. search or referral to an expert in syndrome recognition. The
When adjusted for race, sex, and maternal age, the prev- search for a specific diagnosis is optimally accomplished by
alence of renal agenesis, cystic kidney, exstrophy of the identifying the least common and most distinctive anoma-
bladder, and obstruction of the bladder or urethra do not lies, thereby limiting the list of possible differential diag-
show differences between geographic areas (1). In contrast, noses. To aid in this effort, refer to Tables 6.1 through 6.3
there appears to be a higher prevalence of obstruction of the in addition to tables listing the differential diagnosis that
kidney or ureter in California and Washington compared accompanies the description of each of the major urinary
with Atlanta. It has been suggested that these differences tract anomalies below (see Tables 6.5 through 6.15). For
are due to ascertainment bias; however, differences in pre- example, it is preferable to search for syndromes with ure-
natal teratogenic exposures (e.g., alcohol and cocaine) and thral agenesis (22 syndromes) rather than renal dysplasia
differences in the genetic background of individuals in (more than 80 syndromes) when the two anomalies coexist.
these geographic areas cannot be discounted. Overall, renal A search based on the more common anomalies can be per-
anomalies are found in approximately 3 to 6 per 1000 formed if the first search does not reveal a match. Even after
births (1,3). Genitourinary anomalies are responsible for careful evaluation, a substantial number of children with
approximately 6.8% of infant deaths in the United States multiple congenital anomalies remain undiagnosed.
(March of Dimes Perinatal Data Center 2002; http:// A chromosome analysis is indicated in any child who has
www.marchofdimes.com/aboutus/1523.asp/). at least two major congenital anomalies or one isolated
anomaly that is particularly associated with a defined chro-
mosomal abnormality [e.g., aniridia (microdeletion 11p)].
APPROACH TO THE CHILD WITH A URINARY Growth or developmental delay, dysmorphic features, or
TRACT ANOMALY lack of familial resemblance should also prompt a chromo-
somal analysis. Chromosome abnormalities are found in
The clinical approach to the child with a urinary tract approximately 10 to 12% of all renal anomalies (3,8). Table
anomaly is similar to that for other birth defects. The initial 6.3 lists common and distinct chromosomal disorders with
step is to establish a specific diagnosis if possible based on their reported urinary tract anomalies.
history, physical examination, and laboratory investigation. For a child with no known urinary tract anomaly, find-
A thorough family history for both urinary tract anomalies ings that should prompt an evaluation of the urinary tract
 6. Syndromes and Malformations of the Urinary Tract 107

include oligohydramnios, undefined abdominal mass, (anterior) and the anorectal membrane (posterior) by the
abnormal genitalia, aniridia, hypertension, preauricular pits end of the seventh week. The primitive perineal body forms
or tags, branchial cleft cyst or sinus, imperforate anus, at the site of fusion.
symptoms indicative of renal dysfunction, urinary tract The primitive urogenital sinus develops primarily into
infection, or obstructive uropathy (3). For patients with the urinary bladder. The superior portion, originally con-
known syndromes, the types of associated urinary tract tinuous with the allantois, later becomes a solid fibrous
anomalies are listed in Tables 6.1 and 6.2. cord (the urachus or median umbilical ligament), which
Ultrasonography is recommended in screening for uri- connects the bladder to the umbilicus. The inferior portion
nary tract anomalies. It is noninvasive and provides excel- of the urogenital sinus in the male divides into a pelvic por-
lent anatomic information. It is also the only method tion, containing the prostatic and membranous urethra,
routinely used for the prenatal diagnosis of urinary tract and the long phallic portion, containing the penile urethra.
anomalies. Additional radiographic studies and specialized The inferior portion in the female forms a small portion of
genetic testing may then follow as part of a staged diagnos- the urethra and the vestibule. At the same time, the distal
tic evaluation. Corrective or reparative treatments are avail- portion of the mesonephric ducts is incorporated into the
able for many anomalies (stenosis or atresia, bladder endodermal vesicoureteral primordium, forming the tri-
exstrophy, duplication, diverticula, and tumors). gone of the bladder. A part of the distal end of both meso-
Once a child with a urinary tract anomaly is identified, a nephric ducts just proximal to the trigone develops into the
search for related anomalies in first-degree relatives is indi- seminal vesicles and ductus deferens in the male. Finally, at
cated only when the proband has renal agenesis (9). Other- the end of the twelfth week, the epithelium of the superior
wise, the decision to investigate family members should be portion of the prostatic urethra proliferates to form buds
based on a thorough family history and whether the identi- that penetrate the surrounding mesenchyme. In the male,
fied anomaly is part of a well-described inherited syn- these buds form the prostate gland; in the female, they
drome. Genetic counseling should be provided to the form the urethral and paraurethral glands.
family, and reproductive options should be discussed in a
nondirective fashion. For an isolated anomaly without a
family history of similar or related anomalies, an empiric URINARY TRACT ANOMALIES
risk can be provided. Accurate risk figures can be deter-
mined for mendelian disorders, and estimated risks are Kidney Defects
available for associations.
Renal Agenesis
All children with congenital anomalies need long-term
follow-up. This is especially the case for children with undi- Renal agenesis refers to complete absence of one or both
agnosed multiple congenital anomalies, for whom follow- kidneys without identifiable rudimentary tissue. Renal
up examination may lead to a specific diagnosis. Additional agenesis is usually associated with agenesis of the ipsilateral
relevant family information should be specifically sought ureter. The pathogenesis of renal agenesis is failure of for-
for any newly affected member. Finally, for patients with a mation of the metanephros. Causal heterogeneity has been
urinary tract anomaly who reach reproductive age, the shown, both by animal studies and by human observations
recurrence risk for their offspring and reproductive options (10–12), including failure of ureteric bud formation, failure
should be discussed. of the bud to reach the metanephric blastema, or failure of
the bud and the metanephric blastema, to create mutual
inductive influence on one another (see Chapter 1). In
DEVELOPMENT OF THE URINARY TRACT addition, interruption in vascular supply and regression of a
multicystic kidney can lead to renal agenesis in the fetal
Renal organogenesis is reviewed in Chapter 1. Embryogen- period (11).
esis of the lower urinary tract includes development of the Unilateral renal agenesis is usually asymptomatic and
mesonephric duct and urogenital sinus. The mesonephric detected incidentally, whereas bilateral renal agenesis results
duct inserts into the lower allantois, just above the terminal in severe oligohydramnios and fetal or perinatal loss. Sev-
part of the hindgut, the cloaca. During the fourth to sev- eral studies have demonstrated that unilateral renal agenesis
enth weeks, mesoderm proliferates and forms the transverse is associated with an increased frequency of anomalies in
mesodermal ridge, the urorectal septum. The urorectal sep- the contralateral kidney (9,13). Renal agenesis is often asso-
tum divides the cloaca into the anterior portion (the primi- ciated with anomalies of other organ systems. These anom-
tive urogenital sinus) and the posterior portion (the cloacal alies can occur both in contiguous structures (e.g.,
sinus or anorectal canal). The mesonephric ducts open into vertebrae, genital organs, intestines, and anus) and in non-
the urogenital sinus and later become the ureters. The contiguous structures (e.g., limbs, heart, trachea, ear, and
urorectal septum develops caudally and fuses with the cloa- central nervous system). The diagnosis of renal agenesis is
cal membrane, dividing it into the urogenital membrane made by abdominal ultrasound. Care must be taken to
108 I. Development

TABLE 6.5. SYNDROMES ASSOCIATED WITH TABLE 6.6. SYNDROMES ASSOCIATED WITH
UNILATERAL RENAL AGENESIS UNILATERAL OR BILATERAL RENAL AGENESIS

Acrocallosal syndrome Acrorenal, Johnson-Munson type

Acrorenal syndrome, Dieker type Alkylating agent, maternal use
Acro-renal-mandibular syndrome Caudal duplication syndrome
Acro-renal-ocular syndrome Caudal regression syndrome
Adrenogenital syndrome CHARGE association
Aglossia-adactylia syndrome Cocaine, maternal use
Alagille syndrome (arteriohepatic dysplasia) Diabetic mother, infant of
Branchio-oto-renal syndrome DiGeorge syndrome
C-trigonocephaly syndrome Fraser (cryptophthalmos) syndrome
Campomelic dysplasia Holzgreve syndrome
Cat-eye syndrome Pallister-Hall syndrome
Chondroectodermal dysplasia Potter (oligohydramnios) sequence
Coffin-Siris syndrome Sirenomelia sequence
Cornelia de Lange syndrome Thalidomide embryopathy
Ectrodactyly-ectodermal dysplasia-clefting syndrome Urogenital adysplasia
Femoral hypoplasia–unusual facies syndrome Velocardiofacial syndrome
Fetal alcohol syndrome Winter syndrome
Goldenhar syndrome
Ivemark syndrome CHARGE, coloboma, heart disease, atresia choanae, retarded growth
Kallmann syndrome and development, genital hypoplasia, and ear anomalies.
Klippel-Feil anomaly
Lacrimo-auriculo-dento-digital syndrome
Larsen syndrome
an empiric risk of 3% can be used for families in which
Lenz microphthalmia syndrome
LEOPARD syndrome (multiple lentigines) renal anomalies in first-degree relatives (siblings, parents)
Limb–body wall complex have been excluded (3). First-degree relatives of patients
Miller-Dieker syndrome with nonsyndromic renal agenesis have an increased preva-
MURCS association lence of related urogenital anomalies. In one study, 9.0% of
Neu-Laxova syndrome
first-degree relatives of infants with agenesis or dysgenesis
Oro-facio-digital syndrome, types IV and VI
Pfeiffer syndrome of both kidneys had a related urogenital anomaly, and
Poland anomaly 4.4% had an asymptomatic renal malformation (13).
Renal dysplasia Therefore, renal sonography is recommended for the first-
Roberts syndrome degree relatives of the proband, unless renal agenesis in the
Rokitansky-Mayer-Kuster-Hauser syndrome
proband is clearly sporadic or a specific cause without an
Rubella syndrome, congenital
Rubinstein-Taybi syndrome increased recurrence risk is identified.
Russell-Silver syndrome Tables 6.5 and 6.6 list the syndromes commonly associ-
Short rib polydactyly syndrome, types 1–3 ated with unilateral and bilateral renal agenesis, respec-
Smith-Lemli-Opitz syndrome tively. For more information about these disorders and
Sorsby coloboma-brachydactyly syndrome
other less common conditions with renal agenesis, see
Spondylocostal dysostosis
Townes-Brocks syndrome Tables 6.1 through 6.3.
Trisomy 22
Turner syndrome
Ulnar-mammary syndrome Ectopic Kidney
VATER (VACTERL) association
Ectopic kidneys result from an error(s) of ascent. Most are
Zellweger syndrome
pelvic kidneys that fail to ascend out of the pelvic cavity. Rare
LEOPARD, lentigines, electrocardiographic abnormality, ocular case reports of thoracic kidneys exist (14). Ectopic kidneys
hypertelorism, pulmonic stenosis, abnormal genitalia, retardation of can be unilateral or bilateral. Bilateral pelvic kidneys often
growth, and deafness; VACTERL, vertebral, anal, cardiac, tracheal,
esophageal, renal, limb; VATER, vertebral, anus, tracheoesophageal,
fuse into a midline mass of renal tissue, with two pelvises and
radial, and renal. a variable number of ureters, which is referred to as a pancake
or discoid kidney. Crossed renal ectopia refers to an ectopic kid-
ney whose ureter crosses the midline. Ectopic kidneys are
exclude the possibility of ectopic kidney. Intravenous pyel- usually hypoplastic and rotated and contain numerous small
ography, computed tomography, and radionuclide studies blood vessels and ureteric anomalies. Ectopic kidneys may be
can be helpful in equivocal cases. asymptomatic, but complications from ureteral obstruction,
The recurrence risk for renal agenesis can be estimated if infection, and calculi are common. Table 6.7 provides a list
the pattern of inheritance is known or if the proband has a of syndromes that include ectopic kidney, also described in
recognizable syndrome. For nonsyndromic renal agenesis, Tables 6.1 through 6.3.
 6. Syndromes and Malformations of the Urinary Tract 109

TABLE 6.7. SYNDROMES ASSOCIATED WITH TABLE 6.8. SYNDROMES ASSOCIATED WITH
ECTOPIC KIDNEY HORSESHOE KIDNEY

Acromelic frontonasal dysplasia Acro-facial dysostosis syndrome

Acrorenal syndrome, Dieker type Agnathia-holoprosencephaly syndrome
Acrorenal syndrome, Siegler type Antley-Bixler syndrome
Acro-renal-ocular syndrome Bowen-Conradi syndrome
Baller-Gerold syndrome Caudal regression syndrome
Beckwith-Wiedemann syndrome Diabetic mother, infant of
Branchio-oto-renal syndrome Fanconi anemia syndrome
Caudal regression syndrome Fetal alcohol syndrome
CHARGE association Focal dermal hypoplasia
Crossed ectopia–pelvic lipomatosis syndrome Juberg-Hayward syndrome
DiGeorge syndrome Kabuki syndrome
Drash (Denys-Drash) syndrome Pallister-Hall syndrome
Fanconi anemia syndrome Pyloric stenosis
Fetal alcohol syndrome Roberts syndrome
Floating-Harbor syndrome Thalidomide embryopathy
Frontonasal dysplasia Trisomy 13, 18, 21, and 22
Goldenhar syndrome Turner syndrome
Kaufman-McKusick syndrome VATER (VACTERL) association
Klippel-Feil anomaly Weyers syndrome
Limb–body wall complex Wilms’ tumor
MURCS association
Pallister-Hall syndrome VACTERL, vertebral, anal, cardiac, tracheal, esophageal, renal, limb;
Penoscrotal transposition VATER, vertebral, anus, tracheoesophageal, radial, and renal.
Renal adysplasia
Rokitansky-Mayer-Kuster-Hauser syndrome
Rubinstein-Taybi syndrome
Table 6.8 lists syndromes associated with horseshoe kidney.
Schinzel-Giedion syndrome
Sirenomelia sequence For more details of these disorders, see Tables 6.1 and 6.2.
Turner syndrome
VATER (VACTERL) association
Velocardiofacial syndrome Dysplasia and Polycystic Kidney
Williams syndrome
Renal dysplasia (17–19) and polycystic kidney disease are
CHARGE, coloboma, heart disease, atresia choanae, retarded growth
extensively covered in Chapters 5 and 36, respectively. Table
and development, genital hypoplasia, and ear anomalies; MURCS, 6.9 summarizes well-known syndromes with renal dysplasia/
müllerian, renal, cervicothoracic somite abnormalities; VACTERL, ver- cystic kidney. (See Tables 6.1 through 6.3.)
tebral, anal, cardiac, tracheal, esophageal, renal, limb; VATER, verte-
bral, anus, tracheoesophageal, radial, and renal.
Obstruction and Hydronephrosis
Horseshoe Kidney Urinary obstruction is a complication of a primary anom-
aly, which can be stenosis or atresia of the ureteropelvic
Horseshoe kidney refers to a condition in which the kidneys are
junction, ureter, or urethra; a poorly functional bladder
fused at the lower poles with a renal parenchymal or, less com-
causing stasis or reflux; a malformed dilated ureteral end
monly, fibrous isthmus. The embryopathogenesis of horseshoe
(ureterocele); or extrinsic compression by other structures
kidney with parenchymal isthmus is believed to be abnormal
(e.g., anomalous blood vessels or tumors). Hydronephrosis
migration of nephrogenic cells across the primitive streak
and pelvocaliectasis are the most common urinary tract
before the fifth gestational week. Horseshoe kidney with
abnormalities detected by prenatal ultrasound examination
fibrous isthmus is believed to originate from fusion after the
(20). (See Chapter 4.)
fifth week, before renal ascent (15). Most horseshoe kidneys
Table 6.10 provides a list of syndromes commonly asso-
are located in the pelvis or at the lower lumbar vertebral level,
ciated with obstruction and hydronephrosis. (See Tables
as ascent is further prevented when the fused kidney reaches
6.1 through 6.3.)
the junction of the aorta and inferior mesenteric artery.
Complications of horseshoe kidneys include obstructive
uropathy, calculi, and urinary tract infection. Horseshoe Ureter Defects
kidneys are associated with other genitourinary anomalies as
Duplication
well as renal tumors (16). Wilms’ tumor is the most com-
mon, but renal cell carcinoma, adenocarcinoma, transitional Double ureters or collecting systems are caused by duplica-
cell carcinoma, malignant teratoma, oncocytoma, angiomy- tion of the ureteric bud. Early duplication results in a
olipoma, and carcinoid have all been reported. duplicated kidney, which is usually smaller and fused with
110 I. Development

TABLE 6.9. SYNDROMES ASSOCIATED WITH TABLE 6.10. SYNDROMES ASSOCIATED WITH
RENAL DYSPLASIA/CYSTIC KIDNEY HYDRONEPHROSIS OR HYDROURETER

Acro-renal-mandibular syndrome Acro-cephalo-polysyndactylous dysplasia

Alagille syndrome (arteriohepatic dysplasia) Acrorenal syndrome, Dieker and Johnson-Munson types
Baller-Gerold syndrome Barbet-Biedl syndrome
Bardet-Biedl syndrome Branchio-oto-renal syndrome
Beckwith-Wiedemann syndrome Campomelic dysplasia
Branchio-oto-renal syndrome Caudal duplication and regression syndromes
Campomelic dysplasia CHARGE association
Carbohydrate deficient glycoprotein syndrome Cloacal exstrophy
CHARGE association Coffin-Siris syndrome
Chondrodysplasia punctata, nonrhizomelic Cornelia de Lange syndrome
Cloacal exstrophy Crossed ectopia-pelvic lipomatosis syndrome
Cornelia de Lange syndrome Diabetic mother, infant of
Diabetic mother, infant of Ectrodactyly-ectodermal dysplasia-clefting syndrome
Ectrodactyly-ectodermal dysplasia-clefting syndrome Fanconi anemia syndrome
Fanconi anemia syndrome Fetal alcohol syndrome
Fetal alcohol syndrome Goldenhar syndrome
Fraser (cryptophthalmos) syndrome Hydrolethalus syndrome
Fryns syndrome Kabuki syndrome
Glutaric aciduria, type II Kaufman-McKusick syndrome
Goldenhar syndrome Megacystis-microcolon syndrome
Hajdu-Cheney syndrome Noonan syndrome
Ivemark syndrome Ochoa syndrome
Jeune syndrome OEIS complex
Joubert syndrome Pallister-Hall syndrome
Kaufman-McKusick syndrome Polydactyly–obstructive uropathy syndrome
Lenz microphthalmia syndrome Pyloric stenosis
Leprechaunism (Donohue) syndrome Roberts syndrome
Limb–body wall complex Schinzel-Giedion syndrome
Marden-Walker syndrome Sirenomelia sequence
Marfan syndrome VATER (VACTERL) association
Marshall-Smith syndrome
Meckel-Gruber syndrome CHARGE, coloboma, heart disease, atresia choanae, retarded growth
MURCS association and development, genital hypoplasia, and ear anomalies; OEIS,
Noonan syndrome omphalocele-cloacal exstrophy-imperforate anus-spinal dysraphism;
OEIS complex VACTERL, vertebral, anal, cardiac, tracheal, esophageal, renal, limb;
VATER, vertebral, anus, tracheoesophageal, radial, and renal.
Oral-facial-digital syndrome, types I and VI
Pallister-Hall syndrome
Pallister-Killian syndrome
Potter (oligohydramnios) sequence the ipsilateral kidney and has ureters that enter into the
Prune-belly syndrome bladder separately. Duplication that occurs later results in
Renal adysplasia
double ureters that may have separate openings into the
Roberts syndrome
Rokitansky-Mayer-Kuster-Hauser syndrome bladder. On rare occasions, one of the ureters may have an
Rubella syndrome, congenital ectopic opening into the vagina, vestibule, or urethra. Most
Short rib–polydactyly syndrome double ureters are crossed, and the ureter from the higher
Smith-Lemli-Opitz syndrome pelvis enters the bladder more caudally. Duplication anom-
Thalidomide embryopathy
alies are common but usually asymptomatic; therefore, they
Trisomy 8, 9, 13, 18, 21, and 22
Tuberous sclerosis often remain undetected. One autopsy study reported the
VATER (VACTERL) association prevalence of duplication anomalies to be as high as 1 in
von Hippel–Lindau disease 25, with females approximately four times more likely to be
Zellweger and pseudo-Zellweger syndromes affected than males (21). Unilateral duplication is five to six
times more common than bilateral duplication (3). Double
CHARGE, coloboma, heart disease, atresia choanae, retarded
growth and development, genital hypoplasia, and ear anomalies;
ureters are commonly associated with vesicoureteral reflux
MURCS, müllerian, renal, cervicothoracic somite abnormalities; due to their ectopic opening into the urinary bladder or the
OEIS, omphalocele-cloacal exstrophy-imperforate anus-spinal dys- ureterocele (22). In addition, ureteric obstruction can occur
raphism; VACTERL, vertebral, anal, cardiac, tracheal, esophageal,
renal, limb; VATER, vertebral, anus, tracheoesophageal, radial, at the level of the vesicoureteric junction or that of the ure-
and renal. teropelvic junction.
Table 6.11 summarizes syndromes associated with dupli-
cation, and Tables 6.1 through 6.3 provide clinical infor-
mation about these disorders.
 6. Syndromes and Malformations of the Urinary Tract 111

TABLE 6.11. SYNDROMES ASSOCIATED TABLE 6.12. SYNDROMES ASSOCIATED WITH

WITH DUPLICATION OF URETERS OR BLADDER EXSTROPHY
COLLECTING SYSTEMS
Axial mesodermal dysplasia
Achondrogenesis Caudal duplication syndrome
Acromelic frontonasal dysplasia Caudal regression syndrome
Adrenogenital syndrome Cloacal exstrophy
Antley-Bixler syndrome Frontonasal dysplasia
Bardet-Biedl syndrome OEIS complex
Bowen-Conradi syndrome Sirenomelia sequence
Branchio-oto-ureteral syndrome Syndactyly, type V
Braun-Bayer syndrome Trisomy 18
Caudal duplication syndrome
Diabetic mother, infant of OEIS, omphalocele-cloacal exstrophy-imperforate anus-spinal dys-
Drash (Denys-Drash) syndrome raphism.
Ectrodactyly-ectodermal dysplasia-clefting syndrome
Fanconi anemia syndrome
Fetal alcohol syndrome
Frontometaphyseal dysplasia Bladder exstrophy refers to a urinary bladder that is
G (Opitz-Frias) syndrome open anteriorly because of the lack of abdominal wall
Goldenhar syndrome closure. It is usually associated with anomalies of the
Kabuki syndrome contiguous structures including epispadias and separa-
Kaufman-McKusick syndrome
tion of the pubic rami. Exstrophy is believed to result
Mammo-renal syndrome
Noonan syndrome from an overdeveloped cloacal membrane that interferes
Ochoa syndrome with inferolateral abdominal mesenchymal closure.
Perlman syndrome Therefore, when the cloacal membrane ruptures, the
Poland anomaly inferior abdominal wall has not completely closed and
Prune-belly syndrome
the bladder cavity is exposed. It has been suggested that
Robinow syndrome
Rubinstein-Taybi syndrome bladder exstrophy belongs to the spectrum of omphalo-
Trisomy 8, 9, 13, 18, and 21 cele-cloacal exstrophy-imperforate anus-spinal dysra-
Turner syndrome phism complex (23,24). The extent of anomalies is
Weyers syndrome determined by the timing of the cloacal membrane rup-
ture. Rupture that occurs after the separation of cloaca
by the urorectal septum results in bladder exstrophy,
Hydroureter whereas one that occurs before the separation results in
the more severe cloacal exstrophy and omphalocele-cloa-
Hydroureter, or megaloureter, is caused by distal obstruc-
cal exstrophy-imperforate anus-spinal dysraphism com-
tion and is usually found with hydronephrosis, except in
plex. Bladder exstrophy is six times more common in
ureteropelvic junction obstruction. Hydroureter has the
males.
same etiology as hydronephrosis (see Obstruction and
Table 6.12 lists syndromes associated with bladder
Hydronephrosis).
exstrophy, and Tables 6.1 through 6.3 provide information
about these disorders.
Bladder Defects
Anomalies of the bladder are rare. These include agenesis, Urethral Defects
hypoplasia, diverticulae, and dilatation or megacystis
Agenesis and Atresia
caused by distal obstruction or by nonobstructive causes.
Agenesis of the bladder is usually associated with severe Urethral agenesis is rare. It predominantly occurs in
developmental anomalies of the urinary tract, such as in males, reflecting the complex embryogenesis of the male
sirenomelia and caudal regression syndrome. Hypoplastic urethra. Urethral agenesis is often associated with the
bladder can be found in conditions associated with bilateral bladder obstruction sequence. Table 6.13 lists syndromes
renal agenesis because no urine is produced. Bladder diver- associated with urethral agenesis, and clinical informa-
ticulae have heterogeneous causes. They may result from an tion about these disorders is summarized in Tables 6.1
intrinsic defect in the bladder wall (e.g., in cutis laxa or through 6.3.
Ehlers-Danlos, Ochoa, occipital horn, and Williams syn-
dromes). They can also be caused by increased intravesicu-
Duplication
lar pressure from distal obstruction or by persistent
urachus. For information about specific syndromes associ- Duplication refers to complete or partial duplication of the
ated with bladder diverticulae, see Tables 6.1 through 6.3. urethra, which is a rare anomaly, found only in a few syn-
112 I. Development

TABLE 6.13. SYNDROMES ASSOCIATED WITH TABLE 6.15. SYNDROMES ASSOCIATED WITH
URETHRAL AGENESIS POSTERIOR URETHRAL VALVES

Aase syndrome Acrorenal syndrome, Johnson-Munson type

Acrorenal syndrome, Dieker and Johnson-Munson types Caudal regression syndrome
Adrenogenital syndrome Diabetic mother, infant of
Caudal regression syndrome Kaufman-McKusick syndrome
Cocaine, maternal use Limb–body wall complex
Hydrolethalus syndrome Neurofibromatosis, type I
Kaufman-McKusick syndrome Ochoa syndrome
Limb–body wall complex OEIS complex
Meckel-Gruber syndrome Polydactyly–obstructive uropathy syndrome
Occipital horn syndrome Potter (oligohydramnios) sequence
Ochoa syndrome Prune-belly syndrome
OEIS complex Renal adysplasia
Potter (oligohydramnios) sequence Rubinstein-Taybi syndrome
Prune-belly syndrome Sirenomelia sequence
Renal adysplasia Townes-Brocks syndrome
Russell-Silver syndrome VATER (VACTERL) association
Short rib–polydactyly syndrome, types 1–3
Sirenomelia sequence OEIS, omphalocele-cloacal exstrophy-imperforate anus-spinal dysra-
Sotos syndrome phism; VACTERL, vertebral, anal, cardiac, tracheal, esophageal, renal,
Townes-Brocks syndrome limb; VATER, vertebral, anus, tracheoesophageal, radial, and renal.
Trisomy 21

OEIS, omphalocele-cloacal exstrophy-imperforate anus-spinal dys- ASSOCIATIONS AND SEQUENCES INVOLVING

raphism.
THE URINARY TRACT

dromes. Those syndromes associated with urethral duplica- A number of associations and sequences involve anomalies
tion are listed in Table 6.14, and their findings are provided of the urinary tract that are important for both diagnosis
in Tables 6.1 through 6.3. and clinical management. For this reason, such conditions
are described in more detail in this section in addition to
the information presented in Tables 6.1 and 6.2.
Posterior Urethral Valves
Posterior urethral valves refer to abnormal mucosal folds that
Association of Vertebral, Anus,
obstruct urine flow. Posterior urethral valves can be sus-
Tracheoesophageal, Radial, and
pected prenatally when a dilated bladder is seen in associa-
Renal Anomalies
tion with obstructive uropathy. Recently, the “keyhole sign”
has been demonstrated by prenatal ultrasonography in VATER is an acronym used to designate a nonrandom
fetuses with subsequently confirmed posterior urethral occurrence of vertebral defects, imperforate anus, tracheoe-
valves (25). A voiding cystourethrogram or endoscopy is sophageal fistula, and radial and renal anomalies (26,27).
usually required for a definitive diagnosis. The embryogen- VACTERL (vertebral, anal, cardiac, tracheal, esophageal,
esis of posterior urethral valves is unknown. Proposed renal, limb), another acronym, has been proposed to
hypotheses include an overdeveloped posterior urethral broaden the spectrum of VATER to include cardiac defects
fold, a remnant of the mesonephric duct, and an anoma- and limb anomalies. The term VATER is not a diagnosis per
lous opening of the ejaculatory duct. Table 6.15 lists syn- se, but the designation provides clues for potentially associ-
dromes in which posterior urethral valves can be seen, and ated anomalies and for recurrence risk counseling when no
the other findings in these disorders are provided in Tables specific syndromic diagnosis can be made. Causes of
6.1 through 6.3. VATER association include chromosomal disorders (e.g.,
trisomy 18), genetic syndromes (e.g., Goldenhar and Holt-
Oram syndromes), and teratogenic exposures (e.g., infants
TABLE 6.14. SYNDROMES ASSOCIATED WITH
URETHRAL DUPLICATION
of diabetic mothers and fetal alcohol syndrome). Recently, a
family with a mitochondrial DNA mutation was identified
Amniotic band disruption sequence in which the daughter was born with VACTERL associa-
Limb–body wall complex tion, and her mother and sister had classic mitochondrial
OEIS complex
cytopathy (28). Thus, all patients suspected to have VATER
Prune-belly syndrome
association should have a chromosome analysis, a careful
OEIS, omphalocele-cloacal exstrophy-imperforate anus-spinal dys- family and prenatal exposure history, and a thorough exam-
raphism. ination for dysmorphic features. The spectrum of anomalies
 6. Syndromes and Malformations of the Urinary Tract 113

seen in VATER is broad. Associated renal anomalies are and butterfly vertebrae), and short stature. Additional
usually agenesis, ectopy, or obstruction (26,27). anomalies are common, including rib defects, facial asym-
Because there is apparent causal heterogeneity for metry, limb anomalies, hearing loss, and brain anomalies
VATER association, the inheritance pattern and recurrence (e.g., encephalocele and cerebellar cysts) (35).
risk vary with the cause. VATER association is usually spo- The pathogenesis of MURCS association is unknown,
radic, with an empirical recurrence risk of 1 to 3% when a but it is believed to be related to defects in the paraxial meso-
specific cause cannot be identified (29). Autosomal reces- derm, which gives rise to the cervicothoracic somites and the
sive and X-linked inheritance have been reported for sub- adjoining intermediate mesoderm. Most patients are diag-
sets of patients (e.g., for VATER with hydrocephalus), in nosed because of primary amenorrhea or infertility associated
which recurrence risk can be as high as 25% (28). with normal secondary sexual characteristics, followed by
recognition of reproductive organ atresia. MURCS associa-
tion is usually sporadic. A report of vertebral and renal
Association of Coloboma, Heart Disease,
anomalies associated with azoospermia was proposed to rep-
Atresia Choanae, Retarded Growth and
resent the male version of MURCS association (36).
Development, Genital Hypoplasia,
and Ear Anomalies
Oligohydramnios Sequence
CHARGE is an acronym used to designate an association of
coloboma of iris, choroid or retina, heart defects, atresia choa- Oligohydramnios of whatever cause leads to a recurrent pat-
nae, retarded growth and development, genital anomalies or tern of abnormalities that has been called the oligohydramnios
hypogonadism, and ear anomalies or deafness (30–33). In sequence (3,5). Oligohydramnios may be caused by decreased
addition, unilateral facial palsy is a common finding. Renal production of fetal urine from bilateral renal agenesis or dys-
anomalies found in CHARGE association include ectopy, plasia or by urinary obstruction, or it can result from amni-
dysplasia, renal agenesis, and ureteric anomalies. The presence otic fluid leakage. When oligohydramnios is prolonged and
of two or more anomalies associated with CHARGE associa- severe, the condition is lethal because of pulmonary hypopla-
tion should prompt a search for the others. To prevent over- sia. Moderate oligohydramnios from amniotic fluid leakage
use of the term, it was suggested that at least three anomalies may result in a liveborn child with multiple congenital
are required for the term CHARGE to be applied, and one of anomalies. These anomalies are both malformations and
the anomalies should be either coloboma or choanal atresia deformations. Intrauterine constraint leads to mechanical
(31). CHARGE association has phenotypic overlap with compression that leads to the characteristic flat facial profile
VATER association. Similar to VATER, a specific cause (Potter’s facies), limb deformities (e.g., talipes equinovarus),
should be sought, although familial forms of CHARGE have and intrauterine growth retardation. Decreased fetal move-
been reported more often than for VATER, with recessive, ment as a result of intrauterine constraint causes multiple
dominant, and X-linked inheritance patterns identified. Con- joint contractures (arthrogryposis). Breech presentation is
ditions with anomalies in the spectrum of CHARGE include common. Pulmonary hypoplasia can be the consequence of
trisomy 13, trisomy 18, Wolf-Hirschhorn (deletion 4p), cat- compression of the chest cavity coupled with decreased inspi-
eye, Treacher-Collins, velocardiofacial, Apert, Crouzon, and ration of amniotic fluid. Because the initial defect has many
Saethre-Chotzen syndromes. Therefore, a careful physical causes, recurrence risk is based on the underlying defect.
examination for malformations and dysmorphic features When oligohydramnios is due to nonsyndromic bilateral
should be conducted, and chromosome analysis including renal agenesis or dysgenesis, related renal malformations
specific fluorescence in situ hybridization probes for velocar- occur at an increased frequency in first-degree relatives (13),
diofacial syndrome (deletion 22q) and 4p deletion should be and recurrence risk can be as high as 4 to 9%. The recurrence
performed. Because most cases of CHARGE association are risk can be as high as 25% for an autosomal-recessive disor-
sporadic, the empirical recurrence risk is low (30,33). In der causing bilateral renal agenesis or dysplasia.
familial forms of CHARGE association, the recurrence risk is
substantially higher and can be as much as 25%.
Urethral Obstruction Sequence
The initial defect in this sequence is obstruction of the ure-
Association of Müllerian, Renal, Cervicothoracic
thra leading to dilation of the proximal urinary tract, bladder
Somite Abnormalities
distension, and hydroureter (3,5,37). Obstruction of urine
MURCS refers to the rare association of müllerian duct flow interferes with normal nephrogenesis, resulting in renal
aplasia, renal aplasia, and cervicothoracic somite dysplasia dysplasia. Other potential anomalies related to bladder dis-
(34). Anomalies include absence of the proximal two-thirds tension include cryptorchidism, malrotation of colon, persis-
of the vagina, uterine hypoplasia or aplasia, unilateral renal tent urachus, and limb deficiency caused by iliac vessel
agenesis, ectopic kidney, renal dysplasia, C5-T1 vertebral compression. In addition, oligohydramnios results from lack
anomalies (hypoplasia of vertebrae, fusion, hemivertebrae, of urine and leads to the oligohydramnios sequence.
114 I. Development

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The most common cause of urethral obstruction is poste- and neck, 3rd ed. New York: Oxford University Press, 1990.
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 S E C T I O N

I I

HOMEOSTASIS
 7

SODIUM AND WATER

HOWARD TRACHTMAN

You may not discontinue the salt of your God’s covenant from water compartments, such as the gastrointestinal lumen
upon your meal offerings. . . or cerebrospinal fluid, which need to be considered in a
Leviticus 2:13 distinct category. They are not in direct contact with the
rest of the fluid spaces and are separated by an epithelial
For the life of any creature—its blood represents its life. . . membrane. Water and electrolytes enter these spaces via
active transport processes.
Leviticus 17:14

This chapter reviews the physiologic mechanism involved Composition of Body Water Compartments
in the control of sodium and water homeostasis. Using this
All of the major fluid compartments in the body are sepa-
knowledge as a basis, the common diseases that arise when
rated by semipermeable membranes (2,3). This type of bar-
these systems malfunction are analyzed, and a discussion of
rier permits free passage of the solvent but may limit the
the optimal therapy for these conditions is presented.
movement of selective solutes across the membrane. There-
fore, the solvent will always move down its concentration
gradient to ensure that the osmolality of the solution is the
BODY FLUID COMPARTMENTS AND
same on both sides of the membrane. Despite marked dif-
THEIR COMPOSITION
ferences in the makeup of the cationic and anionic solutes
in the various body water compartments, under equilib-
Total Body Water and Its Compartments
rium conditions, osmolality or tonicity is always equal in all
On average, water comprises 60% of total body weight in body fluids.
adults. This proportion is higher in infants and even greater Because of the presence of active transporters and selec-
in babies born prematurely and very low birth weight neo- tive channels for various solutes within the cell membrane,
nates. Total body water (TBW) declines during early there is an uneven distribution of solutes in the ICW and
infancy and reaches the adult proportion by the end of the ECW compartments. The presence of the Na-K–adenosine
first year (1). The percentage of body weight that is water is triphosphatase (ATPase) pump in the cell membrane
higher in individuals who have lower adipose levels and ensures that potassium is the principal cation in the intra-
higher muscle levels. In addition, TBW may be altered in cellular compartment, and sodium is the primary ECW
disease states that are associated with altered salt handling, cation (4). Similar limitations in membrane permeability to
such as cystic fibrosis. chloride and bicarbonate result in these anions being found
In general, the TBW is divided into two principal com- almost exclusively in the ECW space, whereas proteins and
ponents: the intracellular water (ICW) and the extracellu- phosphate constitute the major intracellular anions. The
lar water (ECW) spaces (2). These spaces are divided in a differences in cell permeability and binding characteristics
2:1 ratio. In those states in which there is an increase in of specific ions are reflected in the coefficients that are used
the TBW, the increase is manifested clinically by the to determine the volume of distribution for individual sol-
increase in the ECW space. The ECW compartment is utes. For example, because of the permeability of cell mem-
further divided into the interstitial and plasma spaces, branes to water, the volume of distribution for sodium is
which are separated in a 3:1 ratio. A component of the equal to the TBW compartment, although sodium is con-
ECW, namely the interstitial fluid in skin and connective fined to the ECW space. In contrast, the volume of distri-
tissue, may serve as a reservoir that can mobilize water bution of bicarbonate is 0.3 × TBW. These considerations
into the plasma volume to sustain circulation during con- are important in formulating therapeutic regimens to treat
ditions of hypovolemia (3). Finally, there are transcellular specific disorders of sodium and water homeostasis (5).
126 II. Homeostasis

Besides the presence of distinctive membrane permeabil- weight premature infants (9). This is a reflection of imma-
ity characteristics for specific solutes, the unequal distribu- turity in renal tubular function coupled with the increased
tion of ions across the membrane is, in part, due to the need for sodium to achieve the high rate of growth that is
Gibbs-Donnan effect, which arises because of the presence normally observed during the first few years of life. The
of impermeant, negatively charged proteins, primarily albu- sodium requirement is exaggerated by intrinsic (diarrhea,
min, in the intravascular space (3). Nonetheless, there is increased losses in children on chronic peritoneal dialysis,
electroneutrality in all body fluids, and the osmolality, or genetic defect in tubular sodium transport) or exogenous
the sum of all osmotically active particles, is equal in all (administration of diuretics) factors that enhance losses of
body water compartments. sodium. In most developed countries, the daily sodium
Under normal conditions, the serum osmolality is 286 ± 4 intake is well in excess of the amount needed to promote
mOsm/kg water. Because sodium is the major cation in the growth or maintain body function. Under normal circum-
ECW, osmolality can be estimated by the following formula: stances, the principal anion that accompanies sodium in
the diet is chloride. In certain disease states, such as renal
Serum osmolality ≈ 2 × [serum sodium concentration]
tubular acidosis, metabolic acidosis associated with chronic
A reflection coefficient of 1.0 indicates a totally non-permeant renal insufficiency, or urolithiasis, it may be advisable to
solute, whereas freely permeable molecules have a reflection provide a portion of the daily sodium requirement as the
coefficient of zero. The reflection coefficient for urea is bicarbonate or the citrate salt.
approximately 0.4. Similarly, in the absence of insulin, the
reflection coefficient for glucose is 0.5. Thus, in circum-
Water
stances in which there is a pathologic elevation in the serum
urea nitrogen (e.g., acute renal failure or glucose concentra- The daily requirement for water has traditionally been
tion) (diabetic ketoacidosis), these solutes will also contrib- expressed as a quantity of mL per metabolic kg (10). How-
ute to osmolality, and the following formula should be used ever, in clinical practice, this is a very cumbersome and
to calculate serum osmolality: impractical method, and all calculations are based on body
weight and size. Currently, three methods are used to esti-
Serum osmolality = 2 × [serum sodium concentration] +
mate the daily fluid requirement. The first is a direct exten-
[serum urea nitrogen]/2.8 + [serum glucose concentration]/18
sion of the use of metabolic kg and uses the following
This formula is based on the molecular weights of urea nitro- formula:
gen (28 d) and glucose (180 d) and the standard practice of
Daily water requirement = 100 mL/kg for a child weighing
reporting these serum concentrations as mg/100 mL. The
less than 10 kg
calculated serum osmolality is normally within 1 to 2% of
+ 50 mL/kg for each additional kg up to 20 kg
the value obtained by direct osmometry in clinical chemistry
+ 20 mL/kg for each kg in excess of 20 kg
laboratories. However, there are circumstances in which the
calculated serum osmolality is significantly lower than the The second method is based on body surface area and uses
value obtained by measurement with an osmometer. This the following formula:
“osmolal gap” reflects the accumulation of unmeasured
Daily water requirement = 1500 mL/m2 body surface area
osmoles such as organic solutes that are produced after an
ingestion of ethanol or ethylene glycol (antifreeze) (6,7). The last method is a refinement of the second and uses the
following formula:
Daily water requirement = Urine output + insensible water
MAINTENANCE SODIUM AND losses
WATER REQUIREMENTS
Based on clinical experience, under normal circumstances,
urine output is approximately 1000 mL/m2/day, and insen-
Sodium
sible losses amount to 500 mL/m2/day.
It is established that sodium is an essential component of the Thus, consider a child weighing 30 kg and 123 cm in
diet. Not only is this cation necessary for the maintenance of height with a body surface area of 1 m2. According to the
the size of the extracellular fluid (ECF) space (see later), it is first method, the daily water requirement is 1700 mL,
also required for normal growth. Wassner (8) has demon- whereas the second method yields 1500 mL/day. This
strated that somatic growth of experimental animals is example illustrates the relative benefits of these two meth-
impaired if they are fed a sodium-deficient diet. This effect is ods. Although the first is easier to apply, it tends to overesti-
independent of protein or calorie content of the diet. mate the water requirement as body weight increases. The
Balance studies indicate that the daily sodium require- third method is the most precise and should be applied in
ment is 2 to 3 mmol/kg body weight. This quantity is more unusual circumstances, such as when the patient has
nearly two- to threefold higher in term and very low birth oliguria secondary to acute renal failure or increased insen-
 7. Sodium and Water 127

sible losses (e.g., diarrhea, increased ambient temperature, turbances are not associated with any obligatory changes in
tachypnea, burns, or cystic fibrosis) (11). In addition to the the serum sodium concentration.
daily energy requirement and insensible losses that are rep- Water homeostasis is a prerequisite for the normal distribu-
resented in the formulas, the amount of water excreted tion of fluid between the ICW and ECW compartments. Cell
daily is dependent on the solute load. Because the urine has function is dependent on stabilization of cell volume to keep
a minimum osmolality—approximately 50 mOsm/kg the cytosolic concentration of enzymes, cofactors, and ions at
H2O, even in the absence of arginine vasopressin (AVP)— the appropriate level. Perturbations in water balance result in
increased dietary intake of solute will result in a larger fluctuations in serum osmolality. Because cell membranes are
obligatory urine volume to accommodate the larger solute semipermeable and generally permit free movement of water
load (12). down an osmolal gradient, changes in osmolality will cause
The daily sodium and water requirement are generally obligatory shifts in water between the cell and the ECW space.
provided enterally. Intravenous administration of fluids and Any disorder that alters the 2:1 ratio of water volume in the
electrolytes should be used only under clinical circum- ICW:ECW spaces will be reflected in changes in cell size and
stances that interfere with normal feeding, such as persis- subsequent cellular dysfunction. Thus, under hypoosmolal
tent vomiting or gastrointestinal tract surgery. conditions, water will move from the intravascular compart-
ment into the cell, causing relative or absolute cell volume
expansion. Conversely, if the serum osmolality is elevated,
DISTINCT ROLES OF SODIUM AND WATER water will exit from the cell to the ECW space, resulting in
IN BODY FLUID HOMEOSTASIS absolute or relative cellular contraction (14).
Disturbances in cell function related to abnormalities in cell
Sodium and water are inextricably linked in the determina- size occur throughout the body. However, this problem is espe-
tion of the serum sodium concentration. However, it is crit- cially prominent in cerebral cells. There are two reasons for this
ical to recognize that sodium and water serve two distinct phenomenon. First, the blood-brain barrier, which is consti-
functions within the body. Sodium is instrumental in the tuted by tight junctions between adjacent endothelial cells,
maintenance of the size of the ECF space, whereas water is limits the movement of solute between the ICW and ECW
critical to the maintenance of the size of individual cells. compartments, while permitting unrestricted flow of water
The regulation of sodium and water homeostasis represents down an osmolal gradient (15). Second, because the brain is
two distinct processes with discrete sensing and effector contained within the skull, which is a closed, noncompliant
mechanisms. Although these systems may overlap from a space, and is tethered to the cranial vault by bridging blood
physiologic and clinical perspective, a complete under- vessels, it has limited tolerance of cell swelling or contraction.
standing of body fluids and electrolytes mandates separate Thus, alterations in water balance and serum osmolality are
evaluation of sodium and water. dominated by clinical findings of central nervous system dys-
As mentioned earlier, sodium is confined primarily to the function, including lethargy, seizures, and coma (14).
ECW compartment as a consequence of active transport Similar to the situation noted earlier in which distur-
mechanisms in the cell membrane. Because sodium is the bances in sodium balance do not necessarily imply specific
principal cation in this space, disturbances in total body abnormalities in serum sodium concentration, the presence
sodium content are reflected by expansion or contraction in of a disturbance in water balance and serum osmolality is
the ECW compartment. Adequacy of the ECW compart- not linked to a specific abnormality in the ECW compart-
ment is essential to maintain the intravascular space and sus- ment. The independent nature of disturbances in sodium
tain perfusion of vital organs. The primary step in the and water balance is illustrated in Table 7.1. It is apparent
pathogenesis of disturbances in ECW compartment size is a that alterations in ECW size can occur in patients with
perturbation in sodium balance. Under normal circum- hypoosmolality, isotonicity, or hypertonicity. Similarly,
stances when TBW and sodium content are within the nor- each alteration in serum osmolality can develop in patients
mal range, sodium balance is zero. This means that the daily with contraction or expansion of the ECW compartment.
intake of sodium is matched by the net losses in the urine, Disturbances in sodium and water homeostasis need to
stool, and other insensible losses. Provided kidney function is be addressed separately in the clinical evaluation of patients
normal, the dietary sodium intake can be as low as 0.1 with abnormalities in sodium and/or water. The clinical
mmol/kg or in excess of 10 mmol/kg without any derange- approach to these problems is outlined below.
ment in ECF compartment size. If the alterations in diet are
not abrupt, then sodium balance is maintained, even when
kidney function is markedly impaired (13). In contrast, if the SENSOR MECHANISMS:
daily input of sodium exceeds the normal losses, then there is SODIUM AND WATER
expansion of the ECF space that may manifest as edema. If
the input does not match the daily losses, there will be symp- For both sodium and water homeostasis, the sensor mecha-
toms and signs related to ECW space contraction. These dis- nisms that maintain the equilibrium state are primarily
128 II. Homeostasis

TABLE 7.1. CLINICAL DISEASES OF SODIUM AND WATER HOMEOSTASIS:

RELATIONSHIP BETWEEN EXTRACELLULAR WATER (ECW) SIZE AND TONICITY
Tonicity

ECW volume Low Normal High

Low Addison’s disease Isotonic dehydration Hypertonic diarrheal

Salmonella diarrhea dehydration
Mannitol infusion Diabetes insipidus
Normal SIADH No disease Acute sodium bicarbon-
ate infusion
High Acute renal failure Nephrotic syndrome Salt intoxication
Nephrotic syndrome Saltwater drowning
Cirrhosis
Congestive heart failure

SIADH, syndrome of inappropriate secretion of antidiuretic hormone.

designed to be responsive to the secondary consequences of the total ECW space size. Neural receptors that respond to
abnormalities in sodium or water balance (i.e., changes in mechanical stretch or changes in right or left atrial pressure
ECW and cell size, respectively) rather than measuring the convey the signal via the vagus nerve (17,18).
primary variable. They operate using negative feedback
loops in which deviations from normal are detected, coun-
Hepatic Receptors
terregulatory mechanisms are activated that antagonize the
initiating event, and the system is restored to its original The enhanced effect on renal sodium excretion of saline infu-
state. sions directly into the hepatic vein versus the systemic circula-
tion suggests that there are also low-pressure sensors within the
portal vein or hepatic vasculature. The hepatic responses to
Sodium
changes in sodium balance have been divided into two catego-
The detection of abnormalities in sodium balance is based ries (19). The “hepatorenal reflex” involves direct activation of
on systems that sense the consequences of these changes. sodium chemoreceptors and mechanoreceptors in the hepato-
Thus, net sodium deficit is detected as a decrease in ECW portal region via the hepatic nerve and causes a reflex decrease
space size, whereas net sodium excess is perceived as an in renal nerve activity. The “hepatointestinal reflex” uses
obligatory expansion of the ECW space. These receptors, chemoreceptors to respond to changes in sodium concentra-
which are influenced by the filling pressure within the cir- tion and modulate intestinal absorption of sodium via signals
culation, are called baroreceptors or mechanoreceptors. It is conveyed along the vagus nerve. Activation of these hepatic
important to note that these signals are supplemented in volume sensors may contribute to the sodium retention and
certain instances by chemoreceptors that respond directly edema states that develop secondary to chronic liver disease
to changes in the serum sodium concentration and trigger and cirrhosis with their associated intrahepatic hypertension.
adaptive modifications in renal sodium handling. These
receptors may effect change by altering nervous system
Pulmonary Receptors
activity or by activating upstream promoter elements and
stimulating the expression of relevant genes (16). There may also be pressure sensors within the pulmonary cir-
culation that are activated by changes in pulmonary perfu-
sion or mean airway pressure (20). The receptors in the lung
Atrial Receptors
may be located in the interstitial spaces and may influence
There are ECW volume receptors on the venous (low pres- the physical forces that modulate paracellular absorption of
sure) and arterial (high pressure) sides of the circulation. sodium and water. Similar receptors in the renal interstitium
Within the right atrium, there are sensors that possess the may also influence paracellular absorption of fluid and sol-
distensibility and compliance needed to detect alterations in utes along the nephron, especially in the proximal tubule seg-
intrathoracic blood volume by maneuvers such as negative ment (21).
intrathoracic pressure or head-out water immersion. Both of
these maneuvers, which increase the central blood volume
Carotid Arch Receptors
and raise central venous and right atrial pressures, are fol-
lowed by a brisk natriuresis and diuresis (17). These changes There are also volume-dependent sensors on the high-pressure
are triggered even in the absence of concomitant change in side of the circulation, including the carotid arch, the brain,
 7. Sodium and Water 129

and the renal circulation. Thus, occlusion of the carotid to alterations in cell size (26). These osmoresponsive cells
leads to increased sympathetic nervous system activity and are located in the anterolateral regions of the hypothala-
alterations in renal sodium handling (18). The responsive- mus, adjacent to but distinct from the supraoptic nuclei.
ness of the carotid arch receptors may be modulated by They shrink or swell in response to increases or decreases in
chronic changes in ECF volume. For example, head-down plasma tonicity, and this change in cell size triggers the
bed rest and a high-salt diet blunt carotid baroreceptor release of AVP and/or the sensation of thirst.
activity and lead to natriuresis (22). AVP is a peptide containing nine amino acids and has a
molecular weight of 1099 d. It is synthesized by the cells in
the hypothalamus, transported down the axon, and stored
Cerebral Receptors
in the posterior pituitary in conjunction with larger pro-
Alterations in the sodium concentration of the cerebrospi- teins, called neurophysins (27). The gene for AVP is located
nal fluid or brain arterial plasma lead to increased renal on chromosome 20 and has a cyclic adenosine monophos-
sodium excretion (23). Various lesions in discrete anatomic phate response element in the promoter region. Prolonged
areas of the brain, such as the anteroventral third ventricle, stimulation of AVP release leads to upregulation of the AVP
alter renal sodium reabsorption and buttress the contention gene; however, synthesis does not keep up with the need for
that there are central mechanisms of sensing changes in the peptide, because pituitary levels of AVP are usually
sodium balance and ECF volume. Derangements in the depleted in states such as chronic salt loading and hyper-
sensing system within the brain in patients with long- natremia (28).
standing central nervous system diseases may contribute to The principal solute that provokes the release of AVP is
the cerebral salt-wasting syndrome. sodium. The infusion of sodium chloride to increase plasma
Of interest, if the arterial sensors perceive underfilling of osmolality results in increased secretion of AVP in the
the vascular space, this activates counterregulatory mecha- absence of parallel changes in ECW volume. This under-
nisms to replete the ECW compartment size, even if the scores the primary role of fluctuations in plasma osmolality
receptors in the venous system detect adequate or even over- per se in stimulating AVP release (26). Mannitol, an exoge-
filling of the venous tree. Despite normal or even excess total nous solute that is used in clinical practice to treat increased
body sodium and net positive sodium balance, there are con- intracranial pressure, is nearly as effective as sodium in stim-
ditions in which the body perceives an inadequate circulating ulating AVP release. Urea and glucose are less than 50% as
plasma volume. This has given rise to the notion of the effective as sodium in provoking AVP secretion, presumably
“effective” intravascular volume, a concept that is applicable because they are more permeable than sodium and cause less
in the edema states such as congestive heart failure, cirrhosis, pronounced changes in osmoreceptor cell volume. However,
and nephrotic syndrome (24). For example, in patients with in disease states such as acute renal failure or diabetic ketoac-
cardiac pump failure, the perceived underfilling of the arte- idosis in which urea or glucose act as osmotically active mol-
rial tree may occur despite significant venous distention and ecules or after the exogenous administration of mannitol,
TBW overload (25). Similarly, women who develop edema these solutes can also stimulate increases in AVP release.
during pregnancy may have primary peripheral vasodilata- Likely there is coupling between mechanical changes in
tion and excess total body sodium (24). membrane structure and hormone release. However, the
In summarizing the sensor mechanisms that are involved exact mechanism and the neurotransmitters that mediate
in the regulation of sodium balance, there are those that are the actions of the osmoreceptors on the cells of the posterior
directly linked via mechanoreceptors to the status of the pituitary have not been identified.
ECF volume. These sensor systems are activated by There are a variety of nonosmotic stimuli to AVP release
decreased size of the ECW compartment and respond to that may contribute to water handling in various disease
“underfilling” of the vasculature tree. However, there are states (26). Vomiting and acute hypoglycemia promote AVP
other mechanisms that may be activated by chemoreceptors release by neural-hormonal pathways that are not well
or localized intra-organ disturbances in perfusion that are defined. Stress associated with pain or emotional anxiety,
dissociated from the ECF volume. These sensors can cause physical exertion, high body temperature, acute hypoxia, and
overfilling of the vascular compartment by stimulating acute hypercapnia are other conditions that lead to increased
renal sodium reabsorption. Correct interpretation of the secretion of AVP in the absence of a primary disturbance
balance between these two processes involved in sodium in water balance. There are numerous drugs that directly
balance is critical to the proper diagnosis and management influence the hypothalamic release of AVP, including carba-
of the edema states (see later). mazepine, cyclophosphamide, and vincristine. Finally, hemo-
dynamic changes arising from primary alterations in sodium
balance and the ECW space can trigger AVP release. If the
Water: Arginine Vasopressin
ECW volume disturbance is mild, then the stimulation of
The receptors that are responsible for regulating water AVP release is modest. However, in the face of severe ECW
homeostasis are primarily osmoreceptors and are sensitive volume contraction, there is marked secretion of AVP. Under
130 II. Homeostasis

TABLE 7.2. FACTORS THAT INCREASE ARGININE these elements are linked in a large feedback loop involving the
VASOPRESSIN RELEASE liver, kidney, and lung, as well as smaller loops within individ-
↑ Plasma osmolality ual organs. This complex system accounts for the often-dispa-
Hemodynamic rate data that have been accumulated about plasma renin
↓ Blood volume activity and the expression of these components within the
↓ Blood pressure kidney during disturbances in ECW compartment size.
Emesis
Hypoglycemia
Angiotensin II is the major signal generated by this axis (3).
Stress The peptide interacts with two different receptors, and most of
Elevated body temperature its biologic activity is mediated by the angiotensin type 1
Angiotensin II (AT1) receptor. The AT2 receptor is expressed more promi-
Hypoxia nently in the fetal kidney; however, interaction of angiotensin
Hypercapnia
Drugs
II with the AT2 receptor postnatally may stimulate the release
of molecules, such as nitric oxide, that counteract the primary
↑, increased; ↓, decreased. action of the peptide (31). The best-known effects of angioten-
sin II include peripheral vasoconstriction to preserve organ
perfusion and stimulation of adrenal synthesis of aldosterone
these circumstances, the imperative to protect the effective
to enhance renal sodium reabsorption. These two actions serve
circulating blood volume takes precedence over the need to
to restore the ECW space to normal. However, angiotensin II
maintain plasma osmolality, and ECW volume is restored at
also has direct actions on tubular function and stimulates both
the expense of hypoosmolality. This clinical observation indi-
proximal and distal sodium reabsorption. The proximal tubule
cates that despite rigorous intellectual attempts to separate
cells contain all of the elements needed to synthesize angioten-
sodium and water homeostatic mechanisms, these two fac-
sin II locally, and the peptide increases the activity of the
tors are closely linked in vivo, and there can be significant
sodium-hydrogen exchanger (32). In the distal tubule, angio-
overlap in sensor and effector mechanisms in the regulation
tensin II modulates this exchanger as well as the amiloride-
of ECW and ICW compartment size. Table 7.2 lists the fac-
sensitive sodium channel (33). The effects of aldosterone on
tors that modulate AVP release.
the renal tubule include both an immediate effect to increase
In addition to AVP release, the osmoreceptor cells also
apical membrane permeability to sodium and more extended
respond to the changes in serum osmolality in an indepen-
effects that involve enhanced gene transcription and de novo
dent manner to stimulate thirst and increase drinking (29).
synthesis of Na-K-ATPase. Aldosterone may also stimulate the
The stimuli for thirst are generally the same as those for AVP
synthesis of other enzymes involved in renal cell bioenergetics
release, among which hypernatremia is the most potent trig-
(e.g., citrate synthase) that are needed to sustain maximal
ger. The osmotic threshold for thirst in humans appears to be
tubular sodium transport (34).
higher than for AVP secretion, namely 295 mOsm/kg. The
sensing mechanism that leads to this increase in water intake
is even more obscure than that for AVP release. It is likely Endothelin
that changes in ECW volume are also involved in this pro-
This vasoactive molecule is part of a family of three pep-
cess, because angiotensin II, which rises in states of ECW
tides of which endothelin-1 is the most important in
volume contraction, is a potent dipsogen (30).
humans (35). It is converted in two steps from an inactive
precursor to a biologically active 21–amino acid peptide.
Endothelins react with two receptors, ETA and ETB, and
EFFERENT MECHANISMS:
cause vasoconstriction, which result in a decrease in renal
SODIUM AND WATER
blood flow (RBF) and glomerular filtration rate (GFR).
With regard to sodium balance, the primary effect of endo-
The efferent mechanisms involved in maintaining sodium
thelin is sodium retention mediated by the reduction in
and water balance include a variety of neural and endo-
GFR. This would suggest that endothelin acts in concert
crine-humoral systems. There often is an overlap in the
with angiotensin II to protect ECW compartment size
action of these effectors, with an individual effector having
under conditions of sodium deficit. However, the situation
distinctive effects on sodium and water balance.
may be more complicated, because direct exposure of prox-
imal tubule and medullary collecting duct cells to endothe-
Sodium lin in vitro inhibits sodium absorption.
Renin-Angiotensin-Aldosterone Axis
Renal Nerves
The major components of this system—renin, angiotensino-
gen, and angiotensin-converting enzyme—are found within There is abundant sympathetic nervous innervation of the
the kidney and within the vasculature of most organs. Thus, renal vasculature and all tubular segments of the nephron
 7. Sodium and Water 131

(36). The efferent autonomic fibers are postganglionic and antinatriuretic signals in the kidney, including sympathetic
originate in splanchnic nerves. The renal innervation is pri- nervous system activity, angiotensin II, and endothelin.
marily adrenergic and involves α1 adrenoreceptors in the The overall effects of ANP to counteract increases in ECW
blood vessels and both α1 and α2 receptors along the baso- compartment have been demonstrated by short-term stud-
lateral membrane of the proximal tubule. Renal sympa- ies in which acute infusions of ANP improved cardiac sta-
thetic nervous system activity contributes to preservation of tus in patients with congestive heart failure and promoted a
ECF volume by promoting renal vasoconstriction and low- diuresis in patients with acute renal failure (40).
ering GFR and by increasing sodium reabsorption. Among
the catecholamines involved in adrenergic transmission,
Prostaglandins
norepinephrine exerts an antinatriuretic effect. The fact
that dopamine, another sympathetic nervous system neu- The kidney contains the enzymes required for constitutive
rotransmitter, promotes a natriuresis suggests that there (COX-1) and inducible (COX-2) cyclooxygenase that are
may be internal regulation of the effect of nerve activation necessary for the conversion of arachidonic acid to prosta-
on renal sodium handling (37). glandins (PGs) (41). The major products of these pathways
Renal sympathetic nervous activity is inversely propor- are PGE2, PGF2α, PGD2, prostacyclin (PGI2), and throm-
tional to dietary salt intake (36). Drug-induced sodium boxane (TXA2). In the cortical regions, PGE2 and PGI2
retention and volume-dependent hypertension (e.g., with predominate, whereas PGE2 is the major PG metabolite in
the use of cyclosporine) is mediated in part by activation of the medulla. These two compounds increase GFR and pro-
the sympathetic nervous system (38). In addition, increased mote increased urinary sodium excretion. In addition, they
adrenergic nervous signaling within the kidney is instru- antagonize the action of AVP. These actions may mediate
mental in the initiation of hypertension in experimental the adverse effects of hypercalcemia and hypokalemia on
animals by causing a right-shift in the pressure-natriuresis renal tubular function (41). The natriuretic effects of PGs
curve (36). However, the observation that sodium balance in response to alterations in dietary sodium intake under
is normal and ECF volume is maintained in the denervated normal circumstances are unclear. However, the role of PGs
transplanted kidney implies that the role of the sympathetic as efferent signals is more apparent in conditions associated
nervous system in maintaining sodium homeostasis is with increased vasoconstrictor tone, such as congestive
redundant and can be taken over by other regulatory mech- heart failure or reduced renal perfusion. In these condi-
anisms (36). tions, PGs act to counteract the vasoconstrictor and
sodium-retaining effects of angiotensin II and norepineph-
rine. Inhibition of PGs with cyclooxygenase inhibitors can
Atrial Natriuretic Peptide
be associated with dramatic declines in GFR and profound
Atrial natriuretic peptide (ANP) is a 28–amino acid pep- sodium retention and edema (42).
tide that is a member of a group of proteins that includes
brain natriuretic peptide and C-type natriuretic peptide
Kinins
(39). It is synthesized as a prohormone that is stored in
granules in the cardiac atria. Increases in right trial pressure Kinins are produced within the kidney and act via β2 recep-
provoke cleavage and release of the mature peptide. For tors. Their principal action is to promote renal vasodilata-
each 1-mm Hg rise in central venous pressure, there is a tion and natriuresis. The kinins act in the distal tubule to
corresponding 10- to 15-pmol/L increase in circulating reduce sodium reabsorption (43).
ANP levels. Conversely, declines in atrial pressure sec-
ondary to sodium depletion or hemorrhage reduce ANP
Nitric Oxide
release. There are two receptors for ANP, and both are cou-
pled to guanylate cyclase. The activation of this enzyme The kidney contains all three isoforms of nitric oxide syn-
results in cytosolic accumulation of cyclic guanosine mono- thase (NOS) that are involved in NO synthase—neuronal
phosphate, which, in turn, appears to diminish agonist- NOS in the macula densa, inducible NOS in renal tubules
stimulated increases in intracellular calcium concentration. and mesangial cells, and endothelial NOS in the renal vas-
The principal effects of ANP are to promote an increase in culature. The neuronal and endothelial isoforms are cal-
GFR, diuresis, and, most important, natriuresis. The cium-dependent enzymes and produce small, transient
increased renal sodium excretion is, in part, mediated by an increases in NO production. The inducible isoform is
increase in filtered load secondary to the rise in GFR. How- upregulated by various cytokines and inflammatory media-
ever, ANP also exerts direct actions on renal tubular cells to tors, resulting in large, sustained elevations in NO release.
diminish sodium reabsorption, including inhibition the Activation of eNOS within the kidney increases the
Na-K-Cl cotransporter in the loop of Henle and the activity of soluble guanylate cyclase and causes vasodilata-
amiloride-sensitive sodium uptake in the medullary collect- tion and an increase in GFR. In addition to its effect on
ing duct. Finally, ANP antagonizes the action of several RBF and GFR, NO has direct effects to inhibit Na-K-
132 II. Homeostasis

ATPase in cultured proximal tubule and collecting duct cells between the ECW and ICW compartments is undisturbed,
(44,45). The specific isoform of NOS that is responsible for these patients are clinically unaffected by their abnormally
modulating urinary sodium excretion is not as well known. high-serum sodium concentration. Although there may be
Recent studies using inducible NOS, neuronal NOS, and gender-related differences in AVP secretion in response to
endothelial NOS knock-out mice suggest that only the first abnormal water homeostasis with increased sensitivity in
two isoforms are involved in the regulation of sodium and women, this is not a relevant clinical concern in prepuber-
water reabsorption in the proximal tubule (44). There is evi- tal children.
dence to support a role of NO in maintaining sodium bal-
ance under normal conditions, because alterations in dietary
Angiotensin
salt intake are associated with parallel changes in urinary
excretion of nitrite, the metabolic byproduct of NO release Angiotensin II serves as an efferent system in water homeo-
(46). In normotensive Wistar-Kyoto rats and spontaneously stasis primarily by stimulating drinking (30). Its role in
hypertensive rats, increased dietary sodium intake is associ- water handling within the kidney is minor and may be
ated with a modest increase in urinary nitrite excretion (47). related to modulation of the renal response to AVP.
This effect is less well documented in pediatric patients.
Along with ANP and bradykinin, NO is part of the defense
Thirst
system against sodium excess and expansion of the ECW
compartment. Derangements in renal NO synthesis and Thirst, or the consciously perceived desire to drink, is a
responsiveness to cyclic guanosine monophosphate may be major efferent system in water homeostasis (29). It is esti-
instrumental in the pathogenesis of salt-dependent hyper- mated that for each 1-pg/mL increase in the circulating
tension in experimental animals (47). plasma AVP level, there is a parallel rise of 100 mOsm/kg
H2O in urinary concentration. If the basal plasma osmolal-
ity and AVP concentration are approximately 280 mOsm/
Adrenomedullin
kg H2O and 2 pg/mL, respectively, and the steady state
Adrenomedullin is a 52–amino acid peptide that was iso- urine osmolality is 200 mOsm/kg H2O, then as soon as the
lated from human pheochromocytoma cells (48). It reacts plasma osmolality and AVP concentration reach 290
with a G-protein cell receptor and causes vasodilatation, an mOsm/kg H2O and 12 pg/mL, respectively, the urine is
effect that may be mediated by increased synthesis of NO. maximally concentrated. Beyond this point, the only oper-
The resultant natriuresis secondary to the increase in GFR is ational defense against further increases in plasma osmolal-
accompanied by direct inhibition of tubular sodium reab- ity is increased free water intake. This underscores the
sorption. Its role in sodium balance is under investigation. essential role of thirst as an efferent mechanism in water
homeostasis. Moreover, it highlights the increased risk of
hyperosmolality developing in patients who do not have
Water free access to water such as infants, those who are physically
or mentally incapacitated, or the elderly (49).
Arginine Vasopressin
Thirst is a very difficult biologic function to quantitate,
The primary efferent mechanism in the maintenance of because it is an expression of a drive rather than an actual
water homeostasis is AVP. This peptide fosters water reten- behavior. At present, visual analog scales using colors or
tion by the kidney and stimulates thirst. The plasma AVP faces are the most useful tools for quantitating thirst under
concentration is approximately 1 to 2 pg/mL under basal controlled condition. There can be dissociation between
conditions (26). It is not known whether there is tonic water intake and the sensation of thirst, as in patients with
release of AVP or whether there is pulsatile secretion in psychogenic polydipsia (e.g., schizophrenia, neurosis). It is
response to minute fluctuations in plasma osmolality. The not known whether specific drugs directly stimulate the
set point, or osmotic threshold for AVP release, ranges from dipsogenic response. The role of diet (e.g., high salt intake)
275 to 290 mOsm/kg H2O. The circulating hormone con- in the regulation of thirst is unknown. The osmotic control
centration rises approximately 1 pg/mL for each 1% of thirst may be suboptimal in both newborn infants and in
increase in plasma osmolality. The sensitivity of the the elderly (49).
osmoreceptors in promoting AVP release varies from per- Thirst and drinking behavior are stimulated by signifi-
son to person, with some individuals capable of responding cant contraction of the ECF space or by hypotension. In
to as small as a 0.5 mOsm/kg H2O increase in osmolality addition, thirst and drinking behavior are modulated by
and others requiring greater than a 5 mOsm/kg H2O incre- signals that originate in the oropharynx and upper gas-
ment to stimulate AVP release. Patients with essential trointestinal behavior. Thus, in animals with hypernatre-
hypernatremia possess osmoreceptors that have normal sen- mia, those that are given free access to water as the sole
sitivity, but the osmotic threshold for AVP release is shifted means of correcting the hyperosmolal state will stop drink-
to the right. Because the relative distribution of water ing sooner than animals, which are corrected in part with
 7. Sodium and Water 133

supplemental intravenous fluid. This is most likely due to predominantly on the vascular tone of the afferent arteriole
oropharyngeal stimuli that curtail drinking before complete and cause substantial reduction in RBF and the filtration
normalization of the plasma osmolality (50). fraction. Agents in this category include adrenergic nerve
stimulation and endothelin. In contrast, angiotensin II acts
primarily on efferent arteriolar tone and tends to preserve
EFFECTOR MECHANISMS:
GFR more than RBF. Therefore, filtration fraction (GFR/
SODIUM AND WATER
RBF) is increased. This pattern is most evident in states of
compromised effective perfusion such as congestive heart
The kidney is the principal organ that acts in response to
failure, cirrhosis, and nephrotic syndrome (24,25,53). The
sensory input, delivered via neural or humoral signals, to
critical role of angiotensin II in maintaining GFR and
restore ECW volume size to normal after the full range of
sodium excretion in these conditions is manifest during the
clinical problems. These are supplemented by specific ana-
functional decline in GFR that occurs after the administra-
tomic features of the effector organ to achieve normal
tion of angiotensin-converting enzyme inhibitors (54). This
homeostasis. Although absorption of sodium and water
phenomenon also explains the reversible reduction in kid-
across the intestinal epithelium may be modulated by
ney function and sodium retention that are observed in
chemoreceptors in the hepatic vasculature, the role of the
patients with a critical renal artery stenosis in a kidney trans-
gastrointestinal tract in the control of sodium balance is
plant after initiation of angiotensin-converting enzyme
clearly secondary to the function of the kidney.
inhibitor therapy (54).

Sodium
Proximal Tubule
Glomerular Filtration Rate
Nearly 60 to 70% of the filtered sodium and water load are
In children with normal kidney function, changes in GFR are reabsorbed in the proximal tubule. Sodium and fluid reab-
generally associated with parallel alterations in sodium bal- sorption are isosmotic in this segment of the nephron. These
ance. This is accomplished by glomerular-tubular balance, in processes are driven by Na-K-ATPase activity along the baso-
which proximal tubule sodium absorption and delivery of fil- lateral membrane surface with secondary active transport of
trate to the distal tubule is modulated in response to GFR solute across the apical membrane. The bulk of sodium reab-
(51). Thus, tubular sodium reabsorption increases in parallel sorption is driven by the sodium-hydrogen exchanger, with a
with an increase in GFR. This, in part, reflects the load- lesser contribution by other cotransport systems for glucose,
dependent nature of sodium reabsorption in the proximal phosphate, organic anions, and amino acids. The linkage
tubule. In addition, changes in GFR lead to changes in the between disturbances in ECF volume and sodium reabsorp-
oncotic pressure within the peritubular capillaries that also tion in the proximal tubule is created, in part, by changes in
influence sodium reabsorption (52). Thus, an increased GFR the physical forces that govern fluid and solute movement.
is associated with higher hydrostatic pressures in the peritubu- These include changes in peritubular capillary hydrostatic
lar capillary network that retard fluid and solute reabsorption pressure, peritubular capillary protein concentration and
in the proximal tubule changes. Finally, tubuloglomerular oncotic pressure, and changes in renal interstitial pressure
feedback is activated by alterations in solute delivery to the that modulate water and solute movement across cells (trans-
distal nephron to bring GFR in line with alterations in tubu- cellular) and along the paracellular pathway.
lar function. Many of the efferent signals, including renin, It is now evident that sympathetic nervous stimulation,
angiotensin, NO, and PGs, participate in this particular path- norepinephrine release, and both filtered and locally syn-
way. The release of these effector molecules is activated via thesized angiotensin II stimulate the activity of the sodium-
myogenic stretch receptors and chemoreceptors located in the hydrogen antiporter and promote sodium reabsorption in
macula densa region of the distal nephron. Even in children conditions associated with decreased ECF volume. Con-
with compromised renal function (GFR <20 to 30 mL/min/ versely, ANP and the kinins act on proximal tubular cells to
1.73 m2) in whom there are adaptive changes in tubular func- inhibit sodium reabsorption and limit expansion of the
tion [e.g., increased fractional excretion of sodium (FENa)], ECW space.
glomerulotubular balance is maintained in the face of gradual
changes in GFR. However, patients with chronic renal failure
Distal Nephron Including Collecting Duct
are unable to respond to abrupt changes in sodium balance
and ECF volume changes as rapidly as healthy children. This portion of the nephron is responsible for the reabsorp-
Therefore, they are susceptible to volume contraction or tion of approximately 10 to 25% of the filtered sodium and
hypervolemia if sodium intake is substantially reduced or water load. Under most circumstances, it can adapt to
increased over a short period of time (13). changes in delivery arising from alterations in proximal
Most of the neural and humoral factors described previ- tubule function. This segment of the nephron is responsive
ously can modulate GFR. Agents that lower GFR may act to virtually all of the humoral efferent signals and accom-
134 II. Homeostasis

plishes the final renal homeostatic response to fluctuations in ous mutations in the gene and corresponding abnormalities
sodium balance. Sodium reabsorption in the distal tubule in protein structure in children with X-linked congenital
and connecting segment is responsive to circulating levels of nephrogenic diabetes insipidus (57).
aldosterone (34). In the collecting tubule, there are mineralo- The water channels that mediate increased transmem-
corticoid-responsive sodium reabsorptive pathways that are brane movement of water across the collecting tubule in
responsible for the final modulation of sodium excretion in response to AVP have been identified and are called aquapor-
response to alterations in sodium intake. The most promi- ins (58). There are nine known members of this group of
nent of these is the epithelial sodium channel (ENaC). This transmembrane water channels, all of which contain six
transepithelial protein is composed of three distinct chains— membrane-spanning domains. The first member to be iden-
α, β, and γ—each of which is encoded by a separate gene tified was aquaporin-1 (AQP-1; originally called channel-
(55). The complete protein has two membrane-spanning forming integral membrane protein of 28 kDa or CHIP-28).
domains with an amino and carboxyl terminus within the It is a transmembrane protein that mediates water movement
cell. The α-chain appears to constitute the actual sodium- across the erythrocyte membrane and along the proximal
conducting pathway, whereas the β- and γ-chains may repre- tubule. Mice that do not express AQP-1 have a normal phe-
sent regulatory components that control the open/closed sta- notype and concentrate their urine normally. Aquaporin-2
tus of the channel. Genetic defects in each of the individual (AQP-2) is the major AVP-sensitive water channel in the col-
components have been described and linked to human dis- lecting tubule (59). Immunogold electron microscopy stud-
ease. Thus, pseudohypoaldosteronism has been mapped to ies have confirmed that AQP-2 represents the water channel
mutations in the α-, β-, and γ-chains, and Liddle’s syndrome that is present within the cytosolic vesicles that fuse with the
has been attributed to truncation in the β-chain (55). apical membrane after exposure of principal cells in the col-
lecting duct to AVP. The contribution of AQP-3 and AQP-4
to the normal urinary-concentrating mechanism has been
Water confirmed in mice that have been genetically manipulated
and do not express these two proteins (60).
Arginine Vasopressin
The importance of AQP-2 in mediating the normal
AVP acts along several segments of the nephron. However, response to AVP has been verified by the discovery of muta-
its primary site of AVP action for maintenance of water tions in the AQP-2 gene in children with non–X-linked,
homeostasis is the collecting tubule (56). In the segment autosomal recessive forms of nephrogenic diabetes insipidus
of the nephron, AVP reacts with a V2 receptor—a 371– (61). Moreover, alterations in AQP-2 protein expression have
amino acid protein that is coupled to a heterotrimeric G- been documented in other states associated with a urinary-
protein—along the basolateral membrane of cells along the concentrating defect, such as lithium exposure, urinary tract
distal tubule and the collecting duct. The V2 receptor gene obstruction, hypokalemia, and hypercalcemia (58).
has been localized to region 28 of the X chromosome. This It is important to acknowledge that water reabsorption
epithelial cell receptor is distinct from the V1 receptor in in the collecting duct is not completely dependent on the
the vasculature that is linked to Ca-activation of the inosi- presence of AVP. Thus, even in animals that are genetically
tol triphosphate cascade, which mediates vasoconstrictor deficient in AVP (Brattleboro rats) or in patients with cen-
response to the hormone (56). tral diabetes insipidus, the urinary osmolality does increase
Binding of AVP to the V2 receptor activates basolateral slightly above basal levels in the face of severe ECF volume
adenylate cyclase and stimulates the formation of cyclic contraction. This concentration may be the consequence of
adenosine monophosphate within the cytosol. This intra- reduction in urinary flow rate along the collecting duct that
cellular second messenger then interacts with the cytoskele- enables some passive equilibration to occur between the
ton, specifically microtubules and actin filaments, and luminal fluid and the hypertonic medullary interstitium.
promotes fusion of intramembrane particles that contain Although the collecting duct is the primary site of reg-
preformed water channels with the apical membrane of ulation of net water reabsorption, the proximal tubule
principal cells in the collecting duct. The AVP-induced may contribute to water balance under circumstances of
entry of preformed water channels involves clathrin-coated decreased ECW compartment size. Whereas the proximal
pits. Withdrawal of AVP is followed by endocytosis of the tubule normally reabsorbs approximately 60% of the fil-
membrane segment containing the water channels into ves- tered water load, this proportion may exceed 70% when
icles that can be localized to the submembrane domain of the ECF volume is diminished. Furthermore, by decreas-
the cell. This results in termination of the hormone signal. ing fluid delivery to the distal nephron, this may enhance
The recycling of water channels from vesicles to the apical the AVP-independent reabsorption of water along the col-
membrane and then back into vesicles has been demon- lecting tubule. These combined effects may explain the
strated in freeze-fracture studies of cells exposed to AVP beneficial effects that have been observed after administra-
(56). The importance of the V2 receptor in water homeo- tion of thiazide diuretics to patients with nephrogenic
stasis has been confirmed by the documentation of numer- diabetes insipidus (62).
 7. Sodium and Water 135

Countercurrent Mechanism that counteract the adverse effects of altered cell size on
protein function (14). This immediate response is followed
The primary locus of the urinary-concentrating mechanism
in short order by the uptake or extrusion of electrolytes as
is the medulla and involves the thin descending limb of
an acute response to the altered size cell. Because there are
Henle, the medullary thick ascending limb of Henle, the
inherent limits on the ability to regulate cell volume exclu-
cortical thick ascending limb of Henle, and the collecting
sively with inorganic electrolytes, this is followed by a more
duct (63). Sodium and water reabsorption are isosmotic in
extended response involving the membrane transport or
all segments of the nephron proximal to the loop of Henle.
synthesis/degradation of a variety of compatible solutes,
To concentrate or dilute the urine, water and solute must
called osmolytes, whose cytosolic concentration can be safely
be separated to enable excretion of free water or urine that
altered within the cell without perturbing cell function.
is hyperosmolal relative to plasma. This process begins in
These osmoprotective molecules include various carbo-
the medullary and cortical thick ascending limb of Henle
hydrates (sorbitol, myo-inositol), amino acids (taurine,
in which NaCl is reabsorbed independently of water, gener-
glutamate), and methylamines (betaine, glycerophospho-
ating a hypotonic luminal fluid. This action is linked in
rylcholine) (14). The osmolytes accumulate in the cytosol
series to the low water permeability of the distal tubule and
to preserve cell function during chronic osmolal distur-
the connecting segment, which together with continued
bances. The cell volume regulatory response can be acti-
sodium reabsorption enhances the hypotonicity of the
vated by electrolytes such as sodium or neutral molecules
urine in this segment. In a secondary step, the permeability
(e.g., urea and glucose) (14). The adequacy of the cell vol-
to water along this segment of the nephron is much lower
ume regulatory response and the accumulation of osmo-
than in the descending limb of the loop of Henle. This
protective molecules in cerebral and renal cells may be
enables water to move down its osmolal gradient from the
dependent on the rate of rise in osmolality, as well as the
tubule lumen into the interstitium as it enters the medulla
magnitude of the absolute change (64).
in the descending limb. Finally, the third critical compo-
Experimental data in animals and clinical experience in
nent of the countercurrent mechanism is the presence of
premenopausal women suggest that estrogens may impair
vasa recta, which perfuse the inner medulla via vascular
the cell volume regulatory response to disturbances in
bundles that contain hairpin loop–shaped blood vessels.
plasma osmolality. This increases the risks associated with
This facilitates the efficient removal of the water that exits
both the untreated abnormalities and therapy (65).
in the descending limb of Henle from the medullary inter-
Although there are some who assert that adaptive control of
stitium without washing out the solute gradient that pas-
cell size is inadequate during development, it has been
sively drives water reabsorption in the collecting tubule.
demonstrated that the accumulation of osmoprotective
The final effector mechanism is the alteration in the water
molecules during chronic hypernatremia is normal in
permeability of the collecting tubule in response to AVP
preweanling rats and, in fact, there is a higher set-point to
and the generation of a concentrated urine or the excretion
preserve the increased brain cell water content (66).
of solute free water in the absence of AVP (Table 7.3).
Failure to adequately account for the cell volume regula-
tory response to osmolal disorders contributes to some of
Osmoprotective Molecule the adverse effects associated with inappropriate correction
(Compatible Osmolytes) of abnormalities in plasma osmolality. These effects include
neurologic dysfunction, specifically seizures, during the
Besides the presence of effector mechanisms to maintain
treatment of hypernatremia; osmotic demyelinating syn-
water balance, cells possess a wide range of adaptive mecha-
drome during rapid reversal of hyponatremia; dialysis dyse-
nisms to counteract the undesirable movement of water
quilibrium syndrome that occurs during the initiation of
between the cell and the ECW during hypotonic and
dialysis in patients with acute or chronic renal failure; and
hypertonic states and prevent neurologic dysfunction.
cerebral edema and brain herniation in patients with dia-
These mechanisms include early response genes that result
betic ketoacidosis (67,68).
in the immediate accumulation of chaperone molecules

TABLE 7.3. FACTORS THAT CONTRIBUTE TO THE LABORATORY ASSESSMENT OF SODIUM

COUNTERCURRENT MECHANISM AND WATER BALANCE
Na-Cl-K–mediated solute absorption in the medullary thick
ascending limb of Henle There are no normal values for sodium and water intake or
Low water permeability of the distal tubule and connecting seg- excretion, reflecting the wide range of normal daily intake for
ment both sodium and water as a consequence of differences in
High water permeability in the descending limb of Henle
dietary composition. Healthy individuals are in balance, and
Vasa recta and elimination of interstitial water volume
Arginine vasopressin responsiveness of the collecting tubule the excretion of sodium and water matches the daily intake.
Therefore, laboratory assessment of sodium and water homeo-
136 II. Homeostasis

stasis is confined to disease states in which the clinicians must If the free water clearance is a positive number, then the
determine whether renal sodium and water handling are urine/plasma osmolality ratio is less than 1, the urine is
appropriate for the clinical circumstances, will maintain bal- dilute, and the kidney is in a diuretic mode. When a water
ance, and will prevent disturbances in ECF volume or water diuresis is maximal, the free water clearance measures the
distribution between the ICW and ECW compartments. capacity of the kidney to excrete free water. In contrast,
patients who are in an antidiuretic mode with urine:plasma
osmolality ratio greater than 1 and who are able to concen-
Sodium
trate their urine will have a negative free water clearance. As
The urine sodium concentration is not a valid index of the solute excretion rate increases, both the maximum val-
sodium balance, because the value may vary depending on ues for free water clearance and free water reabsorption
the volume and concentration of the sample. Therefore, the increase. At any given solute excretion rate, the free water
renal handling of sodium is best evaluated using the FENa. clearance greatly exceeds the free water reabsorption. This
After obtaining a random urine sample and a simultaneous indicates that the renal water homeostatic mechanisms
blood sample and measuring the sodium and creatinine designed to protect against overhydration and dilution of
concentrations in both specimens, the FENa is calculated the ECW are more robust than those used to defend
using the following formula: against water deficit and dehydration.
FENa = Excreted sodium/Filtered sodium
= Urinary sodium concentration × urine flow rate/Plasma OVERVIEW OF THE EVALUATION OF FLUID
sodium concentration × GFR AND WATER ABNORMALITIES
= Urine sodium concentration/Plasma sodium concentration
Urine creatinine concentration/Plasma creatinine concen- In practice, the clinical information and laboratory data
tration that are used to evaluate patients overlap with one another.
This formula is based on the insertion of the creatinine However, in view of the different physiologic roles of
clearance as a measurement of GFR in the second equation sodium and water balance in body fluid homeostasis, the
and the cancellation of the urine flow rate term in the distinct regulatory mechanisms used to control these fac-
numerator and denominator. Therefore, the determination tors, and the varied therapeutic strategies that must be used
of the FENa is a particularly useful test in clinical practice, to restore sodium and water balance to normal in disease
because the measurement does not require a timed urine states, it is essential that disturbances in sodium and water
collection. In healthy individuals, this value varies depend- balance be evaluated separately. In realtime, these separate
ing on the daily sodium intake. However, in patients with assessments are done in parallel, a reflection of the body’s
ECF volume contraction who are responding appropriately own method of operation.
to retain sodium, the FENa is less than 1% (less than 3% in When confronted with a child with a sodium and water
neonates). Conversely, in patients with expansion of the disturbance, the first question that must be addressed is
ECW compartment, the FENa typically will exceed 3% whether there is a life-threatening compromise in the size
unless there is concomitant renal disease. of the ECF volume. Thus, it is necessary to clarify whether
the ECF volume is decreased or expanded. By implication,
this is an assessment of sodium balance. Such patients have
Water a life-threatening disease and may require emergency ther-
Determination of the urine-specific gravity or osmolality in apy such as volume resuscitation or acute ultrafiltration,
a random sample will vary depending on the water intake reflecting the vital importance of a normal ECF volume to
in the past 2 to 4 hours. Therefore, the assessment of water maintain intravascular volume within the normal range to
handling is best judged by determining these values under sustain perfusion of vital organs.
more controlled conditions such as in the water-deprived After making this emergency determination and insti-
state or after administration of a water load (10 to 20 mL/ tuting appropriate therapy, it is important to grade the
kg body weight) to evaluate the urinary concentrating or magnitude of the disturbance in ECF volume. Unfortu-
diluting capacity, respectively. nately, there are no laboratory tests that can reliably substi-
The functional aspects of renal water handling are best tute for clinical judgment. Because acute changes in body
assessed by determining the free water clearance. This rep- weight always reflect alterations in sodium balance and
resents the amount of solute-free water excreted by the kid- ECW compartment size, serial measurements in body
ney. It is calculated using the following formula: weight are the most reliable indicator of the presence and
severity of disturbances in ECF volume. However, these
Free water clearance = Urine volume – osmolal clearance measurements are often unavailable and, therefore, the
= Urine volume – [Urine osmolality × urine flow rate]/ evaluation of sodium balance is based on a wide range of
Plasma osmolality clinical findings, including changes in mental status, level
 7. Sodium and Water 137

of alertness, irritability, presence of thirst, pulse rate, blood SODIUM BALANCE DISTURBANCES:
pressure, orthostatic changes, fullness of the anterior fon- DEFICIT AND EXCESS
tanelle in infants, the presence of tears, dryness of the
mucus membranes, skin color, elasticity of the skin or tent- Sodium Deficit
ing, capillary refill or turgor, peripheral edema, shortness of
Diagnosis and Evaluation
breath, and the presence of rales on auscultation of the
chest. Some investigators have documented that capillary In children with normal kidney function, consumption of a
refill may be the most useful test to rapidly and accurately diet that is low or high in sodium does not generally cause a
assess ECF volume and the response to treatment (69). net negative or positive sodium balance, because the kidney
Other findings on clinical examination include urinary- can adaptively modify sodium reabsorption to parallel fluc-
specific gravity and central venous pressure. Laboratory tuations in salt intake if the changes are not too abrupt or
investigations include BUN, serum creatinine, and bicar- massive in nature.
bonate concentrations. Sodium deficits and ECF volume contraction are danger-
It is important to emphasize that assessment of ECF vol- ous because decreased size of the intravascular fluid can lead
ume is a clinical determination. There is no laboratory test to reduced perfusion and ischemia in organs such as the
that is a valid surrogate marker. Moreover, despite the fre- brain, heart, and kidneys. In children, the absence of con-
quency of clinical disturbances in sodium balance, espe- comitant atherosclerosis disease or endothelial dysfunction
cially ECF volume contraction, no suitable scoring has secondary to essential hypertension, smoking, hyperlipid-
been devised based on any combination of the previously emia, and diabetes decreases this risk. However, there are
mentioned elements that can be used to accurately and reli- groups of pediatric patients who may be more susceptible to
ably distinguish different degrees of ECF volume contrac- the adverse consequences of hypovolemia These include
tion or expansion. This deficiency is in contrast to the newborn babies in whom high circulating levels of vasocon-
Glasgow coma score or APACHE (acute physiology and strictor hormones and impaired autoregulation render the
chronic health evaluation) score that have been successfully glomerular microcirculation particularly sensitive to reduced
applied to the initial assessment of patients with acute neu- perfusion (70). In addition, underlying diseases or medica-
rologic or multisystem illnesses. tions may hinder the counterregulatory responses to ECF
The third step in the evaluation of a patient with a sodium volume contraction and enhance the risks of hypovolemia.
and water disturbance is to evaluate the plasma osmolality. Diseases that cause sodium deficiency can originate out-
This evaluation indicates whether there will be an abnormal side the kidney or within the kidney. It is unfortunate that
distribution of water between the ECW and ICW compart- the word dehydration is often used to describe these states
ments. The most likely symptoms that will be present in because it deflects attention from the primary defect,
affected patients will arise secondary to central nervous dys- namely a net negative sodium balance, and suggests that
function. These include confusion, irritability, lethargy, water deficit is the major pathophysiologic problem in
obtundation, and seizures. These manifestations overlap sig- these conditions (71,72). The critical role of the ECF space
nificantly in patients with hyperosmolality or hypoosmolality. and sodium balance in the pathogenesis of states of volume
Moreover, there is no obligatory change in ECF volume. contraction is highlighted by comparing them with diabe-
Therefore, unlike disorders of ECF volume, disturbances in tes insipidus. When sodium balance is perturbed, >60% of
water balance and distribution require a laboratory determi- the fluid loss is derived from the ECW compartment, pro-
nation for confirmation and grading. The steps involved in voking the rapid onset of symptoms. In contrast, only 8%
the initial evaluation of a child with a disturbance in sodium of the pure water loss that occurs in diabetes insipidus is
and water balance are outlined in Table 7.4. derived from the ECF, accounting for the rare evidence of
ECF volume contraction. Use of the term denaturation may
provide a more accurate depiction of what is occurring in
TABLE 7.4. STEPS IN THE INITIAL EVALUATION AND patients with primary deficits in sodium balance and con-
TREATMENT OF A CHILD WITH A DISTURBANCE IN traction of the ECF volume (71).
SODIUM AND/OR WATER BALANCE Extrarenal causes can be attributed to losses of sodium
Step 1: Determine whether there is a life-threatening alteration
in any body fluid or across any epithelial surface, including
in ECF volume. the cerebrospinal fluid, pleural fluid, biliary tree, gas-
Volume resuscitation if there is ECF volume contraction. trointestinal losses, or the skin. They can represent the
Consider ultrafiltration if there is ECF volume expansion. result of a disease process, or they may be iatrogenic in
Step 2: Grade severity of defect in sodium balance. nature. Renal diseases can cause sodium deficit as a result of
Clinical determination of ECF volume.
Step 3: Determine whether there is a defect in water balance.
limited homeostatic capacity caused by a compromise in
Laboratory measurement of plasma osmolality. GFR. Alternatively, there may be primary renal sodium loss
that is not the consequence of a decrease in kidney func-
ECF, extracellular fluid. tion. Finally, renal sodium reabsorption may be diminished
138 II. Homeostasis

TABLE 7.5. CAUSES OF NET SODIUM DEFICIT abnormalities of the kidney, such as dysplasia or obstruc-
tive uropathy.
Renal causes
Compromised glomerular filtration rate
Acute decrease in sodium intake or increased losses
Treatment
Tubular disorders
Osmotic diuresis In the unusual circumstance in which the ECW is so
Diabetic ketoacidosis
severely contracted that vital organ perfusion is compro-
Renal tubular acidosis
Pseudohypoaldosteronism mised, based on an altered mental status, orthostatic
Obstructive uropathy changes, and azotemia, then fluid resuscitation must be ini-
Bartter’s syndrome tiated on an emergent basis. This treatment is necessary to
Renal dysplasia/hypoplasia prevent the development of acute tubular necrosis, which
Central nervous system
may occur if there is hypotension and renal ischemia secon-
Cerebral salt wasting
Cerebrospinal fluid drainage procedures dary to ECF volume contraction. The risk of acute renal
Hepatobiliary system failure is higher in children with preexisting renal disease,
Biliary tract drainage children who are receiving nephrotoxic medications, or
Gastrointestinal tract children who have concomitant hemoglobinuria or myo-
Infectious diarrhea
globinuria. If there is no evidence of cardiac or pulmonary
Chloride diarrhea
Laxative abuse disease, then the optimal therapy under these conditions is
Malignancy (carcinoid, tumor-related) infusion of isotonic crystalloid (0.9% NaCl, Ringer’s lac-
Adrenal diseases tate), 20-mL/kg body weight. Although transfusions of
Salt-losing congenital adrenal hyperplasia whole blood are optimal for treatment of hemorrhagic
Addison’s disease
shock, infusion of crystalloid solutions avoids difficulties
Skin losses
Cystic fibrosis caused by extravasation of the colloid into the interstitial
Neuroectodermal diseases compartment. Moreover, a systematic review of the litera-
Burns ture does not support the use of colloid solutions for vol-
ume replacement in critically ill patients (74). This fluid is
appropriate regardless of what the initial serum sodium or
because of reduced circulating levels of aldosterone or unre- osmolality is, and concerns about the advisability of infus-
sponsiveness to the hormone. The major causes of sodium ing Ringer’s lactate are misplaced in view of the low potas-
deficiency are summarized in Table 7.5. sium concentration (4 mmol/L) in this solution. A catheter
The diagnosis of the cause of a disturbance in sodium should be placed in the bladder to facilitate monitoring of
balance is made based on the history and physical examina- urine output. The infusion should be as rapid as possible,
tion. In most cases, this information is adequate to identify and the fluid dose should be repeated as often as necessary
the source of the sodium losses. Previously, the degree of to achieve some evidence of clinical improvement, such as
ECF volume contraction was categorized as mild, moder- improved mental status, decrease in pulse, rise in blood
ate, or severe if the changes in body weight were estimated pressure, or improved capillary refill.
to be less than 5%, 5 to 10%, or more than 10%, respec- After addressing and correcting the underlying disease, a
tively. Life-threatening ECF volume contraction was fluid repletion plan should be initiated as soon as possible.
thought to represent a more than 15% decrease in weight. Preference should be given to correcting sodium and elec-
Recent data, based on systematic body weights at the time trolyte deficits with oral rehydration solutions (ORS). In
of hospitalization and immediately after correction of the general, patients can be repleted with a rapid (1 to 2 hour)
sodium deficit, suggest that these numbers overestimate the intravenous infusion to restore ECF volume followed
degree of sodium deficit and that the ECF volume contrac- immediately by initiation of ORS (75). The only condi-
tion is better estimated to be less than 3%, 3 to 6%, and tions that represent contraindications to the use of ORS
greater than 6% with a greater than 9% change in body are impaired neurologic status, persistent vomiting, or dis-
weight representing an emergency (73). eases associated with mucosal damage in the gastrointesti-
If the losses are primarily extrarenal in nature, then nal lumen.
renal sodium retentive mechanisms will be activated, and ORS fluids introduced by the World Health Organiza-
the specific gravity of the urine will be more than 1.015 tion contain sodium 90; potassium 20; bicarbonate 30;
and the FENa will be low, specifically less than 1%. Failure chloride; and glucose (20 g/L), 111 mmol/L. The sodium
to increase the urine concentration and a high FENa in the and glucose are present in a molar ratio that maximizes the
face of clinical signs of ECF volume contraction points secondary active uptake of these solutes via the sodium-glucose
toward a renal or adrenal cause for the disorder. A renal cotransporter across the gastrointestinal epithelium. Water
ultrasound documenting the presence of small, misshaped is absorbed passively down its osmolal gradient. The pres-
kidneys or hydronephrosis may be indicative of congenital ence of other solutes, such as potassium and bicarbonate,
 7. Sodium and Water 139

are not critical to the successful use of ORS. These fluids Sodium Excess
have been used for more than 30 years. They can be admin-
Diagnosis and Evaluation
istered ad libitum in response to the child’s own thirst, and
they are effective and safe with minimal occurrence of These conditions, which generally are less common than
hypernatremia or hyperkalemia. There is evidence that vari- sodium deficit, are evidenced by clinical signs of ECF vol-
ous alternatives to glucose, such as rice-syrup or amylase- ume expansion. The causes of net total body sodium excess
resistant starch, may facilitate sodium and water reabsorption are noted in Table 7.6. They can arise secondary to exoge-
from ORS, decrease fecal fluid loss, and shorten recovery nous addition of sodium or abnormal retention of endoge-
time after an episode of cholera (76,77). However, further nous sodium. Because the normal kidney can rapidly
clinical studies are needed to confirm the use of these addi- excrete a sodium load, for the excess sodium to cause clini-
tives because they may increase the cost and decrease the cal symptoms and signs, there must be a concomitant fac-
shelf life of ORS. These are important considerations in tor that limits natriuresis.
developing countries where there is a high incidence of Common causes of excess exogenous sodium are salt-
infectious diarrhea in infants and children. water drowning, ingestion of a diet abnormally high in
In specific circumstances when parenteral therapy is sodium, or therapeutic infusion of sodium-containing
required to correct sodium and water deficits, the following intravenous solutions, such as sodium bicarbonate during
guidelines can be applied when devising a therapeutic plan. the resuscitation after a cardiopulmonary arrest. Exam-
First, in the absence of reliable data regarding the acute ples of dietary excess of sodium include errors in the
weight loss, it is easiest to calculate the maintenance and preparation of infant formulas. The widespread use of
deficit therapy based on the clinical estimate of the percent- premixed baby formulas may decrease the incidence of
age decrease in body weight. Second, it is advisable to dis- these accidents.
count any emergency fluid therapy, such as bolus infusions The conditions associated with excessive endogenous
of isotonic saline, in computing the fluid prescription. sodium retention include acute renal failure and the edema
Third, if the clinical problem has developed in less than 48 states, namely nephrotic syndrome, cirrhosis, and conges-
hours, then it should be considered an acute process, and tive heart failure. In the first condition, which may occur
the sodium and fluid losses are derived from the ICW and owing to glomerulonephritis or tubular necrosis, the
ECW in the ratio of 80 to 20%. If the patient has been ill sodium retention is directly related to the decrease in GFR
for more than 48 hours and the process is chronic, then the and diminished filtration of sodium. In the latter three
sodium and fluid losses are derived from the ICW and states, renal sodium and water reabsorption are activated by
ECW in the ratio of 60 to 40%. Under most conditions in a combination of mechanisms that are termed underfill and
which the sodium and water losses are isotonic, the ECW overfill. A critical review of available evidence in patients
portion of the loss, in liters, can be multiplied by 140 with nephrotic syndrome suggests that those diseases that
mmol/L to determine the sodium loss. Similarly, the ICW are associated with an inflammatory infiltrate in the kidney
portion of the loss, in liters, can be multiplied by 140 develop primary sodium retention and overfilling of the
mmol/L to determine the potassium deficit. If the ECW ECF volume. This histopathologic feature is absent in most
and ICW fluid losses together with the respective sodium cases of minimal change nephrotic syndrome (78). Thus, in
and potassium deficits are added to the maintenance nephrotic syndrome, the total body sodium excess may be
requirements, then a total fluid volume can be determined. coupled with a diminished, normal, or expanded effective
After selecting a fluid that most closely approximates the ECF volume, explaining the normal distribution of plasma
total sodium and water losses, the total fluid volume is renin activity values and the variability in measurements of
divided by the time frame of the correction to determine plasma volume in these patients.
the intravenous infusion rate. The clinical problems that accompany these conditions
As an example, if a 10-kg child is judged to have a 5% are mainly related to pulmonary venous congestion, impaired
decrease in body weight over 36 hours, then the total fluid
deficit of 500 mL can be divided into two components— TABLE 7.6. CAUSES OF NET SODIUM EXCESS
400 mL ICW with 56 mmol potassium and 100 mL ECW
containing 14 mmol sodium. The daily maintenance water Exogenous sodium
Saltwater drowning
and sodium requirements are 1000 mL and 30 mmol
Errors in formula preparation
sodium. Adding these two quantities together results in a Infusion of hypertonic sodium solutions (e.g., after cardio-
fluid that closely resembles 0.25% saline (37 mmol NaCl/ pulmonary arrest)
L) containing 30 mmol KCl/L, and the infusion rate is Endogenous sodium
approximately 60 mL/hr if the correction is designed to Acute renal failure (glomerulonephritis, acute tubular necrosis)
Nephrotic syndrome
occur over 24 hours. In all cases, it is important to monitor
Cirrhosis
and replace ongoing losses to insure that there is full resolu- Congestive heart failure
tion of the underlying clinical problem.
140 II. Homeostasis

gas exchange in the lung, and difficulty breathing. In idio- nonspecific. Therefore, laboratory confirmation is required
pathic nephrotic syndrome, the intraalveolar pressure is to diagnose this abnormality. Moreover, the presence of
often sufficient to prevent frank pulmonary edema. How- hyponatremia has no predictive value about the status of
ever, in other circumstances, there may be intrinsic capil- the ECF volume. Therefore, this later issue must be
lary leak together with lowered plasma oncotic pressure, assessed clinically to determine whether the child has hypo-
augmenting the development of pulmonary interstitial volemic hypotonicity, isovolemic hypotonicity, or hyper-
fluid. Peripheral edema may be associated with skin infec- volemic hypotonicity.
tion, peritonitis, or thromboembolic events. It is important to recognize that the laboratory measure-
All of the states associated with sodium excess should be ment of serum sodium concentration is no longer suscepti-
evident on physical examination. The FENa is high if there ble to technical errors. In the past, laboratory analyzers
is sodium loading and renal function is normal. In contrast, measured sodium concentration in the total supernatant
in the states associated with abnormal retention of endoge- phase obtained after centrifugation of blood samples,
nous sodium, the FENa is very low. The FENa is more useful including lipids and proteins. Because these molecules are
than the urinary-specific gravity because it is likely to be not water soluble, they displace sodium into a smaller aque-
elevated in all cases of sodium excess. ous phase, leading to a spurious reduction in the serum
sodium concentration. All biochemical analyzers that are
currently in use assay sodium concentration only in the
Therapy
aqueous phase and result in an accurate determination of
The optimal therapy is targeted at correcting the underly- the serum sodium level. Therefore, the entity called pseudo-
ing disease. This is most important in patients with cirrho- hyponatremia is no longer clinically relevant. In contrast,
sis or congestive heart failure. Ancillary therapies include the reduced serum sodium concentration noted in patients
administration of diuretics to facilitate urinary sodium with increased circulating levels of an impermeant solute,
excretion. Although thiazide diuretics may be adequate, a such as mannitol, urea, contrast media, or glucose in
more potent loop diuretic may be required if the GFR is patients with diabetic ketoacidosis, is valid and reflects
diminished. Supplemental oxygen may be necessary to alle- osmotic redistribution of water from the ICW to the ECW
viate shortness of breath and hypoxemia. In more severe space. The phenomenon is reflected in the following for-
cases, acute dialysis may be necessary to foster rapid clear- mula, which enables adjustment of the serum sodium in
ance of the excess sodium. Patients with nephrotic syn- patients with severe hyperglycemia:
drome may require combinations of diuretic agents that act
“Physiologic” sodium concentration =
in the proximal (e.g., metolazone) and distal (e.g., furo-
measured serum sodium concentration + 1.6 ×
semide) segments of the nephron to promote an adequate
[Each 100 mg/dL increment in serum glucose >100 mg/dL]
natriuresis and diuresis. Infusions of albumin (1 g/kg body
weight) may be necessary to promote and augment the The most common clinical causes of hyponatremia are classi-
medication-induced diuresis, especially in those with severe fied by the concomitant ECF volume status in Table 7.7. In
ECF volume contraction, reduced GFR, and azotemia some diseases, such as congestive heart failure, the degree of
(79). Finally, hemofiltration may be a safe and effective hyponatremia may be a reflection of circulating AVP levels
means to rapidly remove sodium and water in severely and sympathetic nervous system activation and provides a
ECF-overloaded children with nephrotic syndrome (80). marker of disease severity. This relationship has not been dem-
onstrated in patients with nephrotic syndrome or cirrhosis.
The syndrome of inappropriate AVP release causes
WATER BALANCE DISTURBANCES: DEFICIT hyponatremia with mild to modest ECF volume expansion.
AND EXCESS It can occur as a consequence of central neurologic lesions,
pulmonary disease, or tumors. In addition, numerous
Hyponatremia drugs can result in abnormal secretion or action of AVP
and lead to chronic hyponatremia. A list of these agents is
Diagnosis and Evaluation
provided in Table 7.8. The diagnosis of this problem
Patients with hyponatremia have relative or absolute expan- requires confirmation that the urine is excessively concen-
sion of the ICW compartment. If renal function is normal, trated relative to the plasma osmolality without any evi-
excess free water is eliminated within 2 to 4 hours. There- dence of ECF volume contraction or adrenal or thyroid
fore, for this problem to occur, two conditions must be sat- insufficiency. The two hormones are required to maintain
isfied—there must be continued AVP release that is the low water permeability of the collecting duct in the
inappropriate to the serum sodium concentration, and the absence of AVP. Deficiencies of either hormone impair free
patient must have continued access to free water. The water clearance, leading to euvolemic hyponatremia. In
symptoms and signs of hyponatremia, which generally practice, this requires comparison of the urine-specific grav-
involve central nervous system dysfunction, are vague and ity or osmolality with the concurrent serum osmolality. The
 7. Sodium and Water 141

TABLE 7.7. CAUSES OF HYPONATREMIA TABLE 7.8. DRUGS THAT CAUSE WATER RETENTION
AND THE SYNDROME OF INAPPROPRIATE ARGININE
Hypovolemic: ECF volume contraction VASOPRESSIN (AVP) RELEASE ACCORDING TO MODE
Renal OF ACTION
Mineralocorticoid deficiency
Mineralocorticoid resistance Increasing water permeability of the nephron
Diuretics AVP (arginine or lysine vasopressin)
Polyuric acute renal failure Vasopressin analogs (e.g., 1-deamino, 8- D-AVP)
Salt-wasting renal disease Oxytocin
Renal tubular acidosis Promoting AVP release
Metabolic alkalosis Barbiturates
Bartter’s syndrome/Gitelman’s syndrome Carbamazepine
Gastrointestinal Clofibrate
Diarrheal dehydration Colchicine
Gastrointestinal suction Isoproterenol
Intestinal fistula Nicotine
Laxative abuse Vincristine
Transcutaneous Inhibition of prostaglandin synthesis
Cystic fibrosis Salicylates
Heat exhaustion Indomethacin
“Third space” loss with inadequate fluid replacement Acetaminophen (paracetamol)
Burns Other nonsteroidal antiinflammatory drugs
Major surgery, trauma Potentiation of the action of AVP
Septic shock Chlorpropamide
Euvolemic: Normal ECF volume Cyclophosphamide
Glucocorticoid deficiency
Hypothyroidism
Mild hypervolemia: ECF volume expansion
Reduced renal water excretion lead to seizures or cardiopulmonary collapse before initia-
Antidiuretic drugs tion of therapy (81). This risk may be especially prominent
Inappropriate secretion of ADH in premenopausal women. In contrast, there are others who
Hypervolemic: ECF volume expansion
emphasize the cerebral cell volume regulatory response to
Acute renal failure (glomerulonephritis, ATN)
Chronic renal failure hyponatremia and highlight the risk of brain cell dehydra-
Nephrotic syndrome tion and osmotic demyelinating syndrome in patients who
Cirrhosis are corrected too quickly (82).
Congestive heart failure Taking into account the entire literature on the subject,
Psychogenic polydipsia/compulsive drinking
current evidence suggests that the risk of hyponatremia is
ADH, antidiuretic hormone; ATN, acute tubular necrosis; ECF, extra-
more closely related to the acuity of the change rather than
cellular fluid. the absolute size of the drop in serum sodium concentra-
tion (83). Thus, therapy should be guided by the clinical
assessment of the time frame in which hyponatremia has
urine should normally be maximally dilute if the serum developed. If the hyponatremia is acute—that is, less than
sodium concentration is <130 mmol/L or the plasma 12 hours in duration—then the brain will behave as a per-
osmolality is <270 mOsm/kg H2O. In addition, if the uri- fect osmometer, leading to potentially life-threatening cere-
nary sodium concentration is >40 mmol/L, there is ade- bral cell swelling. Under these circumstances, there is an
quate evidence against ECF volume contraction. urgent need to reverse the hyponatremia to counteract cell
swelling. Clinical experience indicates that infusion of a
3% NaCl solution (513 mmol/L) in a volume designed to
Treatment
raise the sodium concentration by 3 to 5 mmol/L is suffi-
In all cases, the first line of therapy should be directed at cient to halt central nervous system dysfunction (84). The
the underlying cause of the low serum sodium concentra- benefits and lack of adverse effects of acute correction have
tion. However, hyponatremia often warrants specific cor- been confirmed in a series of 34 infants and children with
rective treatment. Much ink has been spilled in detailing acute water intoxication caused by the administration of
the appropriate therapy of this electrolyte abnormality. At dilute infant formula (85). After partial correction is
times, this issue has been quite contentious, and the neph- achieved, the hyponatremia can be corrected more slowly.
rology community has been divided into two supposedly For example, if a 6-year-old child weighing 20 kg (TBW =
distinct camps—those who advocate “rapid” versus “slow” 0.6 L/kg × 20 kg = 12 L) develops a seizure after a tonsil-
correction of hyponatremia. The former group asserts that lectomy and is noted to have a serum sodium concentra-
hyponatremia has direct adverse effects on central nervous tion of 115 mmol/L, then 36 to 60 mmol of sodium are
system function, including impaired oxygenation that can needed to raise the sodium concentration by 3 to 5 mmol/L.
142 II. Homeostasis

This correction is accomplished by infusing 72 to 120 mL TABLE 7.9. CAUSES OF HYPERNATREMIA

of the hypertonic saline.
Hypovolemic: ECF volume contraction
If hyponatremia has developed over more than 12 hours Gastrointestinal (diarrhea and vomiting)
or the duration of the problem is unclear, especially if the Evaporative (high fever, high ambient temperature)
patient has no signs of neurologic dysfunction, then slow Hypothalamic diabetes insipidus (ADH deficiency)
correction is the prudent course of action (82,83). The cur- Head trauma
Infarction (Sheehan’s syndrome)
rent definition of low correction includes two features:
Tumors (e.g., craniopharyngioma)
Histiocytosis
1. The rate of rise in serum sodium concentration should be Degenerative brain diseases
less than 0.6 mmol/L/hr throughout the correction phase. Infections
2. The total increment and/or the final serum sodium Hereditary central diabetes insipidus (usually dominant)
Idiopathic
concentration after 48 hours of treatment should not
Nephrogenic diabetes insipidus (ADH resistance)
exceed 25 mmol/L or 130 mmol/L, respectively. Chronic renal failure
Hypokalemia
The more cautious criterion should be applied depending Hypercalcemia
on the initial serum sodium level. If the child develops Damage to renal medulla
Sickle cell disease
acute changes in mental status or new neurologic findings,
Nephronophthisis
during or shortly after the fluid treatment, then a serum Renal papillary necrosis
sodium concentration should be checked. Imaging studies, Chronic pyelonephritis (reflux nephropathy)
specifically an MRI of the brain, may reveal the changes of Euvolemic: normal ECF volume
osmotic demyelinating syndrome. Unconscious patients
Infants
In patients with syndrome of inappropriate secretion of
Lack of access to water (lost in the desert)
antidiuretic hormone, if the underlying cause cannot be Primary adipsia
corrected, then there are several therapeutic options. Essential hypernatremia (osmoreceptor destruction or mal-
Restriction of free water intake to match insensible losses function)
and urine output may be adequate to stabilize the serum Hypervolemic: ECF volume expansion
Inappropriate IV fluid therapy
sodium concentration. If this is not well tolerated, then
Salt poisoning
administration of furosemide, 1 to 2 mg/kg/day, to pro- Mineralocorticoid excess
mote a hypotonic diuresis together with oral administra-
tion of NaCl, 1 to 2 g/day, may correct the hyponatremia. ADH, antidiuretic hormone; ECF, extracellular fluid.
If these measures fail, consideration can be given to treat-
ment with lithium or demeclocycline, two drugs that inter-
fere with AVP action in the collecting tubule to foster dramatic decrease in the incidence of hypernatremic dehy-
excretion of free water and raise the serum sodium concen- dration. Nonetheless, recent changes in medical practice with
tration (86). Finally, orally active nonpeptide vasopressin early discharge of newborn infants after delivery have
antagonists have been developed that may be introduced resulted in a steady occurrence of hypernatremic dehydration
into clinical practice for the treatment of chronic hypona- in breast-fed babies (90). Patients with hypernatremia have
tremia (87,88). relative or absolute contraction of the ICW compartment.
Similar to the situation with hyponatremia, the clinical clues
to the presence of hypernatremia are nonspecific. Therefore,
Hypernatremia laboratory confirmation is required to diagnose this abnor-
mality. Moreover, the presence of hyponatremia must be
Diagnosis and Evaluation
evaluated in light of the status of the ECF volume.
The causes of hypernatremia are listed in Table 7.9. Hyper- Those patients with hypernatremia and ECF volume
natremia may arise owing to excessive intake of sodium and expansion are easy to diagnose. The children who represent
ECF volume expansion. However, excessive water loss rela- a serious problem are those with hypovolemia. They may
tive to the sodium deficit with hypovolemia is far more com- have some distinct features, including marked irritability, a
mon. In a recent survey of hypernatremia in hospitalized high-pitched cry, and a doughy skin texture. Because the
children, the vast majority had significant underlying medi- hyperosmolality of the ECW compartment provokes move-
cal problems, and 76% of the cases were secondary to inade- ment of water from the ICW down its osmolal gradient,
quate water intake (89). The prevalence of this electrolyte these patients tend to preserve ECF volume until late in the
abnormality is much lower than hyponatremia. One of the disease course. Thus, their illness is usually chronic, and
common causes is diarrheal illness in infants; however, the there is a greater contribution of the ICW to the water and
reduction in the sodium concentration of most baby formu- electrolyte deficits. Assessment of the FENa is useful in
las to match the level in human breast milk has resulted in a assessing the ECF volume in these patients.
 7. Sodium and Water 143

Treatment 5. Trachtman H, Futterweit S, Tonidandel W, et al. The role of

organic osmolytes in the cerebral cell volume regulatory
Because children with hypovolemic hypernatremia are usu- response to acute and chronic renal failure. J Am Soc Neph-
ally very ill, they often require substantial infusions of iso- rol 1993;12:1913–1919.
tonic saline to restore organ perfusion. Once adequate 6. Glaser DS. Utility of the serum osmolal gap in the diagnosis
perfusion is accomplished, the fluid regimen should of methanol or ethylene glycol ingestion. Ann Emerg Med
include the maintenance fluids, the estimated deficit with 1996;27:343–346.
the assumption that 60% is derived from the ECW and 7. Schelling JR, Howard RL, Winter SD, et al. Increased
40% from the ICW. In addition, there is a free water deficit osmolal gap in alcoholic ketoacidosis and lactic acidosis.
that can be calculated from the following formula: Ann Intern Med 1990;113:580–582.
8. Wassner SJ. Altered growth and protein turnover in rats fed
Water deficit (in L) = 0.6 × Body weight × sodium-deficient diets. Pediatr Res 1989;26:608–613.
[(Actual serum sodium concentration/140) –1] 9. Sulyok E, Varga F, Nemeth M, et al. Furosemide-induced
alterations in electrolyte status, the function of the renin-
There is some evidence that this formula may overestimate angiotensin-aldosterone system, and the urinary excretion
the water deficit, and it has been recommended that the of prostaglandins in newborn infants. Pediatr Res 1980;
following alternative equation (91) be used to estimate the 14:765–768.
change in serum sodium concentration that will be 10. Holliday M. The evolution of therapy for dehydration: Should
achieved after the infusion of 1 L of a given solution: deficit therapy still be taught? Pediatrics 1996;98:171–177.
11. Bell EF, Neidich GA, Cashore WJ, et al. Combined effect of
Change in sodium concentration = radiant warmer and phototherapy on insensible water loss
[Infusate Na+ – serum Na+]/(TBW +1) in low-birth weight infants. J Pediatr 1979;94:810–813.
Finally, with regard to the rate of correction, it is standard 12. Schoorlemmer GH, Evered MD. Water and solute balance
in rats during 10 h water deprivation and rehydration. Can
practice to correct hypovolemic hypernatremia gradually
J Physiol Pharmacol 1993;71:379–386.
over at least 48 hours. After the groundbreaking work of 13. Danovitch GM, Bourgoignie J, Bricker NS. Reversibility of
Finberg et al. (92), the risk of cerebral edema after rapid the “salt losing” tendency of chronic renal failure. N Engl J
correction of chronic hypernatremia is now attributed to Med 1976;296:14–19.
the inability of brain cells to extrude the osmoprotective 14. Trachtman H. Cell volume regulation: a review of cerebral
solutes that accumulate during sustained hyperosmolal adaptive mechanisms and implications for clinical treatment
conditions in parallel with the decline in plasma osmolality of osmolal disturbances. Pediatr Nephrol 1991;5:743–752
during fluid therapy (93,94). Therefore, the osmolal gradi- (Part I) and 1992;6:104–112.
ent is reversed with plasma osmolality lower than cerebral 15. Kleeman CR. Metabolic coma. Kidney Int 1989;36:1142–
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of cerebral edema. This experimental observation was con- 16. Morita H, Tanaka K, Hosomi H. Chemical inactivation of
the nucleus tractus solitarius abolished hepatojejunal reflex
firmed in a randomized trial, which demonstrated that the
in the rat. J Autonom Nerv Syst 1994;48:207–212.
safest and most effective fluid therapy for hypovolemic 17. Firth JD, Raine AEG, Ledingham JGG. Raised venous
hypernatremia is 0.18% NaCl given slowly over 48 hours, pressure: a direct cause of renal sodium retention in edema.
compared to 0.45% saline given slowly or rapidly over the Lancet 1988;331;1033–1035.
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 8

POTASSIUM
GEORGE J. SCHWARTZ

Potassium (K+) is the most abundant intracellular cation. approximately 8 mEq/cm body height at birth to more than
High concentrations of K+ in the cytosol are required for 14 mEq/cm body height by 18 years of age (9). In absolute
many normal cellular functions, including metabolism and terms, total body K+ in infants (approximately 42 mEq/kg) is
growth, cell division, optimal enzyme function, protein less than in older children (approximately 51 mEq/kg), and
and DNA synthesis, volume regulation, and intracellular it increases more rapidly during the first 1.5 years of life than
acid-base balance (1). In addition, a steep concentration after age 3 (9–11) (Fig. 8.1).
gradient of K+ across the cell membranes is required for The average intake of potassium in adults is quite vari-
nerve excitation and muscle contraction. These functions able, ranging from 50 to more than 500 mEq/day (12,13).
are achieved by maintaining high intracellular and low In the healthy adult, K+ output balances intake, so that
extracellular K+ concentrations. The intracellular and extra- constancy of body K+ content is maintained. For a typical
cellular fluids (ECFs) are separately controlled to maintain adult K+ intake of 1.5 mEq/kg body weight, 90 to 95% is
K+ homeostasis. Whereas homeostasis in adults seeks to excreted in the urine and 5 to 10% in the stool (Fig. 8.2).
maintain zero K+ balance, growing infants and children The process of growth requires that the growing organ-
must accumulate K+ for growth. ism be able to conserve K+ and exist in a state of positive K+
The intracellular K+ concentration, 100 to 150 mEq/L, balance (14) to facilitate incorporation of K+ into dividing
far exceeds its concentration in ECF, 3.5 to 5 mEq/L. This cells. Infants fed human milk receive only 13 mEq of K+/L
transcellular K+ gradient is maintained by the action of the (corresponding to 1.8 mEq/100 kcal or 1.8 mEq/kg/day),
enzyme Na+-K+-adenosine triphosphatase (Na+-K+-ATPase) whereas other milk formulas may provide 1.5 to 3 times
in the cell membrane, which pumps Na+ out of and K+ into this amount (15). Under normal physiologic conditions the
the cell in a 3:2 ratio (2–4). Thus, serum potassium level K+ content of breast milk is sufficient, because there is little
provides only an indirect estimate of total body K+ status urinary excretion of K+. In addition, the renal K+ clearance
because it represents only 2% of total body K+. (CK), even corrected for the low absolute glomerular filtra-
tion rate of infants (that is, the fractional excretion of K+, or
FEK*), is less than in the older child (Table 8.1) (16). This
BODY COMPOSITION AND INTAKE tendency to retain K+ early in postnatal life is observed in
infants, who tend to have higher plasma K+ concentrations
Total body K+ in adult men, estimated from total body than older children and adults. With maturation of renal
counting of potassium-40, is approximately 50 mEq/kg, and K+ excretion, however, adjustments of K+ intake are essen-
98% is within cells, mainly muscle (5–8). In proportion to tial. Maintenance K+ intake in children is 2 to 3 mEq/100
muscle mass, total body K+ is lower in females than in males kcal, approximately twice that in adults (17).
(8), and the levels in all adults decline gradually after age 40. The 2% of total body K+ that is located within the ECF
During growth, total body K+ increases linearly with either is tightly regulated. Because of the dependence of so many
height or weight without sex-related differences for height vital processes on K+ homeostasis and the ratio of intracell-
and weight below 135 cm and 30 kg, respectively (9,10) (Fig. ular to extracellular K+, many mechanisms to regulate
8.1). By the onset of puberty, gender-related differences in serum K+ concentration and total body K+ homeostasis
total body K+ content develop, even when values are cor-
rected for body weight or height. The rate of increase in body *Fractionalexcretion of K+ is the percentage of filtered K+
K+ as a function of growth diminishes in girls (9,10). This
that appears in the urine:
change from the male pattern reflects the deposition of adi-
pose tissue, which contains little K+ (6). In boys postnatal FEK = urinary K+ (mEq/L)/plasma K+ (mEq/L) × 100%/
growth is accompanied by an increase in total body K+ from [urinary creatinine (mg/dL)/plasma creatinine (mg/dL)]
148 II. Homeostasis

FIGURE 8.1. Total body potassium as a

function of weight in males and females
3 to 18 years of age. The shaded regions
represent the mean ± 1 standard devia-
tion for 68% of the population of 432
children. (From Flynn MA, Woodruff C,
Clark J, et al. Total body potassium in
healthy children. Pediatr Res 1972;6:239,
with permission.)

have evolved. The cellular volume of distribution for K+ Na+-K+-Adenosine Phosphatase Activity
greatly exceeds that of the ECF volume, and the potential
The high ratio of intracellular to extracellular K+ concentra-
for intracellular redistribution provides protection against
tion is sustained primarily by the action of Na+-K+-ATPase,
abrupt changes in serum K+ concentrations.
the sodium pump. This pump, generally present on the
Direct chemical analysis of various nonsecreting mature
basolateral membranes of polarized epithelial cells, con-
tissues (e.g., skeletal muscle) has provided estimates of
sumes energy (adenosine triphosphatase, or ATP) in the
intracellular fluid (ICF) K+ concentration of 100 to 160
process of transporting three Na+ ions out of and two K+
mEq/L (18–20). Comparable studies in the tissues of new-
ions into the cell, which generates a negative intracellular
born humans and animals have revealed ICF K+ concentra-
tions that are 15 to 30% lower than those found in adults
(19,21) (Fig. 8.3). The more rapid accretion of K+ in
infants probably reflects both a maturation of intracellular Potassium Intake
(100 mEq/day)
chemical composition and actual cellular growth.

RBC
(250 mEq)
Muscle ECF
DISTRIBUTION BETWEEN INTRACELLULAR (2650 mEq) (70 mEq) Liver
AND EXTRACELLULAR FLUID Bone
(260 mEq)

(300 mEq)
Potassium is readily absorbed from the gastrointestinal tract
and enters the ECF. Relative constancy of plasma K+ concen-
tration during entry of K+ into the ECF is maintained by
hormonally mediated temporary shifts of this cation into Potassium Excretion
(Kidney = 90 mEq/day; Stool = 10 mEq/day)
cells, such as muscle, liver, red cells, and bone (Fig. 8.2) (22).
To maintain zero K+ balance, all of the dietary K+ must be FIGURE 8.2. Potassium homeostasis depends on the mainte-
ultimately excreted, primarily by the kidney. However, the nance of external and internal K+ balance. External balance of K+
in adults is determined by the rate of K+ intake (approximately
renal excretion of K+ is rather slow, requiring several hours. 100 mEq/day) and the rate of excretion in the urine (approxi-
Only approximately 50% of an oral load of K+ is excreted mately 90 mEq/day) and feces (approximately 10 mEq/day). Inter-
during the first 4 to 6 hours after ingestion (23–25). Protec- nal K+ balance depends on the distribution of K+ between muscle,
bone, liver, red blood cells (RBC), and extracellular fluid (ECF).
tion is achieved within minutes by hormonally mediated (From Giebisch G. Renal potassium transport: mechanisms and
translocation of extracellular K+ into cells (22–27). regulation. Am J Physiol 1998;274:F817, with permission.)
 8. Potassium 149

TABLE 8.1. PLASMA K+ AND RENAL K+ CLEARANCES IN GROWING CHILDREN

Age (yr) n PK CCr CK FEK UNa/K

0.0–0.3 13 5.2 ± 0.8a,b 62 ± 26a,b,c 5 ± 3b,c 8.5 ± 3.8a,b 1.1 ± 1.1

0.4–1.0 10 4.9 ± 0.5a,b 99 ± 38a,b 14 ± 6a 14.6 ± 5.0 0.8 ± 0.9
3–10 19 4.2 ± 0.5 141 ± 30 20 ± 11 14.5 ± 8.9 1.5 ± 1.1
11–20 17 4.3 ± 0.3 137 ± 21 21 ± 8 16.2 ± 8.2 1.4 ± 0.8

CCr, creatinine clearance in mL/min/1.73 m2; CK, clearance of potassium in mL/min/1.73 m2; FEK, fractional excretion of potassium in percent; n,
number of infants or children; PK, plasma potassium in mEq/L; UNa/K, sodium to potassium ratio in urine.
Note: Mean plus/minus the standard deviation. Patients were on regular diets and were free of renal disease.
ap <.05 vs. 11–20 yr.
bp <.05 vs. 3–10 yr.
cp <.05 vs. 0.4–1.0 yr.

Data from Satlin LM, Schwartz GJ. Metabolism of potassium. In: Ichikawa I, ed. Pediatric textbook of fluids and electrolytes. Baltimore: Williams
& Wilkins, 1990:90.

voltage. It is comprised of an α (catalytic) and a β (glyco- plasma K+ results in a two- to threefold increase in insulin
protein) heterodimer. The activity of Na+-K+-ATPase in red levels (24,25,39–42).
cells (28,29), kidney (30–33), and intestine (34) is low Catecholamines influence K+ distribution, with α recep-
early in life, perhaps mediating lower cellular K+ concentra- tors inhibiting and β2 receptors stimulating cellular K+
tions (19,21). uptake. This results in a biphasic response manifested first by
Na+-K+-ATPase activity is regulated by several hor- an initial increase followed by a prolonged decrease in plasma
mones, namely, insulin, thyroid hormone, catecholamines, K+ concentration to below baseline (22). The transient initial
and aldosterone. Long-term stimulation of Na+-K+-ATPase rise is due to α-adrenergic receptor stimulation, which causes
by aldosterone and thyroid hormone is mediated by release of K+ from cells, whereas the β2-receptor stimulation,
changes in gene expression, whereas short-term regulation via stimulation of adenylate cyclase and generation of cyclic
may be mediated by altered phosphorylation of the pump adenosine monophosphate, activates Na+-K+-ATPase and
(catecholamines) or by changes in the surface distribution mediates increased K+ uptake by liver and muscle cells (22).
of pumps (insulin, aldosterone) (35,36). A small rise in Basal epinephrine levels are permissive in stimulating K+
ECF K+ (from dietary intake, exercise, or acute hyperosmo- uptake by liver, skeletal muscle, adipose tissue, and cardiac
lality) stimulates secretion of these hormones, activating muscle (22,23,43). Indeed, the increment in plasma K+
Na+-K+-ATPase to transport more K+ into cells. concentration after a K+ load or exercise is greater and more
prolonged if the subject has been pretreated with the beta-
blocker propranolol (23,44,45). The administration of a
Hormone Activity
β2-adrenergic agonist (albuterol) can reduce plasma K+ con-
Insulin is the most important hormonal regulator of inter- centration by stimulating K+ uptake by cells and by stimulat-
nal K+ balance. Insulin reduces serum K+ by promoting the ing insulin release, which independently stimulates K+ uptake.
cellular uptake of K+, probably by directly stimulating Na+- Blood levels of aldosterone are sensitively influenced by
K+-ATPase activity (3,22,37,38). Basal insulin levels per- plasma K+ concentration (46–48). The secretion of aldoster-
missively facilitate K+ entry into cells. An acute rise in one protects against hyperkalemia primarily by increasing uri-

FIGURE 8.3. Comparison of cellular K+ con-

centration (mEq/L) in the skeletal muscle
(slashed bars) and cortical collecting duct
(CCD, solid bars) of newborn (NB), 1-month-
old, and adult rabbits. Mean values plus
standard error are given for each age group.
*, p <.05 versus adult; #, p <.05 versus 1-
month-old. (Data from Satlin LM, Evan AP,
Gattone VH III, et al. Postnatal maturation of
the rabbit cortical collecting duct. Pediatr
Nephrol 1988;2:135.)
150 II. Homeostasis

nary and colonic K+ excretion but also by promoting cellular ization and the reuptake of K+ by the Na+-K+-ATPase
K+ uptake (13,25,49). Thyroid hormone, glucocorticoids, and sodium pump (58,59). With strenuous exercise, the reduc-
growth hormone may also promote the cellular uptake of K+ tion in ATP within muscle cells stimulates the opening of
by chronically stimulating Na+-K+-ATPase (22,35,36). ATP-dependent K+ channels, which further promotes an
increase in K+ release from cells (60). Finally, the release of
K+ from the myocyte takes place with H+ exchange, and the
Plasma K+ Concentration exercise-induced hyperkalemia may result from a reduction
An increase in plasma K+ concentration, independent of in nondiffusible intracellular anions, which accompanies
aldosterone and other factors, promotes K+ entry into cells, phosphocreatine hydrolysis (59).
probably by diminishing the K+ concentration gradient
against which the Na+-K+-ATPase–dependent sodium Other Conditions
pump must function (24,25,50). Thus, plasma K+ concen-
tration generally varies directly with body K+ stores (51). In Any condition resulting in cell breakdown leads to the
those cells of the kidney and colon responsible for K+ secre- release of K+ into the ECF and potentially to hyperkalemia
tion, the resulting increase in cellular K+ concentration (61,62). An acute increase in effective plasma hyperosmola-
enhances secretion by favoring increased K+ diffusion into lity can also cause K+ to leave cells, so that the plasma K+
the tubular fluid down its concentration gradient. concentration may rise by 0.3 to 0.8 mEq/L for every 10
mOsm/kg elevation (63–67). Hyperglycemia is a common
clinical example of this phenomenon, especially because the
Acid-Base Balance rise in ECF K+ concentration may not be effectively modu-
Acute changes in acid-base balance may have important lated in a diabetic patient lacking insulin secretion (64).
effects on K+ distribution. During mineral-acid–induced With permeant solutes, such as urea or ethanol, plasma
acidosis, excessive protons are buffered in the cells and K+ hyperosmolality does not result in hyperkalemia, because
moves into the ECF, which results in an increase in plasma there is no transcellular osmotic gradient.
K+ concentration; hyperkalemia may ensue (13,52,53). A
common rule of thumb states that, for every 0.1-U decrease
in blood pH, the plasma K+ concentration increases by RENAL POTASSIUM EXCRETION
approximately 0.6 mEq/L (13,22,51,53).
Increases in plasma K+ concentration are generally much Overview of the Renal Contribution
smaller in acute organic acid acidosis, such as lactic acidosis to Potassium Homeostasis
and diabetic ketoacidosis (13,51,53–55). The concurrent Renal regulation of urinary K+ excretion allows for adjust-
entry into the cell of the anion with the proton may reduce ment of output to equal intake over a wide range. The kid-
the necessity for K+ redistribution. Also, the hyperkalemia ney excretes 90 to 95% of the daily K+ intake (7). Extreme
often observed in ketoacidosis reflects a larger flux of K+ adjustments cannot be achieved as rapidly as for sodium,
from cells to ECF due to insulin deficiency and hyperos- nor are they as complete. Maximal rates of K+ excretion
molality rather than to acidemia per se (13,26,51,53). may not be observed until several days after K+ intake is
Acute metabolic alkalosis causes K+ to shift into cells, increased (68). Whereas urinary Na+ can be reduced to less
with a resulting decrease in plasma K+ concentration. The than 1 mEq/day within 3 to 4 days of Na+ restriction, a
effect on serum K+ concentration is generally smaller (0.2 minimum urinary K+ loss of 5 to 15 mEq/day persists in
to 0.4 mEq/L), probably due to the smaller degree of intra- the adult on K+ restriction (12,26,69–71).
cellular buffering and transcellular movement of protons There is a circadian rhythm of renal K+ excretion in the
(53,56). More prolonged alkalosis usually results in adult that is independent of changes in serum aldosterone
hypokalemia (13,51), due to a shift of K+ into collecting or K+ concentration (72–74). The cause of this rhythmic
duct cells that results in enhanced urinary excretion of K+. K+ excretion is unclear, but it may be due to hypothalamic
Contributing to the variability of proton load and serum oscillators characterized by maximum output during times
K+ concentration, the concentration of plasma HCO3– of peak activity. In children, one study has shown no diur-
itself can reciprocally affect plasma K+ values, independent nal variation in urinary K+ excretion (75).
of a pH effect (13,53,57). Large changes in plasma K+ con- Growing individuals maintain a state of positive K+ bal-
centration are rarely seen with respiratory acid-base disor- ance (14,18). The relative conservation of K+ early in life is
ders (13,52,53,56). generally associated with higher plasma K+ values* than in

Exercise *Serum K+ concentration runs approximately 0.5 mEq/L

Exercise can cause the release of K+ from muscle, due in higher than in plasma, due to the release of K+ from cells
part to the discrepancy between the K+ exit during depolar- during clotting (78).
 8. Potassium 151

TABLE 8.2. FACTORS INFLUENCING K+ DISTRIBUTION

BETWEEN CELLS AND EXTRACELLULAR FLUID
Effect on extracellular
Factor K+ concentration

Physiologic
Insulin ↓
Na-K–adenosine triphosphatase ↓
Catecholamines
α receptor ↑
β receptor ↓
Mineralocorticoids ↓
↑ Plasma K+ concentration ↑
Exercise ↑
Pathologic FIGURE 8.4. Schematic of K+ transport along the nephron.
Metabolic acidosis ↑ Reabsorption occurs in the proximal tubule and thick ascending
Metabolic alkalosis ↓ limb of the loop of Henle. The excretion of K + during varied K+
Hyperosmolality ↑ intake is mainly regulated by the distal nephron (areas indicated
Excessive cell breakdown ↑ by the dotted lines in the lumens). The handling of K+ in the col-
lecting duct during K+ depletion is also influenced by reabsorp-
tive processes mediated by H+-K+-ATPase in the collecting duct, a
subject of active investigation. CCD, cortical collecting duct; DCT,
distal convoluted tubule; G, glomerulus; MCD, medullary collect-
the adult (14,76–78). Our own study of healthy human ing duct; PCT, proximal convoluted tubule; TAL, thick ascending
infants shows that plasma K+ concentrations during the limb of loop of Henle.
first 4 months of life average 1 mEq/L higher than in
infants older than 1 year of age (Table 8.1).
FEK (Table 8.2) provides an additional assessment of lost each day in the stool (5 to 15 mEq) (83,84) and sweat
renal K+ handling under various physiologic and patho- (0 to 10 mEq) (26), K+ homeostasis is regulated in large
physiologic states. Normally, FEK ranges from 10 to 30%, part by the rate of K+ secretion in the distal nephron.
but FEK usually exceeds 40% and urinary Na+/K+ ratio is
less than 1 with enhanced mineralocorticoid activity. Dur-
Segmental Potassium Transport
ing K+ conservation FEK is less than 10% with urinary
Na+/K+ ratios exceeding 2 or 3. The renal handling of K+ requires filtration at the glomeru-
Children and adults ingesting a regular diet containing lus, reabsorption in the proximal nephron, and secretion by
Na+ in excess of K+ excrete urine with an Na+/K+ ratio the distal tubule and cortical collecting duct (1,85). Micro-
higher than 1 (Table 8.1) (76,77). Although the Na/K puncture studies in adult rats by Malnic et al. (86) (Fig.
ratios of breast milk and commercial infant formulas are 8.5) examined the clearance of K+ factored by that of inu-
approximately 0.5 (15), the urinary Na/K ratio of the new- lin. After being freely filtered at the glomerulus, K+ is nearly
born also generally exceeds 1, due in part to a physiologic completely reabsorbed in the proximal tubule and thick
natriuresis, as well as preferential retention of K+ as a func- ascending limb of the loop of Henle, as seen by the progres-
tion of growth. Premature infants may show urinary Na+/ sive decrease in K/inulin (K/In) along the proximal neph-
K+ ratios above 4, which reflects a relative hyporesponsive- ron (Fig. 8.5). Proximal K+ reabsorption is passive after that
ness of the distal tubule and collecting duct to mineralocor- of Na+ and water; 60 to 65% of the filtered K+ load is reab-
ticoid activity in addition to the substantial natriuresis that sorbed in the proximal tubule of the adult rat (86) (Fig.
occurs during this period of gestation (14). 8.6A) (87), a fraction similar to that observed in the imma-
The renal adaptation to chronic K+ loading is due pri- ture rat (88–91).
marily to an enhanced capacity for individual nephrons to In the adult rat, only 5 to 15% of the filtered load of K+
secrete K+. Infants can excrete K+ at a rate that exceeds its reaches the superficial distal tubule, which reflects further
filtration, which indicates the capacity for net tubular secre- significant net reabsorption by the intervening nephron seg-
tion (79); however, they cannot do this as rapidly or as effi- ments (86). Indeed, an additional 20 to 30% of the filtered
ciently as can adults (80–82). load of K+ is reabsorbed along the thick ascending limb of
the loop of Henle, so that by the time the tubular fluid
reaches the distal convoluted tubule, as little as 10% of the
Sites of Potassium Transport in the Kidney
filtered K+ load remains (K+/In of <0.1; Fig. 8.5). The avid
The nephron sites of K+ handling have been ascertained by reabsorption of K+ in the thick ascending limb is mediated
micropuncture and in vitro microperfusion studies (Fig. by apical uptake through a secondary active electroneutral
8.4). Filtered K+ is reabsorbed mainly in proximal segments Na+-K+-2Cl– co-transporter driven by the electrochemical
of the nephron, whereas the urinary K+ is derived primarily gradient for Na+ that is generated by the basolateral Na+-K+-
from distal K+ secretion. Although small amounts of K+ are ATPase activity (Fig. 8.6B). There is passive K+ exit across the
152 II. Homeostasis

FIGURE 8.5. Determination of tubular fluid to plasma

concentration ratios of potassium to inulin [K/In (TF/P)]
as a function of tubular length along the nephron in
rats undergoing hypertonic NaCl administration. (From
Malnic G, Klose RM, Giebisch G. Micropuncture study of
distal tubular potassium and sodium transport in rat
nephron. Am J Physiol 1966;211:529, with permission.)

basolateral membrane by diffusion through K+ channels or Na+-K+-2Cl– co-transporter and generates a positive poten-
by co-transport with chloride or bicarbonate (85). There is tial in the lumen. The molecular identification of this apical
also K+ recycling back across the apical membrane through a channel is ROMK (rat outer medullary K+ channel) (92–94).
K+ channel, which assures adequate amounts of K+ for the Mutations in ROMK have been identified in patients with

FIGURE 8.6. Cell models of K+ transport pathways across apical and basolateral membranes and
paracellularly through the tight junction. A: Proximal tubule. B: Thick ascending limb. C: Princi-
pal cell. D: Type A intercalated cell or outer medullary collecting duct cell. The circles enclosing
the “~” indicate primary active transport; open circles represent exchangers. Channels are
denoted by the pentagonal figures in the membranes. Transcellular transport is denoted by the
long arrow. CA, carbonic anhydrase (isoform II or IV).
 8. Potassium 153

Bartter’s syndrome, an inherited disease characterized by lateral extrusion (via Na+-K+-ATPase), promotes both the
hyperreninemic hypokalemic metabolic alkalosis with secon- secretion of K+ and the paracellular reabsorption of Cl–.
dary hyperaldosteronism and normal blood pressure (95). Thus, the major determinants of passive K+ secretion are
Significant amounts of K+ are also reabsorbed via the paracel- the difference between cell and tubular fluid K+ concentra-
lular pathway across the thick ascending limb, driven by the tions, the transepithelial negative voltage driven by Na+
electropositive potential. The net reabsorption of K+ in this reabsorption through apical Na+ channels, and the number
segment can be changed to net K+ secretion if the Na+-K+- of open K+ channels in the luminal membrane.
2Cl– co-transporter is inhibited by loop diuretics. Potassium reabsorption has been localized morphologi-
The elevation of the K+/In ratio along the distal tubule cally to collecting duct intercalated cells in the cortex and
indicates that K+ has been secreted into the tubular fluid. medulla (111–113). Distal K+ secretion can be partially off-
Potassium is secreted by the late distal tubule (initial col- set by this K+ reabsorptive process in the cortical and med-
lecting tubule), connecting segment, principal cells of the ullary collecting ducts (112,114) and in the inner
cortical and medullary collecting duct (Fig. 8.6C), and, medullary collecting duct (96,115). This process is proba-
under certain circumstances, the inner medullary collecting bly due to a luminal H+-K+-ATPase (Fig. 8.6D) in the col-
duct (7,86,96–100). Under conditions of excessive K+ lecting duct, an enzyme that exchanges a single K+ ion for a
intake or depressed glomerular filtration rate, the rate of K+ proton while consuming ATP and thereby mediating both
secretion may become so high that the net K+ excretion rate H+ secretion and K+ reabsorption (116–120). It is likely
exceeds the filtered load (86,101–103). that both α and β intercalated cells express H+-K+-ATPase
Secretion of K+ in the distal nephron occurs in principal (1,112,121). Several isoforms of H+-K+-ATPases have been
cells by Na+-K+-ATPase–mediated active uptake of K+ identified in the kidney, and they share some identity with
across the basolateral membrane (4) and passive diffusion the gastric and colonic ATPases (121–123). The activity of
through secretory channels across the apical membrane into H+-K+-ATPase is increased by K+ depletion and reduced by
the luminal fluid (Fig. 8.6C). The magnitude of K+ secre- K+ loading (111,119–121,124). The adaptation with severe
tion depends on its electrochemical gradient and apical per- K+ depletion results in net K+ reabsorption along the distal
meability. The electrochemical gradient is generated by the nephron (86,111,113).
cell to lumen ratio of K+ concentrations and the lumen- Medullary K+ recycling occurs when the K+ reabsorbed
negative potential, which is driven by apical Na+ entry in the thick ascending limb enters the medullary intersti-
through epithelial Na+ channels (ENaCs) and transcellular tium and is then secreted into the late proximal tubule or
Na+ flux. The active cellular uptake of two K+ ions in thin descending limb of the loop of Henle; this additional
exchange for the extrusion of three Na+ ions (via Na+-K+- K+ is reabsorbed when it enters the outer medullary collect-
ATPase at the basolateral membrane) generates a negative ing duct (125–128). This recycling of K+ in the medulla
intracellular voltage with respect to the blood. Cell K+ is results in a relatively high concentration of K+ in the inter-
then secreted passively down a favorable electrochemical stitium that tends to favor K+ excretion by decreasing the
gradient into the lumen through apical K+-selective chan- gradients for passive K+ efflux out of the collecting duct and
nels (104–106). The small conductance channel has a high thick ascending limb (7,129).
open probability and is regulated by ATP. Its molecular
identification is ROMK (93,94,107), and it is considered
Factors Affecting K+ Secretion in the
to mediate basal K+ secretion (85,105,106). It has two
Cortical Collecting Duct
potential membrane-spanning helices flanking a region that
forms part of the channel pore in the voltage-gated K+ Whereas 90% of filtered K+ is reabsorbed in the proximal
channel (108). The channel has a unitary conductance of tubule and thick ascending limb, total body K+ homeostasis
30 to 40 picosiemens, high K+/Na+ selectivity, weak inward is maintained by regulation of K+ secretion along the distal
rectification, sensitivity to external Ba2+, and marked inhi- nephron. The cortical collecting duct is the main regulatory
bition to decreases in cytosolic pH within the physiologic site of K+ secretion, capable of generating luminal K+ con-
range (108). The inward rectification permits significant centrations in excess of 100 mmol/L in the adult (98). The
outward K+ current, as for K+ secretion from principal cells. principal cell of this cortical collecting duct secretes K+ and
This same ROMK channel (105,106,108) is present in the absorbs Na+. The major regulators of distal K+ secretion are
apical membrane of thick ascending limb cells and therein listed in Table 8.3.
allows the Na+-K+-2Cl– co-transporter to function opti-
mally by providing readily available substrate, K+ (109)
Aldosterone
(Fig. 8.6B). There is also a high-conductance, stretch- and
calcium-activated maxi-K channel that is likely to mediate Aldosterone secretion rises after a K+ load (130). Mineralo-
flow-stimulated K+ secretion (110). corticoids stimulate net sodium reabsorption and potassium
The lumen-negative potential in the cortical collecting secretion in the principal cells of the cortical collecting duct
duct, generated by apical Na+ entry and electrogenic baso- and adjacent cells of the connecting segment (97,131–134).
154 II. Homeostasis

TABLE 8.3. FACTORS AFFECTING K+ SECRETION is distributed into cells (22). The rise in plasma K+ concen-
IN THE CORTICAL COLLECTING DUCT tration is transient, because only a small fraction of the K+
Aldosterone activity load remains in the ECF. After the initial cell sequestration
Cell K+ concentration of K+, the kidneys continue to excrete K+ at an accelerated
Tubular flow rate rate until the excess is eliminated and balance is restored
Luminal Na+ concentration (1,22,68). Most of the increase in urinary K+ excretion is
Luminal nonreabsorbable anion
Acid-base balance
mediated by a rise in K+ secretion by the distal tubule
Other hormones (99,155–158) and cortical collecting duct (159–163). An
elevation of plasma K+ concentration may also be modu-
lated by K+ uptake into cells of the proximal tubule, which
Aldosterone binds to mineralocorticoid receptors of principal results in decreased cellular H+ concentration and thereby
cells; the hormone-receptor complex is translocated to the decreased HCO3– reabsorption. This proximal effect serves
nucleus, which results in activation of transcription and syn- to increase tubular flow rate, which, in combination with
thesis of physiologically active proteins (135). Cellular effects increased cellular K+ activity and aldosterone-induced
of aldosterone include an early enhancement in apical Na+ effects, serves to increase urinary K+ excretion (99).
permeability; this in turn increases the basolateral Na+-K+- Habitual ingestion of a high-K+ diet leads to an acquired
ATPase turnover rate, which results in increased cell K+ con- tolerance to K+ in the distal nephron, an adaptation that
centration (132,136–138). Later effects include increases in augments the capacity of each individual nephron to
basolateral membrane infolding, insertion of additional Na+- secrete K+ (164). In the principal cell, the cellular mecha-
K+-ATPase pumps into the membrane, and an increase in the nisms underlying this adaptation include increases in baso-
apical K+ conductance as K+-channel density increases lateral surface area, Na+-K+-ATPase activity, and density of
(105,134,136,137,139–142). Stimulation of Na+ reabsorp- apical membrane K+ and Na+ channels; increased cellular
tion leads to an increase in lumen negative transepithelial K+ concentration; and increased transepithelial voltage
voltage, which further favors K+ secretion (97,132,134,142). along the distal nephron (150,152,154,164,165–169)—
Mineralocorticoids stimulate both the electrochemical gradi- events that together enhance K+ movement from cell to
ent and luminal membrane permeability across the principal lumen. The morphologic correlate of this adaptation is the
cell, which results in increased K+ secretion and Na+ reabsorp- major increase in basolateral membrane surface area, the
tion (97,132,133,143,144). Prolonged administration of site at which the Na+-K+-ATPase pumps are inserted (113).
mineralocorticoids leads to a persistent kaliuresis but only a Distal K+ secretion at comparable levels of serum K+ con-
transient phase of Na+ retention (143–145). After a signifi- centration is approximately three times higher in K+-
cant extracellular volume expansion to a new steady state, adapted rats (99,113). A similar adaptive response seen in
there is a subsequent diuresis, which returns plasma volume chronic renal insufficiency (170,171) allows K+ balance to
toward normal (143,146). This “escape” phenomenon is be relatively well maintained during the course of many
probably due to decreased Na+ reabsorption in some other forms of progressive renal disease. Finally, gastrointestinal
nephron segment (147). excretion of K+ can increase in response to K+ loading or
renal insufficiency to help maintain K+ balance (172,173).
When there is chronic K+ depletion, there is less K+
Plasma K+ Concentration
secretion (86,113,174), due in part to decreased release of
The serum K+ concentration directly affects renal K+ excre- aldosterone and, later, diminished cellular K+ concentra-
tion (130). An increase in dietary K+ intake results in a tran- tions (175). There is also an increase in active K+ reabsorp-
sient rise in plasma K+ concentration (50), which in turn tion by cortical and medullary intercalated cells expressing
stimulates renal potassium excretion and the adrenal release the luminal H+-K+-ATPase pump (111,113,124). This
of aldosterone (68,99,130,148). The elevation in serum K+ response is associated with morphologic alterations to
concentration favors the entry of K+ into principal cells increase luminal surface area in the intercalated cells (112).
across the basolateral membrane and thereby drives K+ secre-
tion across the luminal membrane (149,150). Aldosterone
Tubular Flow Rate
enhances the electrochemical gradient, driving K+ secretion
in the distal tubule and collecting duct. In addition, plasma K+ secretion is strongly influenced by the rate of tubular
K+ elevation, through unknown mechanisms, increases Na+ fluid flow and the concentration of K+ in the tubular fluid
reabsorption, luminal Na+ permeability, and Na+-K+-ATPase (151,176–178). The higher the distal flow rate, the slower
activity independently of aldosterone (105,151–153). Also, a the rate of rise in tubular fluid K+ concentration and the
high-K+ diet can induce increases in K+ channel density in larger the driving force favoring additional K+ secretion
the apical membranes of principal cells (105,154). (149,177,178). Thus, volume expansion and diuretics,
In the first 4 hours after an exogenous K+ load, approxi- which increase distal delivery rates, enhance K+ secretion in
mately one-half is excreted by the kidneys, whereas the rest the distal nephron. By causing a concomitant reduction in
 8. Potassium 155

extracellular volume, diuretics also stimulate aldosterone and K+ secretion, which results in a downstream attenua-
secretion, which further favors K+ secretion. tion of the diuretic-induced natriuresis and an increase in
Increased distal flow is usually associated with increased the kaliuresis.
distal Na+ delivery. The increased availability of Na+ at dis-
tal sites results in two additional factors favoring K+ secre-
Nonreabsorbable Anion
tion (177,179). First, the increase in Na+ entry across the
apical channels makes the luminal potential more negative, Anions are generally reabsorbed in the cortical collecting
which creates a more favorable electrochemical gradient for duct via the paracellular route, after the active transport of
K+ secretion. Second, the increase in Na+ transport makes Na+. The delay in the reabsorption of the anion creates the
more K+ available to the cell via the basolateral Na+-K+- negative transepithelial difference (186). Distal delivery of
ATPase pump; the higher cell K+ concentration stimulates Na+ accompanied by an anion that is less reabsorbable than
distal K+ secretion. chloride, such as sulfate, bicarbonate, β-hydroxybutyrate,
Relevant to flow dependence, the high-conductance or carbenicillin, leads to an increase in luminal electronega-
maxi-K channel, which is not open at physiologic mem- tivity in the cortical collecting duct; this further stimulates
brane potentials, can be activated by membrane depolariza- K+ secretion (187–189), occasionally with clinical conse-
tion, elevation of intracellular Ca2+ concentration, quences (190).
membrane stretch, or hypoosmotic stress (180). One study
shows that high urinary flow rates and hydrostatic pressure
Acid-Base Balance
alter intracellular Ca2+ or the membrane stretch to which
renal epithelial cells are exposed, thereby stimulating apical Acute metabolic acidosis causes the urine pH and K+ excre-
stretch- and Ca2+-activated maxi-K channels (110). Stimu- tion to decrease, whereas both acute respiratory alkalosis
lation of these maxi-K channels is likely to mediate flow- and metabolic alkalosis result in increases in urine pH and
dependent K+ secretion in the cortical collecting duct, K+ excretion. Chronic metabolic acidosis has variable
whereas baseline K+ secretion occurs through the small- effects on urinary K+ excretion. Metabolic changes in ECF
conductance ROMK channel. pH produce reciprocal H+ and K+ shifts between cells. Dur-
ing acute acidemia, H+ is taken up by cells while K+ moves
out of cells, and this tends to reduce K+ secretion; in acute
Na+ Concentration
alkalemia, on the other hand, H+ comes out of cells and K+
The effect of luminal Na+ concentration on the magnitude of moves into cells, and this tends to increase K+ secretion
K+ secretion is determined by the entry of Na+ into the cell (150,191–194). Moreover, a decrease in the pH of tubular
across the apical membrane (149,178,181). In addition to fluid, as would be expected during metabolic acidosis, in
increasing luminal electronegativity, enhanced Na+ move- itself inhibits K+ secretion in the cortical collecting duct
ment from the tubular fluid into the principal cell depolar- (192,195,196). Data indicate that cell acidification sup-
izes the apical cell membrane, stimulates Na+-K+-ATPase presses and cell alkalinization stimulates activity of the api-
activity, and accelerates basolateral K+ uptake (149,181). The cal pH-sensitive K+ secretory channels (176). Furthermore,
net effect of these events is creation of a more favorable elec- low pH inhibits Na+-K+-ATPase activity (1), which reduces
trochemical gradient driving passive diffusion of K+ from cell K+ uptake and K+ secretion in the distal nephron.
to lumen. Maneuvers that decrease active Na+ transport (e.g., The effect of chronic acid-base disturbances on K+ secretion
blockers of Na+ channels, such as the K+-sparing diuretic is more complex and may be overridden by modifications of
amiloride) attenuate the lumen negative potential difference the glomerular filtrate (e.g., Cl– and HCO3– concentration),
and thereby reduce K+ secretion (177). tubular fluid flow rate, and circulating aldosterone levels (197–
Once the luminal Na+ concentration falls below 35 199). Indeed, chronic metabolic acidosis is a potent stimulus
mEq/L, K+ secretion declines and the transepithelial voltage of renal K+ excretion (192,193,200–203). Presumably, the aci-
becomes less negative (162,178,179). The decrease in Na+ dosis causes hyperkalemia, which stimulates aldosterone secre-
uptake into the principal cell hyperpolarizes the apical tion, and this results in the high rate of urinary K+ excretion in
membrane and decreases the electrochemical gradient this acid-base disorder. In addition, a reduction in plasma
favoring K+ secretion into the lumen. A reduction in cell HCO3– inhibits fluid and Na+ reabsorption in the proximal
Na+ concentration also reduces the activity of the basolat- tubule, which leads to increased fluid and Na+ distal delivery
eral Na+-K+-ATPase pump, thereby further reducing K+ (192). The development of volume depletion that also results
secretion. from this proximal inhibition stimulates the secretion of aldos-
Prolonged increases in Na+ delivery resulting from long- terone, which further increases K+ secretion. Thus, in contrast
term diuretic treatment will augment the basolateral mem- to acute metabolic acidosis in which K+ secretion is reduced,
brane area of distal convoluted tubule cells, connecting chronic acidosis overrides this inhibition by inducing aldoster-
tubule cells, and principal cells (182–185). This structural one- and flow-dependent K+ secretion. The handling of K+ in
adaptation is accompanied by increased Na+ reabsorption the organic acidurias (lactate, β-hydroxybutyrate) is domi-
156 II. Homeostasis

nated by the increased distal delivery of Na+ with a poorly addition, glucocorticoids stimulate maturation of neonatal
reabsorbable anion, plus volume depletion that stimulates colon K+ secretion by opening up apical K+ channels and
aldosterone secretion—effects that offset those of acute acido- increasing the activity of Na+-K+-ATPase (221,222). On the
sis and stimulate net distal K+ secretion and ultimately K+ other hand, colonic K+ secretion is inhibited by indometha-
depletion in the body (26). cin and dietary K+ restriction. Diarrheal illnesses are typically
associated with hypokalemia, despite an adaptive reduction
in renal K+ excretion. Mucosal inflammation reduces K+
Other Hormones
absorption, whereas cyclic adenosine monophosphate–stim-
Glucocorticoids stimulate K+ excretion through their action ulated secretion during rotaviral enteritis may contribute to
to enhance glomerular filtration, distal Na+ delivery, and rate inappropriate colonic K+ excretion (223).
of urinary flow (133,204,205). The administration of vaso- Potassium adaptation in the colon is demonstrated by
pressin causes a small increase in urinary K+ excretion despite increased fecal K+ excretion under conditions of K+ loading
a reduction in urinary flow rate, due in large part to an (trauma, renal tubular K+ secretory disorders, excessive K+
increase in the electrochemical driving force for K+ secretion intake), especially when exacerbated by renal insufficiency
across the luminal membrane (206–208). Specifically, there (164). Whereas stool K+ content averages 5 to 10% of
is a vasopressin-induced initial increase in apical Na+ perme- dietary intake in healthy adults, fecal K+ excretion may tri-
ability that leads to apical membrane depolarization, basolat- ple in patients with severe renal insufficiency (22,172).
eral Na+-K+-ATPase activation with enhanced K+ uptake,
and increased K+ secretion into the tubular fluid (155,209).
In addition, the vasopressin-induced stimulation of K+ secre- MATURATION OF RENAL K+ TRANSPORT
tion may also reflect an increase in density of apical K+ secre-
tory channels in the principal cell (210). Catecholamines Newborns fed breast milk or formula with an Na+/K+ ratio of
decrease urinary K+ excretion both directly and indirectly 0.5 to 0.6 demonstrate significant renal K+ retention with an
through catecholamine-induced alterations in renal hemody- average urine Na+/K+ higher than 1 (Table 8.1). This relative
namics and cell K+ uptake in the liver and muscle (43,211). conservation of K+ in the newborn and immature animal is
In addition, β-adrenergic agonists directly inhibit K+ secre- generally associated with a higher plasma K+ concentration
tion in the cortical collecting duct via modifications in chlo- than in the adult and is a requirement for somatic growth
ride transport and transepithelial voltage (212,213). (14,76,77,88,224). Clearance and micropuncture studies in
the newborn consistently demonstrate low rates of urinary
K+ excretion under basal conditions (88,225,226) and an
CONTRIBUTION OF THE inability to excrete an exogenously administered potassium
GASTROINTESTINAL TRACT load as efficiently as the adult (14,79–82). Acute K+ loading
of experimental animals results in significantly lower rates of
In the adult under normal conditions, approximately 10% of urinary K+ excretion in weaning than in older rats (Fig. 8.7)
daily K+ intake is excreted in the stool (Fig. 8.2). The gas-
trointestinal tract normally absorbs approximately 85% of
dietary K+. This process is substantially more efficient in the
neonate, probably related to a higher activity of K+-absorptive
pumps (214,215). The general pattern of K+ handling by the
intestine parallels that of the nephron, and the primary site of
regulation of intestinal K+ transport is the colon. Most dietary
K+ is absorbed along with water in the small intestine
(84,214,216), so that a low volume of fluid with a high K+
concentration is delivered to the colon. The direction of net
K+ transport in the colon, as in the distal nephron, is deter-
mined by the balance between K+ secretion and K+ absorption
(172,217). In the colon, K+ secretion requires basolateral
uptake of K+ via the Na+-K+-ATPase pump and Na+-K+-2Cl–
co-transporter and its secretion across the apical membrane
via K+ channels (218). The absorption of K+ is mediated by
apical K+-dependent ATPases, including a colonic H+-K+-
ATPase (214,215). FIGURE 8.7. Renal K+ excretion after intragastric KCl loading
Hormones (aldosterone, glucocorticoids, epinephrine, (0.7 mmol/100 g body weight) in unanesthetized infant (PN20)
and adult (PN50) rats. Values are means ± the standard error of
and prostaglandins) stimulate K+ colonic secretion (22,219). the mean; asterisks indicate statistically significant difference
A high-K+ diet also stimulates colonic K+ secretion (220). In between infant and adult rats. bw, body weight.
 8. Potassium 157

FIGURE 8.8. Maturation of net K+ secretion in cortical

collecting ducts obtained from 1-, 2-, 3-, 4-, 6-, and >8-
week-old (adult) rabbits. Mean values plus standard
error are given for each age group. *, p <.05 versus >8-
week-old (adult). (Data from the Satlin LM. Postnatal
maturation of potassium transport in rabbit cortical
collecting duct. Am J Physiol 1994;266:F57.)

(215). Whereas infants, like adults, can excrete K+ at a rate K+ secretion are mediated by distinct channels with different
that exceeds its filtration (79), the rate of K+ excretion developmental patterns of expression (229).
expressed per unit of body or kidney weight is less than that Ultrastructural comparison of principal cells from new-
observed in older subjects (80,82,214). born and adult cortical collecting ducts lends support to
Micropuncture studies have shown that approximately the observation of relative functional immaturity of the K+
50% of the filtered load of K+ is reabsorbed in the proximal secretory epithelium early in life (21,230). Whereas the
tubule of both suckling and older rats (88,90). However, principal cell of the neonatal cortical collecting duct pos-
up to 35% of the filtered load of K+ reaches the superficial sesses few organelles, smooth apical and basolateral sur-
distal tubule of the 13- to 15-day-old rat, which far exceeds faces, varying amounts of intracellular glycogen, and a low
the distal delivery measured in older animals and indicates volume of mitochondria, the mature principal cell is devoid
functional immaturity of the loop of Henle (88,227). The of glycogen and possesses twice the volume percentage of
fractional reabsorption of K+ along the loop of Henle, mitochondria, has more organelles and basolateral infold-
expressed as a percentage of delivered load, increases from ings, and has a 35% larger apical perimeter (230).
57% at 13 to 15 days to 79% by 30 to 39 days of age (88).
Because the fractional excretion of K+ in the immature
Factors Limiting K+ Secretion in
rat is approximately the same as the percentage of the load
the Cortical Collecting Duct
delivered to the superficial distal tubule (88), it is likely that
there is little net K+ secretion along the immature distal Several factors may limit urinary K+ secretion in the neona-
nephron. Evidence indicates that the limitation in K+ excre- tal principal cell, including an unfavorable electrochemical
tion is due, at least in part, to a greatly reduced K+ secretory gradient (low cell K+, low Na+-K+-ATPase activity, and/or
capacity of the cortical collecting duct early in life. Clear- low transepithelial voltage), high backleak of K+ through
ance experiments in saline-loaded puppies have provided paracellular routes, limited membrane permeability to K+,
indirect evidence for diminished capacity of the immature low tubular fluid flow rates, and diminished principal cell
distal nephron to secrete K+ (225). Comparison of early sensitivity to mineralocorticoids. Alternatively, enhanced
distal tubular fluid and final urine in the newborn suggests K+ absorption by intercalated cell-rich distal nephron seg-
that the immature distal tubule and cortical collecting duct ments (e.g., inner cortical collecting duct, outer medullary
secrete less K+ than do more mature segments (88). collecting duct) may offset much of the distal K+ secretion.
Microperfusion experiments in rabbit cortical collecting
ducts have conclusively shown that net K+ secretion is absent
Na+-K+-ATPase
at birth, first becomes evident at the fourth postnatal week,
and increases sharply to reach adult levels by 6 weeks of age The high cellular potassium concentration is generated and
(Fig. 8.8) (228). These data indicate that the low rates of K+ maintained by the activity of Na+-K+-ATPase. Na+-K+-
excretion characteristic of the newborn kidney are due, at ATPase activity in the neonatal cortical collecting duct is
least in part, to a low capacity for K+ secretion by the imma- only 50% of that measured in the mature segment, when
ture cortical collecting duct. Whereas K+ secretion in the expressed per unit of dry weight (32), in keeping with the
mature cortical collecting duct is strongly stimulated by an smaller number of basolateral infoldings observed in neo-
increase in luminal flow rate, this is not observed in early life, natal principal cells (230). This lower level of Na+-K+-
at least until 6 weeks postnatally. Indeed, the developmental ATPase activity in the immature cortical collecting duct
appearance of flow dependence 2 weeks after the appearance results in a reduced electrochemical K+ gradient across the
of basal K+ secretion suggests that basal and flow-stimulated apical membrane. If one assumes that principal cell K+
158 II. Homeostasis

activity is similar in neonatal and mature collecting ducts that observed in 5-week-old animals. The increase in the
(21), it is likely that the low rate of Na+-K+-ATPase activity number of conducting channels is due to both an increase
observed early in life must be accompanied by a propor- in the number of channels present per patch plus an
tionally low rate of passive K+ efflux from immature princi- increase in open probability (234). Thus, the presence of
pal cells. The efflux of K+ may be limited by an unfavorable conducting Na+ channels in the maturing cortical collect-
electrochemical gradient opposing secretion, reduced apical ing duct approximately 1 week before net K+ secretion is
membrane K+ permeability, or low tubular flow rates pre- observed suggests that a low rate of Na+ entry into the prin-
vailing in the neonate. cipal cell does not limit K+ secretion early in life, unless lit-
tle Na+ is delivered to the distal nephron. In vivo
measurements of distal tubular Na+ concentrations in both
Flow Rate
adult and maturing rats generally exceed 30 mEq/L
The low distal flow rates characteristic of the newborn (86,88,179,232), which rules out the possibility that very
(88,231,232) could limit K+ secretion early in life. To low luminal Na+ concentrations contribute to the reduced
address the role of flow rate on K+ secretion, Satlin mea- rate of distal K+ secretion in the newborn.
sured the rates of K+ transport in cortical collecting ducts.
Flow-dependent stimulation of K+ secretion was not
Backleak
observed until 6 weeks postnatally (228). These studies rule
out a low in vivo tubular flow rate per se as a factor limiting Paracellular K+ conductance may be increased through
K+ secretion in the neonatal kidney. leaky tight junctions in immature proximal tubules and
Micropuncture studies in maturing rats show that the cortical collecting ducts compared with mature segments
early distal tubular fluid concentration of K+ decreases dur- (235,236). Thus, at the lower flow rates found in the
ing maturation from 4.5 to 2.3 mmol/L (88,90). The higher immature kidney, enhanced tubular backleak of K+ could
K+ concentration in the youngest rats reflects, in part, imma- severely limit net K+ secretion. A cortical collecting duct
turity of K+ reabsorption by the thick ascending limb of the microperfusion study showed that the difference between
loop of Henle. These data suggest that only a small increase perfused and collected K+ concentrations was less than 1.5
in distal nephron K+ secretion could be accounted for by this mol/L during the first 3 weeks of life (228). However, in
maturational decrement in chemical gradient along the col- the same age group, a lack of active secretion of K+ over a
lecting duct. Thus, the chemical driving force for K+ secre- wide range of flow rates suggests that backleak may not be a
tion across the apical membrane of the principal cell appears limiting factor early in life. Moreover, despite the absence
to remain relatively constant after birth (228). of net K+ secretion, active Na+ absorption was evident as
early as 2 weeks of age, at a rate that approached 50% of
the mature level. Because the difference between perfused
Electrical Gradient
and collected Na+ concentrations was nearly the same in
The luminal electronegativity of the cortical collecting duct, immature and mature segments (228), it is not likely that
driven by electrogenic Na+ absorption (97,162), favors K+ postnatal development of the tight junctions comprising
secretion from principal cell to lumen. Measurements of the paracellular pathway plays a major role in the matura-
transepithelial voltage in cortical collecting ducts early in life tion of K+ secretion in the cortical collecting duct. On the
have shown maturational increases from a few millivolts neg- other hand, data to suggest that backleak may be important
ative at birth to approximately 10 mV negative in the adult in limiting K+ transport come from the isotopic Na+ studies
(228,233). These findings suggest that a smaller electrical performed by Vehaskari in maturing cortical collecting
gradient slightly limits K+ secretion in the neonatal cortical ducts (237), which showed high rates of passive Na+ perme-
collecting duct. Microperfused cortical collecting ducts iso- ability during the first 2 weeks of postnatal life before a
lated from 1-week-old rabbits show no significant Na+ decrease to adult levels. Thus, a role for backleak in limiting
absorption (228). By 2 weeks of age the rate of net Na+ K+ secretion in the immature cortical collecting duct is still
absorption is approximately half that observed in cortical col- a significant possibility.
lecting ducts taken from mature animals. Concomitant with
the postnatal increase in Na+ absorption is a parallel increase
Aldosterone
in negativity of transepithelial voltage (228).
Finally, to secrete K+, the principal cell requires func- Plasma aldosterone concentrations in the fetus and newborn
tioning apical Na+ channels to provide Na+ for the Na+ are high compared with those in the adult (14,238–241).
pump. The rate-limiting step for Na+ absorption appears to Yet, clearance studies demonstrate that young animals are less
be the expression of the apical Na+ channel. Using patch- sensitive to mineralocorticoids than their mature counter-
clamp analysis of the maturing rabbit principal cell, Satlin parts (14,242). Both the density of aldosterone binding sites
and Palmer (234) found that by 2 weeks of age the number and receptor affinities are similar in mature and immature
of open Na+ channels per patch reached a value similar to rats, which suggests that the early hyposensitivity to aldoster-
 8. Potassium 159

one is likely to be a postreceptor phenomenon (242). In principal cell substantially contributes to the maturational
addition, Vehaskari (237) showed that the administration of surge in the rate of K+ secretion.
mineralocorticoid for 48 hours to 1- and 2-week-old rabbits The secretory K+ channel characterized by Satlin and
had no effect on the sodium-22 absorptive flux in cortical Palmer (243) is generally believed to have a major functional
collecting ducts isolated from these animals. Thus, the bio- subunit called ROMK, based on similarities in biophysical
logical responsiveness to aldosterone is minimal early in life properties (92,107,244,245). Using reverse transcriptase
and increases with age; this development of aldosterone polymerase chain reaction, Benchimol et al. (246) showed that
responsiveness may contribute to the maturation of K+ secre- ROMK messenger RNA (mRNA) was regularly expressed by
tion in the cortical collecting duct. cortical collecting ducts isolated from animals 3 weeks of age
and older. Only 30% of ducts from 2-week-old animals
expressed ROMK mRNA and none expressed ROMK during
Apical K+ Channels
the first week of life. Immunodetectable (247) and functional
The most likely explanation for both the low rate of K+ secre- (243) apical low (ROMK) conductance channels, absent in
tion and the mature level of intracellular K+ concentration neonatal principal cells, are present in principal cells from
despite the low activity of the Na+-K+-ATPase pump is a low maturing rabbits, which indicates that the increase in ROMK
apical permeability of the principal cell to K+. The passage of mRNA contributes to the development of low-conductance
K+ across the apical membrane from the principal cell into secretory K+ channels in the maturing cortical collecting duct.
the luminal fluid is likely mediated by a low conductance
inwardly rectifying K+-selective channel (105,106,108,154).
K+ Absorption by Intercalated Cells
Using patch-clamp technology, Satlin and Palmer (243)
found a progressive increase with age in mean number of Because net K+ transport represents the sum of K+ secretory
open K+ channels per patch in rabbit principal cells (Fig. and K+ reabsorptive processes, it is possible that some K+
8.9). This increase was due primarily to a developmental absorption offsets the limited K+ secretion in the neonatal cor-
increase in the number of channels per patch, because the tical collecting duct. Clearance studies in saline-expanded dogs
open probability remained constant after the second week of showed that newborn dogs reabsorbed 25% more of the distal
life. Because a major increase in net K+ secretion occurs after K+ load than did adult animals (80,225). Because micropunc-
the first 3 weeks of life and a major increment in mean num- ture studies of immature rats indicated little K+ reabsorption in
ber of open channels per patch occurs after the second week, the loop of Henle (88,90), the collecting duct was considered a
it is likely that the increase in apical K+ conductance of the likely segment for K+ reabsorption. This K+ reabsorption is
probably mediated by an apical H+-K+-ATPase that couples K+
reabsorption to H+ secretion identified in collecting duct inter-
calated cells (117,118,121,248). Indeed, K+ deficiency in adult
animals is associated with selective hypertrophy of the apical
membranes of medullary collecting duct intercalated cells
(114). These anatomical changes are accompanied by func-
tional data that show increased K+ absorption (249) and H+-
K+-ATPase protein and activity (124,250–252).
Functional studies indicate that neonatal cortical collect-
ing duct intercalated cells possess an apical H+-K+-ATPase
with activity comparable to that of mature intercalated cells
(253). In addition, the higher tubular fluid K+ concentration
of the immature animal (88) should facilitate lumen to cell
K+ absorption mediated by the H+-K+-ATPase. Although
direct studies of K+ absorption by the immature collecting
duct have not been performed, the preceding data suggest
that parts of the neonatal collecting duct may absorb K+
(233,253), which is essential for conserving some of the
excessive K+ presented to the immature distal nephron.
FIGURE 8.9. Maturation of K+ conductance in split-open cortical
collecting ducts obtained from 1-, 2-, 3-, 4-, and 5-week-old rab-
bits. Mean values of number of open channels per patch plus DISORDERS OF POTASSIUM METABOLISM
standard error are given for each age group. Segments from rab-
bits older than 5 weeks were not examined because the rate of
successful patch-clamp experiments was too low. *, p <.05 versus Hypokalemia
5-week-old. (From Satlin LM, Palmer LG. Apical K+ conductance in
maturing rabbit principal cell. Am J Physiol 1997;272:F397, with K+ is predominantly an intracellular cation, yet clinical estima-
permission.) tion of K+ balance usually depends on the measurement of
160 II. Homeostasis

extracellular (serum or plasma) K+ concentration. Accurate TABLE 8.4. CLINICAL DISORDERS ASSOCIATED
assessment of body potassium stores can be performed only by WITH HYPOKALEMIA
total-body counting methods, laborious measurements not Inadequate intake
readily available to the clinician. Hypokalemia, defined as a Dietary insufficiency
serum K+ concentration below 3.5 mEq/L, usually indicates a Chloride deficiency syndrome
deficit in total body potassium but may also represent a shift of Increased entry of K+ into cells
Acute alkalosis (metabolic, respiratory)
K+ from the extra- to the intracellular space in the setting of Insulin administration
normal total body K+ stores. To a first approximation, each 1 Elevated β-adrenergic activity
mEq/L decrease in serum K+ concentration below 3 mEq/L Hypokalemic periodic paralysis
corresponds to a 200- to 400-mEq (approximately 10%) deficit Increased number of cells in plasma
in total body potassium stores in the 70-kg adult. Total body K+ Barium poisoning
Pseudohypokalemia
depletion causing hypokalemia results from inadequate intake Increased renal losses
or from excessive loss of K+ from the body via the kidney or gas- Renal K+ wasting disorders with low plasma renin activity
trointestinal tract. A list of clinical disorders associated with K+ Disorders of excess mineralocorticoid activity
redistribution and depletion is found in Table 8.4. Primary hyperaldosteronism
Aldosterone-producing adenomas
Bilateral adrenal hyperplasia
Clinical Disorders Associated with Hypokalemia Cushing syndrome
Primary adrenal disease
Insufficient K+ Intake Effect of pharmacologic doses of corticosteroids
Dietary Insufficiency. Potassium is virtually impossible to Secondary effect of nonendocrine tumor
eliminate from the average diet except by limiting intake to Congenital adrenal hyperplasia
11β-hydroxylase deficiency
foods containing a high percentage of carbohydrate or 17α-hydroxylase deficiency
refined sugar or by administering K+-free parenteral fluids Excessive licorice ingestion
for a prolonged period. The K+ content of a variety of foods Inhibition of 11β-hydroxysteroid dehydrogenase
and beverages in the American diet are listed in Table 8.5. Liddle’s syndrome (pseudoaldosteronism type 1)
In the adult a sharp reduction in dietary K+ intake is fol- Pseudoaldosteronism type 2 (mineralocorticoid receptor
defect)
lowed within several days by a decrease in urinary K+ excretion Glucocorticoid-remediable aldosteronism
but only to a minimum rate of approximately 10 mEq/day, as Syndrome of apparent mineralocorticoid excess
the kidney is unable to produce a K+-free urine (69). The com- Renal K+-wasting disorders with high plasma renin activity
bination of the limited ability to maximally conserve urinary Excessive renin production
K+ and ongoing obligatory gastrointestinal losses may lead to Bartter’s syndrome
Gitelman’s syndrome
significant K+ depletion if dietary deprivation is prolonged. Diuretic use
Thiazides and loop diuretics
Chloride Deficiency Syndrome. Dietary chloride deficiency Osmotic diuretics (mannitol, glucose)
syndrome, first recognized in infants fed chloride-deficient Carbonic anhydrase inhibitors
batches of commercial cow’s milk formula, is characterized Renal tubular acidosis
Other conditions associated with increased distal nephron
by hypochloremic metabolic alkalosis, hypochloruria, and flow
hypokalemia (254,255). Clinically, these babies presented with Renal diseases (interstitial nephritis, obstructive uropa-
failure to thrive, anorexia, muscular weakness, lethargy, vomit- thy, dysplasia, hypercalcemia)
ing, and dehydration. Renal K+ excretion was lower in chloride- Antibiotic use (carbenicillin, nafcillin, penicillin, gentami-
deficient patients than in age-matched controls offered formula cin, amphotericin B)
Leukemia
of comparable K+ content (254). The pathogenesis of the Miscellaneous
hypokalemia may be related to volume contraction due to lack Hypomagnesemia
of adequate chloride and resulting in secondary hyperaldoster- Vitamin D intoxication
onism, reduced intake of formula, pH-induced redistribution Thyrotoxicosis
of extracellular K+, and provision of nonreabsorbable anions. Primary polydipsia
Cardiopulmonary bypass
Increased gastrointestinal loss
Increased Entry of K+ into Cells Gastric (vomiting, nasogastric suction, pyloric stenosis)
Acute Alkalosis. Either metabolic or respiratory alkalosis Large intestinal (diarrhea, VIPoma syndrome, villous ade-
can be associated with hypokalemia by promoting K+ entry noma, chronic laxative abuse, biliary drainage, ureterosig-
into cells in exchange for protons. The initial small trans- moidostomy)
Integumental loss
cellular shift of K+ is exacerbated by volume contraction– Excessive sweating
induced hyperaldosteronism and the delivery of nonreab- Full-thickness burns
sorbable anions (bicarbonate) to potassium-secretory sites
in the distal nephron. VIPoma, vasoactive intestinal polypeptide tumor.
 8. Potassium 161

TABLE 8.5. POTASSIUM CONTENT OF VARIOUS FOODS

Food K+ (mg) Food K+ (mg)

Beverages Cookies/desserts
Cola, 12 oz 4 Gingersnaps, 1 oz, 4 cookies 98
Pepsi, 12 oz 10 Doughnut, 1 60
Gingerale, 12 oz 4 Chocolate ice cream, 0.5 cup 164
Diet Coke, 12 oz 18 Vanilla ice cream, 0.5 cup 131
Coffee, 6 oz 96 Chocolate ice cream bar with chocolate coating, 1 bar 247
Cranberry-apple juice, 6 oz 50 Apple turnover, 1 33
Hawaiian punch, 8 oz 50 Orange sherbet, 0.5 cup 92
Orange juice, 6 oz 210 Jello, 0.5 cup 239
Black tea, brewed, 6 oz 66 Meals
Citrus fruit juice drink, 8 oz (from frozen concentrate) 278 Scrambled egg (1) with milk 84
Apple juice, 8 oz 295 Macaroni and cheese, 1 cup 190
Grapefruit juice, 8 oz 378 Cheese pizza, 1 slice 85
Orange juice, 8 oz 436 Chicken pot pie, 1/3 of 9-in. pie 343
Tomato juice, 6 oz 400 Spaghetti with tomato sauce, 1 cup 408
Chocolate shake, 10 oz 566 Cheeseburger, 1 large 644
Candy Hotdog, 1 143
Golden Almond Hershey’s Chocolate 3.2-g bar 429 Mashed potatoes, 1/3 cup 235
Lollipop, 1 0 Fruit, fresh
Marshmallow, 1 25 Apple, 1 medium 159
Milk chocolate, 1.55-oz bar 169 Banana, 1 medium 451
Snickers, 2.16-oz bar 206 Cantaloupe, 1 cup 494
Twizzlers, 2.5 oz 45 Grapefruit, one-half, medium 175
Cereals Peach, 1 medium 171
Bran, 100%, 1 oz 277 Raisins, 2/3 cup 825
Cornflakes, 1.06 oz 35 Watermelon, 1 cup 186
Granola, Kellogg’s lowfat, 1.7 oz 122 Bread
Raisin bran, 2.08 oz 380 White, 1 slice 30
Special K, 1.09 oz 54 Whole wheat, 1 slice 71
Shredded Wheat, 1 oz 93 English muffin, with butter 69
Cheese Vegetables
American processed, 1 oz 46 Potato, with skin 903
Cream cheese, 1 oz 34 Tomato, 1 273
Chips Green beans, 0.5 cup 185
Corn chips, 1 oz 40 Carrot, 1 medium 233
Potato chips, 1 oz 361 Lima beans, 1 cup boiled 955
Pretzels, 1 oz 41 Lentils, 1 cup boiled 731
Broccoli, 0.5 cup raw 143

Data from Pennington JAP. Bowes & Church’s food values of portions commonly used, 17th ed. Philadelphia: Lippincott–Raven, 1998.

Insulin Administration. Insulin promotes the entry of K+ ness and episodes of flaccid paralysis of the limbs and thorax
into skeletal muscle and liver cells (37,256), as observed lasting 6 to 24 hours (261). Serum K+ concentration is normal
most prominently in the treatment of diabetic ketoacidosis. between attacks. In severe cases death from respiratory failure
or cardiac arrhythmia may occur. Both sporadic and familial
Elevated β-Adrenergic Activity. Nonselective β-adrenergic cases have been reported with a male to female preponderance
agonists (isoproterenol, epinephrine) and selective β2-adren- of 3:1 and generally autosomal dominant inheritance. Familial
ergic agonists (albuterol, terbutaline) promote K+ entry forms are due to point mutations in the gene encoding the α-1
into cells (37). Thus, transient hypokalemia can be pro- subunit of the L-type calcium channel, or voltage-gated dihy-
duced by stress-induced release of epinephrine, theophyl- dropyridine receptor (262,263). The dihydropyridine receptor
line intoxication, treatment of asthma with albuterol (257), functions as a voltage-gated calcium channel and is critical for
or treatment of premature labor with terbutaline (258). excitation-contraction coupling in a voltage-sensitive and cal-
Arrhythmias may result from this hypokalemia when there cium-independent manner (264). The defect in hypokalemic
is volume contraction induced by concomitant diuretic periodic paralysis is associated with a reduced sarcolemmal
therapy (259) or by poor oral intake (260). ATP-sensitive K+ current (265).
The disorder usually becomes symptomatic during the
Hypokalemic Periodic Paralysis. Hypokalemic periodic paral- first or second decade of life. Acute attacks generally occur at
ysis is a rare disorder characterized by attacks of muscle weak- night, and the patient awakens paralyzed. Episodes are asso-
162 II. Homeostasis

ciated with sudden movement of K+ into cells, which lowers lecting duct and/or excessive mineralocorticoid. In the setting
plasma K+ concentration by 1.5 to 2.5 mEq/L. Attacks are of adequate distal Na+ delivery, the urine K+ concentration is
often precipitated by a carbohydrate meal, rest after exercise, helpful in differentiating among the causes of hypokalemia.
or stressful events causing hypokalemia by a shift of K+ into
cells via the release of insulin or epinephrine. The paralysis is Renal K+ Wasting Disorders with Low Plasma Renin Activity
characterized by failure of propagation of the muscle action Disorders of Excess Mineralocorticoid Activity. Because min-
potential (266). After the attack, sequestered K+ is released eralocorticoids stimulate K+ secretion in the distal tubule and
from cells and plasma K+ concentration becomes normal. cortical collecting duct, conditions characterized by excessive
Treatment with oral KCl usually aborts symptomatic mineralocorticoid activity are often associated with hypokale-
attacks; intravenous K+ may be required for severe attacks. mia. The classic disorder of endogenous overproduction, pri-
Nonselective beta-blockers can reduce the number and severity mary hyperaldosteronism, may be associated with an adrenal
of attacks, and limit the fall in plasma K+ concentration (261). adenoma or bilateral adrenal hyperplasia and is accompanied
In most patients acetazolamide (20 to 40 mg/kg/day divided by hypertension, a variable degree of hypokalemia, and meta-
into four doses) may be more effective than K+ supplementa- bolic alkalosis (273,274).
tion or K+-sparing diuretics in preventing severe attacks. The Hypokalemia along with metabolic alkalosis may be
metabolic acidosis induced by acetazolamide may decrease the observed in some patients with increased cortisol production
rate of K+ entry into cells and thereby diminish the severity of (Cushing syndrome) or in those receiving pharmacologic doses
the disease (267). Hyperthyroidism may be associated with of corticosteroids, presumably related to the mineralocorticoid
hypokalemic periodic paralysis, probably because thyroid hor- effect of such hormones or to the fact that the rate of delivery
mone stimulates Na+-K+-ATPase activity and sensitivity to of cortisol exceeds its rate of inactivation via 11β-hydroxy-
catecholamines, and thereby drives K+ into cells. Restoration steroid dehydrogenase (11β-HSD) (275,276). Tumors of the
of euthyroidism in thyrotoxic patients combined with beta- lung, thymus, and pancreas that produce an adrenocortico-
blockade generally prevents the hypokalemic attacks. tropic hormone (ACTH)–like substance are less prevalent in
children than in adults but may lead to increased production
Barium Poisoning. Ingestion of soluble barium salts may of cortisol, desoxycorticosterone (DOC), and corticosterone,
result in a severe decrease in plasma K+ concentration sec- which results in severe hypokalemia (277).
ondary to intracellular shifts of K+ from the plasma (268). Among the multiple forms of congenital adrenal hyperplasia,
Barium acts on muscle cells to decrease K+ conductance, deficiencies of 11β-hydroxylase and 17α-hydroxylase are asso-
thereby impairing outward diffusion of K+ (269). Thus, the ciated with hypertension and hypokalemic alkalosis. The 11β-
K+ pumped into the cell via Na+-K+-ATPase cannot exit. hydroxylase deficiency leads to a block in synthesis of cortisol,
enhanced secretion of ACTH, and subsequent stimulation of
Increased Number of Cells in Plasma. A rapid increase in DOC and adrenal androgen production (278). The excess
hematopoietic cell production associated with new cell K+ mineralocorticoid (DOC) activity results in hypokalemia and
uptake, such as resulting from the administration of folic acid hypertension. The 17α-hydroxylase deficiency leads not only
or vitamin B12 to patients with megaloblastic anemia, can lead to a block in cortisol synthesis but also to defective production
to hypokalemia and potentially to cardiac arrhythmias (270). of adrenal androgens (278,279).
Similarly, after the administration of granulocyte-macrophage Ingestion of large quantities of European licorice, which
colony-stimulating factor to correct neutropenia, a marked contains glycyrrhetinic acid, a plant steroid with weak mineral-
increase in white blood cell production may lead to hypokale- ocorticoid activity, can lead to a syndrome similar to primary
mia (271). hyperaldosteronism with sodium retention, edema, hyperten-
sion, and hypokalemia, except that plasma aldosterone levels
Other Cellular Shifts. Hypothermia has been associated are suppressed (280). Licorice and its analogues are also effec-
with hypokalemia as K+ enters cells and is reversible on tive inhibitors of 11β-HSD, which converts cortisol to corti-
rewarming. sone in the distal nephron (275,281). Cortisol binds to the
mineralocorticoid receptors present in the cortical collecting
Pseudohypokalemia. Because metabolically active cells con- duct with an avidity equal to that of aldosterone but circulates
tinue to take up K+ after blood has been drawn, patients with at much higher concentrations in plasma. However, cortisol is
very high white blood cell counts may become apparently converted locally by 11β-HSD to inactive metabolites such as
hypokalemic if their blood has been allowed to stand for pro- cortisone. This conversion is impaired with licorice-induced
longed periods at room temperature (271,272). inhibition of 11β-HSD, which allows cortisol to activate the
mineralocorticoid receptors and produce a clinical picture of
Increased Renal Losses primary hyperaldosteronism.
Urinary K+ excretion is determined primarily by K+ secretion
in the cortical collecting duct. Increased urinary losses usually Liddle’s Syndrome. Liddle’s syndrome is a rare autosomal
result from increased flow of water and Na+ to the cortical col- dominant disorder that presents a clinical picture similar to
 8. Potassium 163

that of primary hyperaldosteronism with severe hypokalemic to correlate with the level of aldosterone secretion, and gluco-
metabolic alkalosis and hypertension accompanied by Na+ corticoids suppress this hypertension (284). The mechanism
retention and substantial urinary K+ excretion (282). Plasma for urinary K+ wasting is similar to that observed in Liddle’s
renin and aldosterone levels are low, however, which gives syndrome.
rise to the clinical descriptor of pseudoaldosteronism (283).
This disease is caused by mutations in the carboxyterminal Syndrome of Apparent Mineralocorticoid Excess. In the syn-
of the β or γ subunits of the ENaC (282,284). These gain-of- drome of apparent mineralocorticoid excess, inactivating muta-
function mutations result in enhanced channel activity due to tions in the 11β-HSD type II gene allow cortisol to act as the
increased cell surface expression (and lack of downregulation) of major endogenous mineralocorticoid (284,288). This enzyme
functional Na+ channels in the cortical collecting duct. The con- normally converts cortisol to cortisone, thereby preventing cor-
sequent increase in Na+ reabsorption leads to severe hyperten- tisol from activating the type I mineralocorticoid receptor in
sion. The hypokalemia reflects urinary K+ wasting due to the the kidney. In this rare autosomal recessive disorder character-
secondary effects of increased apical Na+ reabsorption, which ized by pre- and postnatal growth failure, juvenile hypertension,
results in an increased electrochemical driving force favoring K+ hypokalemic metabolic alkalosis, and hyporeninemic hypoal-
secretion from the principal cell into the urinary space. The uri- dosteronism, reduced metabolism of cortisol to cortisone
nary Na+ and K+ abnormalities are not affected by spironolac- results in local cortisol excess and increased mineralocorticoid
tone, a competitive aldosterone antagonist, but rather by the response, even in the absence of elevations in plasma cortisol
Na+ channel blockers triamterene and amiloride (285). level (289). The underlying mechanism for urinary K+ wasting
is similar to that in Liddle’s syndrome. Treatment usually
Pseudoaldosteronism Type 2. This disorder results from a includes spironolactone (which competes for the mineralocorti-
mutation in the hormone-binding domain of the mineralo- coid receptor), K+ supplements, and a low-salt diet.
corticoid receptor (286). The phenotype resembles that of
Liddle’s syndrome with autosomal dominant inheritance, Renal K+ Wasting Disorders with
hypertension, hypokalemia, suppressed plasma renin activ- High Plasma Renin Activity
ity, and aldosterone secretion. However, a very severe pre- Bartter’s and Gitelman’s Syndromes. Bartter’s and Gitel-
sentation during pregnancy is also seen (284). man’s syndromes are rare autosomal recessive disorders associ-
A serine to leucine mutation occurs at position 810 in the ated with urinary K+ wasting, hypokalemia and metabolic
hormone-binding domain of the mineralocorticoid receptor alkalosis, hyperreninism, and hyperaldosteronism, but with
that changes the affinity of the receptor for a variety of steroids. normal blood pressure (282,290). Hypokalemia, chronic vol-
This mutation results in constitutive mineralocorticoid receptor ume contraction, and high plasma concentrations of angio-
activity and alters receptor specificity, so that progesterone and tensin, kallikrein, kinins, and vasopressin all stimulate the
other steroids lacking 21-hydroxyl groups, normally mineralo- production of prostaglandin E2. The absence of hypertension
corticoid receptor antagonists, become potent agonists (286). rules out primary mineralocorticoid excess, and the finding of
Progesterone has a very high affinity for the mutated recep- high urinary chloride excretion rules out secondary hyperal-
tor, which accounts for the severe presentation during preg- dosteronism due to extrarenal fluid loss. Such findings occur
nancy. Because the receptor has constitutively stimulated basal only in Bartter’s syndrome and with long-term diuretic ther-
activity and may respond to other endogenous steroids, the apy (282). The pathogenesis of Bartter’s and Gitelman’s syn-
clinical phenotype can present in males and nonpregnant dromes is similar to that in long-term treatment with loop and
females as well. Spironolactone is an agonist for the mutated thiazide diuretics, respectively. The genetic defect of Bartter’s
receptor, and its use is therefore contraindicated in this form of syndrome involves transporters in the thick ascending limb of
pseudoaldosteronism. the loop of Henle and collecting duct. Clinical disease is attrib-
utable to defective NaCl reabsorption in the thick ascending
Glucocorticoid-Remediable Aldosteronism. In glucocorticoid- limb, where approximately 30% of the filtered salt is reab-
remediable aldosteronism, aldosterone and the weak min- sorbed. Gitelman’s syndrome presents with milder salt wasting
eralocorticoids 18-oxo-cortisol and 18-hydroxy-cortisol are due to abnormal salt transport in the distal convoluted tubule.
produced in excess (287). This is a single-gene disorder that At least three phenotypically different subgroups of patients
causes hypertension. It results from a relatively common auto- with Bartter’s and Gitelman’s syndromes can be identified. The
somal dominant mutation that creates a chimeric gene com- neonatal variant, previously called hyperprostaglandin E syn-
prised of 5' regulatory sequences of the 11β-hydroxylase gene drome, presents with dehydration, failure to thrive, dysmor-
fused to the 3' coding sequences of the gene encoding aldoster- phic facies, and a history of polyhydramnios and premature
one synthase, the rate-limiting enzyme for aldosterone synthe- delivery (291,292). There is significant Na+ wasting, polyuria,
sis. This ectopic aldosterone synthesis in the zona fasciculata is hypercalciuria, osteopenia, and nephrocalcinosis with normal
under the control of ACTH, rather than the renin-angiotensin serum magnesium levels. The pathophysiology is likely due to
system, and results in salt and water retention, hypertension, mutations in the gene encoding the bumetanide-sensitive Na-
and dramatic hypokalemia. The level of hypertension appears K-2Cl co-transporter in the thick ascending limb (293) or in
164 II. Homeostasis

the gene encoding the ATP-regulated K+ channel (ROMK1) tion when dietary intake of Na+ and K+ is adequate (295).
(291) in the thick ascending limb and cortical collecting duct. However, flooding of the distal nephron segments with exces-
The second type is classic Bartter’s syndrome, which pre- sive bicarbonate ion will exacerbate urinary K+ losses.
sents in childhood (younger than 5 years of age), often as Proximal or type II RTA, characterized by a depression
failure to thrive with signs of severe intravascular volume in the renal HCO3– threshold, may exist as an isolated
depletion. Compared to the neonatal form, urine calcium is proximal defect of HCO3– reabsorption or may be associ-
elevated less severely and nephrocalcinosis is generally absent ated with other proximal tubular transport abnormalities as
(260). The pathogenesis is generally a loss-of-function muta- a Fanconi syndrome, in which patients excrete large amounts
tion of the renal basolateral Cl– channel (ClC-Kb). of HCO3–, phosphate, amino acids, and glucose. Distal
The third type of hypokalemic metabolic alkalosis is deliveries of Na+ and the poorly reabsorbed anion HCO3–
known as Gitelman’s syndrome, which presents in childhood are high, which results in excessive K+ secretion. Secondary
or later and is not associated with salt craving or bouts of hyperaldosteronism due to Na+ wasting and volume deple-
dehydration. The presentation is much milder than Bartter’s tion further increases K+ secretion, as does treatment with
from a clinical and biochemical viewpoint. Gitelman’s syn- sodium bicarbonate. On the other hand, correction of the
drome is due to mutations in the thiazide-sensitive NaCl co- acidosis and hypokalemia can usually be accomplished by
transporter (NCCT or TSC), which is localized in the distal replacing some of the sodium bicarbonate with potassium
convoluted tubule (294). This subgroup comprises the Bart- citrate (296–298).
ter’s-type patients with hypomagnesemia and hypocalciuria.
Defective NaCl reabsorption in the distal convoluted tubule Diuretic Use. The most common cause of renal K+ wasting
results in increased delivery to the cortical collecting duct, is the administration of diuretics, particularly loop and thia-
with consequent mild volume contraction and aldosterone- zide diuretics. The major mechanisms for K+ wasting include
stimulated K+ and H+ secretion, which leads to a mild increased delivery of loop or distal tubular fluid to the corti-
hypokalemic metabolic alkalosis. The extent of volume con- cal collecting duct, where K+ secretion is flow dependent,
traction and K+ depletion appear to be less marked than in and secondary hyperaldosteronism resulting both from the
Bartter’s syndrome and are not sufficient to raise prostaglan- underlying disease and from the volume contraction induced
din E2 production. by the diuretics (298–300). In addition, the metabolic alka-
Treatment focuses on correcting the electrolyte abnormali- losis that accompanies the use of such diuretics is likely to
ties and preventing dehydration. Potassium chloride therapy is promote additional K+ secretion.
required to fully address the K+ imbalance and NaCl helps Diuretic-induced hypokalemia may be associated with an
manage the defect in Cl– reabsorption. Spironolactone is often increased incidence of arrhythmias (301,302) and should be
helpful. Indomethacin inhibits prostaglandin production and treated in patients with hypomagnesemia or cardiac disease,
decreases renin and aldosterone production; this results in less especially during digitalis therapy (303). Treatment is recom-
renal K+ wasting but can have adverse long-term effects on renal mended with KCl supplements or K+-sparing diuretics (such
function. Despite treatment, patients with Bartter’s syndrome as spironolactone or triamterene); the latter will also partially
often have persistent hypokalemia. Patients with Gitelman’s correct diuretic-induced magnesium depletion by diminishing
syndrome need magnesium chloride supplements but rarely magnesium excretion (304).
need volume resuscitation or prostaglandin synthase inhibition.
Excessive Renin Production. Activation of the renin-angio-
Renal Tubular Acidosis. The mechanisms responsible for tensin system provides a major stimulus for adrenal aldos-
hypokalemia and renal K+ wasting differ for proximal (type II) terone synthesis with the development of hypertension and
and distal (type I) renal tubular acidosis (RTA). Despite the K+ hypokalemia. Such findings may be observed in patients
depletion, however, the plasma K+ concentration may be nor- with poor oral intake, diuretic use, vomiting or diarrhea,
mal because the acidemia promotes K+ movement out of cells. renal vascular disease, malignant hypertension, and renin-
Failure of intercalated cells in the distal nephron to secrete producing tumors, including hemangiopericytomas and
protons into the urinary fluid results in distal RTA. The defect Wilms’ tumor (298).
in H+ secretion increases tubular electronegativity, thereby Secondary hyperaldosteronism in edema-producing states
enhancing K+ secretion and limiting Na+ reabsorption in the does not usually result in hypokalemia (305). Edematous
cortical collecting duct. Volume depletion and consequent sec- patients do not experience increased urinary K+ losses or
ondary hyperaldosteronism further aggravate urinary K+ losses. become hypokalemic when administered Na+ loads to ensure
Hypokalemic variants of distal RTA may arise from abnormal- adequate distal delivery and flow (306). Thus, the development
ities of the luminal H+-K+-ATPase, which mediates H+ secre- of significant K+ depletion in the presence of Na+ retention
tion and K+ reabsorption in the distal nephron, or a defect in (nephrotic syndrome, cirrhosis, congestive heart failure) sug-
the membrane permeability of the distal nephron. Treatment gests that additional kaliuretic factors are involved; for example,
with alkali to correct the acidosis and volume depletion may the administration of potent diuretics or the presence of meta-
diminish urinary K+ losses and normalize serum K+ concentra- bolic alkalosis.
 8. Potassium 165

Other Conditions Associated with Increased Distal Nephron monly found in hypomagnesemic patients due to decreased
Flow. Conditions associated with increased flow of water and secretion of parathyroid hormone and skeletal resistance to its
salt to the distal nephron may result in hypokalemia. As effect. Indeed, the combination of hypokalemia plus hypocal-
described for diuretic use and Bartter’s and Gitelman’s syn- cemia is highly suggestive of underlying magnesium depletion
dromes, the initial renal Na+ wasting leads to a secondary rise (316). Correction of the hypokalemia frequently requires the
in aldosterone release, which further contributes to the ten- restoration of magnesium balance (314–316).
dency to hypokalemia. A variety of renal diseases, including Other conditions have been associated with hypokale-
chronic interstitial nephritis, urinary tract obstruction, and dys- mia, including vitamin D intoxication, thyrotoxicosis, lith-
plasia, can cause hypokalemia by these mechanisms (271). ium toxicity, primary polydipsia, and cardiopulmonary bypass
Similarly, hypercalcemia is frequently associated with a urinary (298). Hypokalemia can also follow transfusion of previ-
concentrating defect and high distal flow, because calcium- ously frozen washed erythrocytes, which reflects avid
induced tubular damage may impair Na+ reabsorption (307). uptake of K+ by these K+-depleted red cells (317).
Hypokalemia and urinary K+ wasting may follow
administration of various antibiotics, including high-dose Increased Gastrointestinal Losses
penicillin and penicillin analogues, such as carbenicillin and Gastric Loss. Vomiting and nasogastric suction often lead
nafcillin. The penicillins behave as nonreabsorbable anions, to hypokalemia despite the fact that gastric secretions con-
enhancing transepithelial electronegativity in the distal tain only 10 to 20 mEq/L of K+ (318). The major site of K+
nephron, and are often administered as sodium salts, so loss is, in fact, the kidney. Volume contraction after pro-
that they enhance distal Na+ delivery and thus K+ secretion longed vomiting or nasogastric suction and consequent sec-
(298). Hypokalemia associated with prolonged or high- ondary hyperaldosteronism are primarily responsible for
dose administration of aminoglycosides (e.g., gentamicin) increased distal K+ and H+ losses. The metabolic alkalosis
has been attributed to development of secondary hyperal- that accompanies significant vomiting causes an increase in
dosteronism and drug-induced renal magnesium wasting both distal tubular cell K+ concentration and distal delivery
(308,309). Polyene antibiotics, such as amphotericin B, fre- of HCO3–, a nonreabsorbable anion; these two factors, in
quently induce renal K+ wasting by increasing K+ perme- addition to secondary hyperaldosteronism, stimulate uri-
ability of the luminal membrane and by causing a distal nary K+ excretion.
acidification defect (310).
Renal K+ wasting and hypokalemia can be seen in Large Intestine Loss. Fecal excretion of K+ usually amounts
patients with various forms of leukemia. The elevated uri- to between 5 and 15 mEq/day. Because gastrointestinal flu-
nary excretion of lysozyme (a low-molecular-weight protein ids (e.g., from stomach, ileum, cecum) contain approxi-
present in leukocytes), which is characteristic of these leu- mately 10 mEq/L of K+ and because colonic mucus is rich
kemias, may be toxic to the proximal tubule (311,312). in K+ (100 to 140 mEq/L) (319), diarrhea can induce sig-
The hypokalemia observed in childhood leukemia (acute nificant K+ loss. In cholera, daily stool losses may average 8
lymphocytic leukemia, acute myelogenous leukemia) and L of water, 1000 mEq of Na+, and 130 mEq of K+ (320).
other adult leukemias (chronic myelogenous leukemia, Comparable losses may occur in patients with VIPoma syn-
chronic lymphocytic leukemia) is probably multifactorial drome, a severe, watery diarrhea and histamine-fast achlor-
in origin and may be attributed to causes such as reduced hydria that is usually but not always due to a vasoactive
K+ intake, antibiotic-induced renal K+ wasting, the miner- intestinal peptide–producing non–β-cell islet cell tumor
alocorticoid effect of high-dose steroid therapy, increased (321). When volume contraction ensues, secondary hyper-
K+ entry into metabolically active leukemic cells, and the aldosteronism further stimulates renal and colonic K+ secre-
osmotic diuresis (and nephrotoxicity) encountered in the tion (322).
administration of some chemotherapeutic agents (e.g., cis- A similar picture may be seen early in the course of con-
platinum) (271,298,313). genital chloride diarrhea, a rare autosomal recessive condi-
tion characterized by mutations in the downregulated
Miscellaneous Factors. Hypomagnesemia is a relatively com- adenoma gene (DRA) that codes for an intestinal Cl–-
mon finding in hypokalemic patients (314,315). Hypo- HCO3– exchanger in the terminal ileum and colon (323).
magnesemia can lead to K+ depletion (314,316). The Infants with the disorder have distended abdomens and
mechanism for the increased kaliuresis is not clear, but it may absence of meconium, and fail to thrive. They develop met-
reflect decreased reabsorption of K+ in the loop of Henle and abolic alkalosis, dehydration, secondary hyperaldosteronism,
possibly the cortical collecting duct. Magnesium deficiency and severe electrolyte disturbances, including hypochlore-
may result in loss of cell K+, either by impairment of Na+-K+- mia, hyponatremia, and hypokalemia. Fecal Cl– concentra-
ATPase activity or alteration of cell membrane permeability to tion always exceeds 90 mEq/L in patients with corrected
K+. Magnesium depletion may enhance renin and thus aldos- electrolyte balance (324).
terone secretion, with the resulting hyperaldosteronism in turn Severe diarrhea and dehydration (10 to 15%) in children
promoting urinary K+ excretion. Hypocalcemia is also com- can incur losses of between 7 and 14 mEq of K+ per kilo-
166 II. Homeostasis

gram of body weight (325). Evaluation of such patients exchange for the lost K+ ions. Hypokalemia results in a high
usually reveals a hyperchloremic metabolic acidosis, despite intracellular to extracellular concentration ratio for K+,
the secondary hyperaldosteronism, due to the marked bicar- which induces a state of electrical hyperpolarization and
bonate and organic anion (potential alkali) losses in diar- thereby slows nerve impulse conduction and muscle con-
rheal fluid. The metabolic acidosis may induce K+ to shift traction. Neuromuscular dysfunction is most commonly
from the cells to the ECF, which thereby masks total body manifest as skeletal muscle weakness. Usually the muscles
K+ depletion. of the lower extremities are affected first, followed by the
Profound large intestine losses of K+ are commonly quadriceps, then those of the trunk and upper extremities,
observed in patients with villous adenomas of the colon and and ultimately of respiration (271,301).
rectum (326). Ureterosigmoidostomies, surgical diversions Potassium deficiency can also reduce skeletal muscle
performed to relieve chronic urinary tract obstruction, are blood flow. During exercise and muscular contraction,
typically associated with a hypokalemic hyperchloremic met- muscle cells normally release K+ to the interstitial fluid. The
abolic acidosis arising from colonic secretion of K+ and increasing concentration of interstitial K+ is believed to act
HCO3– in exchange for Na+ and Cl–. This complication can as a vasodilator (329). It has been suggested that severe K+
be mitigated by implantation of the ureter into the ileum. depletion (plasma K+ concentration lower than 2 mEq/L)
Those who habitually abuse laxatives frequently develop leads to failure of the vasodilatory response to exercise,
marked K+ depletion (298). These patients, typically overly which results in muscle ischemia with subsequent muscle
concerned with body image, may also abuse diuretics. With cramps, rhabdomyolysis, and myoglobinuria (330).
habitual use of either laxatives or diuretics, Na+ depletion, Hypokalemia-induced smooth muscle dysfunction is usu-
volume contraction, secondary aldosteronism, and metabolic ally manifest as paralytic ileus with abdominal distention,
alkalosis may develop. Thus, these patients may present with anorexia, nausea, vomiting, and constipation. Impaired pres-
a picture mimicking Bartter’s syndrome. The habitual laxa- sor responsiveness to angiotensin and catecholamines may
tive abuser who has not recently taken diuretics usually lead to postural hypotension (331).
shows a urinary Cl– concentration below 25 mEq/L.
Cardiac
Excessive Integumental Losses Hypokalemia contributes to cardiovascular morbidity and
The concentration of K+ in sweat is approximately 5 to 10 mortality by its effect on cardiac conduction and arterial
mEq/L (327). Vigorous exercise or heat stress usually results in blood pressure. The electrical disturbances of conduction
a significant increase in the volume of sweat and its Na+ con- and rhythm, manifest on the electrocardiogram as depres-
centration (though rarely exceeding 60 mEq/L), which leads sion of the ST segment, diminished T-wave voltage, and
to salt losses and volume contraction. The secondary hyperal- appearance of U waves, are generally observed at plasma K+
dosteronism that ensues results in augmented K+ secretion into concentrations below 3 mEq/L (Fig. 8.10). Premature atrial
both exocrine gland products (327) and urine, and thereby and ventricular contractions and atrial arrhythmias may be
produces additional K+ deficits. Excessive loss of sweat and its seen. In patients who have congestive heart failure, cardiac
salts in patients with cystic fibrosis may lead to hypokalemia by ischemia, or left ventricular hypertrophy, or who are taking
similar mechanisms (328), with patients occasionally present- digoxin therapy, hypokalemia significantly increases the
ing with a picture resembling Bartter’s syndrome. risk of cardiac arrhythmias (332).
Patients with full-thickness burns who develop volume A diet that is low in K+, especially in the presence of
contraction and consequent secondary hyperaldosteronism high salt intake, has been implicated in the genesis of high
from massive integumental fluid losses often demonstrate blood pressure (333). The mechanism for hypokalemia-
an enhanced kaliuresis by 48 hours (298). induced hypertension may relate to intravascular volume
expansion as a result of renal Na+ retention (334). The asso-
ciation between hypokalemia and hypertension is marked
Clinical Manifestations of Hypokalemia
in African Americans, a population in whom a reduction in
An acute fall in plasma K+ concentration generally results in Na+ intake and supplementation with K+ may be particu-
a more severe manifestation than does a chronic K+ loss, larly beneficial.
which is often well tolerated.
Metabolic
Neuromuscular Potassium depletion interferes with both carbohydrate and
Hypokalemia can induce skeletal muscle weakness and, in protein metabolism. Potassium deficiency suppresses pancre-
severe cases, paralysis (301). Excitable tissues require a spe- atic insulin release, which results in increased glucose intoler-
cific transmembrane K+ gradient for optimal function. ance (335). However, a 10% reduction in body K+
Briefly, depolarization of excitable cells is characterized by (corresponding roughly to a decrease in plasma K+ concen-
the rapid entry of Na+ ions and exit of K+ ions. Repolariza- tration of 1 mEq/L) in healthy subjects results in only a mild
tion of the membrane involves extrusion of the Na+ ions in impairment as measured by the glucose tolerance test, which
 8. Potassium 167

The most common renal functional abnormality associated

with K+ depletion is impaired urinary concentration, a deficit
arising from a reduced osmolar gradient in the medullary
interstitium (339). This concentrating defect is resistant to the
administration of exogenous vasopressin and prolonged water
deprivation (337). Hypokalemia inhibits activation of renal
adenylate cyclase, which prevents vasopressin-stimulated uri-
nary concentration in the inner medullary collecting duct. In
addition to this direct tubular effect, K+ depletion stimulates
the central thirst center via increased production of angioten-
sin II (340). Enhanced proximal tubular Na+ reabsorption
(341) in the face of K+ depletion leads to Na+ retention, expan-
sion of the ECF, and even frank edema (301).
Potassium depletion would be expected to result in increased
net acid excretion because of enhanced cellular uptake of H+ as
a substitute for K+; this leads to intracellular acidosis (and alkale-
mia) and stimulation of both H+ secretion and renal ammonia
production by renal tubular cells (342). Indeed, moderate K+
depletion induced by restriction of dietary K+ (substituting with
Na+ without changing dietary anion content) results in a mild
systemic metabolic alkalosis unaccompanied by chloride deple-
tion or hyperaldosteronism (343). The metabolic alkalosis is
likely due to enhanced proximal reabsorption of HCO3–, medi-
ated by an increase in the activity of the luminal Na+-H+
FIGURE 8.10. Relationship between plasma K+ concentration and exchanger and basolateral Na+-HCO3– co-transporter. This is
electrocardiographic changes. (From Winters RW. The body fluids manifest by an elevated plasma HCO3– concentration and is
in pediatrics. Boston: Little, Brown, 1973:134, with permission.)
associated with the paradoxical excretion of an acid urine, possi-
bly via the stimulation of H+-K+-ATPase isoforms.
suggests that more severe glucose intolerance in the presence
of hypokalemia is associated with an underlying disorder, for
Approach to the Patient with Hypokalemia
example, subclinical diabetes. In diabetic patients hypokale-
mia impairs both insulin release and end-organ sensitivity to Diagnosis
insulin, which aggravates the hyperglycemia. The evaluation of a patient with hypokalemia should begin
Growth failure is commonly observed with chronic K+ with taking a detailed history that includes the pattern of
depletion (298). Although the mechanism is unclear, it childhood growth, occurrence of chronic illness, family history
may be due in part to a disturbance in protein metabolism. of similar disease, use of medications and special diets, and
Because K+ is the predominant intracellular cation, body K+ symptoms associated with the present electrolyte disturbance.
depletion results in enhanced cellular uptake of other posi- Physical examination must include measurement of growth
tively charged cations, specifically Na+, H+, and cationic indices and blood pressure and assessment of any evidence of
amino acids (301). Thus, K+ depletion results in an increase edema and altered neuromuscular function.
in the ratio of ECF volume to total body water volume, a The initial laboratory evaluation should include measure-
decrease in the ratio of total body K+ to body water, and a ment of serum electrolytes and acid-base status and a complete
decrease in body noncollagen protein (8). blood count. These routine laboratory studies will identify
whether or not the hypokalemia is due to K+ uptake by abnor-
Renal mal leukocytes or is associated with the redistribution of K+
Potassium depletion, regardless of cause, produces a charac- from the extra- to the intracellular space. If the laboratory data
teristic picture of structural and functional abnormalities in do not support either of these two possibilities, the hypokale-
the kidney, known collectively as potassium-depletion neph- mia likely represents total body K+ depletion due to K+ losses
ropathy (301). One function that is preserved is the ability to via the gastrointestinal tract, skin, or kidney.
conserve K+ (336), mediated by decreased K+ secretion as To evaluate these possibilities, a urine collection (preferably
well as increased K+ absorption via the H+-K+-ATPase in the timed) on a controlled Na+ and K+ intake of 2 to 3 mEq/kg/
collecting duct (111,124). A specific reversible vacuolar day for each cation should be obtained to yield information on
lesion of proximal tubular cells has been described in humans renal function and renal K+ handling (urinary Na+/K+ ratio,
(336,337). Interstitial fibrosis, tubular atrophy, and cyst for- absolute and fractional urinary K+ excretion). A low urinary K+
mation in the medulla also may be seen (338). concentration (less than 15 mEq/L) in the absence of recent
168 II. Homeostasis

diuretic use implies near-maximal urinary K+ conservation, TABLE 8.6. ORAL POTASSIUM PREPARATIONS
which suggests that the K+ deficit is secondary to extrarenal Potassium chloride (40 mEq K = 3 g KCl)
losses (gastrointestinal system, skin) or inadequate intake. The Solution 10% (6.7 mEq/5 mL)
measurement of total body K+ is not readily available to the cli- 15% (10 mEq/5 mL)
nician. Gastrointestinal K+ losses occur from either vomiting, 20% (13.3 mEq/5 mL)
diarrhea, fistulas, nasogastric suction, or a villous adenoma. Powder 15, 20, 25 mEq/packet
Sustained-release tabs 6, 7, 8, 10, 20 mEq/tablet
Laxative abuse should be considered in patients overly con- Sustained-release cap- 8, 10 mEq/capsule
cerned with body image. sules
A child with a high rate of urinary K+ excretion (greater Potassium gluconate (40 mEq K = 9.4 g K gluconate)
than 15 mEq/L or exceeding intake by 0.5 to 1.0 mEq/kg/day, Elixir 20 mEq/15 mL
a fractional urinary K+ excretion exceeding 30%, or a urinary Tablets 500 mg (2.15 mEq)
Na+/K+ ratio consistently less than 1 in the absence of renal 595 mg (2.56 mEq)
Potassium citrate
failure) has renal K+ wasting. Inappropriate urinary K+ wasting
Liquid Na+ K+ Citrate (HCO3–) each
is often associated with hypertension and/or abnormalities of given as mEq/mL
acid-base status. Hypokalemia associated with hypertension Polycitra 1 1 2
and metabolic alkalosis is classically found in hyperreninemic Polycitra-K 0 2 2
states, such as renal vascular stenosis, and primary hyperaldos- Bicitra 1 0 1
teronism. The finding of hypokalemia and metabolic acidosis Oracit 1 0 1
Extended-release tablets 5 mEq/tablet Urocit-K (wax matrix)
in a normotensive patient should suggest RTA or diabetic 10 mEq/tablet Urocit-K (wax matrix)
ketoacidosis. Bartter’s and Gitelman’s syndromes should be
considered in patients with hypokalemic metabolic alkalosis, Data from Gunn VL, Nechyba C. The Harriet Lane handbook: a man-
chronic salt wasting with volume depletion, and hyperrenine- ual for pediatric house officers, 16th ed. Philadelphia: Mosby, 2002.
mia and hyperaldosteronism.

Therapy dosis, more aggressive K+ replacement may be appropriate.

The choice of K+ replacement therapy depends primarily on Under these circumstances, adults have been shown to tolerate
the magnitude of the K+ deficit. Treatment of the deficit, par- K+ administered in concentrations of 40 to 100 mEq/L and
ticularly in the presence of complicating factors that affect the infused at a rate not to exceed 40 mEq/hr, while serial electro-
transcellular distribution of K+, must be performed cautiously, cardiograms and frequent measurements of plasma K+ concen-
especially in patients with renal or cardiac disease. tration are monitored (334,346).
Mild hypokalemia will often respond simply to dietary sup- In patients with diuretic-induced hypokalemia who require
plementation with foods having a high K+ content (Table 8.5). continued use of their diuretic, addition of a potassium-
Oral K+ supplementation is necessary for moderate K+ deple- sparing diuretic such as amiloride, triamterene, or spironolac-
tion (Table 8.6). Among the oral preparations available in liq- tone should be considered. Hypomagnesemia can lead to renal
uid, powder, and slow-release preparations are potassium K+ wasting and refractoriness to K+ replacement. Magnesium
chloride, potassium citrate, and potassium gluconate. Potas- repletion facilitates correction of the coexisting K+ deficit.
sium chloride is most commonly used; the citrate and gluco-
nate formulations are ideal for patients with concomitant
Hyperkalemia
acidosis in whom the organic anion provides potential alkali.
The adverse effects associated with oral therapy, especially after Hyperkalemia is defined as a serum K+ concentration higher
administration of KCl, include gastrointestinal irritation, rang- than 6.0 mEq/L in the newborn and 5.5 mEq/L in the older
ing from vomiting to ulceration, and K+ intoxication. child and adult. Hyperkalemia is an uncommon finding in
Intravenous K+ supplementation therapy should be reserved healthy subjects, because cellular and renal adaptations pre-
for patients with severe hypokalemia, including those patients vent significant accumulation of K+ in the ECF. Because the
demonstrating neuromuscular or cardiac disturbances, those kidney provides the primary means of eliminating excess K+,
receiving amphotericin B, and diabetic patients in ketoacidosis. hyperkalemia is frequently observed with major disturbances
Administration of K+ in dextrose-containing solutions may of renal excretory function. A classification of disorders asso-
worsen the hypokalemia, because the sugar stimulates insulin ciated with hyperkalemia is presented in Table 8.7.
secretion, which results in movement of K+ into cells. Conven-
tionally, parenteral K+ should be infused in a solution contain-
Clinical Disorders Associated with Hyperkalemia
ing no more than 40 mEq/L and infused at a rate not to exceed
0.5 to 1.0 mEq/kg of body weight per hour in children Pseudohyperkalemia
(298,344) or 10 to 20 mEq/hr in adults (334,345). When life- Pseudohyperkalemia represents an in vitro elevation of serum
threatening paralysis or ventricular arrhythmias are present, or K+ concentration in the absence of any clinical evidence of
there are excessive ongoing losses such as with diabetic ketoaci- hyperkalemia and is attributable to K+ movement out of cells
 8. Potassium 169

during or after the drawing of the blood specimen. The serum for every 0.1 unit reduction in arterial blood pH (53). In
K+ concentration is normally up to 0.5 mEq/L higher than the contrast, organic acids such as lactic acid, β-hydroxybu-
plasma value due to release of K+ by platelets and leukocytes tyric acid, and methylmalonic acid and respiratory aci-
during the in vitro clotting process. Potassium leakage by red dosis usually provoke a lesser rise in the plasma K+
blood cells, coincident with in vitro hemolysis, is readily concentration (352).
demonstrable because of simultaneous release of hemoglobin
into the plasma or serum. Hematologic disease associated with Hormones. Cellular uptake of K+ is influenced by several
marked leukocytosis, thrombocytosis [especially with hormones, including insulin, catecholamines, and mineralo-
Kawasaki disease (347)], or familial disorders of erythrocyte corticoids. Impairment of hormonally mediated uptake pro-
membrane permeability (348,349) can lead to increases in cesses may result in hyperkalemia. Thus, hyperkalemia may
serum, but not plasma, K+ concentration. Inappropriate blood complicate insulin deficiency (353), exercise during treatment
drawing can raise the apparent blood or plasma K+ concentra- with beta-blockers such as propranolol and metoprolol (354),
tion by more than 2 mEq/L (350,351). and hypoaldosteronism, either hyporeninemic or that which
follows administration of angiotensin-converting enzyme
Transcellular Shift of K+ from Cells to the Extracellular Fluid (ACE) inhibitors (355) or angiotensin receptor antagonists.
Metabolic Acidosis. The relationship between acid-base
balance and transcellular distribution of K+ predicts that Severe Exercise. Potassium is normally released from mus-
acute metabolic acidosis will often be accompanied by an cle cells during exercise (51), and this local release of K+
increase in plasma K+ concentration as excess protons plays a vasodilatory role to maintain enhanced blood flow
enter the cell to be buffered intracellularly. Metabolic aci- to the exercising muscle (60,356). With more severe exer-
dosis due to mineral acids such as hydrochloric acid or cise and reduction of ATP levels in the cells, the ATP-
ammonium chloride is generally accompanied by an dependent K+ channels open, so that K+ is released from
increase in plasma K+ concentration of up to 1.5 mEq/L muscle cells.

TABLE 8.7. CLINICAL DISORDERS ASSOCIATED WITH HYPERKALEMIA

Pseudohyperkalemia Decreased renal excretory capacity
Hematologic disorders: leukocytosis, thrombocytosis, test tube Renal failure
hemolysis Acute
Improper collection of blood Chronic
Transcellular shift of K+ from cells to the extracellular fluid Effective circulating volume depletion
Metabolic acidosis Hypoaldosteronism
Impaired hormone activity Hyporeninemia
Insulin deficiency Congenital adrenal enzymatic defects
Exercise (beta blockade) 21-hydroxylase defects
Severe exercise Aldosterone synthase defects
Hyperosmolality Primary adrenal insufficiency
Increased tissue catabolism Impaired renal tubular secretion without abnormalities in miner-
Drug effects alocorticoid production
Succinylcholine Pseudohypoaldosteronism type I
Digoxin (overdose) Mutations in mineralocorticoid receptor (consider new
β-adrenergic blockers and α-adrenergic agonists pseudohypoaldosteronism type II)
Arginine hydrochloride Familial hyperkalemia with hypertension (originally pseudohy-
Familial hyperkalemic periodic paralysis poaldosteronism type II)
Increased potassium load Includes Spitzer-Weinstein and Gordon’s syndromes
Exogenous Acquired forms with normal mineralocorticoid production
Oral or parenteral potassium supplements Sickle cell disease
Salt substitutes Renal transplantation
Potassium penicillin in high doses Systemic lupus erythematosus
Blood transfusion Lead nephropathy
Addition of KCl to parenteral fluid bags with poor mixing Papillary necrosis
Endogenous Obstructive and reflux uropathy
Intravascular hemolysis Distal renal tubular acidosis (voltage-dependent defect)
Rhabdomyolysis Drug effects
Exercise Potassium-sparing diuretics
Infection Prostaglandin synthesis inhibitors
Trauma Angiotensin-converting enzyme inhibitors
Burns Miscellaneous (trimethoprim-sulfamethoxazole, pentami-
Tumor lysis dine, heparin, cyclosporine A, tacrolimus)
Severe starvation
170 II. Homeostasis

Hyperosmolality. Hyperosmotic infusion of mannitol or hyperkalemia with the attacks, differ from those of the
saline, or acute hyperglycemia may lead to an acute increase entity hypokalemic periodic paralysis. Attacks begin at a
in plasma K+ concentration, as K+ leaks from cells younger age (first or second decade of life) and are more
(63,65,67). With the rise in osmolality, there is a fluid shift frequent and of shorter duration (usually less than 2 hours)
from intracellular to extracellular space and a resulting than those of the hypokalemic disorder.
increase in intracellular K+ concentration. K+ accompanies The disease is associated with a point mutation of the
the fluid shift out of the cells via solvent drag. The higher gene encoding the α subunit of the voltage-gated Na+
cellular K+ gradient stimulates K+ efflux via K+ channels in channel of human skeletal muscle (SkM1), which results in
the membrane. a single amino acid substitution of a highly conserved resi-
due (369,370). The mutation leads to increased activity of
Increased Tissue Catabolism. When an increased rate of the Na+ channel (371) or disruption of fast inactivation of
tissue breakdown occurs, large amounts of K+ may be these channels after the action potential; this allows Na+
released into the ECF to cause hyperkalemia. Clinical entry into the cells, which depolarizes the membrane and
examples of hypercatabolism include the use of cytotoxic favors K+ diffusion out of the cells with the development of
drugs causing tumor lysis syndrome (61), extensive trauma hyperkalemia.
(357), and massive hemolysis (358). Attacks can be aborted by treatment with β-adrenergic
agonists such as albuterol, which drive K+ back into cells
Drug Effects. Certain drugs alter the normal distribution (372). Other long-term treatment modalities include lim-
of K+ in the body. Depolarizing muscle relaxants used in iting exercise, consuming a low-K+, high-carbohydrate
general anesthesia such as succinylcholine cause release of diet, inducing mild K+ depletion with a thiazide diuretic
K+ from muscle during cellular depolarization (359). In or mineralocorticoid, and adding a carbonic anhydrase
healthy subjects the increment in plasma K+ concentra- inhibitor (373).
tion is small, but in patients with neuromuscular disease,
trauma, burns, tetanus, or renal insufficiency succinyl- Increased K+ Load
choline can induce an increase in plasma K+ concentra- Exogenous K+ Load. The healthy individual is easily able
tion of as much as 6 mEq/L (360–362). Patients who to tolerate an acute K+ load due to both early cellular
have taken an overdose of digoxin, a drug that inhibits uptake, the predominant means of short-term disposal of
cellular Na+-K+-ATPase so that intracellular K+ leaks K+, and, later, renal excretion (22). However, oral K+ loads
from cells, can also acutely develop marked hyperkale- exceeding 160 mEq in adults can cause fatal increases in
mia (363–365). plasma K+ concentrations to above 8 mEq/L, even in
Nonspecific β-adrenergic blockade and α-adrenergic stim- patients with normal renal function (374). Severe hyper-
ulation impair extrarenal disposal of K+. Although beta- kalemia is more likely to occur with a rapid intravenous
blockers normally produce only a small increment in infusion or in infants, because of their small size (373).
plasma K+ concentration, they should be used with caution Infants, as well as adults, have become hyperkalemic after
in patients with renal insufficiency (366). Commonly used the administration of potassium penicillin in an intrave-
α-adrenergic agonists such as phenylephrine can also con- nous bolus (1.7 mEq K+ per 1 million U) (375,376) or
tribute to hyperkalemia. A relatively pure β1-selective adren- after the use of stored blood (more than 5 days old) in
ergic blocker (metoprolol, atenolol) is safer in these settings exchange transfusions (377).
(367). Most salt substitutes contain significant amounts of K+
Arginine hydrochloride may cause hyperkalemia as the (10 to 13 mEq/g or 50 to 65 mEq per level teaspoon)
cationic amino acid enters cells and displaces intracell- (378) and can cause severe hyperkalemia in infants (379)
ular K+ (368). Severe hyperkalemia has been reported or those who concurrently use ACE inhibitors. Many
in patients with renal and hepatic failure; patients in enteral nutrition products, including Boost and Ultracal
the latter group are unable to metabolize the arginine (Mead Johnson), Ensure Plus (Nestle), Promote (Ross)
(352). and Scandishake (Scandipharm) contain K+ in concentra-
tions exceeding 40 mEq/L. Intravenous KCl preparations
Hyperkalemic Periodic Paralysis. Hyperkalemic periodic added to parenteral fluid bags without adequate mixing
paralysis is a rare familial disorder with autosomal domi- may layer out at the injection port, so that the initial solu-
nant inheritance characterized by recurrent episodes of tion administered to the patient has a markedly elevated
muscle weakness that occur in association with hyperkale- K+ concentration (380,381).
mia (369). Episodes are precipitated by rest after strenuous
exercise, exposure to cold or glucocorticoids, or ingestion of Endogenous K+ Load. Massive cellular breakdown with
small amounts (0.5 to 1 mEq/kg) of exogenous K+ (266), endogenous K+ release after intravascular coagulation, sickle
and are often aborted by carbohydrate feeding. Several cell hemolytic crisis, rhabdomyolysis, trama, burns, massive
characteristics of this disorder, aside from the association of gastrointestinal bleeding, tumor lysis syndromes associated
 8. Potassium 171

with chemotherapeutic destruction of lymphoproliferative patient with chronic renal failure suggests the presence
malignancies (e.g., lymphoma), and, less frequently, solid of an increased excretory burden, possibly due to inges-
tumors (e.g., neuroblastoma), as well as severe starvation, can tion of K+-rich foods and medications.
rapidly induce hyperkalemia (62,353). Acute renal failure is often accompanied by metabolic
acidosis secondary to accumulation of organic acids, con-
Decreased Renal Excretory Capacity comitant lactic acidosis, or ketoacidosis, factors that
Potassium excretion is normally so efficient that even a reduce cellular uptake of K+ and exacerbate hyperkalemia
massive long-term increase in K+ intake will not produce (53,397). Although most patients with nonoliguric renal
hyperkalemia in healthy subjects (382). Thus, for hyper- failure who maintain a reasonable urine output do not
kalemia to persist, urinary K+ excretory capacity must be develop hyperkalemia, plasma K+ concentrations exceed-
reduced. Three major causes of reduced urinary K+ excre- ing 7 mEq/L have been observed in very-low-birth-
tion include renal failure, decreased effective circulating weight neonates with nonoliguric renal failure (398). The
volume, and hypoaldosteronism. hyperkalemia that develops in these infants, provided no
exogenous K+ source exists, likely reflects a shift of K+
Renal Failure. Hyperkalemia is a routine complication from the intracellular to the extracellular space during
of acute renal failure. Not only does an acute decrease in the transition from fetal to postnatal life, as well as a lim-
glomerular filtration rate limit net urinary K+ losses, but ited urinary K+ excretory capacity (399).
also the diminished urinary flow rate in the distal neph-
ron, where K+ secretion is flow dependent, markedly Effective Circulating Volume Depletion. The ability to
impairs further K+ excretion (383,384). Hyperkalemia handle an acute K+ load is diminished in hypovolemia, in
may develop in nonoliguric patients because of an acute which there is usually a low glomerular filtration rate and
increase in K+ load requiring excretion, resulting from increased proximal Na+ and fluid reabsorption; this results
enhanced tissue breakdown, increased K+ intake (see in decreased distal fluid delivery and impaired K+ secre-
Table 8.5 for K+ content of various foods), hypoaldoster- tion, despite secondary hyperaldosteronism (86,400). Vol-
onism, or therapeutic maneuvers such as blood transfu- ume depletion from salt-losing nephropathy can also result
sion. Although the rate at which endogenous hyperkalemia in poor renal K+ excretion and hyperkalemia (401).
develops is variable, patients with uncomplicated abrupt
cessation of renal function are expected to demonstrate a Hypoaldosteronism. Mineralocorticoids stimulate K+
rise in plasma K+ concentration at a rate of approxi- secretion in both the colon and the kidney and so are of
mately 0.5 mEq/L/day (357,385). primary importance in maintaining K+ homeostasis.
In addition to decreased kaliuresis, K+ uptake by cells Deficiencies in mineralocorticoid production or end-
is impaired in chronic renal failure (386,387). Cells have organ responsiveness can thus result in hyperkalemia.
reduced K+ concentrations in the basal state as well as The most common causes are hyporeninemic hypoaldos-
after K+ loading (22,388). Total body K+ stores are nor- teronism and the use of K+-sparing diuretics in adults
mal or slightly reduced in stable chronic renal insuffi- and adrenal enzyme deficiencies in children (298,373).
ciency (389–391). The reduction in K+ content in In addition to hyperkalemia, varying degrees of Na+
leukocytes and muscle cells in uremic patients can be wasting and metabolic acidosis are present in hypoaldos-
normalized by hemodialysis, which suggests that accu- teronism (because aldosterone normally stimulates Na+
mulation of a uremic toxin inhibits cellular Na+-K+- reabsorption and H+ and K+ secretion; see earlier). The
ATPase activity and results in impaired K+ uptake into metabolic acidosis is a type IV RTA.
cells (392–394). Hyporeninemic hypoaldosteronism may account for
Plasma K+ concentration in chronic renal failure may more than half the cases of initially unexplained hyper-
be maintained within normal limits by both renal and kalemia in adults (402,403), after elimination of obvious
extrarenal adaptive mechanisms (170,395,396). Despite causes. Most patients are adults who have a mild to mod-
a marked decrease in glomerular filtration rate, renal erate impairment of renal function that complicates dia-
adaptation provides for a rapid increase in K+ excretion betic glomerulosclerosis, or other chronic renal disease such
per surviving nephron to the extent that K+ excretion as obstructive uropathy, sickle cell disease, tubulointersti-
may exceed filtered load in patients with a significant tial nephritis, or even glomerulonephritis (298,404).
reduction in functional renal mass (101,170,391). How- Typically, the hyperkalemia and hyperchloremic meta-
ever, the capacity of the surviving nephrons to excrete an bolic acidosis found in these patients is disproportionate
additional acute K+ load is generally impaired (391). to the degree of renal insufficiency (353,402). A similar
Extrarenal adaptive mechanisms include increased excre- clinical picture has been observed in hyporeninemic chil-
tion of K+ by the colonic mucosa and increased cellular dren, who often have primary renal interstitial disease
uptake due to high circulating levels of insulin and cate- (405). Because angiotensin II and K+ are the main physi-
cholamines. The finding of hyperkalemia in a stable ologic stimuli to aldosterone secretion, hyporeninemia is
172 II. Homeostasis

clearly an important cause of the hyperkalemia. The grow the need for therapy (414–417). The defect appears
mechanisms responsible may include a primary renal to be a mutation in any one of the three ENaC subunits
defect leading to decreased renin secretion, a primary leading to a loss of function (418,419). Although many
adrenal defect in aldosterone biosynthesis or release, or a patients outgrow the need for therapy by age 4 years
mixed disturbance (402,404). (298), the disorder has been reported in children as old as
Congenital adrenal enzymatic defects in mineralocorti- 7 years of age (420).
coid production lead to depressed aldosterone synthesis. Low-birth-weight infants may demonstrate a similar
Whereas certain of these disorders are accompanied by transient disorder with salt wasting and hyperkalemia,
an increased synthesis of other compounds with miner- abnormalities that usually resolve spontaneously by approx-
alocorticoid-like properties, such as DOC, other imately 6 weeks of age (421,422). The underlying disorder
enzyme deficiencies may not allow production of alter- appears to be a hyporesponsiveness of the immature distal
nate mineralocorticoid-like hormones. The most com- tubule and cortical collecting duct to aldosterone (421).
mon of the latter disorders is 21-hydroxylase deficiency, Unlike those with the ENaC mutations, these affected
characterized by diminished mineralocorticoid and glu- patients do not have pulmonary or other organ system
cocorticoid production with consequent salt wasting, involvement. In keeping with the hyporesponsiveness to
hyperkalemia, virilization, and failure to thrive (278, aldosterone, studies have identified in affected patients and
406,407). Children with a defect in aldosterone synthase kindreds mutations in the mineralocorticoid receptor gene
have isolated hypoaldosteronism (408–410). Infants (MLR) (423).
affected by this rare autosomal recessive disorder have Carbenoxolone, which inhibits 11β-HSD type II (the
salt wasting, recurrent dehydration, hyperkalemia and enzyme that converts cortisol to cortisone), can partially
failure to thrive (278,411). correct the apparent mineralocorticoid resistance in these
Treatment varies with the type of enzyme deficiency. patients (424). By slowing the conversion of cortisol to cor-
Mineralocorticoid replacement with fludrocortisone is suf- tisone, carbenoxolone raises the intracellular cortisol con-
ficient in patients with isolated hypoaldosteronism, whereas centration sufficiently to maintain high activation of the
those with 21-hydroxylase deficiency require replacement wild-type mineralocorticoid receptor and thus overcome
of both mineralocorticoid and glucocorticoid (as hydrocor- the functional defect in the mutant receptor in this autoso-
tisone or dexamethasone) (373). mal dominant disorder (284).
In primary adrenal insufficiency there is diminished glu- All index cases had presented with neonatal salt wast-
cocorticoid as well as mineralocorticoid secretion, but the ing with hyperkalemic acidosis despite high aldosterone
clinical picture is dominated by the complications of salt levels, and all improved with age, so that none was symp-
wasting, even though there may also be symptoms and tomatic by 10 years of age. This mild disease contrasts
signs of hyperkalemia (412). Severe hyperkalemia is more with the more severe form of pseudohypoaldosteronism
likely to occur in patients receiving inadequate mineralo- type I, which is mediated by mutations in ENaC genes,
corticoid replacement or in those with significant intravas- and might be considered as a new class of pseudohypoaldo-
cular volume depletion and reduced glomerular filtration steronism type II (284). A suggested nomenclature for
rate in whom low distal urinary flows further limit renal K+ these genetic disorders in which blood pressure and aldos-
excretion (413). terone secretion are inversely related is shown in Table
8.8. Similar improvement with age is seen in patients with
Impaired Tubular Secretion without Abnormalities in Min- congenital hypoaldosteronism due to aldosterone synthase
eralocorticoid Production. Pseudohypoaldosteronism type I deficiency, which probably reflects a reduced dependence
is an autosomal recessive disorder of end-organ resistance on aldosterone action with increasing age (425). Therapy
to aldosterone associated with renal salt wasting; elevated with Na+ supplementation has been shown usually to cor-
NaCl concentrations in sweat, stool, and saliva; hyper- rect the biochemical and clinical abnormalities, although
kalemia; and markedly elevated plasma renin activity and plasma aldosterone levels remain elevated. High-dose
aldosterone concentrations (414,415). These findings fludrocortisone may be beneficial if a high-salt diet is inef-
resemble the aldosterone resistance caused by K+-sparing fective or not tolerated (426).
diuretics, except that all aldosterone target organs are Patients with sickle cell disease, renal transplants, sys-
involved, including colon and sweat and salivary glands. temic lupus erythematosus, lead nephropathy, papillary
The disorder is especially marked in the neonatal period, necrosis, or reflux or obstructive uropathy may present with
with vomiting, hyponatremia, failure to thrive, and occa- a similar clinical picture that resembles hyporeninemic
sionally respiratory distress syndrome. These infants do hypoaldosteronism but is characterized by normal levels of
not respond to exogenous mineralocorticoids, but with aldosterone in the plasma (435,437,438).
vigorous salt replacement and control of hyperkalemia, Familial hyperkalemia with hypertension (formerly
they can survive. The severity of the disorder appears to pseudohypoaldosteronism type II) is manifest by a pri-
lessen with maturation, and most patients ultimately out- mary defect in renal distal nephron K+ secretion. Rather
 8. Potassium 173

TABLE 8.8. SUGGESTED NOMENCLATURE FOR DISORDERS OF POTASSIUM

HOMEOSTASIS IN WHICH BLOOD PRESSURE IS INVERSELY PROPORTIONAL
TO ALDOSTERONE SECRETION
Descriptor Inheritance Mutations Phenotype OMIM

Pseudoaldosteronism
PA1 AD γ and β ENaC subunits ↑ BP, ↓ K, ↓ pH 177200
PA2 AD S801L MR ↑ BP, ↓ K, ↓ pH 605115
Pseudohypoaldosteronism
PHA1 AR α, β, γ ENaC subunits ↑ K, ↑ pH, ↓ BP (neonatal) 264350
PHA2 AD MR ↑ K, ↑ pH, ↓ BP (mild) 177735

↑, increased; ↓, decreased; AD, autosomal dominant; AR, autosomal recessive; BP, blood pressure; ENaC,
epithelial sodium channel; K, plasma potassium concentration; MR, mineralocorticoid receptor; OMIM,
Online Mendelian Inheritance in Man; PA, pseudoaldosteronism; pH, plasma pH; PHA, pseudohypoal-
dosteronism; S801L, serine to leucine mutation at position 801 of the MR.
From Warnock DG. Renal genetic disorders related to K+ and Mg2+. Annu Rev Physiol 2002;64:845, with
permission.

than being volume depleted due to salt wasting, these kinases may serve to increase transcellular or paracellular
patients present with hyperkalemia, volume expansion– Cl– conductance in the collecting duct, thereby increas-
mediated hypertension, normal glomerular filtration ing salt reabsorption and intravascular volume, while
rate, low or low-normal plasma renin activity and aldos- concomitantly dissipating the electrical gradient and dis-
terone concentrations, metabolic acidosis, and short sipating K+ secretion. Another possibility is that the
stature; all have normal dietary K+ intake and Na+ han- mutant kinases stimulate constitutive activity of the Na-
dling (Spitzer-Weinstein syndrome) (427–430). This Cl co-transporter in the cortical collecting duct or a
syndrome is probably similar to Gordon’s syndrome and marked increase in the activity of this transporter in the
the chloride shunt syndrome (431,432). distal tubule (436).
Despite the hyperkalemia, the fractional excretion of
K+ (2.8%) is an order of magnitude less than in controls Distal Renal Tubular Acidosis—Hyperkalemic Form. Hypo-
studied similarly (427). Low-renin hypertension appears kalemia is generally observed with distal RTA, in part
to become more severe with aging in this autosomal because the decrease in H+ secretion requires that Na+ reab-
dominant disorder (433). The lack of Na+ wasting and a sorption occur in exchange for K+ secretion. However,
normal antinatriuretic response to exogenous mineralo- hyperkalemia may result when the underlying mechanism
corticoids indicate that the underlying abnormality is is a decrease in distal Na+ reabsorption (439); this is the
not simply resistance to aldosterone. Enhanced NaCl voltage-dependent defect. Hyperkalemic RTA most often
reabsorption by the distal tubule leads to both volume occurs in patients with obstructive uropathy (440) and
expansion and decreased distal Na+ and fluid delivery to sickle cell disease (441). Unlike in hyporeninemic hypoal-
the K+-secreting cells of the cortical collecting duct, dosteronism, which is also observed in these disorders, in
which thereby reduces K+ secretion and causes hyper- RTA the plasma aldosterone level is normal.
kalemia. Administration of a mineralocorticoid leads to
an appropriate reduction in Na+ excretion but does not Drug Effects. Distal nephron K+ secretion is inhibited by
appreciably affect that of K+. On the other hand, urinary the K+-sparing diuretics, which are used primarily to mitigate
K+ excretion increases dramatically after the infusion of diuretic-induced K+ depletion but are also administered to
Na+ with a nonchloride anion, such as sulfate or bicar- patients with cirrhosis. Spironolactone acts as a competitive
bonate (431). inhibitor of aldosterone. Triamterene and amiloride dimin-
Therapy consists of a high-Na+, low-K+ diet and usu- ish K+ secretion independently of mineralocorticoids by
ally a thiazide diuretic; the latter suggests that the primary directly closing the luminal Na+ channel in cells of the distal
defect in this disorder may be increased activity of the nephron and thereby decreasing the negative luminal volt-
thiazide-sensitive NaCl co-transporter in the luminal age. Severe hyperkalemia can develop in patients with com-
membrane of cells of the distal tubule and adjacent con- promised renal function and occasionally in patients with
necting segment (432,434,435). diabetes in whom cellular K+ uptake is impaired in the
Genetic studies of patients with this disorder (Online absence of insulin (442).
Mendelian Inheritance in Man No. 145260) have iden- Inhibitors of prostaglandin synthesis such as nonster-
tified probable gain-of-function missense mutations in oidal antiinflammatory drugs also cause hyperkalemia.
members of the WNK family of serine-threonine kinases Inhibition of prostaglandin synthesis produces iatrogenic
that are expressed in the distal nephron (436). These hyporeninemic hypoaldosteronism (443). Prostaglandins
174 II. Homeostasis

also inhibit NaCl reabsorption in the loop of Henle, oped acutely or is accompanied by other metabolic
thereby augmenting distal tubular delivery of Na+. derangements such as acidosis or hypocalcemia (373). The
ACE inhibitors such as captopril or enalapril increase characteristic electrocardiographic changes accompanying
plasma K+ levels by blocking the conversion of angiotensin hyperkalemia are depicted in Figure 8.10. The first and
I to angiotensin II, the latter of which is a potent vasocon- most specific abnormality seen is development of tall,
strictor and stimulator of aldosterone release. Patients on peaked T waves, which usually become apparent with
long-term ACE inhibitor therapy do not routinely develop plasma K+ levels above 6 mEq/L. The PR interval lengthens
clinically significant hyperkalemia unless their course is and the QRS complex then widens. At K+ concentrations
complicated by renal or adrenal insufficiency, congestive above 8 mEq/L, the P-wave amplitude decreases and may
heart failure with decreased renal perfusion, or administra- in fact disappear with atrial standstill. As ventricular con-
tion of nonsteroidal antiinflammatory drugs. duction time continues to lengthen, the QRS complex
Other drugs that are associated with hyperkalemia merges with the peaked T wave, producing a “sine-wave”
include trimethoprim-sulfamethoxazole, pentamidine, hep- pattern. Finally, ventricular fibrillation or asystole may
arin, and calcineurin inhibitors (cyclosporin A, tacrolimus). occur with K+ levels above 10 mEq/L.
High-dose trimethoprim therapy, commonly used for the
treatment of Pneumocystis carinii infection, frequently Neuromuscular
causes a significant increase in plasma K+ levels (by approx- Hyperkalemia is clinically associated with changes in neuro-
imately 1 mEq/L). Like amiloride, trimethoprim and pen- muscular conduction. Persistent depolarization inactivates Na+
tamidine cause hyperkalemia by blocking the luminal Na+ channels in the cell membrane, thereby producing a net
channel, which decreases luminal electronegativity and decrease in membrane excitability (373). This is manifested
thereby reduces K+ secretion (444–446). Heparin reversibly clinically by skeletal muscle weakness, paresthesias, and
suppresses adrenal aldosterone production and can thus be ascending flaccid paralysis. Such muscle weakness does not
associated with a natriuresis and reduced urinary excretion usually develop until the plasma K+ concentration exceeds 8
of K+ (447). Therefore, plasma K+ concentration should be mEq/L (452). Generally, the trunk and head muscles as well as
monitored in patients taking heparin for 3 or more days, muscles of respiration are spared.
particularly if these patients are at risk for development of
hyperkalemia due to complicating renal insufficiency, dia-
Approach to the Patient with Hyperkalemia
betes, or use of other K+-retaining drugs. Cyclosporine A
(448) and tacrolimus (449), immunosuppressive drugs rou- Diagnosis
tinely used by transplant recipients, have been associated The diagnostic workup of a patient with hyperkalemia
with hyperkalemia, even in patients with normal renal should begin with a complete history taking similar to that
function and no evidence of transplant rejection. described earlier for hypokalemia. The physical examina-
tion must include measurement of growth indices and
blood pressure, assessment of volume status, and evaluation
Clinical Manifestations of Hyperkalemia
for signs of chronic illness.
The clinical consequences of hyperkalemia are related to An electrocardiogram must be obtained promptly for
the adverse electrophysiologic effects of an altered trans- any patient with hyperkalemia. Because renal failure is a
membrane K+ gradient on excitable tissues. Specifically, an common cause of hyperkalemia, additional laboratory
increased extracellular K+ concentration reduces the resting evaluation should include a repeat set of plasma, not
membrane potential, initially enhancing but ultimately serum, electrolyte levels including glucose, calcium,
suppressing tissue excitability (“depolarizing block”). blood urea nitrogen, and creatinine. Note that sequen-
tial measurements of plasma K+ concentrations are
Cardiac expected to vary little from day to day. Increased K+ load
Cardiac toxicity generally develops when the plasma K+ due to increased tissue breakdown can be assessed from
concentration rises above 7 mEq/L, a concentration that the complete blood count and levels of uric acid, lactate
should immediately signal the need for initiation of specific dehydrogenase, and creatine phosphokinase enzymes.
treatment for hyperkalemia (450,451). Regardless of the Renal function can be further assessed with a urinalysis.
degree of hyperkalemia, however, treatment should be initi- An abnormal urinary specific gravity or pH may suggest
ated in any patient with suspected hyperkalemia if electro- a renal tubular defect, such as obstructive uropathy or
cardiographic abnormalities characteristic of hyperkalemia RTA, whereas the presence of casts or large amounts of
are noted. It is important to remember that the extracellu- protein or red blood cells is consistent with glomerular
lar K+ concentration demonstrates at best a rough correla- disease. Information regarding renal K+ conservation can
tion with the onset and degree of cardiotoxicity. For be obtained from a timed collection on a constant Na+
example, adverse effects may be seen with a plasma K+ con- and K+ intake (2 to 3 mEq/kg/day), which will allow the
centration of less than 7 mEq/L if hyperkalemia has devel- calculation of creatinine clearance, absolute K+ excretion
 8. Potassium 175

TABLE 8.9. PHARMACOLOGIC TREATMENT OF ACUTE HYPERKALEMIAa

Mechanism Duration
of action Drug Dosage Route of administration Onset of action of action

Antagonism Calcium gluconate (10%) 100 mg/kg/dose (= 1 mL/kg/dose) i.v. over 3–5 min 1–3 min 30 min
Redistribution Glucose (25%) (insulin) 0.5 g/kg (= 2 mL/kg) i.v. over 15–30 min 30 min 2–4 h
(regular insulin, 0.1 U/kg)
Albuterol (0.5%) 0.1–0.3 mg/kg for <12 yr Nebulized over 10 min Within 30 min 4–6 h
2.5–5.0 mg for >12 yr
Sodium bicarbonate 1–2 mEq/kg i.v. over 5–10 min 10–30 min 2h
Removal Furosemide 1–2 mg/kg i.v. or i.m. 15–30 min 4–6 h
Sodium polystyrene sul- 0.5–1.0 g/kg Orally or rectally 60–120 min 4–6 h
fonate resin

aIn the setting of acute or chronic renal failure or failure to remove adequate amount of potassium, dialytic removal may be necessary.

rate, fractional excretion of K+, urinary Na+/K+ ratio, particularly below 5, in a hyperkalemic patient is highly
and transtubular K+ gradient (TTKG) (see later). suggestive of hypoaldosteronism. Limited urine flow
Hyperkalemia is frequently due to a condition that causes (and distal Na+ delivery), rather than a limitation in the
impairment of urinary K+ excretion. The most common con- secretion of K+, may be seen in advanced congestive heart
ditions are advanced renal failure, severely reduced effective failure resulting in insufficient urinary K+ excretion.
volume depletion, and hypoaldosteronism (because aldoster- The TTKG is normally higher in infants (median
one is the primary hormone mediating urinary K+ excretion). 7.8) than in children (median 6.3) and correlates
If renal function is normal or only modestly impaired and no directly with the fractional excretion of K+ and urine
other causes are obvious, the patient should be evaluated for K+/Na+ ratios (455). In patients with hypoaldoster-
hypoaldosteronism. In infants and children, enzyme defi- onism and pseudohypoaldosteronism, TTKG values
ciencies and type I pseudohypoaldosteronism are most com- varied between 1.6 and 4.1, and all were below the third
mon. Salt wasting may be very severe in infants with percentile established for the age of the subject (455).
hypoaldosteronism, who typically present with volume The TTKG level is lower in preterm neonates than in
depletion, hyperkalemia, hyponatremia, metabolic acidosis, term infants (456), which probably reflects the immatu-
and an elevated urinary Na+ concentration. Ultimately, mea- rity of renal tubular K+ secretion or hyporesponsiveness
surement of morning plasma renin activity and aldosterone to aldosterone.
and cortisol levels should be performed. The patient should
be given 0.5 to 1 mg/kg of furosemide the night before the Therapy
blood specimens are obtained. This regimen enhances aldos- The presence of severe hyperkalemia (plasma K+ concen-
terone secretion (via stimulation of renin) in healthy subjects, tration above 8 mEq/L) with electrocardiographic
but not in patients with hypoaldosteronism (402). abnormalities or severe muscle weakness is a potentially
In addition to measuring plasma aldosterone level, one life-threatening event that requires immediate therapeu-
can estimate the degree of aldosterone activity by measur- tic intervention with almost all of the measures listed in
ing the tubular fluid K+ concentration at the end of the Table 8.9. Such treatment can prevent fatal cardiac
cortical collecting duct. In humans, this determination arrhythmias. Treatment measures used in this medical
can be estimated clinically from the calculation of the emergency can be divided into three general categories:
TTKG, on the assumption that (a) urine osmolality at the (a) those that rapidly antagonize the membrane effects
end of the cortical collecting duct is comparable to that in of hyperkalemia, (b) those that rapidly enhance the trans-
plasma and (b) little or no K+ is transported in the medul- fer of extracellular K+ into cells, and (c) those that remove K+
lary collecting duct. The calculation is relatively accurate from the body. Because the onset and duration of action
as long as the urine is not dilute and urine [Na+] is above of each of these maneuvers differ, and because the first
25 mEq/L (453,454). Thus, two measures are only temporizing, several treatment
methods, including at least one for inducing K+ removal
TTKG = {urine [K+]/(urine osmolality/plasma osmolality)}/
from the body, should be instituted simultaneously for
plasma [K+]
symptomatic hyperkalemia (373). For asymptomatic
Normally, TTKG is 8 to 9 on a regular diet and rises to hyperkalemia, treatment can be solely with a cation
above 11 with a K+ load, which indicates increased K+ exchange resin, because rapid therapy is not necessary.
secretion by the cortical collecting duct (454). Hypokale- The effect of hyperkalemia on the cardiac cell mem-
mia with a TTKG higher than 4 suggests renal K+ loss brane can be rapidly reversed with calcium. A rise in ion-
due to increased distal K+ secretion. A value below 7, and ized calcium makes less negative the threshold potential at
176 II. Homeostasis

which excitation occurs and thereby mitigates the depo- removal by the resin is variable and the cation exchange
larizing blockade resulting from hyperkalemia. Calcium is not entirely specific for K+, plasma concentrations of
gluconate (10%) is the preparation most often used; it is K+, Ca2+, and Mg2+ as well as the electrocardiogram
infused slowly intravenously with continuous electrocar- must be monitored frequently. Administration can be
diographic monitoring. The duration of action is tran- repeated at least six times in 24 hours.
sient, and cardiotoxicity will reappear once the infused Addition of sodium polystyrene sulfonate directly to
calcium is sequestered in bone or is excreted. infant formulas and nutritional supplements (1 g/mEq K+
Somewhat more long-lasting effects (several hours) can in the formula) effectively lowers the K+ content by
be obtained by transferring extracellular K+ into cells and approximately 50% (459). However, caution should be
thereby reestablishing a more physiologic transmembrane exercised in routinely prescribing this mode of therapy,
potential gradient. This can be well accomplished by because pretreatment of the formulas is associated with a
inducing endogenous insulin release through infusion of 200% increase in Na+ concentration, a problem that may
glucose. It has been customary to give insulin as well, be avoided by using calcium polystyrene sulfonate (459).
although this may not be necessary in the absence of dia- Failure of the resin to control hyperkalemia should
betes. The dose of glucose with or without insulin may be prompt the clinician to evaluate whether K+ is being con-
repeated as needed, with monitoring for the complica- tinuously delivered to the ECF, possibly from extensive
tions of hyperglycemia or hypoglycemia. ongoing tissue damage or continued absorption of K+-
Administration via nebulizer of β2-agonists (albuterol, containing gastrointestinal contents.
salbutamol), which promote K+ uptake by cells, has been If the amount of K+ to be removed is of such magni-
widely used in adults for the treatment of hyperkalemia tude that the administration of cation exchange resins
in chronic renal failure (457) and has been shown to be does not suffice, dialysis may be necessary. Hemodialysis
safe and efficacious in children as well (458). A single can remove up to 1 mEq/kg of body weight per hour in
dose of nebulized albuterol can lower plasma K+ concen- children and adults (460). Plasma K+ concentration can
tration by as much as 0.5 mEq/L (457,458); the more be lowered by using a low dialysate K+ concentration.
potent parenteral preparation is not available in the Dialysis against a low-K+ bath must be performed cau-
United States. Transient side effects associated with this tiously, however, because rapid fluctuations in plasma K+
class of drug include elevation of heart rate, tremor, and concentration can induce cardiac rhythm disturbances
mild vasomotor flushing. (461). One approach that is especially useful if frequent
Although alkalinization of the ECF with sodium bicar- measurements of plasma K+ concentration are not possi-
bonate to promote the rapid cellular uptake of K+ was for- ble is to lower the K+ concentration of the dialysate in
merly recommended as a mainstay of therapy, it is no gradual decrements over the course of the dialysis treat-
longer considered to be useful. However, this maneuver ment (461). Continuous venovenous hemofiltration is
remains valuable if metabolic acidosis is at all responsible also effective in removing total body K+ from hemody-
for the hyperkalemia. namically compromised patients. Peritoneal dialysis is
The treatment options discussed thus far are only less efficient in removing K+ (462).
temporizing. To remove K+ from the body, renal K+ Patients with disease characterized by chronic hyper-
excretion must be enhanced, for example, by stimulating kalemia, such as chronic renal insufficiency (glomerular
flow-dependent K+ secretion in the distal nephron filtration rate less than 10% of normal), require indefinite
through administration of loop or thiazide diuretics, a dietary K+ restriction and frequent monitoring of the
response that requires the presence of good renal func- plasma K+ concentration. Because patients differ in their
tion. Diuretics are useful in chronic hyperkalemia due to dietary intake, extrarenal handling of K+, and rates of glo-
hypoaldosteronism, heart failure, or a selective K+ secre- merular filtration and urinary flow, the regimen for K+
tory defect (373). restriction must be tailored to the individual. Occasion-
In patients with renal insufficiency, a cation exchange ally, these patients require the use of supplemental oral
resin may be administered to promote gastrointestinal cation exchange resins (0.1 g/kg three times a day) to
elimination. Sodium polystyrene sulfonate (Kayexalate) maintain normokalemia.
is the resin used most commonly. Within the intestinal Children with hypoaldosteronism often respond to the
lumen the resin binds 1 mEq of K+ in exchange for 1 oral mineralocorticoid fludrocortisone acetate (0.1 to 1
mEq of Na+; 1 g/kg of resin is expected to reduce plasma mg/day). Fludrocortisone may exacerbate preexisting
K+ concentration by 1 mEq/L. When given orally, each hypertension or edema, however, so that better control of
gram of resin is suspended in 3 to 4 mL water and mixed the hyperkalemia is achieved using a low-K+ diet and a
with 70% sorbitol or a 10% aqueous dextrose solution loop or thiazide-type diuretic. Persistent hyperkalemia
to reduce constipation. The resin is most often provided associated with K+ secretory abnormalities in the absence
as a retention enema in sorbitol, to be retained for at of diminished renal function or hypoaldosteronism often
least 15 to 30 minutes. Because the efficacy of K+ improves with a thiazide diuretic.
 8. Potassium 177

ACKNOWLEDGMENTS 17. Winters RW. Maintenance fluid therapy. In: Winters RW,
ed. The body fluids in pediatrics. Medical, surgical, and neona-
The studies in the author’s laboratory have been supported tal disorders of acid-base status, hydration, and oxygenation.
by National Institutes of Health grants HD13232 and Boston: Little, Brown, 1973:113–133.
DK50603 and grants-in-aid from the New York Heart 18. Wilde WS. Potassium. In: Comar CL, Bronner F, eds.
Mineral metabolism. New York: Academic Press, 1962:73–
Association and American Heart Association. The author
107.
was an Established Investigator of the American Heart 19. Dickerson JWT, Widdowson EM. Chemical changes in
Association (1984–1989). Thanks to Ms. W. Lepsch for skeletal muscle during development. Biochem J 1960;74:
assistance in preparing the manuscript. 247–257.
20. Vernadakis A, Woodbury DM. Electrolyte and nitrogen
changes in skeletal muscle of developing rats. Am J Physiol
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48:228–234. 432. Gordon RD. Syndrome of hypertension and hyperkalemia
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 9

ACID-BASE HOMEOSTASIS
JAMES C. M. CHAN
ROBERT H. K. MAK

The importance of acid-base homeostasis is underscored by the hydrogen ion concentration. In 1923, Bronsted and
Hastings (1): Lowry (10) set forth the modern definition of an acid as a
substance that donates hydrogen ion and a base as a sub-
To Faraday, we are indebted for naming the products of disso-
stance that accepts hydrogen ion. At the same time, Van
ciation, ions—and thus we came by hydrogen ion, a term now
synonymous with proton. . . . Tiny though it is, I suppose no Slyke (11), in collaboration with Wu and McLean in the
constituent of living matter has so much power to influence Beijing Union Medical College and Hastings in the Rock-
biological behavior . . . efeller University, perfected the acid-base measuring equip-
ment that has since come to be known as the Van Slyke
Endogenous cellular metabolism produces two kinds of apparatus. In the preinsulin era, these advances had major
hydrogen ion or acid (2). The first class of acid is the volatile, clinical implications in the early detection and treatment of
metabolizable acid that on complete oxidation is converted diabetic ketoacidosis by averting diabetic coma and mark-
to carbon dioxide (CO2) and water, with the CO2 rapidly edly improving the survival rate of patients (12,13).
excreted by the lungs (3). The second category of acid is the
nonvolatile, nonmetabolizable net acid, with the kidneys as
its sole route of elimination (4). With the lungs and kidneys
working in concert to maintain acid-base homeostasis, the PHYSICAL CHEMISTRY OF ACIDS AND BASES
normal blood pH can be maintained close to 7.40 despite
challenges to acid-base homeostasis. This complex subject In accordance with the universally accepted Bronsted and
may seem difficult. A historical prospective on how the field Lowry definition (10), an acid is a donor of proton or hydro-
developed may assist in this understanding (5). gen ion, and a base is an acceptor of proton or hydrogen ion.
HA H + + A–
HISTORICAL PERSPECTIVE As visualized in the above equation, HA is an acid able to
donate a hydrogen ion, and A– is a base able to accept a
The first clinical account of severe disturbances in acid-base hydrogen ion. The acid-base pair is intimately linked, with
homeostasis was O’Shaughnessy’s delineation of the mas- HA being the conjugate acid of A–, and A– being the conju-
sive loss of carbonate of soda in the watery diarrhea of chol- gate base of HA. Acids and bases differ in “strength” contin-
era victims during the 1831 epidemic in London (6). No gent on their readiness to donate or accept a hydrogen ion. A
distinction was made between carbonate and bicarbonate at strong acid yields its hydrogen ion promptly; a weak acid
the time. Physiologic acid-base disturbances were next tends not to (e.g., hydrochloric acid is a strong acid because
noted in 1877 when Walter (7) found that administration it dissociates almost completely into its hydrogen ion and
of hydrochloric acid by stomach tube to rabbits caused a chloride ion; in contrast, a weak organic acid does not readily
virtual depletion of the CO2 in blood. In 1909, Hender- dissociate into hydrogen ion and its conjugate base).
son’s renowned monograph illustrated the interrelation- The blood pH of 7.40 is normally maintained within
ships between the metabolic and respiratory components of the range of 7.35 to 7.45, despite continuous dietary meta-
acid-base equilibrium by an equation that subsequently bolic and endogenous production of acids and bases. Pre-
became known as the Henderson-Hasselbalch equation (8). vention of excess fluctuations of blood pH beyond these
In the same year, Sorensen (9) perfected the first electrode ranges is necessary for optimal cellular enzyme activity and
to measure hydrogen ion concentration in the blood and membrane integrity. Substantial pH fluctuations can pre-
coined the term pH to stand for the negative logarithm of cipitate severe glycolysis and brain dysfunction (14,15).
190 II. Homeostasis

In view of the fact that the term pH stands for the nega- base disturbance is gradual, with stepwise increments of
tive logarithm of hydrogen ion concentration, it is inappro- net acid excretion to a hydrogen ion load over 3 or 4 days
priate to present calculated mean values of pH or to take until maximum renal acidification occurs and the excre-
percentage changes in pH values. The change in pH and the tion of hydrogen ion reaches a plateau (Fig. 9.1). Closer
corresponding hydrogen ion concentration are shown below: examinations of the normal responses show that the titrat-
able acidity component of the renal net acid excretion
rises abruptly, primarily with the phosphate buffering of
pH H+ (nEq/L) the hydrogen ion in the urine. The ammonium compo-
7.35 44.7 nent of the net acid excretion rises more gradually in a
7.40 39.8 stepwise fashion until glutaminase-dependent ammonium
7.45 35.5 formation reaches a plateau.

BICARBONATE AND NONBICARBONATE INTERRELATIONSHIP BETWEEN METABOLIC

BUFFERING SYSTEMS AND RESPIRATORY COMPONENTS

The first line of defense against acid-base disturbances resides Changes of serum bicarbonate concentration correlate
in the blood bicarbonate and nonbicarbonate buffering sys- closely with changes in blood pH. Carbonic acid (H2CO3)
tems. In the plasma, the buffers consist of bicarbonate, inor- concentration stays in balance with the CO2 content of the
ganic phosphate, and protein. In the erythrocytes, the buffers blood because the lungs provide an open system of
consist of hemoglobin, oxyhemoglobin, inorganic and exchange. These relationships between the metabolic com-
organic phosphate, and bicarbonate. At a normal blood pH ponent (as represented by the plasma bicarbonate in mEq/
of 7.40, 40% of the total buffering capacity of the blood L) and the respiratory component [as represented by the
resides in the plasma and erythrocyte bicarbonate buffering plasma partial pressure of CO2 (PCO2) in mm Hg] are gov-
system. As metabolic acidosis develops and the blood pH erned by the Henderson-Hasselbalch equation:
falls to, for example, 7.30, more and more bicarbonate is HCO –
3
used up. Thus, determination of the total blood bicarbonate pH = pK + log ---------------------
-
concentration is a reliable indicator of the degree of meta- S × PCO 2
bolic acidosis.
In addition to the blood buffering system, two addi- where HCO3– is the bicarbonate concentration (24.0 mEq/
tional systems defend the acid-base homeostasis: the respi- L); S is the solubility constant of H2CO3 (S = 0.03 at
ratory system and the renal system. 37°C); PCO2 is the partial pressure of CO2 in blood (PCO2
The respiratory compensation to any acid-base distur- = 40 mm Hg); and pK is the dissociation constant for both
bance is prompt. In contrast, the renal response to acid- CO2 and H2CO3. With pK of 6.1 and the log of

FIGURE 9.1. Net acid excretion of urinary titratable acid (TA) and ammonium (NH 4+). With any acid
load, the renal acidification rises in stepwise fashion until a maximum level is achieved in 3 or 4 days,
after which a plateau is reached. In chronic renal failure, the urinary acidification is inadequate.
 9. Acid-Base Homeostasis 191

FIGURE 9.2. Relationship between the metabolic component (plasma bicarbonate) and the res-
piratory component [plasma partial pressure of carbon dioxide (P CO2)] in clinical acid-base distur-
bances. The diagrams represent 95% confidence limits between changes in plasma bicarbonate
(mEq/L) in relationship with a predictable increase or fall in P CO2.

HCO3–/S × PCO2 = 1.3, the normal pH is 7.4. postulated. Thus, any PCO2 value outside of the ninety-
fifth percentile confidence limits for metabolic acidosis
In the clinical setting, the pH of blood is determined by
would indicate a secondary process other than a normal res-
the interrelationship between the metabolic component
piratory compensation.
and the respiratory component. The metabolic component
in blood and the respiratory component are shown in Fig-
ure 9.2 (2,16–18). There is a narrow confidence limit for
NUTRITION AND ACID-BASE HOMEOSTASIS
the respiratory compensation to acid-base disturbance. The
ninety-fifth percentile confidence limits for respiratory aci-
Hydrogen ion production from metabolism of dietary
dosis and metabolic acidosis are wide and are less useful
intake or endogenous production is eliminated by excretion
clinically (Fig. 9.2).
in the urine. The impact of dietary intakes on the urinary
Let us use the example of a child with severe metabolic
acidity was first described by Claude Bernard (19):
acidosis secondary to diarrhea dehydration to illustrate the
respiratory compensation to metabolic acidosis. The One day, rabbits from the market were brought into my labo-
plasma bicarbonate has fallen to 10 mEq/L due to bicar- ratory. . . . I happened to observe that their urine was clear and
bonate loss in the diarrhea. In this case, in the absence of acid. This fact struck me because rabbits, which are herbivore,
any secondary process, the respiratory response (hyperven- generally have turbid and alkaline urine; while on the other
tilation) brings the PCO2 down to approximately 20 mm hand carnivore, as we know, have clear and acid urine. . . . So I
Hg, within the ninety-fifth percentile confidence limits for had rabbits fed on cold, boiled beef (which they eat very nicely
metabolic acidosis (Fig. 9.2). However, if the plasma PCO2 when given nothing else). My expectation was again verified,
and as long as the animal diet was continued, the rabbits kept
should stay above the ninety-fifth percentile confidence
their clear and acid urine.
limits, an independent secondary process interfering with
CO2 exchange (e.g., a pneumonitis or airway obstruction) As a result of Bernard’s observation, the acidity of a car-
would have to be considered. On the other hand, if the nivorous diet influencing the urinary pH and net acid
PCO2 were to fall below the fifth percentile confidence lim- excretion has become widely accepted (13,20). The urine of
its, an independent secondary process stimulating the respi- vegetarians has an average pH of 6.6 (21). However, some
ratory center (e.g., salicylate intoxication) would have to be confusion remains because of the ambiguity of what consti-
192 II. Homeostasis

TABLE 9.1. INFLUENCE OF DIETARY ALKALINE ASH AND METABOLISM OF

SULFUR-CONTAINING AMINO ACIDS ON RENAL ACID EXCRETION
Urinary Net acid
Alkaline ash sulfate excretion
Diet N (mEq/d) (mEq/d) (mEq/d) Urine pH

Milk 4 131.3 71.4 66.7 <7.00

Lettuces 2 40.1 56.7 60.4 <7.00
Vegan 2 58.8 23.5 29.2 >7.10
DL-methionine 1 — 92.1 92.9 5.10
Beef fillet 1 — 124.5 137.5 5.60

N, number of subjects studied.

Note: Calculated mean values from the original data of Hunt (21) in normal medical students and in
vegan subjects.

tutes a typical vegetarian diet. In some cases, a vegetarian therefore, cannot be criticized as being “unnatural.” The
may simply eschew red meat while continuing to consume pH of the daily outputs of urine showed no increase,
eggs, dairy products, and fish as well as vegetables and fruit. although the diet certainly had an alkaline ash. The total
Those whose alkaline ash diets do have a small effect on net acid excretion was confirmed to be more closely corre-
urine pH are the vegans who consume only food of vegeta- lated with the urinary sulfate excretion (Table 9.1).
ble origin and exclude dairy products, eggs, and fish. However, interpretation of Hunt’s experimental results
was clouded by the fact that the protein content of the diet
was high and protein metabolism was known to promote
ALKALINE ASH IN FOOD acid urine. Therefore, another source of alkaline ash was used
in a second experiment. Hunt (21) persuaded two volunteers
The acidity-alkalinity of dietary ash has been calculated by to eat 4 kg of lettuce each day for 4 days, supplying an alka-
McCance and Widdowson (22) to be the cation content of line ash content of approximately 40 mEq/day. However,
sodium, potassium, calcium, and magnesium, minus the even such large amounts of alkaline ash did not neutralize the
anion content of chloride and phosphorus. If the cations acid excreted through the kidney, whereas the sulfate output
are in excess, as is the case in most vegetables, the foodstuff approximately matched the output of acid. Hunt (21) also
is considered to provide an alkaline ash. collected data on two subjects who had lived on a vegan diet
Such excess of cation implies that the foods with an for several years, showing that their urinary output of acid
alkaline residue must contain a surplus of organic anions was lower but well balanced by the urinary output of sulfate.
(e.g., citrate and bicarbonate). Therefore, it has been Their urinary pH was greater than 7.
assumed that the ingestion of a high alkaline ash diet
should result in an alkaline urine. Consistent with that
assumption is the fact that ingestion of large amounts of CONCEPT OF HYDROGEN ION BALANCE
sodium bicarbonate indeed alkalinizes the urine, the
bicarbonate being recovered more or less quantitatively To determine the influence of diets with a high content of
from the urine. The reasonableness of such an assumption sulfur, Hunt (21) supplemented the diet of one subject
may account for the scarcity of published experiments with 583 mEq of sulfur as DL-methionine. The mean uri-
testing it. Until recently, the dietary and metabolic nary output of acid immediately increased to 92.9 mEq/
sources of acid in the urine do not appear to have been day, and the urinary output of sulfur rose to 92.1 mEq/
thoroughly investigated since the work of Sherman and day—results clearly demonstrating that the sulfur of
Gettler (23). Subsequently, Sherman (24) came to the methionine is oxidized in the body to sulfate, which is then
conclusion that most of the acid in the urine had its ori- excreted in the urine together with a corresponding amount
gin in the oxidation of sulfur from dietary sulfur-contain- of net acid. In further studies, Hunt (21) provided another
ing amino acids. However, confirmatory data were not volunteer with 7 pounds of beef fillet only, most of which
available until the work of Hunt (21). To test the relation- he ate in 2 days. His urinary net acid excretion, however,
ship between an alkaline ash diet and urinary output of did not match his urinary sulfate excretion.
acid, Hunt placed four students on a diet limited to 3 to 5 To reconcile these discrepancies, Lemann and Relman
liters of milk per day for 4 days, supplying a caloric intake (25) conducted additional studies that confirmed Hunt’s
of 2100 to 3400 calories a day. Milk was chosen both work by showing that sulfur-containing amino acids are
because it is a “highly standardized food” with an alkaline oxidized to urea plus CO2, water, sulfate, and hydrogen
ash and because it is a “complete food for infants” and, ion. From the amount of urinary sulfate excreted, the
 9. Acid-Base Homeostasis 193

amount of hydrogen ion produced in this step of inter- insignificant portion of the total dietary anions. Similar
mediary metabolism could be extrapolated. In view of the studies in ten adult volunteers who agreed to consume only
fact that the total net acid excretion was not fully accounted milk for 14 days also revealed insignificant undetermined
for, Relman et al. (26) searched for a second component anion absorption (31).
of endogenous acid production, discovering that incom- Goodman, Lemann, and Lennon (32) demonstrated
pletely oxidized organic acid dissociates in the renal that, despite minor daily fluctuations, the cumulative acid
tubule to organic anion and hydrogen ion. By measure- balance in normal individuals is approximately zero. Typi-
ment of organic anions, the obligatory renal excretion of cal acid production in adults is estimated at 70 mEq/day
fixed, “nonmetabolizable” hydrogen ion produced by dis- by extrapolating from urinary sulfate and organic anion.
sociation of organic acid could be extrapolated. Further- The urinary acid output is also estimated to be 70 mEq/
more, because the hydrogen ion produced by dissociation day (32). Goodman et al. (32) demonstrated that with
of organic acid could be extrapolated from the two meta- chronic renal insufficiency, serum CO2 content reaches
bolic sources combined and approximated the net acid the acidotic level over a period of 6 days after withdrawal
excretion measured, Relman et al. (26) proposed that of bicarbonate therapy. The patient’s net acid production
endogenous acid production from intermediary metabo- is not matched by the compromised net acid excretion;
lism can be estimated by summing urinary sulfate and therefore, a positive balance of 21 to 30 mEq of acid per
organic anion. A typical daily acid production of 70 mEq day can be demonstrated (32). In other words, withdraw-
was thus established. ing the alkaline therapy of a patient with chronic renal
In a later study (27), Lennon, Lemann, and Litzow disease led to a metabolic acidosis resulting primarily
found that the correlation between net acid excretion and from hydrogen ion retention.
endogenous acid production as computed by the sum of In view of the fact that acid retention may continue for
the sulfate and organic anions could be improved by sub- months or years in patients with chronic renal acidosis, the
tracting the quantity of “undetermined anion” in the diet. base-donating tissue must have a large alkaline pool that
As defined by McCance and Widdowson (22), the “unde- can be expended very slowly. Evidence points toward bone
termined anion” in foodstuff is the difference between the as the source of the endogenous base.
cations and the predominant anions, calculated as the sum The acidosis of chronic renal failure is accompanied by
of sodium, potassium, calcium, and magnesium minus the retention of such metabolic wastes as urea and creatinine.
sum of chloride and phosphorus. Lennon et al. (27) further Moreover, in renal insufficiency, vitamin D metabolism is
refined the correlation by taking the undetermined anion defective (33–35). Thus, the effects of metabolic acidosis
in the stool into consideration. The difference between on calcium metabolism are more appropriately examined in
undetermined dietary anion and undetermined stool anion the “pure” state, namely in children with renal tubular aci-
constitutes the amount of undetermined anion absorbed. dosis (RTA) but normal renal function. Most adults with
Despite these refinements, the endogenous acid produc- RTA have some renal impairment resulting from nephro-
tion continues to be estimated as the sum of sulfate and calcinosis and nephrolithiasis; for example, of adult patients
organic anions (27–29). Finally, in view of the fact that with RTA studied by Goodman et al. (32), few had a nor-
phosphorus is present in food as phosphates, phosphorus mal glomerular filtration rate.
oxidation is negligible (21), and phosphoric acid produc- The acid balance data of two young brothers with dis-
tion, once proposed as a third component of endogenous tal type I RTA, studied by Chan, are shown in Figure
acid production (30), is consequently ignored in the com- 9.3A,B. Over a 6-day period, the patient in Figure 9.3B
putation of endogenous acid production (27–29). Rather, maintained a mean net acid balance of 22 mEq/M2/day
phosphate intake is accounted for in the dietary undeter- and manifested persistent metabolic acidosis. In con-
mined anion computations (27–29). trast, with the alkaline therapy increased to over 100
mEq/M2/day, the patient in Figure 9.3A regained a nor-
mal serum CO2 concentration and a zero acid balance
ACID-BASE BALANCES IN INFANTS by the end of the study period (41).
AND CHILDREN The major effects of alkaline therapy on mean daily
calcium balances are shown in Figure 9.4. During the
Chan (31) carried out metabolic balance studies in ten patient’s acidotic period, his mean calcium intake was 44
healthy, normal, 1-week-old infants receiving the milk for- mEq/day, and the stool and urinary calcium outputs
mula Similac as their sole dietary intake. The sum of cat- were 25 and 8 mEq/day, respectively, leaving a mean cal-
ions minus the sum of anions in the milk left a value of 8 cium balance of 11 mEq/day. The dramatic threefold
mEq/kg body weight per day as the undetermined dietary increase in his mean calcium balance to 32 mEq/day (p
anion fraction. Similarly computed, the undetermined <.05) on correction of acidosis by alkaline therapy is
stool anion fraction was 5 mEq/kg body weight per day. clearly shown. Most of the improvement occurred
Thus, the undetermined anion fraction absorbed was an through reduction of calcium losses in the stool; a lesser
194 II. Homeostasis

FIGURE 9.3. A: Serum CO2 content and acid balance in a 4-year-old boy with type I renal tubular
acidosis (RTA) who received alkaline therapy that increased to more than 100 mEg/M 2/day. B: Serum
CO2 content and acid-base balance in a 6-year-old boy with RTA-I. C: Fifty-nine-year-old woman
with distal RTA and normal kidney functions.

FIGURE 9.4. Serum CO2 content and calcium balances in a 10-year-old girl with type I renal
tubular acidosis. Redrawn from data of Chan (28).
 9. Acid-Base Homeostasis 195

FIGURE 9.5. Data from deconvolution analysis of pul-

satile growth hormone (GH) secretion in control (N =
9), acidotic (N = 9), pair-fed-1 (N = 8), and pair-fed-2 (N
= 6) groups. Values for mean GH secretory pulse ampli-
tude (height), pulse area (mass), pulse number, and
total GH secreted as pulses are shown. Data are
expressed as means ± standard error of means. Pair-
fed-1 indicates animals pair-fed in the morning, and
pair-fed-2 indicates pair-fed in the afternoon. In each
panel, values with a, b, or c are significantly different
(p = .05). (From Challa A, Krieg RJ, Thabet MA, et al.
Metabolic acidosis inhibits growth hormone secretion
in rats: mechanism of growth retardation. Am J Physiol
1993;265:E547–E553, with permission.)

effect was attributable to reduction of urinary calcium dosis correction amply demonstrates that metabolic aci-
losses. dosis diminishes intestinal calcium absorption in children
The data of Greenberg et al. (36,37) and of Cooke et al. with classic type I RTA.
(38) for children with RTA also show that their patients’ cal- In a recent study, Lee et al. (39) showed that the enzy-
cium balances underwent improvement with correction of matic hydroxylation of 25-hydroxyvitamin-D to 1,25-
metabolic acidosis. dihydroxyvitamin-D was impaired in rats made acutely
The underlying mechanism of the growth failure of acidotic by ammonium chloride ingestion. Despite some
children with RTA has remained unclear. It is commonly contrary evidence (40), the data of Lee et al. (39) rein-
assumed that because negative calcium balances have force the hypothesis, based on observations of children
been found to accompany positive acid balances and with classic RTA (12), that interference with vitamin D
hypercalciuria in adults with renal acidosis secondary to metabolism is the mechanism by which systemic meta-
renal parenchymal diseases, then negative calcium bal- bolic acidosis provokes malabsorption of calcium through
ances must also occur in children with RTA. The forego- the intestinal tract.
ing calcium balance data therefore contain some interesting
information. First, in contrast to the persistent negative
calcium in adults with stable metabolic acidosis secon- MECHANISMS OF GROWTH RETARDATION IN
dary to parenchymal renal diseases, the children studied CHRONIC ACIDOSIS
clearly maintained positive calcium balances in both the
acidotic and the corrected states. The manifold augmen- Growth retardation is a frequent, long-recognized compli-
tation of their calcium balances through reduction of cation of chronic metabolic acidosis (41). Preliminary stud-
fecal and urinary calcium losses as a consequence of aci- ies in acidotic children with classic RTA have shown a
196 II. Homeostasis

blunted release of growth hormone (GH) in response to TABLE 9.2. DIFFERENTIAL DIAGNOSIS OF NEONATAL
provocative stimuli (42). The growth retardation associated FAILURE TO THRIVE AND NORMAL ANION GAP
METABOLIC ACIDOSIS
with acidosis can be reversed by systemic bicarbonate ther-
apy (43). The release of GH under acidotic conditions has Distal renal tubular acidosis
recently been revisited (44). Exploration in rats with nor- Congenital hypothyroidism
mal anion gap acidosis from ingestion of ammonium chlo- Obstructive uropathy
Early uremic acidosis
ride demonstrated aberrant GH secretion. Significant Bicarbonate loss
inhibition of pulsatile GH secretion was present in the aci- Proximal renal tubular acidosis
dotic rats such that both the amplitude of the GH secretory Diarrhea
pulse and the area under the curve were significantly Intestinal fistula
smaller than in controls (Fig. 9.5). These reductions in Ureterosigmoidostomy
Medications: cholestyramine, magnesium sulfate, calcium
pulse amplitude and area were correlated to decreased chloride
growth (weight) in the acidotic rats (44). Using in situ Acid loading
hybridization histochemistry in combination with immu- Ammonium chloride
nocytochemistry, the expression of GH/insulin-like growth Arginine hydrochloride
factor (IGF)-1 in the tibial epiphyseal growth plate has
Modified from Chan JCM, Scheinman JI, Roth KS. Renal tubular aci-
been examined (45). Evaluation of tibial epiphyseal growth dosis. Pediatr Rev 2001;22:277–286.
plate (IGF-1) gene expression in acidotic and control rats
revealed that IGF-1 messenger RNA abundance was lower
in the acidotic growth plates (45). IGF-1 peptide was pre-
dominantly localized to the hypertrophic zone of chondro- paraldehyde, methanol, or ethylene glycol (“antifreeze”) may
cytes and was weakly detectable in the proliferative zone in give rise to acidosis with large anion gaps. Elevated anion
both the acidotic and control rats’ growth plates (45). gaps, in the absence of intoxication or uremia, are usually
Anthropomorphic measurements demonstrated that aci- encountered in alcoholic ketoacidosis and diabetic ketoaci-
dotic rats grew less than did control rats in both length and dosis, with lactic acid, β-hydroxybutyric acid, and acetoace-
weight, and these physical measurements were reflected in tic acid accumulation. The differential diagnosis of normal
the size of the tibial epiphyseal growth plates being signifi- anion gap associated with acidosis, especially in the neonate,
cantly smaller in the acidotic rats compared with the con- is presented in Table 9.2.
trol group. Taken together, these observations suggest that
metabolic acidosis reduces IGF-1 message abundance and
induces resistance to IGF-1 peptide action within the tibial
epiphyseal growth plate. As demonstrated in two indepen-
dent laboratories (46,47), the use of GH to stimulate
growth in normal anion gap acidosis has been ineffective,
despite enhancement of IGF-1 and IGF binding protein
immunoreactivity within the stem-cell chondrocyte zone of
the tibial epiphyseal growth plate.

CONCEPT OF UNDETERMINED ANION GAP

The serum undetermined anion gap is usually approxi-

mated by the difference between the measured cation,
sodium (140 mEq/L), minus the measured anions, chloride
(105 mEq/L), and bicarbonate (25 mEq/L). This typically
gives a normal anion gap of 10 mEq/L. This anion gap is
made up of the undetermined variables consisting of sul-
fate, phosphates, and others. In view of the variability in
measurements of serum sodium, chloride, and bicarbonate,
the normal anion gap varies between 8 and 15 mEq/L.
Significantly elevated anion gap, not related to laboratory FIGURE 9.6. Acid-base nomogram. Shown are the 95% confi-
errors in measurements of the cation and anions, can be dence limits of the normal respiratory (RESP) and metabolic
found when there is accumulation of endogenous lactic acid compensations for primary acid-base disturbances. (From
Dubose TD Jr, Cogan MG, Rector FC. Acid-base disorder. In: Bren-
and keto acids or in uremic acidosis related to accumulation ner BM, Rector FC, ed. Brenner and Rector’s the kidney, 5th ed.
of sulfate and phosphates. Intoxications with salicylate, Philadelphia: WB Saunders, 1996:929–998, with permission.)
 9. Acid-Base Homeostasis 197

Abnormally depressed anion gap may be related to a

reduction in serum albumin or secondary to overhydration.
In addition, intoxications (e.g., lithium or polymyxin B)
may give rise to significantly reduced anion gap due to
increased undetermined cations. Increased serum cationic
proteins in multiple myeloma should be in the differential
diagnosis of a decreased anion gap.

RENAL REGULATION

The average American diet generates 1 to 3 mEq/kg/day of

net acid in healthy infants and children (48,49) and 50 to
80 mEq/day in healthy adults (50). In addition, HCO3– FIGURE 9.7. Schematic model of HCO3– reabsorption in proxi-
may be lost in urine or stool. The kidney maintains acid- mal convoluted tubule. The processes occurring are H + secretion
at the luminal membrane via a specific Na +-H+ exchanger (NHE)
base homeostasis by reabsorption of HCO3– from the glo- and HCO3– transport at the basolateral membrane via a 1 Na +–3
merular filtrate as well as hydrogen ion secretion into the HCO3– cotransporter. Cytoplasmic carbonic anhydrase (CA II) and
urine. The balance of HCO3–, pH, and PCO2 in acute and membrane-bound carbonic anhydrase IV (CA IV) are necessary
to reabsorb HCO3–. ATP, adenosine triphosphate. (From Rod-
chronic abnormal acid-base states is shown in Figure 9.6. riguez-Soriano J. Renal tubular acidosis: the clinical entity. J Am
Most of the known transporters and exchangers involved Soc Nephrol 2002;13:2160–2170, with permission.)
have now been cloned and characterized. Mutations in the
genes encoding these proteins form the molecular basis of
the known inherited disorders of acid-base balance. NHEs extrude protons from cells and take up sodium
ions into cells. Six isoforms (NHE-1 to NHE-6) have been
cloned. The housekeeping NHE-1 is located at the basolat-
Proximal Mechanisms
eral membrane of most renal tubular cells. NHE-2 is located
The proximal tubule reabsorbs 80 to 90% of the filtered apically in selected nephron segments. Both isoforms have
HCO3– and lowers luminal pH by only 0.5 to 0.7 pH units minor roles in renal salt and water handling. NHE-3 is the
(50). HCO3– reabsorption in the proximal tubule is medi- predominant apical isoform in the proximal tubule and loop
ated primarily by H+ ion secretion by a Na+-H+ exchanger of Henle, whereas NHE-2 is the predominant isoform in
(NHE)-3 at the luminal membrane (51,52) and HCO3– the distal tubule (51,52). Fourteen isoforms of CA have
transport via a Na+-HCO3– cotransporter (NBC)-1 (53). been identified. Cytosolic CA II comprises approximately
H2CO3 is formed within proximal tubular cells by hydra- 95% of renal CA. CA II is expressed in the proximal convo-
tion of CO2, a reaction catalyzed by cytoplasmic carbonic luted and straight tubules, thin ascending and thick ascend-
anhydrase (CA) II (54). H2CO3 ionizes and H+ ion is ing limbs of the loop of Henle, and intercalated cells of the
secreted in exchange for luminal Na+. This mechanism is cortical and medullary cortical collecting duct; it is weakly
electron neutral and is driven by the lumen-to-cell Na+ gra- expressed in principal cells and the inner medullary collect-
dient, which is generated by the basolateral membrane Na+- ing duct. Membrane-associated CA is mostly CA IV, which
K+–adenosine triphosphatase (ATPase). HCO3– exits across is linked to the apical membrane via a glycosylphosphati-
the basolateral membrane by mass action as well as by car- dylinositol anchor. CA IV is also localized on the basolateral
rier-mediated cotransport by the Na+-HCO3– exchanger membranes of proximal tubules (54). There are four NBC
(NBC-1). The secreted H+ combines with filtered HCO3– isoforms and two NBC-related proteins. NBC-1 is
to form luminal H2CO3, which is dehydrated by luminal expressed on the basolateral membrane of the proximal
CA IV to form H2O and CO2. Luminal CO2 can freely dif- tubule, is electrogenic, and is directly stimulated by CA II,
fuse back into the cell to complete the reabsorption cycle. which binds to the carboxy terminal of NBC-1. NBC-2 has
These processes are described in Figure 9.7 (49). Approxi- two variants—NBCn1 and mNBC3—both of which are
mately 20% of the filtered HCO3– is reabsorbed by passive electroneutral transporters. NBCn1 is expressed on the
back-diffusion along the paracellular pathway. Major fac- basolateral membrane of the thick ascending limb of Henle,
tors that regulate the proximal acid-base processes include whereas mNBC3 is expressed on the apical membrane of
the filtered load, peritubular HCO3–, pH, PCO2, and intercalated cells (53). Furthermore, an apical proton-trans-
angiotensin (AT) II. Other less important or less well- locating vacuolar ATPase (H+-ATPase) is also present and
defined factors include extracellular volume, peritubular contributes significantly to proximal HCO3– reabsorption.
protein concentration, NaCl reabsorption, K+ depletion, This is a multisubunit enzyme with a membrane-bound
parathyroid hormone (PTH), Ca2+, and adrenergic nerve component (V0 domain) and an intracellular catalytic com-
activity (49,50). ponent (V1 domain) (55).
198 II. Homeostasis

Distal Mechanisms processes involved in distal acidification are described in

Figure 9.8. H+ is secreted by vacuolar H+-ATPase on the
Urine acidification occurs in the distal tubule by three pro-
luminal surface of α intercalated cells in the cortical collect-
cesses: (a) reabsorption of the remainder of the filtered
ing tubule and outer medullary collecting duct. The
HCO3–, which is left over from the proximal tubule
secreted H+ is generated by intracellular CA on H2CO3.
(approximately 20%); (b) titration of divalent basic phos-
Basolateral Cl–-HCO3– exchanger facilitates exchange of
phate (HPO4–), which is converted to the monovalent acid
Cl– for HCO3–. A gastric-type H+,K+-ATPase is also present
form (H2PO4–) or titratable acid; and (c) accumulation of
on the luminal surface of intercalated cells in the same
ammonia intraluminally, which buffers H+ to form nondif-
regions and contributes to H+ secretion and K+ absorption.
fusible ammonium (NH4+). These processes are described
Buffer systems are present to prevent extreme luminal acid-
in Figure 9.8.
ity. NH3 is produced from glutamine and enters the lumen
The thick ascending limb of the loop of Henle reab-
by nonionic diffusion. NH3 captures H+ to form NH4+.
sorbs approximately 15% of the filtered HCO3– load
Filtered phosphate provides another buffer (Fig. 9.8). These
through the apical NHE-3. It also participates in NH3
acid salts comprise titratable acidity and enable urine acidi-
transport. NH4+ absorption occurs in the apical mem-
fication. NH3 also stimulates the cortical collecting duct
brane of the loop of Henle by both the Na+K+–2 Cl–
net HCO3– reabsorption by activating an apical H+,K+-
cotransport as well as K+-H+ antiport systems. The medullary
ATPase independent of NH3’s known effects on intracellu-
thick ascending limb has low permeability to NH3, which
lar pH. This mechanism is dependent on intracellular
limits back-diffusion. A medullary NH4+ concentration
calcium and a zinc endopeptidase specific for vesicle-associ-
gradient is generated and amplified by countercurrent
ated SNARE protein and appears to involve insertion of
multiplication through NH4+ secretion into the proximal
cytoplasmic vesicles into the apical plasma membrane of
tubule and possibly into the thin descending limb of the
cortical collecting duct intercalated cells (56).
loop of Henle. The accumulation of NH3 in the medul-
Mineralocorticoids, Na transport, buffer delivery, changes
lary interstitium increases the driving force for diffusion
in systemic pH and PCO2, luminal pH, and potassium sta-
of NH3 into the collecting tubule.
tus are important factors in the regulation of distal acidifi-
Distal urinary acidification occurs mainly in the collect-
cation. Aldosterone stimulates H+-ATPase in the intercalated
ing duct. In the cortical collecting tubule, the principal cells
cells and sodium resorption by the principal cells. Distal
are in charge of Na+ reabsorption and K+ secretion, whereas
acidification is subject to different regulatory factors along
the intercalated cells are involved in HCO3– absorption and
different segments. Acidification in the cortical collecting
H+ secretion. There are two types of intercalated cells. The
tubule is regulated by Na+ transport–dependent changes in
α cell is responsible for H+ secretion, and the β cell is
potential difference as well as chronic systemic acid-base
responsible for HCO3– absorption. There is a lack of con-
status. The outer medullary cortical tubule has a high
sensus as to whether α and β intercalated cells are molecu-
capacity for H+ secretion but is not affected by systemic
lar images of each other or separate cell types. The cellular
acid-base status or by sodium transport. The inner medul-
lary duct is regulated by systemic acid-base status and K+
balance (49,50).
H+,K+-ATPases belong to the X+K+-ATPase subfamily of
P-type cation-transporting ATPase. H+,K+-ATPases are
composed of at least 13 different subunits, organized into a
membrane-anchored V0 (stalk) domain through which pro-
tons are moved and a V1 head that hydrolyzes ATP. At least
two of the α intercalated cell apical pump’s subunits, the β
subunit in the V1 domain and the α subunit in the V0
domain, have been found to be different tissue-specific iso-
forms. Several isoforms of H+,K+-ATPase have been identi-
fied in the kidney. Renal tubular apical pumps contain β1
rather than β2 subunits and α4 instead of α1 subunits.
FIGURE 9.8. Schematic model of H+ secretion in cortical collect- Being ubiquitously expressed, β2 and α1 are regarded as the
ing tubule. The main pump for luminal H + secretion in the α- housekeeping isoforms. The colonic isoform, H+,K+α2, has
type intercalated cell is a vacuolar H+-adenosine triphosphatase been localized to the outer and inner renal medulla and
(ATPase). An H+,K+-ATPase is also involved in H+ secretion. Intrac-
ellularly formed HCO3– leaves the cell via Cl-HCO3– exchange, appears to be site-specifically upregulated in response to
facilitated by an anion exchanger (AE1). Cytoplasmic carbonic chronic hypokalemia. H+,K+α2 assembles stably with β1-
anhydrase II (CA II) is necessary to secrete H +. ATP, adenosine Na+K+-ATPase in the renal medulla and in the distal colon
triphosphate. (From Rodriguez-Soriano J. Renal tubular acidosis:
the clinical entity. J Am Soc Nephrol 2002;13:2160–2170, with (57–59). Apical tubular H+,K+-ATPase is functionally cou-
permission.) pled to basolateral HCO3– exit (in exchange for Cl–) via the
 9. Acid-Base Homeostasis 199

anion exchanger (AE) AE1. Na+-independent AEs—AE1, presence of a gastric fistula and result in HCO3– retention
AE2, and AE3—are expressed in the kidney. The AE1 gene and metabolic alkalosis. Hypovolemia from extracellular
encodes erythrocyte eAE1 (band 3), the major intrinsic volume contraction also results in increased levels of renin
protein of the erythrocyte as well as kidney kAE1, the baso- and aldosterone, which enhance renal HCO3– reabsorption
lateral Cl–-HCO3– exchanger of the acid-secreting type A in exchange for Cl–, which further potentiates the meta-
intercalated cell. Mutations in AE1 are responsible for dis- bolic alkalosis (50).
tal tubular acidosis. The widely expressed AE2 participates
in recovery from alkaline load and in regulatory cell volume
increase after shrinkage. AE2 is regulated by NH4+. These MINERALOCORTICOID AND OTHER
properties are not shared by AE1. Less is known about AE3 HORMONAL ACID-BASE REGULATION
in the kidney (60). Two other potential Cl–-HCO3–
exchangers, pendrin and AE4, have been reported and may Mineralocorticoids increase net acid excretion and result in
reside apically in the β intercalated cell (61–63). hypokalemic metabolic alkalosis which is only sustained by
sufficient distal Na+ delivery and, therefore, dependent on
adequate salt intake. The normal feedback is extracellular
RESPIRATORY REGULATION volume expansion and hypertension. There are several con-
genital forms of metabolic alkalosis with low-renin hyper-
The CO2-HCO3– system in the respiratory tract provides tension from conditions of mineralocorticoid excess or
the most important open buffer system for acid-base pseudoaldosteronism: Liddle’s syndrome, glucocorticoid
homeostasis in the body. Its significance can be appreciated remediable alkalosis, and apparent mineralocorticoid
if one considers the rate of development of acidosis during excess. Congenital hyperkalemic metabolic acidosis may be
an acute respiratory tract obstruction versus that during an caused by different forms of pseudohypoaldosteronism.
acute urinary tract obstruction. H+ ions from the addition These congenital disorders are now known to result from
of acid combine with HCO3– to form H2CO3, which dis- genetic mutations, and the underlying molecular patho-
sociates in the presence of CA into H2O and CO2. CO2 is physiology is now well understood (see the section Molec-
freely diffusible across alveolar barriers and cell membranes. ular Genetics of Acid-Base Homeostasis). Renin generates
The efficacy and potency of this system are due to the large AT II, which stimulates aldosterone. Renin is increased by
buffer capacity in an open system, as CO2 can rapidly diminished effective circulating volume, which is sensed by
escape from the fluid and be excreted in the lung. A con- the kidney. AT stimulates H+ transport by both Na+-H+
stant PCO2 is maintained by rapid adjustments in alveolar exchange and vacuolar H+-ATPase in the late distal tubule
ventilation (50). and only in the early distal segments. This mechanism is
mediated through the AT1 receptor (64). AT II, on the
other hand, plays an important role in the adaptive
GASTROINTESTINAL REGULATION enhancement of NH4+ secretion in the proximal tubule
(65). Deficiency of 11α- or 17α-hydroxylase gives rise to
Gastrointestinal disorders are common causes of acid-base hypokalemic metabolic alkalosis related to excess mineralo-
imbalance in the pediatric age group. Diarrhea is the most corticoids (e.g., deoxycorticosterone and corticosterone).
common cause of metabolic acidosis in children and results Glucocorticoids stimulate renal tubule acidification both
from loss of large quantities of HCO3–. Compared with in proximal and distal nephrons by enhancing NH4 pro-
plasma, diarrheal stools contain a higher concentration of duction and net acid secretion. Excess glucocorticoids
HCO3– as well as higher amounts of HCO3– decomposed cause metabolic alkalosis.
by reactions with organic acids. Large quantities of K+ are Hypercalcemia and vitamin D excess may increase renal
also lost in the stools and from the urine as high levels of HCO3– reabsorption. Vitamin D poisoning and excessive
renin and aldosterone result from hypovolemia. Urine pH ingestion of milk and antacids as in the milk alkali syn-
may not be less than 5.5 because metabolic acidosis and drome lead to metabolic alkalosis, nephrocalcinosis, and
hypokalemia enhance renal NH4+ synthesis and excretion. renal insufficiency (50). The effects of PTH on acid-base
Thus, high urinary NH4+ may be the differentiating clinical homeostasis is controversial. Hyperchloremic metabolic
feature for diarrhea-associated metabolic acidosis from acidosis has been reported in both primary and secondary
RTA, which typically presents with low urinary NH4+. The hyperparathyroid states, and PTH inhibits tubular reab-
urinary anion gap is a useful clinical tool for estimation of sorption of HCO3– in rats under conditions of chronic
urinary NH4+. Gastrointestinal HCO3– loss can also result hyperparathyroidism (66). PTH infusion in normal human
from external loss of pancreatic and biliary secretions. subjects causes a biphasic response. An initial transient
Coexistent lactic acidosis is also common in severe diar- renal acidosis develops on the first day of PTH infusion,
rheal diseases. On the other hand, H+ loss through gastric followed by a prompt increase in net acid excretion and an
secretions can be caused by vomiting, gastric aspiration, or increase in plasma HCO3– to result in a steady state of mild
200 II. Homeostasis

metabolic alkalosis for up to 13 days (67). PTH is a potent utes. A second phase of buffering by intracellular pro-
inhibitor of renal proximal tubular NHE-3 (68). GH and cesses then occurs. Approximately two-thirds of this
IGF-I increase plasma HCO3– concentration by increasing intracellular buffering is through Na+-H+ exchange and
renal NH3 production and NH4+ net acid excretion (69). one-third through either K+-H+ or Cl–-HCO3– exchanges
(70). Intracellular processes function as H+ storage and
restore normal arterial pH within 24 hours of an acute
PHYSIOLOGIC RESPONSES TO METABOLIC acid load. Renal mechanisms then increase net acid excre-
ACIDOSIS AND ALKALOSIS tion by efficiently retaining all filtered HCO3– and
increasing both titratable acid and NH4+ excretion. With
An acute, nonvolatile acid load is distributed rapidly and chronic metabolic acidosis, renal acid excretion increases
attenuated by extracellular buffers within 20 to 30 min- over the course of 3 to 5 days. Proximal conservation of

TABLE 9.3. CAUSES OF METABOLIC ACIDOSIS

Mechanism Class of agents Clinical conditions

↑ Production of acid β-Hydroxybutyric acid and Fasting or starvation

acetoacetic acid Insulin deficiency
Ethanol intoxication
Ketotic hypoglycemia with
hypoalaninemia
Lactic acid Hypoxia
Muscular exercise
Ethanol ingestion
Type 1 glycogen storage disease
Fructose-6-diphosphate deficiency
Leukemia
Diabetes mellitus
Pancreatitis
Cirrhosis
Incompletely identified Ethylene glycol ingestion
organic acids Paraldehyde intoxication
Salicylate intoxication
Methanol intoxication
Methylmalonic aciduria
Propionyl coenzyme A carboxylase
deficiency
Acidifying salts Arginine hydrochloride
Ammonium chloride
Lysine hydrochloride
Hyperalimentation
Sulfuric acid Methionine
Nutramigen
High-protein milk formula
↑ Extra renal losses Bicarbonate (or combusti- Diarrhea
of base ble base) Ureterosigmoidostomy
Drainage of pancreatic, biliary, or small bowel
secretion
Ingestion of calcium chloride, cholestyramine,
and magnesium
sulfate
Dilutional acidosis Infusion of bicarbonate— Impaired renal acidification
free isotonic or hyper- Oliguria or salt-retaining states
tonic solutions Renal tubular acidosis
Impaired renal acid- Accumulation of fixed, Polycystic kidney disease
ification nonmetabolizable acids Hyperparathyroidism
Adr enal insufficiency
Pseudohypoaldosteronism
Leigh’s syndrome

↑, increased.
 9. Acid-Base Homeostasis 201

TABLE 9.4. CAUSES OF METABOLIC ALKALOSIS

Mechanism Class of agents Clinical conditions

Excessive loss of acid with Chloride deficiency syndromes Normal blood pressure, high renin and aldosterone, low potassium:
volume contraction vomiting of gastric juices, gastric drainage fistula; diuretic and laxa-
tive abuse; Bartter’s syndrome; chloride-deficient infant formula
Cystic fibrosis; villous adenoma of the colon; congenital alkalosis with
chloride diarrhea (Darrow)
Excessive gain of base Base overload Iatrogenic, especially in the context of renal insufficiency; milk alkali
syndrome
Conversion of lactate, acetate Iatrogenic; dialysis excess
to base
Nonmetabolizable acid into Glucose-induced alkalosis in fasting
cells
Excess proximal tubular bicar- Posthypercapnic state
bonate reabsorption Phosphate excess
Hypoparathyroidism
Increased (distal) bicarbon- Volume expansion, mineralo- Hypertension, high renin and aldosterone: secondary nonedematous
ate reabsorption corticoid excess aldosteronism (e.g., renal artery stenosis, intrarenal vascular disease,
accelerated hypertension)
Renin-secreting tumors
Hypertension, low renin, high aldosterone: primary hyperaldoster-
onism; dexamethasone-suppressible hyperaldosteronism; adrenal
carcinoma
Hypertension, low renin, low aldosterone: adrenocorticosteroid
excess; deficiency of 11-hydroxylation/17-hydroxylation; adrenal
carcinoma; Liddle syndrome; licorice (glycyrrhizic acid) excess

HCO3– is achieved by low filtered load of HCO3– and and conditions that cause metabolic alkalosis are
enhanced proximal acid excretion. However, the secon- described in Table 9.4.
dary hypocapnia induced by metabolic acidosis may An acute base load is rapidly distributed in the extracel-
counteract proximal HCO3– conservation. The greatly lular fluid within 25 minutes (71) followed by cellular buff-
reduced HCO3– delivery to the distal tubule plus stimula- ering of the HCO3– load with a half-life of 3 hours. Only
tion of collecting duct H+ secretion and NH4+ excretion one-third of the base load is buffered cellularly, principally
result in urine with low pH, almost no HCO3–, and high by H+-Na+ exchange and to a lesser extent by Cl–-HCO3–
levels of titratable acid and NH4+. Acidosis increases exchange. Modest hypokalemia also results. There is poorer
NH4+ production through stimulation of glutaminase and stabilization of intracellular pH in the alkaline range than in
phosphoenolpyruvate carboxykinase in the proximal the acid range. Neutralization of HCO3– by buffers results
tubule. The increased distal delivery enhances NH4+ reab- in an increase in PCO2, which stimulates ventilation acutely.
sorption by the thick ascending limb of the loop of Henle, However, if the respiratory system is compromised, danger-
thus increasing the inner medullary NH3 concentration. ous hypercapnia may ensue. The increase in PCO2 is
This, in turn, increases NH4+ excretion in the medullary approximately 0.75 mm Hg/mEq/L in plasma HCO3–. The
collecting duct. The net effect is an increase in both renal kidney excretes HCO3– more rapidly than acid. The proxi-
NH4+ production and excretion. Urinary NH4+ can mal tubule is principally responsible for HCO3– excretion.
increase up to approximately fivefold. The nature of the Glomerular ultrafiltrate HCO3– rises in conjunction with
anion accompanying H+ affects renal acid excretion. The plasma HCO3–, but absolute proximal HCO3– reabsorp-
nonreabsorbable anions enhance the ability to generate a tion does not increase because of suppression of proximal
distal tubule potential difference and augment H+ excre- acidification processes by alkalemia. The most sensitive
tion. Titratable acid excretion can increase by two- or response to alkali input is a decline in the excretion of
threefold with an absorbable anion but by five- to tenfold NH4+. The next level of response is to increase the excretion
with a nonabsorbable anion (e.g., organic anions during of unmeasured anions. This latter rise is quantitatively the
ketoacidosis). In severe cases, renal mechanisms may not most important process in eliminating alkali. The maxi-
account for total acid excretion. Bone may represent a mum excretion of citrate is approximately 70% of its fil-
major site of acute and chronic intracellular acid buffer- tered load. An even higher alkali load augments the
ing. Calcium, phosphorus, and hydroxyproline excretion excretion of 2-oxoglutarate to more than 400% of its fil-
are increased during metabolic acidosis (50). Conditions tered load. Only with the largest alkali load does bicar-
that cause metabolic acidosis are described in Table 9.3, bonaturia become quantitatively important. Thus, renal
202 II. Homeostasis

mechanisms eliminate alkali while minimizing bicar- TABLE 9.5. CAUSES OF ACUTE
bonaturia. This process of limiting changes in urine pH RESPIRATORY ACIDOSIS
without sacrificing acid-base balance lessens the risk of kid- Mechanism Conditions
ney stone formation (72).
Airway obstruction Aspiration of vomitus or foreign
The physiologic adaptation to metabolic acidosis and
body
alkalosis has also been studied at the molecular level. Chronic Laryngospasm and edema
acid loading (7-day) is associated with an increase in apical Bronchospasm
NHE-3 in the renal proximal tubule. Because NHE-3 medi- Obstructive sleep apnea
ates both proton secretion and Na reabsorption, compensa- Neuromuscular impair- Injury of brain stem and high cord
ment Botulism
tory changes in Na handling develop, involving decreases in
Tetanus
the abundance of the thiazide-sensitive Na+-Cl– cotransporter Guillain-Barré syndrome
of the distal convoluted tubule and both the β and γ subunits Myasthenia gravis crisis
of the amiloride-sensitive epithelial Na channel of the collect- Overdose of narcotic, sedatives
ing duct. In addition, the renal cortical abundance of the Toxic agents (curare, succinylcholine)
Aminoglycoids, organophosphate
proximal type 2 Na-dependent phosphate transporter was
Hypokalemic myopathy
markedly decreased. In contrast, abundance of the bumet- Familial hypokalemic periodic paralysis
anide-sensitive Na-K–2 Cl cotransporter of the thick ascend- Thorax or pulmonary Respiratory distress syndrome
ing limb and the α subunit of the epithelial Na channels were disorders Pneumothorax
unchanged. A similar profile of changes is seen with short- Hemothorax
Smoke inhalation
term (16-hour) acid loading. Long-term (7-day) base loading
Severe pneumonitis
with NaHCO3 results in the opposite pattern of response, Inadequate ventilation Large dead space mechanical ventila-
with marked increases in the abundance of the β and γ sub- tion
units of epithelial Na channel and type 2 Na-dependent Erroneous settings for tidal volume
phosphate. These adaptations may play critical roles in the Vascular accidents Massive pulmonary embolism and
edema
maintenance of Na balance when changes in acid-base bal-
Cardiac arrests
ance occur (73). Several targets have also been identified at Central nervous system General anesthesia
the gene expression level to account for the adaptation of depression Tranquilizer overdose
renal NH4+ synthesis and transport in response to an acid Cerebral trauma or infarction
load. These are key enzymes of ammoniagenesis (mitochon- Central sleep apnea
drial glutaminase and glutamine dehydrogenase) and gluco-
neogenesis (phosphoenolpyruvate carboxykinase) in the
proximal tubule, the apical Na+-K+(NH4+)–2 Cl– cotrans- Respiratory alkalosis is initiated by a decrease in PaCO2
porter of medullary collecting ducts. Two major factors con- from different causes (Table 9.7). Respiratory alkalosis
trol the expression of these genes during metabolic acidosis, causes alkalinization of body fluids. The acute response
an acid pH and glucocorticoids, which appear to act in con- consists of a decrease in plasma and is complete within 5 to
cert to coordinate the adaptation of various tubular cell types 10 minutes from the onset of hypocapnia. It occurs by alka-
(74). line titration of nonbicarbonate buffers of the body as well
as increased production of organic acids. When respiratory
alkalosis is chronic, renal adjustments worsen the alkalemia
PHYSIOLOGIC RESPONSES TO RESPIRATORY by an additional decrease in plasma HCO3–. This adapta-
ACIDOSIS AND ALKALOSIS tion takes 2 to 3 days to complete and involves downregu-
lation of renal acidification mechanisms. Ninety-five
Respiratory acidosis (Tables 9.5 and 9.6), which follows percent confidence limits for graded degrees of acute and
hypercapnia, is initiated by an increase in arterial partial pres- chronic respiratory alkalosis are also shown in Figure 9.6.
sure of CO2 (PaCO2) and elicits acidification of body fluids. For the same PaCO2, the degree of alkalemia is lower in
An acute increase in plasma HCO3– occurs and is complete chronic than in acute respiratory alkalosis (75). Conditions
within 5 to 10 minutes. This results from acidic titration of that cause respiratory acidosis are described in Tables 9.5
nonbicarbonate buffers, such as phosphates, hemoglobin, and 9.6, and conditions that cause respiratory alkalosis are
and intracellular proteins. When respiratory acidosis is described in Table 9.7.
chronic, renal adjustments worsen the acidemia by a further Chronic but not acute respiratory acidosis stimulates
increase in plasma HCO3–. This chronic adjustment phase activity of H+-ATPase and H+,K+-ATPase in the proximal
takes 3 to 5 days to complete and involves upregulation of tubule, medullary thick ascending limb, and collecting
the renal acidification mechanisms. Ninety-five percent con- tubule. By contrast, both acute and chronic respiratory
fidence limits for graded degrees of acute and chronic respira- alkalosis decrease both renal proton pumps. The stimula-
tory acidosis are shown in Figure 9.6. tory effect of respiratory acidosis and inhibitory effect of
 9. Acid-Base Homeostasis 203

TABLE 9.6. CAUSES OF CHRONIC PHYSIOLOGIC RESPONSES TO MIXED

RESPIRATORY ACIDOSIS ACID-BASE DISORDERS
Mechanism Conditions
Metabolic acidosis and alkalosis primarily involve plasma
Airway obstruction Chronic obstructive airway disease:
HCO3– disturbances, whereas respiratory acidosis and
bronchitis, emphysema
End-stage interstitial lung disease alkalosis primarily involve PCO2 disturbances. Figure
Respiratory center Chronic narcotic or tranquilizer overdose 9.2 shows the 95% confidence limits of these simple
depression acid-base disorders. Values that fall outside these limits
Primary hypoventilation (Ondine’s curse) imply that a mixed disorder may exist. Table 9.8 shows
Brain tumor
the possible combinations of mixed acid-base distur-
Restrictive lesions Kyphoscoliosis, spinal arthritis
Diaphragmatic paralysis bances. Primary metabolic disturbances invoke secon-
Hydrothorax dary respiratory responses, whereas primary respiratory
Fibrothorax disturbances invoke secondary metabolic responses. Meta-
Interstitial fibrosis bolic acidosis resulting from the addition of nonvolatile
Prolonged pneumonitis
acids lowers the extracellular fluid HCO3– concentra-
Obesity hypoventilation syndrome (Pick-
wickian syndrome) tion and, hence, extracellular pH. Medullary chemore-
Neuromuscular Poliomyelitis ceptors are stimulated by the low pH and invoke an
defects Multiple sclerosis increase in ventilation. The ratio of HCO3– to PCO2 and
Muscular dystrophy subsequent pH are returned toward but not entirely to
Amyotrophic lateral sclerosis
normal. Compensation is a physiologic consequence of
Myxedema
Myopathic polymyositis the primary disturbance and does not represent a secon-
Acid maltase deficiency dary alkalosis or acidosis. However, by definition,
mixed acid-base disturbances exceed physiologic limits
of compensation.
respiratory alkalosis appear to be potassium and aldoster-
one independent. Although the precise mechanisms are not
MOLECULAR GENETICS OF
known, direct effects of PCO2, pH, or HCO3– delivery may
ACID-BASE HOMEOSTASIS
be involved (76).
The advent of molecular biology and molecular genetics
TABLE 9.7. CAUSES OF RESPIRATORY ALKALOSIS
has advanced our understanding of the in vivo functions
of acid-base transport proteins and the clinical syn-
Mechanism Conditions dromes caused by their genetic mutations. A gene fam-
Reflex excitation of respira- Pulmonary edema, cardiopul- ily of Na+-H+ exchanger has been identified. One of its
tory center via pulmonary monary disease members, NHE-3, is identified as the Na+-H+ exchanger
stretch receptors Embolus isoform responsible for transepithelial electroneutral
Interstitial pulmonary disease Na+ absorption in intestine and renal epithelial cells.
Primary excitation of central Anxiety
respiratory center Hyperventilation (voluntary or
The NHE-3 gene has been localized to 5p15.3 (77).
mechanical) The transient neonatal form of distal RTA may be
Encephalitis, meningitis caused by immaturity of the NHE-3 Na+-H+ exchanger
Cerebrovascular incidents, head isoform, which is known to undergo postnatal develop-
trauma, brain tumor or vascu- ment (78). No human mutations of the NHE-3 exchange
lar accidents
Medications: salicylate, nicotine,
have been identified as yet. The human gene for NBC-1
xanthine, pressor agents, has been cloned and maps to 4p-21. A nonsense muta-
progesterone tion in the kidney NBC-1 gene (SLC4A4) has been
Heat exposure, fever, pain identified. It predicts a truncated NBC-1 protein with
Pregnancy loss of function, compatible with the phenotype of iso-
Reflex excitation of respira- Low inspirational oxygen (e.g.,
tory center via peripheral high altitude)
lated proximal RTA. Other features of the syndrome
chemoreceptor Hypotension include ocular abnormalities, mental retardation, and
Tissue hypoxia (e.g., anemia, con- short stature (79).
gestive heart failure, asthma) Inherited defects in two of the key acid-base transport-
Arterial hypoxemia ers involved in distal acidification, as well as mutations in
Multiple mechanisms Hepatic failure
Gram negative sepsis
cytosolic CA gene, can cause distal RTA. Both autosomal-
Shock dominant and autosomal-recessive patterns have been
described. Recessive distal RTA present with either acute
204 II. Homeostasis

TABLE 9.8. MIXED ACID-BASE DISORDERS

Mechanism Disorders Adaptation Blood pH

Inadequate response Mixed metabolic acidosis and respiratory PaCO2 ↑↑ Depressed

acidosis HCO3 ↓↓
Mixed metabolic alkalosis and respiratory PaCO2 ↓↓ Elevated
alkalosis HCO3 ↑↑
Excessive response Mixed metabolic acidosis and respiratory PaCO2 ↓↓ Normal or decreased or
alkalosis HCO3 ↓↓ increased
Mixed metabolic alkalosis and respiratory PaCO2 ↑↑ Normal or increased or
acidosis HCO3 ↑↑ decreased
Triple acid-base disorders Mixed metabolic alkalosis (diuretics or Cl- PaCO2 inappropriate Variable
deficient intake), metabolic acidosis HCO3 inappropriate
(lactic acids of sepsis to hypoxemia, Anion gap exceeds 20 mEq/L
hypotension), and respiratory acidosis
or alkalosis
Chronic respiratory acidosis, obstructive PaCO2 inappropriate Variable
lung disease, superimposed acute res- HCO3 inappropriate
piratory acidosis from pneumonitis or
congestive heart failure, acute respira-
tory alkalosis (intubation) mechanical
ventilation

↑↑, increased; ↓↓ , decreased; PaCO2, partial pressure of CO2.

illness or growth failure at a young age, sometimes accom- salt wasting with dehydration, metabolic acidosis, hyper-
panied by deafness, whereas dominant distal RTA is usu- kalemia, and hypotension. Two forms of PHA-I exist. An
ally a milder disease and involves no hearing loss (80). autosomal-recessive form features severe disease with
The AE1 gene encodes band 3 Cl–-HCO3– exchangers manifestations persisting into adulthood. This form is
that are expressed in the basolateral membranes of the caused by loss-of-function mutations in genes encoding
intercalated cells in the distal tubule as well as erythro- subunits of the amiloride-sensitive epithelial sodium
cytes. Several mutations of the AE1 gene cosegregate with channels (86–88). Autosomal-dominant or sporadic
dominant nonsyndromal distal RTA (81). However, the PHA-I is a milder disease that remits with age. Mineralo-
modest degree of loss of function exhibited in vitro by corticoid receptor gene mutations, including frameshifts,
these mutations does not explain the abnormal distal premature terminations codons, and splice donor muta-
acidification phenotype. Other AE1 mutations have been tions, have been identified with the autosomal-dominant
linked to the recessive syndrome of distal RTA without or sporadic form of PHA-I (89). PHA type II, otherwise
hemolytic anemia, in which loss of function can be dem- known as Gordon’s syndrome, is an autosomal-dominant
onstrated by in vitro studies of the mutations (82). Several disorder with a phenotype of hyperkalemic hyperchlo-
mutations in the CA II gene are associated with the auto- remic acidosis with normal renal function and volume-
somal-recessive syndrome of RTA, osteopetrosis, and cere- dependent, low-renin hypertension, which is responsive
bral dysfunction (83). Mutations in ATP6B1, encoding to thiazide diuretics. Recently, two genes causing PHA-II
the B subtype unit of the apical H+-ATPase, are responsi- have been identified. Both genes encode members of the
ble for a group of patients with autosomal-recessive distal WNK (with no kinase) family of serine-threonine
RTA associated with sensorineural deafness (84). Another kinases and are on chromosomes 1 and 17. Disease-caus-
gene, ATP6N1B, which encodes a noncatalytic accessory ing mutations in WNK1 are large intronic deletions that
of the proton pump, is exclusively expressed on the lumi- increase WNK1 expression. The mutations in WNK4 are
nal surface of the intercalated cells in the collecting duct missense, which cluster in a short, highly conserved seg-
and maps to chromosome 7. Nonsense, deletion, and ment of the encoded protein. Both proteins localize to
splice-site mutations of this gene truncate the protein and the distal nephron. WNK1 is cytoplasmic, whereas WNK4
result in the phenotype of distal tubular acidosis with pre- localizes to tight junctions (90).
served hearing (85). Bartter’s syndrome is an autosomal-recessive disease
Syndromes of aldosterone resistance [pseudohypoal- characterized by diverse abnormalities in electrolyte
dosteronism (PHA)] leading to hyperkalemic or type 4 homeostasis including hypokalemic metabolic alkalosis.
RTA have also been characterized at the molecular level. Mutations in the Na-K–2 Cl cotransporter (NKCC2)
PHA type I has a characteristic phenotype of neonatal cause Bartter’s syndrome with the phenotype of hypokale-
 9. Acid-Base Homeostasis 205

mic alkalosis, salt-wasting, hypercalciuria, and low blood sion of cortisol to inactive metabolites, and result in the
pressure (91). NKCC2 mutations can be excluded in phenotype of apparent mineralocorticoid excess (102).
some Bartter’s syndrome kindreds, prompting examina-
tions of regulators of cotransporter activity. One such
regulator is ROMK, an ATP-sensitive K+ channel that ACID-BASE CHANGES RELATED TO GROWTH
recycles reabsorbed K+ back to the tubule lumen. Exami- AND DEVELOPMENT
nation of the ROMK gene reveals mutations that coseg-
regate with the disease and disrupt ROMK function in Children with chronic metabolic alkalosis (e.g., Bartter’s
Bartter’s kindreds. These findings establish the genetic syndrome) do not grow well. However, the mechanism of
heterogeneity of Bartter’s syndrome and demonstrate the this growth failure has not been well studied. Preliminary
physiologic role of ROMK in vivo (92). The antenatal data have suggested the growth failure of Bartter’s syn-
variant of Bartter’s syndrome is also caused by mutations drome may be related to a primary effect of metabolic alka-
in ROMK (93). Bartter’s syndrome type III phenotype is losis or suppressing the IGF-GH axis (41). The effects of
characterized by hypokalemic alkalosis with salt-wasting, chronic hypokalemia in Bartter’s syndrome on the growth
low blood pressure, normal magnesium, and hyper- or axis have not been examined.
normocalciuria with notable absence of nephrocalcino- Children with chronic metabolic acidosis from RTA also
sis. Linkage of this phenotype has also been demon- suffer from failure to thrive and growth retardation (20). In
strated to a segment of chromosome 1 containing the an abstract presented in 1979, McSherry et al. (42)
gene encoding the chloride channel CLCNKB. Loss-of- reported the blunting of GH release in children who have
function mutations, including large deletions and non- RTA. To determine how metabolic acidosis affects GH
sense and missense mutations of this gene, impair chloride secretion and expression, Challa et al. (44) demonstrated
reabsorption in the thick ascending limb of the loop of that acidosis inhibits the GH pulse amplitude, pulse area,
Henle (94). A variant of Bartter’s syndrome, associated and total GH secretion in acidotic animals compared with
with sensorineural deafness and renal failure, has recently that of control and pair-fed animals. They also demon-
been mapped to chromosome 1 (95). A novel gene that strated that serum IGF, hepatic IGF-1 mRNA, hepatic GH
encodes Bartin is expressed in the thin limb and the receptor mRNA, and gene expression of IGF at the growth
thick ascending limb of the loop of Henle in the kidney plate of the long bone all are suppressed in the presence of
and in the dark cells of the inner ear (96). Bartin acts as metabolic acidosis (45,47,103). Thus, it appears that acido-
an essential β subunit for basolateral CLC chloride sis interferes with major aspects of the IGF-GH axis,
channels, with which it colocalizes in basolateral mem- although reduced nutrition from acidosis may contribute
branes of renal tubules and of potassium-secreting epi- to decreased GH secretion. However, metabolic acidosis
thelia of the inner ear (96). Gitelman’s syndrome represents inhibits IGF-1 mRNA expression in the growth plate of the
the predominant subset of Bartter’s patients having long bone and in the hepatic GH receptor mRNA specifi-
hypomagnesemia and hypocalciuria. Complete linkage cally. These animal experiments showed that metabolic aci-
of Gitelman’s syndrome has been demonstrated to the dosis directly inhibited GH secretion and gene expression
locus encoding the renal thiazide-sensitive Na-Cl cotrans- at target sites—anomalies that contribute to the growth
porter. A wide variety of nonconservative mutations has failure of metabolic acidosis.
been identified, consistent with loss of function alleles,
in affected subjects (97).
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63. Parker MD, Ourmozdi EP, Tanner MJ. Human BTR1, a tubular acidosis is associated with mutations in the red cell
new bicarbonate transporter superfamily member and anion exchanger (band 3, AE1) gene. J Clin Invest 1997;
human AE4 from kidney. Biochem Biophys Res Commun 100:1693–1707.
2001;282:1103–1109. 82. Tanphaichitr VS, Sumboonnanonda A, Ideguchi H, et al.
64. Malnic G, Fernandez R, Cassola AC et al. Mechanisms and Novel AE1 mutations in recessive distal renal tubular acido-
regulation of H+ transport in distal tubule epithelial cells. sis. Loss-of-function is rescued by glycophorin A. J Clin
Wien Klin Wochenschr 1997;109:429–434. Invest 1998;102:2173–2179.
65. Nagami GT. Enhanced ammonia secretion by proximal 83. Roth DE, Venta PJ, Tashian RE, Sly WS. Molecular basis of
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84. Karet FE, Finberg KE, Nelson RD, et al. Mutations in the natal variant of Bartter syndrome: evidence for genetic het-
gene encoding B1 subunit of H+ATPase cause renal tubular erogeneity. Hum Mol Genet 1997;6:17–26.
acidosis with sensorineural deafness. Nat Genet 1999;21: 94. Simon KB, Bindra RS, Mansfield TA, et al. Mutations in
67–68. the chloride channel gene, CLCNKB, cause Bartter’s syn-
85. Smith AN, Skaung J, Choate KA, et al. Mutations in drome type III. Nat Genet 1997;17:171–178.
ATP6N1B, encoding a new kidney vacuolar proton pump 95. Birkenhager R, Otto E, Schurmann MJ, et al Mutations of
116-kD subunit, cause recessive distal renal tubular acidosis BSND causes Bartter syndrome with sensorineural deafness
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86. Strautniekds SS, Thompson RJ, Gardiner RM, Chung E. A 96. Estevez R, Boettger T, Stein V, et al. Bartin is a Cl- channel
novel splice-site mutation in the gamma subunit of the epi- beta-subunit crucial for renal Cl- reabsorption and inner ear
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87. Chang SS, Grunder S, Hanukoglu A, et al. Mutations in variant of Bartter’s syndrome, inherited hypokalemic alkalo-
subunits of the epithelial sodium channel cause salt wasting sis, is caused by mutations in the thiazide-sensitive Na-Cl
with hyperkalemic acidosis, pseudohypoaldosteronism type cotransporter. Nat Genet 1996;12:24–30.
1. Nat Genet 1996;12:248–253. 98. Hansson JH, Schild L, Lu Y, et al. A de novo missense muta-
88. Grunder S, Firsov D, Chang SS, et al. A mutation causing tion of the beta subunit of the epithelial sodium channel
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cine that is involved in the gating of the epithelial sodium proline-rich segment critical for regulation of channel activ-
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89. Geller DS, Rodriguez-Soriano J, Ballor Boadao A, et al. 99. Shimkets RA, Warnock DG, Bositis CM, et al. Liddle’s syn-
Mutations in the mineralocorticoid receptor gene cause drome: heritable human hypertension caused by mutations
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hypertension caused by WNK kinases. Science 2001;293: tension caused by a truncated epithelial sodium channel
1107–1112. gamma subunit: genetic heterogeneity of Liddle syndrome.
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92. Simon DB, Karet FE, Rodriquez-Soriano J, et al. Genetic 102. Wilson RC, Dave-Sharma S, Wei JQ, et al. A genetic defect
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in the K+ channel ROMK. Nat Genet 1996;14:152–156. U S A 1998;95:10200–10205.
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 10

CALCIUM AND PHOSPHORUS

ANTHONY A. PORTALE

CALCIUM tion of acidemia with sodium bicarbonate can precipitate tet-

any, due to increased binding of calcium to albumin and a
Body Composition consequent decrease in the ionized calcium concentration.
The total serum calcium concentration exhibits a circa-
Calcium is the most abundant electrolyte in the human
dian rhythm characterized by a nadir at 1 to 3 a.m. and a
body, and in healthy adults, accounts for approximately
peak at 12 to 1 p.m., with amplitude (nadir to peak) of
2%, or 1300 g, of body weight. Approximately 99% of
approximately 0.5 mg/dL (4–6). This rhythm is thought to
body calcium is in the skeleton, mainly in the form of
reflect hemodynamic changes in serum albumin concentra-
hydroxyapatite crystals ([Ca10(PO4)6(OH)2]); the remain-
tion that result from changes in body posture (7). Prolonged
der is in teeth, soft tissue, and extracellular fluid. By con-
upright posture or venostasis can cause hemoconcentration
trast, at birth, calcium accounts for only approximately
and thus increases of approximately 0.5 mg/dL in serum cal-
0.9% of body weight (1). From birth to approximately 20
cium concentration. There is little difference between values
years of age, when the skeleton reaches its full size and den-
taken in fasting and nonfasting states.
sity, calcium content increases by some 40-fold (2). During
Normal values of serum total calcium concentration dif-
this period, the increase in skeletal weight and calcium con-
fer among clinical laboratories and in general range from
tent requires the net retention of 150 to 200 mg of calcium
9.0 to 10.6 mg/dL. The calcium concentration is higher in
per day. Thus, in growing individuals, calcium balance
children than in adult subjects. It is highest at 6 to 24
must be positive to meet the needs of skeletal growth and
months of age (mean of approximately 10.2 mg/dL),
consolidation. In adults, calcium balance is zero after peak
decreases to a plateau of approximately 9.8 mg/dL at 6 to 8
bone mass is attained and becomes slightly negative as bone
years, and decreases further to adult values at 16 to 20 years
is slowly lost with aging.
(8). In men, the calcium concentration decreases from a
mean of approximately 9.6 mg/dL at age 20 to approxi-
mately 9.2 mg/dL at age 80 years; the decrease can be
Extracellular Metabolism of Calcium
accounted for by a decrease in serum albumin concentra-
Calcium in plasma exists in three fractions: protein-bound cal- tion (9). In women, no change is observed with age.
cium (40%), which is not filtered by the renal glomerulus, and Ionized calcium is the fraction of plasma calcium that is
ionized calcium (48%) and complexed calcium (12%), which important for physiologic processes such as muscle con-
are filtered (3). Complexed calcium is that bound to various traction, blood coagulation, nerve conduction, hormone
anions such as phosphate, citrate, and bicarbonate. Albumin secretion and action (parathyroid hormone [PTH] and
accounts for 90% of the protein binding of calcium in plasma, 1,25-dihydroxyvitamin D [1,25(OH)2D]), ion transport,
and globulins for the remainder. Conditions that affect the and bone mineralization. Measurement of the blood ion-
concentration of albumin in plasma, such as nephrotic syn- ized calcium concentration is most useful in critically ill
drome or hepatic cirrhosis, affect the measurement of total patients, particularly those in whom serum protein levels
serum calcium concentration. A decrease in albumin concen- are decreased or acid-base disturbances are present, or to
tration of 1 g/dL results in a decrease in protein-bound and whom large amounts of citrated blood products are given,
hence total calcium concentration of approximately 0.8 mg/ such as with cardiac surgery or hepatic transplantation. A
dL. Binding of calcium to albumin is strongly pH dependent decrease in the blood ionized calcium concentration can
between pH 7 and pH 8; an acute increase or decrease in pH occur not only due to increased binding of calcium to albu-
of 0.1 pH units will increase or decrease protein-bound cal- min, such as in metabolic alkalosis, but also due to
cium, respectively, by approximately 0.12 mg/dL. Thus, in increased complexing with other anions. For example, in
hypocalcemic patients with metabolic acidosis, rapid correc- severe uremia, the ionized fraction of calcium can decrease
210 II. Homeostasis

due to increased complexing with phosphate, sulfate, and

citrate (10). Based on in vitro studies of human serum (11),
an increase in serum phosphorus concentration of 3.7 mg/
dL was required to induce a decrease in ionized calcium of
0.1 mg/dL, the smallest decrease thought necessary to stim-
ulate release of PTH (12,13).
Determinations of the concentration of ionized calcium
in healthy individuals vary somewhat among laboratories
depending on which technique is used and whether the
measurement is made on serum, plasma, or heparinized
whole blood. In healthy infants, ionized calcium levels
decrease from approximately 5.8 mg/dL (1.4 mmol/L) at
birth to a nadir of 4.9 mg/dL (1.2 mmol/L) at 24 hours of
life (14), and increase slightly during the first week of life
(15). Values in young children are slightly higher (by
approximately 0.2 mg/dL) than those in adults until after
FIGURE 10.1. Calcium fluxes between body pools in the normal
puberty. In adult men and women, normal serum ionized adult human in zero calcium balance. ECF, extracellular fluid.
calcium concentrations range from 4.6 to 5.3 mg/dL (1.0
to 1.3 mmol/L); there are no significant sex differences
(16,17). The blood ionized calcium concentration exhibits and in growing children is less than the net amount
a circadian rhythm characterized by a peak at 10 a.m. and a absorbed due to deposition of calcium in bone. In response
nadir at 6 to 8 p.m., with an amplitude of 0.3 mg/dL (4). to a decrease in extracellular concentration of ionized cal-
Specimens must be obtained anaerobically to avoid spuri- cium, secretion of PTH from the parathyroid gland is
ous results due to ex vivo changes in pH. increased (Fig. 10.2). PTH acts on the kidney to decrease
excretion of calcium, to increase excretion of phosphorus,
Extracellular Calcium Homeostasis and to stimulate the production of 1,25(OH)2D. The
1,25(OH)2D acts on the intestine to stimulate active
Calcium homeostasis is maintained by the interaction absorption of calcium and phosphorus, and, together with
between three major organ systems, bone, intestine, and PTH, acts on bone to stimulate release of calcium and
kidney. It is regulated principally by PTH and 1,25(OH)2D phosphorus into the extracellular fluid. PTH action on
and, to a lesser extent, by calcitonin. In healthy adults, net bone is thought to occur in two phases: an initial rapid
intestinal absorption of calcium is 20 to 25% of dietary mobilization of bone mineral that occurs within hours, is
intake. To meet the demands of rapid skeletal growth, frac- associated with increased metabolic activity of osteoclasts,
tional calcium absorption in infants is higher, 40 to 45%, and does not require protein synthesis, and a later phase
reaching values as high as 80% in low-birth-weight, breast- that occurs after 12 to 24 hours of exposure to PTH, is
fed infants (18,19). The efficiency of calcium absorption associated with an increase in both the activity and num-
also is increased during adolescence, during pregnancy, and
with administration of vitamin D metabolites, and is
decreased in vitamin D deficiency and in the elderly. Cal-
cium is absorbed principally in the duodenum and proximal
jejunum, both by a saturable, active transport mechanism
that requires stimulation by 1,25(OH)2D and by a nonsat-
urable, passive diffusion mechanism. A small amount of cal-
cium is secreted into the intestinal lumen, presumably by
paracellular diffusion. An overall schema of calcium metab-
olism is depicted in Figure 10.1.
Absorbed calcium enters the extracellular calcium pool,
which is in equilibrium with the bone calcium pool; the
latter includes a rapidly exchangeable pool, which plays an
important role in maintaining extracellular calcium con-
centration, and a more stable bone mineral pool. Calcium
is filtered by the renal glomerulus and is nearly completely
reabsorbed by the renal tubule. In individuals in zero cal-
FIGURE 10.2. The homeostatic response to hypocalcemia.
cium balance, the amount of calcium excreted by the kid- 1,25(OH)2D, 1,25-dihydroxyvitamin D; Pi, inorganic phosphate;
ney is equal to the net amount absorbed by the intestine, PTH, parathyroid hormone.
 10. Calcium and Phosphorus 211

bers of osteoclasts, and does require protein synthesis (20). enzyme 25-hydroxyvitamin D-1α-hydroxylase (1α-hydrox-
The combined effects of PTH and 1,25(OH)2D on their ylase or P450c1α). The circulating concentration of
target tissues results in an increase in extracellular calcium 1,25(OH)2D primarily reflects its synthesis in the kidney;
concentration toward normal values, with the serum phos- however, 1α-hydroxylase activity also is found in kerati-
phorus concentration being little changed. nocytes, macrophages, and osteoblasts (25–27). The 1α-
Conversely, in response to an increase in blood ionized hydroxylation is the rate-limiting step in the bioactivation of
calcium concentration, secretion of PTH and production of vitamin D, and enzyme activity in the kidney is tightly regu-
1,25(OH)2D are decreased, and release of calcitonin is stim- lated. The steroid 1,25(OH)2D is one of the principal hor-
ulated. The combined effects of these hormonal changes on monal regulators of calcium and phosphorus metabolism
bone, kidney, and intestine are opposite to those occurring and thus is critically important for normal growth and min-
with hypocalcemia and result in a decrease in calcium con- eralization of bone. The classical actions of 1,25(OH)2D are
centration toward normal values. to stimulate calcium and phosphorus absorption from the
intestine and thereby maintain plasma concentrations of
these ions at levels sufficient for normal growth and miner-
Calcium Receptor
alization of bone. The hormone 1,25(OH)2D also has direct
Changes in the extracellular calcium concentration are actions on bone, kidney, parathyroid gland, and many other
detected by the extracellular calcium-sensing receptor (CaR) tissues unrelated to mineral metabolism (reviewed in 28).
located on the plasma membrane of parathyroid cells. The The other important vitamin D–metabolizing enzyme, 25-
CaR has been cloned from bovine, human, and rat parathy- hydroxyvitamin D-24-hydroxylase (24-hydroxylase), is found
roid tissue (21–23) and from rat kidney (24). The receptor has in kidney, intestine, lymphocytes, fibroblasts, bone, skin, mac-
a predicted molecular weight of approximately 120 kDa and is rophages, and possibly other tissues (29). The enzyme can cat-
a member of the superfamily of receptors that couple with alyze the 24-hydroxylation of 25OHD to 24,25(OH)2D and
guanine-nucleotide-regulatory (G) proteins. Activation of the of 1,25(OH)2D to 1,24,25(OH)3D; both reactions are
CaR results in increased activity of the enzyme phospholipase- thought to initiate the metabolic inactivation of vitamin D via
C, which catalyzes the hydrolysis of the membrane-bound the C24-oxidation pathway. The kidney and intestine are
phospholipid inositol 4,5-bisphosphate to two second messen- major sites of hormonal inactivation of vitamin D by virtue of
gers, inositol 1,4,5-triphosphate (IP3) and diacylglycerol. their abundant 24-hydroxylase activity.
Intracellular accumulation of IP3 induces release of calcium The synthesis of 1,25(OH)2D in the kidney is subject to
from storage pools and thereby a rapid increase in cytosolic cal- complex regulation by PTH, calcium, phosphorus, and
cium concentration, and possibly an increase in movement of 1,25(OH)2D (28,30,31). Synthesis of 1,25(OH)2D can be
calcium from the extracellular to the cellular compartment. In stimulated by PTH, insulin-like growth factor-1, and phos-
parathyroid cells, the increase in cytosolic calcium concentra- phorus deficiency, and can be suppressed by plasma ionized
tion is associated with a decrease in secretion of PTH. calcium and 1,25(OH)2D itself. The renal 1α-hydroxylase
enzyme is a mitochondrial cytochrome P450 mixed-function
oxidase that requires the presence of two electron transport
Vitamin D
intermediates for catalytic activity, a flavoprotein termed ferre-
Vitamin D exists as either ergocalciferol (vitamin D2) pro- doxin reductase and an iron-sulfur protein termed ferredoxin
duced by plants, or cholecalciferol (vitamin D3) produced (32). These two proteins mediate the transfer of electrons from
by animal tissues and by the action of near-ultraviolet radi- the reduced form of nicotinamide adenine dinucleotide phos-
ation (290 to 320 nm) on 7-dehydrocholesterol in human phate to the 1α-hydroxylase. The complementary DNA
skin. Both forms of vitamin D are biologically inactive pro- (cDNA) for the 1α-hydroxylase, designated P450c1α, has
hormones that must undergo successive hydroxylations at been cloned from human, rat, mouse, and pig (33–38). The
carbons 25 and 1 before they can bind to and activate the human P450c1α cDNA is 2.4 kilobases (kb) in length and
vitamin D receptor. The 25-hydroxylation of vitamin D encodes a protein of 508 amino acids with a predicted molecu-
occurs in the liver, catalyzed by one or more enzymes, lar mass of 56 kDa (33). The human gene for 1α-hydroxylase
including the mitochondrial enzyme vitamin D 25-hydrox- (officially termed CYP27B1) is single copy, comprises nine
ylase. The activity of hepatic 25-hydroxylation is not under exons and eight introns, and is located on chromosome 12
tight physiologic regulation, and thus circulating concen- (Fig. 10.3) (34,39). Although, at 5 kb, it is a substantially
trations of 25OHD are determined primarily by dietary smaller gene than those for other mitochondrial P450 enzymes
intake of vitamin D and exposure to sunlight. Although (39), its intron-exon organization is very similar, especially to
25OHD is the most abundant form of vitamin D in the that of P450scc (39,40). This strongly suggests that, although
blood, it has minimal capacity to bind to the vitamin D the mitochondrial P450 enzymes retain only 30 to 40%
receptor and elicit a biologic response. amino acid sequence identity with each other, they all belong
The active form of vitamin D, 1,25(OH)2D, is produced to a single evolutionary lineage. The mouse P450c1α gene also
by the 1α-hydroxylation of 25OHD by the mitochondrial has been cloned (41,42).
212 II. Homeostasis

FIGURE 10.3. Scale diagram of the human

P450c1α gene showing the intron-exon organi-
zation and principal restriction endonuclease
cleavage sites. The lesser-thickness bars at the
beginning and end of the first and last exons
represent the 5' and 3' untranslated regions,
respectively. (From Fu GK, Portale AA, Miller
WL. Complete structure of the human gene for
the vitamin D 1α-hydroxylase, P450c1α. DNA
Cell Biol 1997;16:1499–1507, with permission.)

Loss-of-function mutations in the human P450c1α tubule occurs via either the paracellular (between cells) or
gene result in the autosomal recessive disease vitamin D the transcellular (across cells) pathway, or via both path-
1α-hydroxylase deficiency (33,43–49), also known as ways (Fig. 10.4). Paracellular transport is passive, linked to
hereditary pseudo–vitamin D deficiency rickets (50), vitamin the net paracellular absorption (lumen-to-interstitium) of
D dependency (51), or vitamin D–dependent rickets type I. As water, a process termed solvent drag or convection. Paracellu-
of mid-2003 a total of 31 different mutations have been lar transport of calcium also can occur by passive diffusion
found on 88 distinct chromosomes since the first descrip- driven by a chemical gradient, as occurs in the proximal
tion of gene mutations in 1997 (33,48). convoluted tubule (PCT), or a lumen-positive transepithe-
lial potential difference that results from sodium chloride
reabsorption, as occurs in the thick ascending limb of the
Cellular Metabolism of Calcium
loop of Henle (TALH).
A detailed discussion of intracellular calcium metabolism is Transcellular transport of calcium is a three-step process
found in comprehensive reviews (52–54). Most of the total consisting of passive entry across the apical membrane, dif-
amount of calcium in the cell, estimated at approximately 10–3 fusion through the cytosol facilitated by 1,25(OH)2D-
mol/L, is sequestered in intracellular storage compartments, dependent calcium-binding proteins (calbindins), and
principally the endoplasmic reticulum and mitochondria, or active extrusion across the basolateral membrane mediated
bound to specific cytoplasmic proteins such as calmodulin and by a high-affinity Ca2+–adenosine triphosphatase (ATPase)
calbindin-D. The cytosolic concentration of free ionized cal- and Na+-Ca2+ exchanger (55).
cium is approximately 10–7 mol/L, 10,000-fold lower than that
in extracellular fluid. Thus, a steep gradient exists that favors
Calcium Entry
movement of calcium into the cell. Cytosolic calcium serves as
an intracellular second messenger and plays a critical role in reg- Although calcium entry across the luminal membrane is
ulating physiologic functions, including muscle contraction, favored by both electrical and chemical gradients (56,57),
neurosecretion, hormone secretion and action, cell division and the physical and chemical properties of lipid bilayer mem-
aggregation, membrane permeability, enzyme activity, and gene branes prevent passive diffusion of the positively charged
transcription. The cytosolic calcium concentration is tightly Ca2+ ion across cell membranes. Thus, calcium entry
regulated by interplay between specific mechanisms of calcium across the luminal membranes of the kidney and intestine
entry into and exit from the cell, movement of calcium into is thought to occur through Ca2+ channels. Studies using
and from intracellular storage compartments, and reversible a variety of approaches, including Ca2+ channel agonists
complexing of calcium by high-affinity cytosolic calcium-bind- and antagonists, patch-clamp analysis, and cell-attached
ing proteins. Binding of calcium to such proteins provides for electrophysiologic techniques, have shown the presence of
rapid and precise buffering of cytosolic calcium concentration. calcium channels in proximal and distal nephron seg-
Long-term maintenance of the calcium gradient between the ments and in cultured distal renal tubule cells (58). Using
cytosol and extracellular environment is provided by calcium an expression cloning strategy, Hoenderop et al. (59)
pumps and exchangers, which are intrinsic to the plasma mem- cloned and characterized the cDNA encoding a new epi-
brane and the membranes of intracellular organelles. Mito- thelial Ca2+ influx channel, which is named ECaC by
chondria can buffer large amounts of ionized calcium when its analogy with the amiloride-sensitive, aldosterone-depen-
cytosolic concentration increases to levels sufficient to activate dent epithelial sodium channel, ENaC (60,61). ECaC is
the low-affinity mitochondrial transport systems. expressed exclusively in the distal renal tubule, proximal
small intestine, and placenta. The rabbit ECaC cDNA
encodes a protein of 730 amino acids with a predicted
Transepithelial Transport of Calcium
molecular mass of 83 kDa (59). In rabbit kidney, ECaC is
Transport of calcium across the plasma membrane of cal- present along the apical membranes of the distal convo-
cium-absorbing epithelia such as the intestine and renal luted tubule (DCT), connecting tubule (CNT), and cor-
 10. Calcium and Phosphorus 213

FIGURE 10.4. Possible cellular mechanisms

involved in calcium transport in the proxi-
mal S1 and S2 tubule segments (A) and the
distal convoluted tubule (B). Major trans-
port systems are (a) Na+-K+-adenosine tri-
phosphatase; (b) adenosine triphosphate–
dependent Ca 2+-H+ exchanger; (c) Na +-Ca2+
exchanger; and (d) Mg 2+-dependent Ca 2+-
adenosine triphosphatase. For the distal
tubule, calcium and chloride channels are
depicted by “— — .“ (From Suki WN, Rouse
D. Renal transport of calcium, magnesium,
and phosphate. In: Suki WN, Rouse D, eds.
Brenner and Rector’s the kidney. Philadel-
phia: WB Saunders, 1996:472–509, with
permission.)

tical collecting duct, where it co-localizes with calbindin- lung, and bone (69,70). Much is now known about the
D28K (59,62). This part of the nephron is the major site of amino acid sequence, x-ray crystal structure, and biophys-
regulation of Ca2+ reabsorption by PTH and 1,25(OH)2D, ical and calcium-binding properties of the calbindins.
and ECaC is thought to play a major role in such regula- Calbindin-D28K is highly conserved in evolution, with a
tion. ECaC has been identified in several species, includ- high degree of sequence homology observed among the
ing rabbit, rat, mouse, and human (58,63). various mammalian and avian D28K calbindins (71). By
contrast, calbindin-D9K is not highly conserved, and there
is no amino acid sequence similarity between calbindin-
Transcytoplasmic Calcium Movement
D28K and calbindin-D9K. The genes for both rat calbindin
It is proposed that calcium diffusion through the cyto- species and chicken calbindin-D28K have been cloned and
plasm is facilitated by the calcium-binding protein calbin- sequenced, and their transcriptional regulation by
din-D, whose synthesis is dependent on 1,25(OH)2D. 1,25(OH)2D, glucocorticoids, and other factors has been
Calbindins also are proposed to act as an intracellular investigated (reviewed in 69 and 70).
Ca2+ buffer to keep the otherwise tightly regulated cyto- The calbindins belong to the superfamily of EF-hand
solic calcium concentration within physiologic levels dur- helix-loop-helix, high-affinity calcium-binding proteins
ing periods of stimulated transcellular Ca2+ transport (dissociation constant of 10–8 to 10–6 mol/L), which con-
(64–68). Two forms of calbindin-D have been described: tains more than 250 proteins. Calbindin-D28K binds 4
a 28-kDa protein (calbindin-D28K) found in highest con- mol of calcium per mole of protein and calbindin-D9K
centration in avian intestine and avian and mammalian binds 2 mol of calcium per mole of protein. In the intes-
kidney, brain, and pancreas, and a 9-kDa protein (calbin- tine, 1,25(OH)2D stimulates both the synthesis of calbin-
din-D9K) found in highest concentration in mammalian din and the transfer of calcium across the luminal brush-
intestine, placenta, and uterus but also present in kidney, border membrane (BBM). The rate and time course of
214 II. Homeostasis

active calcium absorption correlate well with the amount Analysis of different nephron segments of rat kidney using
of calbindin-D over a wide variety of physiologic condi- reverse transcription polymerase chain reaction revealed that
tions (72,73), which provides strong support for the role the Ca2+ pump is expressed in both the distal and proximal
of calbindin-D28K and calbindin-D9K in vitamin D– nephron (89). In the intestine, the Ca2+ pump is stimulated
dependent active calcium transport. Calbindin-D may by calmodulin and by 1,25(OH)2D, which acts to increase
play a similar role in mediating active renal tubular reab- pump activity by increasing its Vmax (90).
sorption of calcium (74,75). In several mammalian spe- Four isoforms of the plasma membrane Ca2+ pump have
cies, both calbindin-D28K and ECaC have been localized been identified and their cDNAs cloned (91–95). In
in the DCT and CNT (59,62,76,77), which are the humans and rats, the isoforms are encoded by a family of
major sites of active calcium reabsorption. Calbindin-D9K four genes that have been mapped to chromosomes 12, 1,
also has been localized to the distal nephron in the rat and 3, and X. Additional isoforms of the enzyme are created by
mouse (78,79). alternative RNA splicing of the primary gene transcript.
The isoforms exhibit 81 to 85% amino acid homology
among themselves, and a single isoform exhibits approxi-
Calcium Exit
mately 99% homology among different species (96,97).
At the basolateral membrane, calcium is actively extruded The deduced amino acid sequences of rat and human iso-
from the cell against its electrochemical gradient, a process forms of the plasma membrane Ca2+ pump predict a secon-
mediated via a high-affinity, magnesium-dependent Ca2+- dary structure that contains 10 transmembrane domains,
ATPase or an electrogenic 3Na+-1Ca2+ exchanger. with four main units accounting for most of the pump
mass protruding into the cytoplasm (80,81,97). The pre-
Ca2+–Adenosine Triphosphatase dicted secondary structure of the human plasma membrane
The plasma membrane Ca2+-ATPase (Ca2+ pump) is an Ca2+ pump is depicted in Figure 10.5.
obligatory component of eukaryotic plasma membranes
that mediates efflux of calcium from the cell. It is thought Na+-Ca2+ Exchanger
to play the most important role in maintaining the cyto- The Na+-Ca2+ exchanger is an integral membrane protein
solic calcium concentration within the normal range. The that normally exports calcium from the cell, although
Ca2+ pump belongs to the family of P-type ATPases in under some circumstances it mediates calcium influx. The
that it forms a phosphorylated intermediate (an aspar- exchanger is a low-affinity, high-capacity transport system
tylphosphate) during the reaction cycle. The pump has a for calcium, which is driven by the inwardly directed trans-
high affinity for calcium, with an estimated Michaelis- cellular electrochemical Na+ gradient that is normally
Menton constant of 0.2 mmol/L and an apparent molecu- maintained by the basolateral Na+-K+-ATPase. In excitable
lar weight of 120 to 140 kDa (reviewed in 80–84). The tissue such as heart and neurons, the exchanger is electro-
pump is activated by direct interaction with calmodulin, a genic, mediating the export of one intracellular calcium ion
specific calcium receptor protein present in the cytosol, for the import of three extracellular sodium ions. Activity
which results in an increase in both the pump’s affinity for of the exchanger is regulated by intracellular calcium, by
calcium and its maximum transport velocity (Vmax). The changes in the transmembrane voltage; it is inhibited by
pump also is activated by cyclic adenosine monophos- sodium ionophores and ouabain, which reduce the trans-
phate (cAMP)–dependent and protein kinase C–depen- membrane sodium gradient, and is increased by ATP
dent phosphorylation of the pump protein, by limited (reviewed in 98–100).
proteolysis, and by exposure to acidic phospholipids. Complementary DNAs encoding functional Na+-Ca2+
Transport of calcium from the cell is balanced by counter- exchangers have been isolated from heart, kidney, and brain
transport of hydrogen ion (H+), and thus the activity of in a variety of species (101–107), which suggests that the
the Ca2+ pump can be either electroneutral (Ca2+-2H+) or protein plays an important role in different physiologic pro-
electrogenic (Ca2+-H+). cesses in various cell types. Three mammalian isoforms of the
The Ca2+ pump is located exclusively in the basolateral Na+-Ca2+ exchanger (NCX), designated NCX1, NCX2, and
portion of the plasma membrane of renal tubule cells NCX3, have been cloned and are the products of separate
(85,86). In earlier studies, activity of the Ca2+ pump was genes (108–111). NCX1 is expressed most abundantly in
found along the entire length of the rabbit nephron, with the heart but is found in most tissues, including kidney, whereas
activity highest in the distal tubule where the majority of expression of NCX2 and NCX3 is restricted to brain and
active calcium reabsorption occurs (87). In later studies using skeletal muscle (111,112). A number of alternative splicing
monoclonal antibodies against the erythrocyte plasma mem- variants of NCX1 are expressed in a tissue-specific fashion,
brane Ca2+ pump, an epitope of this enzyme was identified which is a common feature of a variety of transmembrane
in human and rat kidneys in the basolateral portion of only proteins (112). The three NCX proteins share 68 to 75%
the DCT (85,88). The purified Ca2+ pump protein co-local- amino acid sequence identity, and all are predicted to share
izes with calbindin-D28K in this nephron segment (85,88). the same topology of 11 membrane-spanning segments with
 10. Calcium and Phosphorus 215

FIGURE 10.5. Sequence, predicted secondary structure, and topology of the plasma membrane
Ca2+ pump showing ten membrane-spanning domains and four large cytoplasmic regions. CaM is
the calmodulin-binding domain. PL is a sequence that interacts with acidic phospholipids. The
shaded region (ATP) is the domain where adenosine triphosphate becomes bound. (From
Carafoli E, Garcia-Martin E, Guerini D. The plasma membrane calcium pump: recent develop-
ments and future perspectives. Experientia 1996;52:1091–1100, with permission.)

a large hydrophilic cytoplasmic loop located between mem- localized exclusively to the distal tubule in the rabbit and rat
brane-spanning segments 5 and 6. In NCX1, the cytoplas- (114). Using antibodies that recognize the Na+-Ca2+
mic loop is thought to be a regulatory region and contains exchanger in rabbit kidney, immunolocalization was detected
the binding site for Ca2+ and the location of the exchanger predominately along the basolateral plasma membrane of
inhibitory peptide (XIP) sequence (Fig. 10.6). cortical CNTs, with weak staining of principal cells of the
Activity of the Na+-Ca2+ exchanger is found only in baso- collecting duct; no staining was detected in other cell types in
lateral membrane preparations of renal tubules (113) and is either the cortex or medulla (115). When polymerase chain
216 II. Homeostasis

FIGURE 10.6. Topology of the Na+-Ca2+ exchanger isoform NCX1 showing putative transmem-
brane-spanning domains 1 to 11 and a long intracellular loop, f. Loop f contains regions impor-
tant in calcium regulation of exchanger activity (Ca 2+ binding site) and in sodium-dependent
inactivation (endogenous XIP region). The α-repeats in the transmembrane domains appear to
be important in ion translocation. XIP, exchanger inhibitory peptide. (From Doering AE, Nicoll
DA, Lu Y, et al. Topology of a functionally important region of the cardiac Na +/Ca2+ exchanger. J
Biol Chem 1998;273:778–783, with permission.)

reaction was used to localize the exchanger in microdissected sorptive rate exists for calcium within the normal physiologic
segments of rat nephron, NCX1 expression was observed in range (124).
the DCT but little or no expression was seen in other seg- Approximately 70% of filtered calcium is reabsorbed in the
ments (116). Thus, the distal nephron exhibits Ca2+ pump proximal tubule; approximately 20% is reabsorbed between
and Na+-Ca2+ exchanger activity, messenger RNA (mRNA) the late proximal and early distal tubule, primarily in the
expression, and protein expression, which is consistent with TALH; 5 to 10% is reabsorbed in the distal tubule; and less
the important role of this nephron segment in hormone- than 5% is reabsorbed in the collecting duct (Fig. 10.7)
regulated calcium reabsorption. (117,125–127). Thus, 1 to 3% of filtered calcium is excreted
in the urine. As discussed later, physiologic regulation of renal
calcium reabsorption occurs in the distal nephron.
Calcium Transport by the Nephron
Approximately 60% of plasma calcium is freely filtered by the Proximal Nephron
glomerulus, as shown by a glomerular filtrate to plasma ratio The majority of filtered calcium is reabsorbed in the proxi-
for calcium that ranges between 0.63 and 0.70 (117,118). The mal tubule, with approximately 60% being reabsorbed by
fraction of plasma calcium that is filterable represents ionized the end of the accessible portion of the superficial proximal
calcium and complexed calcium. To maintain zero calcium tubule and an additional 10% reabsorbed in the proximal
balance, 98 to 99% of the filtered load of calcium, estimated at straight tubule. In the early PCT (S1 and S2 segments), cal-
approximately 8 g/day in the adult, must be reabsorbed by the cium is reabsorbed passively in parallel with the reabsorp-
renal tubules. Clearance studies in humans and experimental tion of sodium and water principally via the paracellular
animals show that an increase in the filtered load of calcium, as pathway, mediated by convection (solvent drag) across the
occurs with calcium infusion, results in an increase in both tight junctions. Evidence for passive reabsorption of cal-
urine excretion and absolute tubular reabsorption of calcium cium is the finding that, in several species studied by micro-
(119–123). Thus it is thought that no maximum tubular reab- puncture, the ratio of calcium concentration in tubular
 10. Calcium and Phosphorus 217

lumen-positive transepithelial voltage difference in this seg-

ment (131–133) that results from the secondary active
transport of sodium chloride out of tubular fluid. Inhibi-
tion of sodium chloride transport with furosemide reduces
the transepithelial voltage and thus increases calcium excre-
tion. In some studies, however, calcium transport in the
TALH is found to be active (130,134,135). It has been pro-
posed that axial heterogeneity may account for some of the
differences observed in the studies reported (136). Calcium
transport in the cortical TALH can be increased by addi-
tion of PTH to the bath (137) and, in medullary segments,
by addition of calcitonin or cAMP (138).

Distal Convoluted Tubule and Connecting Tubule

Physiologic regulation of calcium excretion occurs in the
DCT, which reabsorbs up to 10% of the filtered calcium load.
The capacity for calcium transport in this segment appears to
be high and to be limited mainly by the availability of trans-
portable ions. Although calcium transport in the DCT nor-
mally occurs in parallel with that of sodium, it is not
dependent on either sodium or the transepithelial voltage and
occurs against an electrochemical gradient; thus, it is active
FIGURE 10.7. Profile of calcium reabsorption along the neph- and presumably transcellular. Reabsorption of calcium in the
ron, as derived from micropuncture data. CCD, cortical collecting DCT can be dissociated from that of sodium by administra-
duct; CNT, connecting tubule; DCT, distal convoluted tubule;
IMCD, inner medullary collecting duct; PCT, proximal convoluted
tion of thiazide diuretics, which increase reabsorption of
tubule; PST, proximal straight tubule; TALH, thick ascending limb calcium and decrease that of sodium. As discussed earlier,
of the loop of Henle. (From Friedman PA, Gesek FA. Calcium luminal calcium entry is thought to be mediated via the apical
transport in renal epithelial cells. Am J Physiol 1993;264:F181–
F198, with permission.)
ECaC. Both vitamin D–dependent calbindin-D and the
Ca2+-ATPase co-localize in the DCT and are thought to facil-
itate transcellular movement and basolateral extrusion of cal-
fluid to that in glomerular ultrafiltrate is approximately 1.0 cium, respectively. The Na+-Ca2+ exchanger also is localized,
in the early PCT (127). In the late S1 segment, reabsorp- perhaps exclusively, to the basolateral membrane of the DCT
tion of calcium lags slightly behind that of sodium, which and CNT (79,114–116), although its physiologic role in
thus creates a favorable chemical gradient for reabsorption these segments remains to be defined.
downstream. In the S2 segment of the PCT, the transepi-
thelial voltage is lumen positive; thus, the electrical gradient Collecting Tubule
is favorable for passive calcium reabsorption. In rabbit early Net reabsorption in the collecting tubule accounts for less
S2 segments, net flux of calcium was zero in the absence of than 5% of filtered calcium (127). In the cortical collect-
both water transport and an electrochemical gradient ing tubule, calcium transport probably is active, because
(128), which provides further evidence for passive calcium both calbindin-D (77,79) and Ca2+-ATPase activity (87)
reabsorption. There also is evidence that calcium reabsorp- are found in this nephron segment. In the medullary col-
tion is active in the proximal nephron, particularly in the lecting tubule, approximately 1% of the filtered load may
earliest segments of the PCT where the transepithelial volt- be reabsorbed (139).
age is lumen negative (127). Calcium reabsorption also
appears to be active in the S3 segment of the proximal
Determinants of Renal Handling of Calcium
tubule, because it is not dependent on sodium, occurs
against an electrochemical gradient, and is not inhibited by Diet
ouabain (129). A number of factors can influence renal tubular reab-
sorption and urine excretion of calcium (Table 10.1)
Loop of Henle (124,127). Expansion and contraction of the extracellu-
In the thin descending and ascending limbs of the loop of lar fluid volume induce an increase and decrease, respec-
Henle, calcium transport is negligible (129,130). In the tively, in excretion of both calcium and sodium. Acute
TALH, however, approximately 20% of the filtered calcium infusion of sodium chloride increases urine calcium
load is reabsorbed (127). In isolated, perfused segments of excretion, an effect attributed to inhibition of calcium
TALH, calcium reabsorption is passive, driven by the large reabsorption in both the proximal and late distal tubule
218 II. Homeostasis

TABLE 10.1. FACTORS AFFECTING nephron (142). Phosphorus loading can reduce intestinal
RENAL CALCIUM EXCRETION calcium absorption and stimulate PTH secretion, both in
Factor Ca excretion Mechanism/nephron site part by decreasing the renal production and serum concen-
tration of 1,25(OH)2D (6,143). Conversely, phosphorus
Diet
restriction increases urine calcium excretion (124,127).
Volume expan- ↑ ↓ Distal reabsorption
sion This effect is independent of vitamin D and is attributed to
Sodium chloride ↑ Undefined a reduction in tubular calcium reabsorption, principally in
Protein ↑ ↑ Net acid and sulfate excre- distal nephron segments, that is independent of PTH.
tion
Phosphorus ↓ ↓ Production of 1,25(OH)2D
↓ Intestinal absorption of Ca Hormones
↑ Reabsorption distal neph- PTH stimulates renal calcium reabsorption and is thought
ron to be the principal hormonal determinant of urine calcium
Metabolism excretion. PTH acts to reduce calcium excretion in part by
Acidosis ↑ ↓ Proximal and distal reab-
sorption
decreasing glomerular filtration rate (GFR) via a reduction
Alkalosis ↓ ↑ Proximal and distal reab- in the glomerular capillary ultrafiltration coefficient Kf
sorption (144), and thus decreasing the filtered load of calcium.
Hypercalcemia ↑ ↑ Filtered load of Ca PTH also increases tubular calcium reabsorption in the cor-
↓ Proximal and distal reab- tical TALH, DCT, and CNT of the rabbit, with the princi-
sorption (PTH)
Hormones
pal effect being on the DCT (131,138,145–149). PTH
PTH ↓ ↑ Reabsorption TALH, DCT, action is attributed to activation of both the cAMP–protein
and CNT kinase A and phospholipase-C–protein kinase C signaling
Vitamin D ↓ ↑ Distal reabsorption, ? pathways (126). The PTH-induced increase in calcium
other sites reabsorption is associated with an increase in the cytosolic
Insulin ↑ ↓ Proximal and distal reab-
sorption
calcium concentrations in cortical TALH, DCT, and CNT,
Glucagon ↑ ? ↑ RBF and GFR which is mediated by an increase in the rate of calcium
Growth hor- ↑ Undefined entry through dihydropyridine-sensitive channels (149–
mone 151). The role of ECaC in PTH-mediated stimulation of
Thyroid hor- ↑ ↑ Filtered load of Ca, ↓ PTH calcium reabsorption remains to be determined.
mone
Calcitonin ↓ ↑ Reabsorption TALH
The effect of vitamin D on renal calcium reabsorption is
Diuretics variable, depending on vitamin D status, the activity of
Mannitol ↑ ↓ Proximal reabsorption PTH, and the species studied (55). Although vitamin D
Furosemide ↑ ↓ Reabsorption TALH has no detectable effect on calcium transport by the proxi-
Thiazides, ↓ ↑ Reabsorption DCT mal tubule (152), 1,25(OH)2D stimulates Ca2+ transport
amiloride
Other
in distal nephron segments, including DCT of the dog and
Glucose ↑ ↓ Proximal and distal reab- CNT and cortical collecting duct of the rabbit (55,153).
sorption The effect of 1,25(OH)2D on expression of ECaC was
Glucocorticoids ↑ ? ↓ Bone resorption, vol- studied in vitamin D–deficient rats. Administration of
ume expansion 1,25(OH)2D induced an increase in the abundance of
Estrogens ↓ ? ↓ Bone resorption
ECaC mRNA and protein in the distal part of the DCT
↑, increased; ↓, decreased; 1,25(OH)2D, 1,25 dihydroxyvitamin D; CNT,
and in CNT (154). The human ECaC promoter contains
connecting tubule; DCT, distal convoluted tubule; GFR, glomerular fil- several putative vitamin D–responsive elements, which sug-
tration rate; PTH, parathyroid hormone; RBF, renal blood flow; TALH; gests that 1,25(OH)2D stimulates ECaC expression at least
thick ascending limb of loop of Henle.
in part at the transcriptional level (154). Thus, it is thought
that 1,25(OH)2D acts on the distal nephron to increase cal-
(140). An increase in dietary sodium chloride induces an cium reabsorption by increasing the expression of ECaC in
increase in urine calcium, although the intrarenal mech- this nephron segment.
anisms responsible have not been defined. Urine calcium excretion is increased by exposure to the
Changes in dietary calcium within the normal range following: insulin, glucose, glucagon, growth hormone,
have only a modest effect on urine calcium excretion. A lin- thyroid hormone, and corticosteroids; urine calcium is
ear relationship exists between dietary protein intake and decreased by calcitonin and estrogens (127).
urine calcium excretion (141), an effect that is exaggerated
in patients with recurrent nephrolithiasis. An increase in Metabolism
either oral or parenteral intake of phosphorus is associated Both acute and chronic metabolic acidosis, as induced by
with a decrease in urine calcium excretion, an effect medi- ammonium chloride loading in humans and experimental
ated in part by increased calcium reabsorption in the distal animals, is attended by an increase in urine calcium excre-
 10. Calcium and Phosphorus 219

tion (155–157). This increase is irrespective of a change in Extracellular Metabolism of Phosphorus

filtered load of calcium or in circulating PTH and is attrib-
Phosphorus exists in plasma in two forms, an organic form
uted to a decrease in calcium reabsorption in the distal
consisting principally of phospholipids and phosphate
nephron (155). Hypercalciuria is reversed when the acido-
esters, and an inorganic form (168). Of the total plasma
sis is corrected with administration of alkali (155,158,159).
phosphorus concentration of approximately 14 mg/dL,
Conversely, metabolic alkalosis is associated with a decrease
approximately 4 mg/dL is in the inorganic form. In clinical
in calcium excretion (160,161). The effects of respiratory
settings, only the inorganic orthophosphate form is rou-
acid-base changes are similar to those of metabolically
tinely measured. Approximately 10 to 15% of total plasma
induced changes (162,163). Hypercalcemia, by increasing
inorganic phosphorus is protein bound; the remainder,
the filtered load of calcium, is associated with an increase in
which is filtered by the renal glomerulus, exists principally
its excretion. This effect is mitigated to some extent by
either as the undissociated or “free” phosphate ions HPO42–
hypercalcemia-induced reduction in the ultrafilterability of
and H2PO4–, which are present in serum in a ratio of 4:1 at
calcium and phosphorus (164) and in GFR (165); the
pH 7.4, or as phosphate complexed with sodium, calcium,
reduction in GFR is attributed to a PTH-dependent
or magnesium.
decrease in Kf. Hypercalcemia can decrease calcium reab-
The terms phosphorus concentration and phosphate con-
sorption in the PCT, TALH, and distal nephron (124); the
centration are often used interchangeably, and for clinical
effect on this latter segment requires the presence of para-
purposes the choice matters little. Phosphorus in the form
thyroid glands (166).
of the phosphate ion circulates in blood, is filtered by the
renal glomerulus, and is transported across plasma mem-
Diuretics
branes. The content of “phosphate” in plasma, urine, tissue,
Diuretic agents such as mannitol, which act on the proxi-
or foodstuffs, however, is measured and expressed in terms
mal nephron, and furosemide or ethacrynic acid, which act
of the amount of elemental phosphorus contained in the
on the TALH, induce an increase in both calcium and
specimen, hence use of the term phosphorus concentration.
sodium excretion, because calcium reabsorption is passive
In healthy subjects ingesting typical diets, the serum
and linked to that of sodium in these nephron segments.
phosphorus concentration exhibits a circadian rhythm,
By contrast, thiazide diuretics and amiloride, which act on
characterized by a rapid decrease in early morning to a
the DCT, decrease the excretion of calcium but increase
nadir shortly before noon, a subsequent increase to a pla-
that of sodium (127). The hypocalciuric effect of thiazide
teau in late afternoon, and a small further increase to a peak
diuretics is due in part to increased calcium reabsorption
shortly after midnight (Fig. 10.8) (4,6). The normal circa-
by the DCT; the blockade of sodium chloride entry may
dian rhythm in serum phosphorus concentration can be
reduce the cell sodium concentration sufficiently to
described as the sum of sinusoidal functions with periodici-
enhance basolateral Na+-Ca2+ exchange (167). The hypo-
ties of 24 and 12 hours (6). The amplitude of the rhythm
calciuria induced by chronic administration of thiazide
(nadir to peak) is approximately 1.2 mg/dL, or 30% of the
diuretics is thought to reflect enhanced calcium reabsorp-
24-hour mean level. Restriction or supplementation of
tion by the proximal tubule (127); this effect can be
dietary phosphorus induces a substantial decrease or
reversed by increased salt intake.
increase, respectively, in serum concentrations of phospho-
rus during the late morning, afternoon, and evening, but
induces less or no change in the morning fasting phospho-
PHOSPHORUS
rus concentration (Fig. 10.8) (6). To minimize the impact
of changes in dietary phosphorus on the serum phosphorus
Body Composition
concentration, one should obtain specimens for analysis in
In the adult, phosphorus accounts for approximately 1% of the morning fasting state. Specimens obtained in the after-
body weight, or 600 to 700 g (1). Approximately 85% of noon are more likely to be affected by diet and thus may be
body phosphorus is in the skeleton and teeth, approxi- more useful to monitor the effect of dietary phosphorus on
mately 15% is in soft tissue, and the remainder is in extra- serum phosphorus concentrations, as in patients with renal
cellular fluid. At birth, phosphorus accounts for only insufficiency receiving phosphorus-binding agents to treat
approximately 0.6% of body weight (1). In growing indi- hyperphosphatemia.
viduals, the balance of phosphorus must be positive to meet Factors other than time of day and diet can affect the
the needs of skeletal growth and consolidation; in the adult, serum phosphorus concentration. Presumably because of
phosphorus balance is zero. Phosphorus not only is an movement of phosphorus into cells, the serum phosphorus
important constituent of bone mineral but also plays a crit- concentration can be decreased acutely by intravenous infu-
ical role in many metabolic processes within the cell, sion of glucose or insulin, ingestion of carbohydrate-rich
including those involved in energy metabolism, protein meals, acute respiratory alkalosis, or infusion or endoge-
phosphorylation, nucleotide metabolism, and phospholipid nous release of epinephrine. The decrease in phosphorus
metabolism. concentration induced by acute respiratory alkalosis can be
220 II. Homeostasis

FIGURE 10.8. Effect of dietary phosphorus on the circadian

rhythm of serum phosphorus concentration in healthy men. Blood
was drawn from an indwelling venous needle at hourly intervals FIGURE 10.9. Phosphorus fluxes between body pools in the
for 24 hours after subjects had received a normal intake (•) (1500 normal human adult in zero phosphorus balance. ECF, extracel-
mg/day) of phosphorus for 9 days and after phosphorus was lular fluid; Pi, inorganic phosphate.
restricted for 1 day ( - - - ) and 10 days (o) (<500 mg/day), and then
supplemented (▲) (3000 mg/day) for 10 days. Spectral analysis of
the variations in serum phosphorus concentration over time (time
series) revealed significant periodicities of 24 hours, 12 hours, or
both in each of the subject studies. Depicted are mean values plus
90% of dietary intake (172,173). Metabolic balance studies in
and/or minus the standard error of the mean. (From Portale AA, normal adult humans reveal that over the customary range of
Halloran BP, Morris RC Jr. Dietary intake of phosphorus modulates dietary phosphorus, net absorption is a linear function of
the circadian rhythm in serum concentration of phosphorus: impli-
cations for the renal production of 1,25–dihydroxyvitamin D. J Clin
intake (174). Phosphate is absorbed primarily in the duode-
Invest 1987;80:1147–1154, with permission.) num and jejunum and to a lesser extent in the ileum and
colon. Intestinal phosphate absorption occurs via two mecha-
nisms: an active, sodium-dependent transcellular process that
as much as 2.0 mg/dL (169). Serum phosphorus concen- has been localized to the mucosal surface, and passive diffusion
tration can be increased acutely by metabolic acidosis and through the paracellular pathway. Under usual dietary circum-
by intravenous infusion of calcium (127). stances, the bulk of phosphate absorption is thought to occur
There are substantial effects of age on the fasting serum via passive diffusion, with active transport playing an impor-
phosphorus concentration. Phosphorus levels are highest in tant role when luminal phosphorus concentration is low,
infants, ranging from 4.8 to 7.4 mg/dL (mean, 6.2 mg/dL) as when dietary phosphorus is restricted (175,176). The
in the first 3 months of life and decreasing to 4.5 to 5.8 active absorption of phosphate across the luminal membrane
mg/dL (mean, 5.0 mg/dL) at age 1 to 2 years (170). In involves a sodium-dependent high-affinity transport system
mid-childhood, values range from 3.5 to 5.5 mg/dL (mean, that is simulated by 1,25(OH)2D (177–180). This process is
4.4 mg/dL) and decrease to adult values by late adolescence mediated by the now cloned intestinal sodium-phosphate co-
(8,171). In adult men, serum phosphorus levels decrease transporter (type IIb), which is expressed in small intestine and
with age from approximately 3.5 mg/dL at age 20 years to other epithelial cells but not in kidney and plays a role in the
3.0 mg/dL at age 70 (9,171). In women, the values are sim- physiologic regulation of intestinal phosphate absorption
ilar to those of men until after menopause, when they (181,182). A small amount of phosphate is secreted into the
increase slightly from approximately 3.4 mg/dL at age 50 intestinal lumen in digestive fluids. An overall schema of phos-
years to 3.7 mg/dL at age 70. Because plasma phosphate is phorus metabolism is depicted in Figure 10.9.
composed of divalent (HPO42–) and monovalent (H2PO4–) Absorbed phosphate enters the extracellular phosphate
ions in a ratio of 4:1, the composite valence of phosphorus pool, which is in equilibrium with the bone and soft tissue
in serum (or intravenous solutions) at pH 7.4 is 1.8. At this phosphate pools. Phosphate is filtered at the glomerulus and
pH, 1 mmol phosphorus is equal to 1.8 mEq. is reabsorbed to a large extent by the renal tubule. In adults
in neutral phosphorus balance, the amount of phosphorus
excreted by the kidney is equal to the net amount absorbed
Extracellular Phosphorus Homeostasis
by the intestine; in growing children it is less than the net
In the adult in zero phosphorus balance, net intestinal absorp- amount absorbed due to deposition of phosphorus in bone.
tion of phosphate (dietary phosphorus minus fecal phospho- The tubular reabsorption of phosphate plays a central role
rus) is 60 to 65% of dietary intake. To satisfy the demands of in the regulation of plasma phosphorus concentration and
rapid growth of bone and soft tissue, net intestinal absorption phosphate homeostasis. In response to a decrease in the extra-
of phosphate is higher in infants than in adults and can exceed cellular phosphorus concentration, urine excretion of phos-
 10. Calcium and Phosphorus 221

gradient by a unidirectional, transcellular, secondary active

process, driven by the inwardly directed electrochemical
sodium gradient that is maintained by the basolateral Na+-
K+-ATPase. The transport of phosphate is dependent on
the presence of luminal sodium and is coupled to that of
sodium. Based on electrophysiologic and tracer studies of
phosphate transport, it is proposed that three sodium ions
and one phosphate ion are transported together via a trans-
porter protein that spans the lipid bilayer of the BBM; both
monovalent and divalent phosphate can interact with the
transporter (188).

FIGURE 10.10. The homeostatic response to hypophosphatemia.

Factors Affecting Sodium-Phosphate
1,25(OH)2D, 1,25-dihydroxyvitamin D; Pi, inorganic phosphate; PTH, Co-Transport by the Renal Tubule
parathyroid hormone.
The rate of phosphate reabsorption by the proximal tubule can
be modulated by several factors, and this modulation reflects
phorus decreases promptly due to an increase in phosphate changes in sodium–inorganic phosphate (Na-Pi) co-transport
reabsorption by the proximal tubule (Fig. 10.10). This acute by the BBM (124,189,190). Indeed, the BBM entry step is
response reflects both a decrease in the filtered load of phos- thought to be the final target of physiologic and pathophysio-
phate and an adaptive response of the nephron to increase logic regulation of proximal tubular phosphate reabsorption
phosphate reabsorption in response to a decrease in plasma (190–193). The rate of phosphate reabsorption by the proxi-
concentration and dietary intake of phosphorus (see later). mal tubule decreases with decreasing pH of tubular fluid
Hypophosphatemia is a potent stimulus for the renal produc- (194). In BBM vesicles isolated from rat kidney cortex, the
tion of 1,25(OH)2D (183–185). In normal humans, moderate rate of phosphate uptake decreased severalfold when extrave-
as well as severe restriction of dietary phosphorus can induce sicular (luminal) pH was decreased from 8.0 to 6.0 or when
within 48 hours a substantial increase in the serum concentra- extravesicular sodium was decreased from 175 to 10 mmol/L
tion of 1,25(OH)2D (143,186,187); the increase is mediated (195). The effect of luminal pH on phosphate reabsorption is
by an increase in renal production of this hormone (143). In sodium dependent; it is greater at lower luminal sodium con-
healthy humans in whom dietary phosphorus was manipu- centrations. The decrease in phosphate reabsorption observed
lated throughout its normal range and beyond, the 24-hour with acidic luminal pH is thought to be due to competition
mean serum phosphorus concentration varied inversely and between protons and sodium ions for a common binding site
significantly with the serum concentration of 1,25(OH)2D on the phosphate transporter, which results in decreased affin-
(187). The increased serum 1,25(OH)2D induced by hypo- ity of the transporter for luminal sodium and thereby for lumi-
phosphatemia acts to stimulate absorption of phosphorus and nal phosphate (196). Phosphate uptake by BBM vesicles is
calcium by the intestine and mobilization of these minerals increased when intravesicular (cytosolic) pH is lowered
from bone. Hypophosphatemia also can directly promote (197,198); this finding is also consistent with an effect of
mobilization of bone phosphate and calcium. The resulting hydrogen ion to decrease the transporter’s affinity for sodium
increase in plasma calcium concentration induces suppression and hence for phosphate at the cytosolic side of the plasma
of PTH release, which leads to a further decrease in urine phos- membrane, which thus accelerates the transport cycle.
phorus excretion and an increase in calcium excretion. These Changes in the lipid composition or fluidity state of the
homeostatic adjustments result in an increase in extracellular BBM are associated with changes in BBM Na-Pi co-trans-
phosphorus concentration toward normal values, with little port (199–201). However, short-term (hours-long) restric-
change in the serum calcium concentration. Conversely, in tion of dietary phosphorus induces an increase in phosphate
response to an increase in plasma phosphorus concentration, uptake before changes in either BBM fluidity or cholesterol
production of 1,25(OH)2D is decreased and release of PTH is content are detectable, which suggests that neither fluidity
increased. The net effects of hyperphosphatemia on bone, kid- nor cholesterol content is critical to the early adaptive
ney, and intestine are opposite to those occurring with hypo- response of the BBM to phosphorus restriction (202).
phosphatemia, the net result being a decrease in phosphorus
concentration toward normal values.
Cellular and Molecular Mechanisms
Attention has focused on the molecular events involved in
Renal Tubular Transport of Phosphate
regulation of renal tubular phosphate reabsorption. Using an
In the proximal renal tubule, phosphate is transported expression cloning strategy, the laboratory of Murer cloned
across the luminal membrane against its electrochemical two different Na-Pi co-transporters, designated type I and
222 II. Homeostasis

by the proximal tubule under physiologic and pathologic con-

ditions, and the rate of transport is principally regulated by
altering the abundance of type IIa protein in the BBM
(188,193,205,216,217). Genetic disruption of the NPT2
gene, which encodes the type IIa transporter, results in hypo-
phosphatemia due to severe renal phosphate wasting (218). A
related Na-Pi transporter, type IIb, is expressed in the BBM of
the small intestine but not in kidney, and plays a role in the
physiologic regulation of intestinal reabsorption of phosphate
(181,182). A type IIa transporter appears to be expressed also
in osteoclasts and may play a role in bone resorption (219).
The type IIa Na-Pi co-transporter cDNA, initially cloned from
rat and human kidney cortex libraries (204), encodes a protein
of 637 amino acids with a predicted secondary structure con-
taining eight transmembrane regions. Type II–related trans-
porters have now been identified in renal cortex from rabbit
(220), mouse (211,221), flounder (222), and opossum kidney
cells (OK cells) (223), and from bovine renal NBL-1 cells
(224). The human NPT2 gene is located on chromosome
region 5q35, and the corresponding mouse gene is located on
FIGURE 10.11. Location of identified and postulated Na-depen- chromosome region 13B (225–227). The structures of the
dent and Na-independent phosphate transporters in the proximal NPT2 genes are similar in both species; they are approximately
tubule cell. Available data indicate that most proximal phosphate
reabsorption occurs via the type IIa co-transporter, which is the 16 kb in length and consist of 13 exons and 12 introns. A high
major target of physiologic regulation of renal phosphate reab- degree of homology is found within the promoter regions of
sorption. Org., organic; Pi, inorganic phosphate. (From Murer H, the human, mouse, rat, and opossum genes. A model of the
Forster I, Hernando N, et al. Posttranscriptional regulation of the
proximal tubule NaPi-II transporter in response to PTH and dietary secondary structure of the rat type IIa Na-Pi co-transporter is
Pi. Am J Physiol 1999;277:F676–F684, with permission.) depicted in Figure 10.12.
The viral receptors for gibbon ape leukemia virus (Glvr-1)
and for mouse amphotropic retrovirus (Ram-1) exhibit Na-
type II (203,204). Currently, three types of Na-Pi co-trans- Pi co-transport activity when expressed in X. laevis oocytes
porters that mediate transport of phosphate from the extra- (228–230). These receptors have been named PiT-1 and
cellular to the intracellular compartment are identified: type PiT-2, respectively, and are classified as type III Na-Pi
I, type II, and type III (Fig. 10.11) (reviewed in 205). co-transporters. Type III transporter mRNA is expressed in
The type I Na-Pi co-transporter is expressed primarily in many tissues, including kidney, parathyroid glands, bone,
apical BBMs of the proximal renal tubule, as determined by liver, lung, striated muscle, heart, and brain (205); in mouse
immunohistochemical analysis in rabbits and mice (206). kidney, mRNA abundance of the type III transporter is two
Conditions that physiologically regulate proximal tubule orders of magnitude less than that of the type IIa transporter
phosphate transport such as dietary phosphorus or PTH do (231). Type III transporter protein is localized to the basolat-
not alter type I Na-Pi co-transporter protein or mRNA eral region of the proximal tubule. The role of the type III
expression. Thus, the type I co-transporter is not thought to transporter is thought to be that of “housekeeping” Na-Pi
be a major determinant of proximal tubule phosphate han- transport; that is, mediating phosphate flux into cells if lumi-
dling. The cDNA for the type I Na-Pi co-transporter was ini- nal phosphate entry is not sufficient for metabolic processes
tially cloned from rabbit kidney, and its phosphate transport The mechanisms of phosphate efflux at the basolateral
activity was expressed in Xenopus laevis oocytes (203); homol- membrane are not well defined. Current evidence suggests
ogous cDNA has now been isolated from human, mouse, and that phosphate can exit the cell down its electrochemical
rat kidney cortex and human brain (207–215). The rabbit gradient via (a) a low-capacity Na-Pi co-transport system
cDNA encodes a protein of 465 amino acids with a predicted that couples flux of one sodium ion with that of one diva-
molecular mass of approximately 52 kDa and a secondary lent phosphate ion, and (b) a high-capacity phosphate-
structure that contains six to eight transmembrane regions. anion exchange mechanism (124).
The human gene encoding the type I Na-Pi co-transporter
(NPT1) is located on chromosome 6 p21.3-p23.
Overall Phosphate Transport
The type IIa transporter is highly expressed in the apical
by the Nephron
region of the proximal tubule BBM and in subapical vesicular
structures. The type IIa Na-Pi transporter is the principal Under normal physiologic conditions, 80 to 97% of the fil-
determinant of overall Na-dependent phosphate reabsorption tered load of phosphate is reabsorbed by the renal tubule
 10. Calcium and Phosphorus 223

FIGURE 10.12. Model of the secondary structure of the rat type IIa Na-Pi co-transporter, derived
from hydropathy predictions and experimentally supported (see reference 205). The two N-glyco-
sylation sites in the secondary extracellular loop are indicated by asterisks. Cysteine residues likely
to form a disulfide bridge and possibly important for function are shown in black. (From Murer H,
Hernando N, Forster I, et al. Proximal tubular phosphate reabsorption: molecular mechanisms.
Physiol Rev 2000;80:1373–1409, with permission.)

(124,127). Clearance studies in humans and experimental capacity for phosphate is the principal determinant of the
animals show that, when the filtered load of phosphate is pro- serum phosphorus concentration. Approximately 80% of fil-
gressively increased, phosphate reabsorption rises until a tered phosphate is reabsorbed in the proximal tubule, 5 to
maximum tubular reabsorptive rate for phosphate, or TmP, is 10% is reabsorbed in the distal tubule, and 2 to 3% is reab-
reached, after which phosphorus excretion increases in propor- sorbed in the collecting tubules (Fig. 10.13).
tion to its filtered load. The measurement of TmP varies
among individuals and within the same individual, due in part
Proximal Tubule
to variation in GFR. Thus, the ratio TmP/GFR, or the maxi-
mum tubular reabsorption of phosphate per unit volume of Approximately 70% of the filtered load of phosphate is reab-
GFR, is the most reliable quantitative estimate of the overall sorbed by the PCT. Under normal physiologic conditions,
tubular phosphate reabsorptive capacity and can be considered reabsorption rates in early convolutions (S1 segment) are as
to reflect the quantity of Na-Pi co-transporters available per much as four times higher than those in late convolutions (S2
unit of kidney mass (175). The serum phosphorus concentra- segment) and the pars recta (S3 segment) (232–234). Due to
tion at which phosphate reabsorption is maximal is called the this axial heterogeneity in phosphate transport, most of the
theoretical renal phosphate threshold; this value is equal to the phosphate reabsorption in the PCT occurs within the first
ratio TmP/GFR and closely approximates the normal fasting 25% of PCT length. Internephron heterogeneity is also seen
serum phosphorus concentration. Thus, the renal reabsorptive in phosphate reabsorption in the proximal tubule; proximal
224 II. Homeostasis

or decrease in dietary phosphorus predictably induces an

increase or decrease, respectively, in urine excretion of phos-
phorus; with severe phosphorus restriction, urine phospho-
rus excretion is negligible. This adaptation is independent of
changes in the filtered load of phosphate; does not depend
on changes in extracellular fluid volume, plasma calcium
level, growth hormone level, vitamin D status, or parathy-
roid activity; and appears to reflect changes in the rate of
phosphate reabsorption by the proximal tubule, specifically,
an increase or decrease in the Vmax of Na-Pi co-transport
activity. In addition, phosphorus restriction in animals can
severely blunt the phosphaturic response to administration of
PTH (196). The adaptation can be demonstrated both in
vivo and in isolated perfused PCT segments and BBM vesi-
cles taken from animals maintained on differing dietary
intakes of phosphorus (244–249).
FIGURE 10.13. Profile of phosphate reabsorption along the
mammalian nephron, as derived from micropuncture data. CCD, Renal tubule adaptation to changes in either dietary
cortical collecting duct; DCT, distal convoluted tubule; IMCD, inner intake or plasma concentration of phosphorus can occur
medullary collecting duct; PCT, proximal convoluted tubule; Pi, rapidly; an increase in BBM vesicle Na-Pi co-transport was
inorganic phosphate; PST, proximal straight tubule; PTH, parathy-
roid hormone; TALH, thick ascending limb of the loop of Henle. observed after 2 to 4 hours of phosphorus restriction in the
(Data from Suki WN, Rouse D. Renal transport of calcium, magne- rat (250–252); conversely, a decrease in phosphate trans-
sium, and phosphate. In: Suki WN, Rouse D, eds. Brenner and Rec- port was induced after 1 hour of phosphorus infusion
tor’s the kidney. Philadelphia: WB Saunders, 1996:472–509.)
(253). The adaptation induced by short-term (hours-long)
phosphorus restriction was preceded by a decrease in serum
phosphorus concentration and was not inhibited by cyclo-
tubules of juxtamedullary nephrons have a greater capacity
heximide or actinomycin D, which suggests that protein
both to reabsorb phosphate (235–238) and to adapt to
synthesis is not required for the adaptation to occur (251).
changes in its filtered load (239,240) than do proximal tubules
By contrast, the adaptation induced after long-term (3 days)
of superficial nephrons. In the absence of PTH, up to an addi-
phosphorus restriction was inhibited by cycloheximide and
tional 10% of filtered phosphate can be reabsorbed in the
actinomycin D, which suggests that new protein synthesis
proximal straight tubule.
is required (254). Similarly, exposure of cultured renal epi-
thelial cells (LLC-PK1) to a low-medium phosphorus con-
Loop of Henle, Distal Convoluted Tubule, centration induced both short-term (minutes-long) and
and Connecting Tubule long-term (hours-long) adaptations in Na-Pi co-transport
(255–257). Thus, adaptation to changes in both dietary
Little or no transport of phosphate is thought to occur in
intake and plasma concentration of phosphorus involves
the loop of Henle except for the proximal straight tubule
two phases with different time courses: a rapid phase that is
segment (127). Up to 10% of the filtered load of phosphate
independent of protein synthesis and appears to involve
is reabsorbed by the DCT in the absence of PTH; an addi-
activation or recruitment of preexisting transporter units,
tional 3 to 7% may be reabsorbed beyond the accessible
and a long-term phase that requires protein synthesis and
late DCT, presumably by the CNT (127).
permits a large increase (>100%) in phosphate transport
rate (190,196,256).
Collecting Tubule Physiologic and pathologic changes in the renal handling of
phosphate can be accounted for by changes in the abundance
Although some investigators have failed to demonstrate phos-
of type IIa Na-Pi co-transporter protein in the renal BBM.
phate reabsorption in isolated perfused cortical collecting
Short-term (hours-long) exposure of rats to a low-phosphorus
tubules (241), others have shown a small but significant net
diet induced an increase in both BBM Na-Pi co-transport
efflux of phosphate in this portion of the nephron (242,243).
activity and protein abundance but no change in Na-Pi–
specific mRNA (258). The changes in transporter activity and
Physiologic Regulation of protein abundance were blocked by prior treatment of the ani-
Renal Phosphate Transport mals with colchicine, a microtubule-disrupting agent, but were
not blocked by treatment with actinomycin D or cyclohexi-
Dietary Phosphorus and Plasma Phosphorus
mide (258). In opossum kidney (OK) cells, exposure to a low-
Dietary intake of phosphorus is one of the most important phosphate medium induced within hours an increase in type
physiologic regulators of renal Na-Pi co-transport. An increase IIa protein abundance in parallel with an increase in Na-Pi co-
 10. Calcium and Phosphorus 225

transport activity but no change in type II co-transporter port (278,279). Based on findings in isolated perfused
mRNA abundance (259,260). Thus, acute regulation of Na-Pi mouse tubules, binding of PTH to its receptor located on
co-transport activity appears to be independent of transcrip- the apical surface primarily activates the phospholipase
tion and translation and is thought to result from retrieval C–protein kinase C pathway, whereas binding to the
(decrease) or insertion (increase) of preexisting transporters receptor on the basolateral surface primarily activates the
from or into the BBM (reviewed in 182, 193, 205, 261, and adenylate cyclase–protein kinase A pathway (280). Studies
262). An intact microtubular network is required for the acute indicate that activation of the extracellular signal-related
adaptation to phosphorus restriction (258). In contrast, long- peptide/mitogen-activated protein kinase (ERK/MAPK)
term (days-long) restriction of dietary phosphorus in mice, pathway also plays a central role in the regulation by PTH
rats, and rabbits leads to an adaptive increase in BBM Na-Pi of renal phosphate transport (281).
co-transport and in the abundance of type II Na-Pi co-trans- The phosphaturic action of PTH is attributed to inhi-
porter protein and mRNA (220,263–268); no change was bition of phosphate transport by the BBM (205) and is
observed in the abundance of type I transporter mRNA or associated with a reduction in the abundance of type IIa
protein (220). The increase in Npt2 gene expression induced Na-Pi co-transporter protein, as determined by Western
by a long-term (4 days) low-phosphorus diet was attributed to blot analysis of isolated proximal tubule BBMs and Na-
transcriptional mechanisms (269). Pi-IIa–specific immunofluorescence in convoluted and
straight segments of proximal tubules (280,282). A pro-
longed increase in PTH also can induce a decrease in type
Hormones
II Na-Pi co-transporter mRNA abundance (282). The
Parathyroid Hormone reduced abundance of type IIa Na-Pi co-transporter pro-
PTH is the principal hormonal regulator of renal phos- tein, induced acutely by infusion of PTH or feeding of a
phate reabsorption (124,270). PTH inhibits renal phos- high-phosphorus diet, is attributed to endocytic internal-
phate reabsorption in the convoluted and straight segments ization of phosphate transporters from the plasma mem-
of the proximal tubule, where the majority of filtered phos- brane (205). The type IIa Na-Pi co-transporters are
phate is reabsorbed. Although PTH also inhibits proximal thought to be internalized via the same endocytic pathway
tubule reabsorption of bicarbonate, sodium, and fluid, the as are soluble proteins (205,283) and, after internaliza-
suppressive effect of PTH on phosphate reabsorption can tion, are directed to the lysosomes for degradation. Stud-
be dissociated from that of sodium and fluid and thus is ies in OK cells suggest that Na-Pi co-transporters, once
specific for phosphate (270). As further evidence of such internalized, are not immediately available for recycling
specificity, in renal BBM vesicles isolated from animals back to the plasma membrane (284,285).
treated with PTH in vivo, Na-Pi co-transport was reduced,
whereas sodium-dependent transport of D-glucose and Vitamin D
other solutes was unchanged (271,272). PTH-sensitive Chronic administration of vitamin D or its metabolites is
phosphate transport also is observed in the DCT and in the associated with a decrease in renal phosphate reabsorp-
cortical collecting duct (135,243,273); however, the local- tion. This effect is thought to be mediated by a vitamin
ization of PTH action along the nephron differs greatly in D–induced increase in intestinal phosphate absorption
the various species studied (270). and thus in phosphate balance, which induces an adaptive
PTH binds to its specific receptors on the apical and decrease in phosphate reabsorption by the proximal
basolateral membranes of proximal tubule cells and acti- tubule (124,127). The effects of acute administration of
vates adenylate cyclase, which results in an increase in vitamin D on phosphate transport appear to depend on
intracellular cAMP and subsequent activation of protein the experimental conditions of study, including the dose
kinase A. The phosphaturic effect of PTH can be mim- of vitamin D administered and the prior vitamin D,
icked by exogenous cAMP in intact animals, isolated per- PTH, and phosphorus status of the organism. In the rat,
fused tubules, and BBM vesicles. PTH binding also activates vitamin D depletion is associated with a decrease in renal
the phosphoinositide-cytosolic calcium-dependent signal- phosphate reabsorption, which is rapidly corrected by
ing pathway. In proximal tubule cells, PTH activates physiologic amounts of 1,25-dihydroxyvitamin D3. This
phospholipase C, which stimulates the production of effect is mediated by an increase in BBM Na-Pi co-trans-
diacylglycerol and IP3 (274); the latter induces an port (286) and is associated with a change in the lipid
increase in the cytosolic calcium concentration (275,276). composition of the membrane (287,288). Vitamin D
The formation of diacylglycerol and increase in cytosolic metabolites also stimulate renal phosphate reabsorption in
calcium result in activation of protein kinase C. In OK hypophosphatemic or phosphorus-deprived states, or when
cells, exposure to PTH activates phospholipase C, stimu- basal phosphorus excretion is increased, as occurs with
lates production of IP3, and inhibits Na-Pi co-transport volume expansion or administration of PTH. By contrast,
(274,277); activation of protein kinase C by phorbol vitamin D induces phosphaturia in hyperphosphatemic or
esters also is associated with inhibition of phosphate trans- phosphorus-replete states (124).
226 II. Homeostasis

Growth Hormone the mechanisms by which FGF-23 or other peptide factor(s)

Growth hormone acts to increase renal phosphate reabsorption, regulates renal phosphate handling or vitamin D metabolism,
independently of PTH (191,289). In growth hormone–defi- and whether such factors play a role in the physiologic regula-
cient subjects, the serum phosphorus concentration and the tion of phosphorus or vitamin D metabolism.
TmP/GFR ratio are reduced; both increase with administration Urine phosphorus excretion can be increased by expo-
of growth hormone (290,291). In patients with acromegaly, sure to the following: calcitonin, atrial natriuretic peptide,
serum phosphorus concentrations are increased (292). Growth glucose, glucagon, and glucocorticoids; urine phosphorus
hormone also increases GFR, renal plasma flow, and renal glu- excretion can be decreased by thyroid hormone and insulin
coneogenesis (289). Growth hormone stimulates proximal (reviewed in 127, 205, and 315). The mechanism of action
tubular Na-Pi co-transport (293–296) and is mediated, at least of such lipophilic substances as thyroid hormone, vitamin
in part, by increased production and release of insulin-like D, and glucocorticoids is unclear, although it is assumed
growth factor-1 (289,297). Receptors for growth hormone that genomic mechanisms are involved (316).
have been identified on the basolateral membrane of proximal
tubule cells and appear to activate the phospholipase C path-
Volume Status
way. Receptors for insulin-like growth factor-I also have been
identified in proximal tubule membranes, and their effects may Expansion of the extracellular fluid volume results in an
involve tyrosine kinase activity (289). increase, and volume contraction in a decrease, in urine
excretion of phosphorus (Table 10.2) (124,127). The effect
Phosphatonin can be attributed in part to changes in the filtered load of
Attention has focused on the role of a circulating humoral fac- phosphate and rate of phosphate reabsorption by the proxi-
tor(s), originally named phosphatonin (298), in the regulation mal tubule as well as to changes in plasma ionized calcium,
of serum concentration and renal handling of phosphorus. the latter affecting secretion of PTH. A direct effect of vol-
Three disorders, autosomal dominant hypophosphatemic ume expansion on tubular phosphate reabsorption also has
rickets (ADHR) (299–301), oncogenic hypophosphatemic been reported.
osteomalacia (OHO) (also known as tumor-induced osteo-
malacia) (302–304), and X-linked hypophosphatemia (XLH) TABLE 10.2. FACTORS AFFECTING RENAL
(305), each are characterized by hypophosphatemia due to PHOSPHORUS EXCRETION
renal phosphate wasting, inappropriately low or normal serum
Mechanism/
concentrations of 1,25(OH)2D, and rickets or osteomalacia Factor Pi excretion nephron site
(305). ADHR is caused by mutations in the FGF23 gene,
which encodes a novel secreted peptide that is processed to Diet
Volume expansion ↑ ↑ Filtered load of Pi,
aminoterminal and carboxyterminal peptides. Affected indi-
↓ proximal and distal
viduals harbor mutations in the peptide’s furin cleavage site, reabsorption
which prevent the processing of mutant fibroblast growth fac- Phosphorus restriction ↓ ↑ Proximal reabsorption
tor-23 (FGF-23) (306), presumably resulting in its accumula- Metabolism
tion in the plasma. FGF-23 is abundantly expressed in tumors Acidosis ↑ ↓ Tubular reabsorption
Alkalosis ↓ ↑ Tubular reabsorption
that cause OHO (307,308), and serum concentrations of
Hormones
FGF-23 are greatly increased in patients with this disorder and PTH ↑ ↓ Proximal and distal
also in patients with XLH (309,310). With surgical removal of reabsorption
the tumor, FGF-23 concentrations can decrease to normal val- Vitamin D (long term) ↑ ↓ Proximal reabsorption
ues and the disorder resolves. Because extracts from these Growth hormone ↓ ↑ Proximal reabsorp-
tion, ↑ GFR
tumors inhibit phosphate transport in renal proximal tubule
Calcitonin ↑ ↓ Tubular reabsorption
cells in vitro (298,311), it has been suggested that FGF-23 is Thyroid hormone ↓ ↑ Tubular reabsorption
responsible for this inhibition. In support of this hypothesis Insulin ↓ ↑ Proximal reabsorption
are the findings that recombinant FGF-23 inhibits phosphate Phosphatonin ↑ ↓ Proximal reabsorption
transport in renal proximal tubule cells in vitro (312) and that Diuretics
Mannitol, loop diuret- ↑ ↑ Tubular reabsorption,
administration of recombinant FGF-23 to mice induces hypo-
ics, thiazides site varies
phosphatemia and increased renal phosphate clearance (308). Other
FGF-23–treated mice also exhibit decreased serum concentra- Growth and develop- ↓ ↓ Proximal and distal
tions of 1,25(OH)2D (308,313,314) due to reduced renal ment reabsorption
1α-hydroxylase mRNA expression (308,314) and increased Glucose ↑ Osmotic diuresis, ↓ reab-
sorption PCT
24-hydroxylase mRNA expression (314). Taken together, these
Glucocorticoids ↑ ↓ Proximal reabsorption
findings suggest that a common circulating factor, perhaps
FGF-23, plays a central pathogenetic role in ADHR, OHO, ↑, increased; ↓, decreased; GFR, glomerular filtration rate; PCT, prox-
and XLH. Further study is required, however, to determine imal convoluted tubule; Pi, inorganic phosphate.
 10. Calcium and Phosphorus 227

Acid-Base Status phosphate is greater, both in the early PCT and in more
distal nephron segments, presumably the pars recta and
Changes in acid-base status can significantly effect the renal
segments beyond the DCT. In BBM vesicles, the Vmax for
handling of phosphate (124,270). Acute respiratory acidosis
Na-Pi co-transport is higher in newborn than in adult
results in a decrease in renal phosphate reabsorption; this
guinea pigs, a finding that cannot be accounted for by dif-
effect may depend on an increase in pCO2 tension, but it
ferences in plasma concentrations of phosphorus, ionized
does not depend on an increase in filtered load of phos-
calcium, PTH, thyroxine, or calcitonin. The higher capac-
phate, expansion of the extracellular fluid volume, or change
ity for phosphate reabsorption by the immature kidney
in PTH or blood bicarbonate concentration (317). Con-
also may reflect its relatively greater number of juxtamedul-
versely, acute respiratory alkalosis induces an increase in
lary nephrons, which have a higher capacity to reabsorb
renal phosphate reabsorption and resistance to the phospha-
phosphate. Newborn animals demonstrate a blunted phos-
turic action of both PTH and cAMP; these effects may
phaturic response both to phosphorus loading and to
depend on changes in pCO2 tension but are independent of
administration of PTH, the latter despite a normal increase
changes in plasma phosphorus concentration (318).
in urine cAMP. In phosphorus-restricted rats, the adaptive
Although acute metabolic acidosis has minimal effects on
increase in phosphate reabsorption is much greater in
urine phosphorus excretion (319), chronic metabolic acidosis
immature animals than in adults. Growth hormone may
can impair renal phosphate reabsorption independently of
play an important role in mediating the increased renal
PTH and even when dietary phosphorus is severely restricted
reabsorption of phosphate during development. The intra-
(320,321). The suppressive effect of metabolic acidosis on
cellular free phosphate concentration, as determined by
Na-Pi co-transport is demonstrable in BBM vesicles and is
phosphorus-31 nuclear magnetic resonance spectroscopy,
attributed to a decrease in the Vmax of the transporter (321).
is lower in the kidneys of growing rats than in those of
The suppression of phosphate reabsorption might be medi-
adult rats; it has been suggested that this finding is due to
ated, in part, by changes in luminal and intracellular pH.
age-related differences in cellular phosphate metabolism
Acute metabolic alkalosis induced by infusion of sodium
and results in part in an adaptive increase in tubular phos-
bicarbonate reduces phosphate reabsorption when the prior
phate transport (330,331). It has also been suggested that
dietary intake of phosphorus is high, but increases phosphate
most of the age-related changes in phosphate reabsorption
reabsorption when dietary phosphorus is normal (124,322–
can be explained by differences in BBM expression of the
325). Chronic metabolic alkalosis predictably increases renal
type II Na-Pi co-transporter (188). Thus, through a variety
phosphate reabsorption (270).
of mechanisms (329), phosphate handling by the imma-
Changes in the pH of luminal fluid can affect tubular
ture kidney is regulated so that phosphate retention is pro-
reabsorption of phosphate. In rat proximal tubules, phos-
moted, presumably to meet the increased needs for
phate reabsorption was stimulated by increasing luminal
phosphorus of the growing organism (329,332).
pH when luminal phosphorus concentration was low
(326). However, the effect of luminal pH on phosphate
reabsorption was reduced or abolished when luminal phos- Diuretics
phorus concentration was high (326), as occurs with intra-
Although their mechanisms and sites of action differ, the
venous infusion of phosphorus (327) or high dietary
following diuretic agents predictably induce phosphaturia:
phosphorus (328). These findings suggest that the sensitiv-
mannitol, acetazolamide, thiazide diuretics, and loop
ity of phosphate reabsorption to luminal pH depends on
diuretics. Urine phosphorus excretion is little affected by
both luminal and peritubular and/or intracellular phospho-
amiloride, spironolactone, and triamterene (127).
rus concentration (196). The effects of luminal pH on
phosphate transport observed in vivo are in good agreement
with those observed in isolated BBM vesicles.
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981. phosphate metabolism in the isolated perfused kidney of
313. Saito H, Kusano K, Kinosaki M, et al. Human fibroblast developing rats. Pediatr Nephrol 1990;4:392–398.
growth factor-23 mutants suppress Na+-dependent phos- 331. Barac-Nieto M, Dowd TL, Gupta RK, et al. Changes in
phate co-transport activity and 1α,25–dihydroxyvitamin NMR-visible kidney cell phosphate with age and diet: rela-
D3 production. J Biol Chem 2003;278:2206–2211. tionship to phosphate transport. Am J Physiol 1991;261:
314. Bai XY, Miao D, Goltzman D, et al. The autosomal domi- F153–F162.
nant hypophosphatemic rickets R176Q mutation in 332. Spitzer A, Kaskel FJ, Feld LG, et al. Renal regulation of
FGF23 resists proteolytic cleavage and enhances in vivo bio- phosphate homeostasis during growth. Semin Nephrol 1983;
logical potency. J Biol Chem 2003;278:9843–9849. 3:87–93.
 11

DISORDERS OF PHOSPHORUS,
CALCIUM, AND VITAMIN D
CRAIG B. LANGMAN

Patients who have disturbances in mineral homeostasis ities occur: (a) an inability to excrete phosphorus and (b) the
exhibit several different clinical pictures: a predominance of resultant hyperphosphatemia. Furthermore, the consequent
bone disease (rickets or hyperparathyroidism), the metabolic hyperphosphatemia does not lead to an appropriate suppres-
effects of disturbances in calcium (neurologic, musculoskele- sion of the production of 1,25(OH)2D, the active vitamin
tal disorders) or phosphorus (cardiovascular, hematologic D metabolite, and thus normal serum levels of the hormone
disorders), or evidence of phenotypic syndromes. Many of are present. The pathophysiologic basis of this disorder is
these disturbances result from or produce abnormalities in unknown but presumably involves abnormalities of tubular
renal tubular divalent mineral handling. phosphorus handling as well as of vitamin D metabolism.
In the evaluation of such patients, the clinician is expected Thus, affected patients have hyperphosphatemia, a normal
to perform serum and urine biochemical analyses that involve blood calcium level, and a reduction in urinary phosphorus
phosphorus, calcium, and vitamin D. Such blood measure- excretion. This is the mirror image of the serum and urine pro-
ments include levels of ionized calcium, serum total calcium, file of a patient with hypophosphatemic rickets. In tumoral
serum phosphorus, intact parathyroid hormone (PTH), and calcinosis, serum levels of intact PTH are suppressed to low
25-hydroxyvitamin D and 1,25-dihydroxyvitamin D [1,25 normal or frankly low values, as is nephrogenous cyclic ade-
(OH)2D]. Urinary studies may include quantitative determina- nosine monophosphate (cAMP) excretion. The physical-
tions of excretion of calcium, phosphorus, and other ions of chemical product of calcium × phosphorus is greater than 70,
interest (oxalate, citrate). The results of such biochemical evalu- and soft tissue calcifications occur. Of importance, a systemic
ations lend themselves to diagnostic schemes that involve the osteopenia is seen in such patients, despite the elevation of the
kidney in a primary or secondary manner. In what follows, product of blood phosphorus × calcium.
these diagnostic schemes are arranged by primary abnormality The calcifications are commonly located along the extensor
in serum calcium or phosphorus and are then further organized surfaces of major joints. Relief of symptoms often requires sur-
by pattern of urinary biochemical findings. Because the changes gical removal of the calcifications. Without adequate control of
in one component of mineral homeostasis often produce the underlying disease, recurrence is universal. Neural com-
abnormalities in the other components of the system, the bio- pression is common. The tumors can ulcerate the overlying
chemical findings of any given disorder can be approached in a skin, leading to draining sinus tracts and chronic infections.
number of different ways that are discussed later. Associated findings include anemia, splenomegaly, low-grade
The reader is referred to Chapter 10 for a discussion of fevers, and regional lymphadenopathy. In some patients, a
the normal physiology and renal handling of calcium and characteristic dental abnormality may occur. Although the dis-
phosphorus and to a published review of the physiology of ease is worldwide in distribution, in North America there is an
vitamin D (1). ethnic bias toward African Americans; there is no gender bias.
The treatment of patients with tumoral calcinosis has
been uniformly unrewarding. Simple surgical removal of
DISORDERS OF PHOSPHORUS HOMEOSTASIS calcifications provides temporary relief of painful symp-
toms. Radiation therapy and corticosteroid therapy have
Hyperphosphatemia Associated with not been successful. The use of phosphate-binding antacids
Reduced Urine Phosphorus Excretion and a low dietary intake of phosphate and calcium has met
with some success, but this regimen is generally difficult for
Tumoral Calcinosis
patients to follow.
Tumoral calcinosis (2–10) is a rare but disabling disorder of In an effort directly to promote phosphaturia and indi-
mineral metabolism in which two pathophysiologic abnormal- rectly to increase suppressed levels of PTH with consequent
238 II. Homeostasis

phosphaturia, we have used long-term calcitonin administra- TABLE 11.1. FEATURES OF THE AUTOIMMUNE
tion (5 U/kg subcutaneously initially, and then repetitively POLYGLANDULAR SYNDROME ASSOCIATED
WITH HYPOPARATHYROIDISM
but with increasing intervals between administrations) in
these patients. Using calcitonin therapy in this manner, com- Feature Frequency (%)
bined with dietary phosphate restriction and/or administra-
Addison disease (adrenal failure) —
tion of oral phosphate binders, we have prevented the Chronic mucocutaneous candidiasis —
recurrence of the tumors in six such patients over the past Alopecia totalis 38
several years. In addition, we have seen an increased bone Steatorrhea 30
density in nonaffected skeletal sites of such individuals. Primary hypogonadism 20
Pernicious anemia 15
Primary hypothyroidism 15
Hypoparathyroidism Chronic active hepatitis 12
Vitiligo —
Absence of PTH biologic action is associated with significant
hyperphosphatemia, hypocalcemia, and inappropriate renal
tubular retention of phosphorus. It may occur as an inherited polyglandular disease (Table 11.1). The disease is inherited
abnormality or be acquired through surgical removal of para- in an autosomal recessive manner, and the other organ dys-
thyroid gland tissue (for a general review, see reference 11). functions may occur years after the diagnosis of one com-
Idiopathic hypoparathyroidism begins in early childhood ponent of the syndrome. PTH antibodies occur in 30 to
and is part of the differential diagnosis of prolonged neona- 40% of cases. Associated clinical findings include the devel-
tal hypocalcemia. In the neonate, hypoparathyroidism is opment of cataracts, basal ganglion calcifications, and stea-
associated with aplasia of the glands, but beyond the neona- torrhea in more than 25% of cases. Repetitive diagnostic
tal age range, glandular hypoplasia is found instead. Idio- testing for the diseases associated with this syndrome is sug-
pathic hypoparathyroidism may be X-linked, especially gested once the diagnosis of autoimmune hypoparathyroid-
when it occurs in the neonate. The DiGeorge anomaly, a ism is made.
migrational disturbance in neural crest cells that results in Other rare causes of isolated hypoparathyroidism in
abnormal development of the structures arising from the children and adults may also be inherited, with patterns
third and fourth branchial pouches, including anatomic car- varying from autosomal recessive to autosomal dominant
diac defects and thymic dysplasia or absence, also is associ- (see earlier), with incomplete penetrances. Hypoparathy-
ated with a spectrum of abnormalities of the parathyroid roidism is a commonly encountered component of mito-
gland ranging from frank hypoparathyroidism to a nor- chondrial myopathies and associated renal (tubular and
mocalcemic disorder termed latent hypoparathyroidism. Both glomerular) dysfunction, which are now understood to
normocalcemic children and adults with the DiGeorge result from either deletions or mutations of mitochondrial,
anomaly may be unable to secrete PTH when faced with a maternally inherited genes related to electron transport
hypocalcemic stimulus such as an infusion of ethylenedi- (18,19). Such diseases include complete or partial absence
aminetetra-acetic acid (EDTA), which suggests that maxi- of cytochrome c oxidase, Kearns-Sayre syndrome (ophthal-
mal secretion of serum PTH is occurring from hypoplastic moplegia, retinitis pigmentosa, and heart block), MELAS
parathyroid tissue (12). Furthermore, children with isolated (mitochondrial encephalopathy, lactic acidosis, and stroke),
conotruncal cardiac defects have a high frequency of latent and MERRF (mitochondrial encephalopathy with ragged
hypoparathyroidism (13) associated with a 22q11 gene dele- red fiber myopathy). Hypoparathyroidism, transient or per-
tion commonly seen in DiGeorge anomaly (see reference 14 manent, is also an integral part of Kenny-Caffey syndrome,
for general review). Evolution of the latent hypoparathy- which is marked by both growth retardation and tubular
roidism associated with the 22q11 deletion into frank hypo- long bones with medullary stenosis (some children with
parathyroidism (hypocalcemia, hyperphosphatemia) has this disorder have been noted to have pseudohypoparathy-
been demonstrated across generations and over time within roidism; see below).
a generation (15).
Hypoparathyroidism (autosomal dominant, occasionally
Pseudohypoparathyroidism
sporadic) may result from heterozygous mutations (16) in
now-cloned extracellular calcium receptor, which sets a sta- Pseudohypoparathyroidism is characterized by peripheral
ble level of hypocalcemia (for a general review, see reference resistance to endogenously secreted PTH (for a general
17). Such patients appear to be uniquely sensitive to ther- review, see reference 11). It may be inherited in an autoso-
apy with calcium and vitamin D analogues and must be mal recessive, autosomal dominant, or X-linked manner. In
closely monitored for the complications of nephrocalcino- addition to the lack of appropriate phosphaturia to endoge-
sis, nephrolithiasis, and renal insufficiency. nously secreted PTH, patients have hypercalciuria, reduced
Hypoparathyroidism diagnosed in the latter part of the bony mobilization of calcium, and reduced renal produc-
first decade of life may be associated with autoimmune tion and subsequent serum levels of 1,25(OH)2D. Systemic
 11. Disorders of Phosphorus, Calcium, and Vitamin D 239

osteopenia is present, and despite the lack of classic renal been associated with hyperphosphatemia. The disorder is
responses to endogenous PTH levels, excess bony resorp- inherited as either an autosomal recessive or autosomal
tion does occur. dominant trait and has its onset in late childhood, often
Patients with pseudohypoparathyroidism may be classi- with asymmetric mandibular enlargement. Nasal bridge
fied by their response to exogenous administration of syn- enlargement and mild frontal bossing are common accom-
thetic PTH. Most have an absence of urinary excretion of panying signs; facial nerve palsies or neural deafness are
cAMP (type I pseudohypoparathyroidism) with administra- seen occasionally from nerve entrapment.
tion of PTH; a few (fewer than 10%) have an intact gener-
ation of cAMP (type II pseudohypoparathyroidism).
Type I pseudohypoparathyroidism is associated with a Hyperphosphatemia Associated with
characteristic clinical phenotype called Albright hereditary Increased Urine Phosphorus Excretion
osteodystrophy, composed of facial dysmorphism, shortened
Disorders of Phosphorus Distribution
metacarpals and metatarsals, short stature, mild mental retar-
and Systemic pH
dation, thickened cranium, and abnormal carrying angles at
the elbow, knee, and hip. Subcutaneous ossification may Hyperphosphatemia with resultant phosphaturia may occur
occur (and is seen with higher frequency in a disorder that because phosphorus is released from intracellular stores into
resembles Albright osteodystrophy phenotypically but has the extracellular fluid (ECF). This occurs commonly during
normal serum phosphorus, calcium, and PTH levels and is acute respiratory acidosis. Aggressive treatment of the elevation
termed pseudopseudohypoparathyroidism; frank hypoparathy- in serum phosphorus is best approached by recognition of
roidism may develop later in life in this rare disorder). The the systemic acid-base disturbance.
disturbance in Albright osteodystrophy is a generalized During the generation of acute metabolic acidosis, phos-
decrease in the function of the stimulatory guanine nucle- phorus is released into the ECF from intracellular stores,
otide–binding protein (Gs) present in all cells (20,21). The and phosphaturia results. However, during the mainte-
decrease in function may be secondary to a point mutation nance phase of metabolic acidosis, ECF phosphorus levels
in the α subunit of Gs (22). Because the G-protein signaling are often reduced, but phosphaturia continues.
system mediates the response of other hormones, it is not
unexpected that associated hormonal disturbances are seen in
Disorders of Phosphorus Distribution
patients with Albright osteodystrophy, including hypergona-
dotropic hypogonadism and hypothyroidism (23). Cell Breakdown
Hyperphosphatemia and phosphaturia occur commonly in
clinical diseases in which there is rapid cell lysis or turnover,
Acute Tubular Necrosis
including tumor lysis syndrome (most commonly seen with
The abrupt cessation of effective glomerular filtration is hematologic malignancies, Burkitt lymphoma, and meta-
associated with many pathophysiologic changes, including static breast or small cell lung carcinomas), rhabdomyolysis
hyperphosphatemia and, usually, hypocalcemia. Levels of (of traumatic and nontraumatic causes, Table 11.2), and
1,25(OH)2D are often reduced and, with the relief of oli- severe hemolytic anemias. The pathophysiology of the resul-
guria, may increase above normal (24). Nonoliguric acute tant abnormality in these diverse groups of diseases is the
tubular necrosis is also associated with hyperphosphatemia same: excessive egress of intracellular phosphate stores into
from insufficient urinary phosphorus excretion. the ECF compartment from cellular destruction exceeds the
kidney’s ability to excrete the released phosphorus load.
Commonly associated findings with either tumor lysis or
Chronic Renal Insufficiency
rhabdomyolysis are severe hypocalcemia and hyperkalemia.
Hyperphosphatemia occurs late in the course of chronic The latter results from the fact that potassium is the major
renal insufficiency, usually when the glomerular filtration intracellular cation released with cell breakdown.
rate (GFR) is less than 20% of normal. Functional neph- The increased cell metabolic and turnover rates that
rons filter more of the filtered phosphorus load, and thus occur in thyrotoxicosis or Addison disease are rare causes of
phosphaturia is common in early chronic renal insuffi- hyperphosphatemia.
ciency. However, as the GFR declines to less than 20%,
despite a low fractional reabsorption of filtered phosphorus, Increased Phosphorus Absorption
quantitative phosphorus excretion falls dramatically. Long-term use of the bisphosphonate etidronate for the
treatment of osteopenias of varying causes has been associ-
ated with mild hyperphosphatemia and phosphaturia. This
Hyperostosis
first-generational bisphosphonate can now be replaced with
Closely linked but pathogenetically distinct from tumoral more potent ones (amino-bisphosphonates) that have not
calcinosis is endosteal hyperostosis (25), which has also been associated with such biochemical findings.
240 II. Homeostasis

TABLE 11.2. CAUSES OF RHABDOMYOLYSIS ASSOCIATED WITH HYPERPHOSPHATEMIA AND PHOSPHATURIA

Hereditary Exertional Metabolic Toxic

Phosphorylase deficiency Anterior tibial syndrome Carbon monoxide poisoning Haff disease
Phosphofructokinase deficiency Status epilepticus Barbiturate, narcotic use Chronic alcoholism
Muscle carnitine palmitoyltrans- High-voltage electrical shock Hypothermia Heroin use
ferase deficiency
Malignant hyperthermia Agitated delirium Diabetic ketoacidosis, nonke- Malayan snakebite poison
totic hyperglycemic coma
Defect unknown, familial distri- Overexercise, often in exces- Systemic infection with fever K+ loss: glycyrrhizate, carbenox-
bution sively hot environment Idiopathic paroxysmal paralytic olone, amphotericin B associated
Crush injury myoglobinuria (Mayer-Batz
Coma (crush by own body mass) disease)

Infusion of inappropriate amounts of sodium or potas- sugar phosphate moieties. Such disorders include acute
sium phosphate salts during parenteral alimentation or intra- hyperventilation (such as with temper tantrums), sepsis,
venous fluid therapies may result in hyperphosphatemia heat stroke, or salicylate intoxication. In addition, patients
and phosphaturia. More common is the severe hyperphos- with chronic bronchospastic pulmonary disorders can
phatemia and phosphaturia caused by the use of sodium demonstrate hypophosphatemia and reduced urine phos-
phosphate enema preparations (26). Tremendous quantities phorus excretion during acute exacerbations.
of phosphate can be absorbed by the dilated colon, and the
use of such enemas in small children has been associated
with renal failure and death. Disorders of Phosphorus Distribution:
As part of the syndrome of vitamin D intoxication, Hormonal and Food-Fuel Effect
whether from the use of the parent compound or one of the The following hormones promote a primary transport of
available vitamin D metabolites, hyperphosphatemia and phosphorus from the ECF into the intracellular fluid
phosphaturia result. Such disturbances commonly have compartment: insulin, glucagon, androgens of adrenal or
associated hypercalcemia. The disturbance in phosphate testicular origin, and β-adrenergic agonists. Endogenous
appears to result in part from increased vitamin D–driven physiologic overproduction or pharmacologic administra-
dietary phosphate absorption. tion may produce the hypophosphatemia and associated
reduction in urine phosphorus excretion. Insulin aug-
Hypophosphatemia Associated with ments renal tubular reabsorption of phosphorus in addi-
Reduced Urine Phosphorus Excretion tion to directing extracellular phosphorus to move to the
intracellular compartment.
Phosphorus Deprivation In addition, infusions of specific food-fuel nutrients pro-
Dietary restriction of phosphorus, if severe, can lead to mote the same translocation of phosphorus into the intra-
phosphorus deprivation and elimination of urine phos- cellular compartment. Such agents include glucose, fructose,
phorus excretion. Severe decreases in phosphorus intake lactate, and amino acid salts. Two other agents closely related
may be simulated by the excessive use of phosphate-bind- to the food-fuels, glycerol and xylitol, also promote such
ing antacids or by the rare occurrence of an enteroenteric phosphorus movement.
fistula that leads to excessive stool phosphorus losses. The
clinical picture may be dominated by the accompanying
Increased Phosphorus Uptake into Bone
caloric or protein deprivations. An elevated serum cal-
cium level is common, and PTH levels are suppressed as a Two additional situations are associated with the produc-
result. Acutely, increased serum 1,25(OH)2D levels are tion of hypophosphatemia and a reduced urine phosphorus
present, but levels subside to subnormal after 10 to 14 excretion. After long-standing primary or secondary hyper-
days of dietary deprivation. parathyroidism is relieved, increased avidity of bone for cal-
cium and phosphorus accumulation is seen. This has been
termed the hungry bone syndrome and is commonly mani-
Disorders of Phosphorus Distribution
fest with both hypophosphatemia and hypocalcemia, which
and Systemic pH
may be symptomatic. Circumstances in which the hungry
Disorders associated with respiratory alkalosis cause an bone syndrome occurs include the immediately postopera-
acute shift of extracellular phosphorus into the intracellu- tive period after parathyroidectomy or after successful renal
lar space because alkalosis stimulates production of the transplantation in cases of previous long-standing secon-
enzyme phosphofructokinase and increases formation of dary hyperparathyroidism. However, the latter condition
 11. Disorders of Phosphorus, Calcium, and Vitamin D 241

may also be associated with a phosphorus-losing tubulopa- termed the PHEX gene for phosphate-regulating with homol-
thy as noted later. ogy to endopeptidase on the X chromosome (32). The disor-
The hungry bone syndrome has been observed also with der is now viewed as involving an abnormality of the
vitamin D treatment of long-standing vitamin D deficiency. In phosphaturic factor fibroblast growth factor-23 (FGF-23). If
this setting, long-standing secondary hyperparathyroidism PHEX is mutated, its natural substrate, FGF-23, is not inacti-
produces hypophosphatemia with phosphaturia. Over a short vated by proteolysis and can exert its phosphaturic action (33).
time after reintroduction of vitamin D, the biochemical pic- A study has documented the excessive levels of FGF-23 in this,
ture worsens with respect to the serum level of phosphorus, and several other, rachitic hypophosphatemic disorders dis-
whereas the excessive urine phosphorus excretion ceases. cussed later (34). It is not yet understood why there is a lack of
appropriately increased synthetic ability of the renal 1α-
hydroxylase enzyme that produces 1,25(OH)2D in the face of
Miscellaneous Conditions
such profound hypophosphatemia. Phenotypically normal
Chronic myelogenous leukemia in a proliferative blast crisis family members of affected children with rickets have normal
has been associated with hypophosphatemia and decreased serum phosphorus levels, but serum levels of 1,25(OH)2D
phosphorus excretion. In this condition, the increased ana- have not been systematically studied.
bolic state of white blood cells necessitates a large amount The untreated disorder in children results in generalized
of intracellular phosphorus transport. rickets and often linear growth failure (35); other features
Refeeding of a starved infant or child may promote appear in adults (36,37). Although there is controversy sur-
increased phosphate transport into the intracellular com- rounding the efficacy of treatment, most clinicians believe
partment and a reduction in phosphaturia. The hypophos- that frequent phosphate supplementation and pharmacologic
phatemia is rarely severe, unlike in the disorders mentioned dosing with 1,25(OH)2D are indicated (38). Despite the
earlier. large doses of phosphate used, hypophosphatemia remains,
and secondary hyperparathyroidism may be induced with
long-term phosphate salt administration (39,40). Thus, a bet-
Hypophosphatemia Associated with ter understanding of the need for phosphate is required. The
Increased Urine Phosphorus Excretion addition of 1,25(OH)2D has improved the healing of the
rickets (41,42). Recommendations for both phosphate ther-
Fanconi Syndrome
apy and 1,25(OH)2D therapy are based on clinical experi-
For information regarding hypophosphatemia and Fanconi ence. Neither the initial dosing nor subsequent modifications
syndrome, see Chapter 41. are based solely on measured serum levels of either phospho-
rus or the hormone, but rather on the clinical course of the
child with respect to rickets, linear growth, or both. A recom-
X-linked Hypophosphatemic
mendation for the starting dosage for toddler-aged children is
(Vitamin D–Resistant) Rickets
elemental phosphorus 1 to 3 g/day in four to five divided
Disorders of vitamin D metabolism and resultant rickets occur doses and calcitriol 30 to 70 ng/kg/day in two or three
commonly in children and are most often related to a simple divided doses (43).
deficiency of the parent vitamin. Lack of subsequent hepatic Two different murine models of the human disease exist.
production of the 25-hydroxyvitamin D substrate leads to In the affected Hyp mouse (44), the renal 25-hydroxyvita-
insufficient amounts of the active, renally produced vitamin D min D–1α-hydroxylase defect is pervasive; it does not
metabolite 1,25(OH)2D. This results in profound hypocalce- respond to PTH or phosphorus by increasing the produc-
mia, secondary hyperparathyroidism, hypophosphatemia, and tion of 1,25(OH)2D. Calcitonin does stimulate hormonal
rachitic bone disease. Provision of modest amounts of the par- synthesis, which suggests that the enzyme is capable of
ent compound, vitamin D, cures the disorder. being upregulated in this animal model (45). Hormone
The most commonly inherited form of rickets is X-linked catabolism also is increased in the Hyp mouse (46,47). In
hypophosphatemic rickets. Hypophosphatemic rickets in chil- the Gy mouse, however, only the PTH-independent arm,
dren is a systemic disorder in which renal phosphate wasting the response to hypophosphatemia, is blunted (48). Thus,
produces hypophosphatemia (27). Normocalcemia and nor- in response to dietary calcium restriction and subsequent
mal levels of PTH are demonstrated. The circulating levels of secondary hyperparathyroidism, the renal production of
the active vitamin D metabolite 1,25(OH)2D also are often 1,25(OH)2D increases in the affected Gy mouse (49).
normal (28,29). The “defect” in vitamin D metabolism in this Which murine model most resembles children with hypo-
disorder is the inappropriately normal levels of 1,25(OH)2D, phosphatemic rickets with respect to renal 1α-hydroxylase
because reduced serum phosphorus levels represent a potent is untested. Evidence from experimental animals suggests
stimulus for increased renal production of the active metabo- that the renal 1α-hydroxylase is regulated inversely by
lite of the vitamin D endocrine system (1,30,31). The gene for phosphorus during chronic metabolic acidosis, a disorder
X-linked hypophosphatemic rickets has been cloned and is characterized by phosphaturia, mild hypophosphatemia,
242 II. Homeostasis

“normal” basal levels of 1,25(OH)2D, and insufficient syn- severely affected nor are levels of 1,25(OH)2D as low as in
thetic capacity in response to PTH-stimulation but intact patients with X-linked hypophosphatemic rickets. FGF-23
response to phosphate restriction (50). Increasing the phos- abnormalities have been implicated in each.
phate concentration of the bathing medium increases the in Frymoyer and Hodgkin described a large kindred with
vitro production of 1,25(OH)2D in kidney tubules from X-linked renal phosphate wasting in whom bone disease
rats with chronic metabolic acidosis; tubules from nonaci- did not manifest as rickets but as osteomalacia (58). Adults
demic controls show the expected decline in production. showed progressive lower extremity bowing but were other-
Similarly, tubules from control animals increase production wise indistinguishable biochemically and clinically from
of 1,25(OH)2D during depletion of extracellular phospho- patients with X-linked hypophosphatemic rickets. Scriver
rus, whereas tubules from the acidotic rats show only basal et al. described autosomal dominant transmission of a simi-
production. The data for humans with metabolic acidosis lar disorder in a different kindred (59,60). Both disorders
are controversial (51,52). Interestingly, administration of have been called nonrachitic hypophosphatemic osteomalacia.
phosphate to the Hyp mouse increases peripheral receptors Phosphaturia, hypophosphatemia, and rickets or osteo-
for 1,25(OH)2D (53) and stimulates uptake of the hor- malacia have been described in a handful of patients with
mone by target cells in the intestine (54). fibrous dysplasia (monostotic or polyostotic, the latter in
McCune-Albright syndrome), epidermal nevus syndrome, or
other soft tissue tumors of mesenchymal origin (61) and has
Other Causes of Hypophosphatemic Rickets
been termed tumor-induced osteomalacia. This is likely a het-
Several other disorders of renal tubular phosphorus trans- erogeneous disorder at the level of renal 1α-hydroxylase,
port are associated with rickets similar to X-linked hypo- because levels of 1,25(OH)2D have been reported to be nor-
phosphatemic rickets. Hereditary hypophosphatemic rickets mal, elevated, or reduced; a minority of patients also have
with hypercalciuria (55,56) is a rare disorder in which the elevated serum PTH levels. Therapy with 1,25(OH)2D may
hypophosphatemia is associated with increased levels of heal or worsen the underlying bone disease, but it generally
1,25(OH)2D. In turn, PTH secretion is suppressed, both improves the hypophosphatemia. Excision of the offending
from an increase in calcium consequent to the elevated tumor, if possible, is curative. Again, FGF-23 has been impli-
levels of 1,25(OH)2D and from direct inhibition of prepro- cated in the pathogenesis (53,54).
parathyroid hormone gene transcription by 1,25(OH)2D.
It is presumed that the inhibition of PTH leads to the
Vitamin D–Dependent Rickets
hypercalciuria. The genetic transmission of the disorder
remains unknown at present. In the few reports of this dis- Children with the rare autosomal recessive disorder vitamin
ease, children first manifest rickets from the first year of life D–dependent rickets, type 1 present with classic vitamin D–
through the end of the first decade of life (55). There are deficiency rickets but do not respond to replacement dos-
also unaffected relatives, children or adults, who manifest ages of the parent vitamin D compound that are used to
only the hypercalciuria portion of the disorder (56). treat the deficient state. Rather, such children remain hypo-
Although clinically well, these unaffected relatives may phosphatemic and severely hypocalcemic. The disease arises
have some or all of the biochemical abnormalities of their from a primary deficiency in the renal 1α-hydroxylase
affected relatives, but with values closer to the normal val- enzyme that produces the active vitamin D metabolite
ues for age. Treatment consists of a high intake of phospho- 1,25(OH)2D (62). This is borne out by the lack of change
rus supplements alone. Unlike the disturbances in X-linked of serum 1,25(OH)2D levels when vitamin D or PTH is
hypophosphatemic rickets, the rickets in this disorder is administered to such patients (63). Children with the dis-
often healed by 6 to 9 months of therapy. Serum phospho- order have elevated levels of the substrate of that reaction,
rus levels increase and 1,25(OH)2D levels decrease to nor- 25-hydroxyvitamin D; children with deprivational rickets
mal. A study has documented that the kidney proximal have reduced levels of 25-hydroxyvitamin D, or it is absent.
tubular sodium–inorganic phosphate co-transporter is not Lifelong administration of the active hormone calcitriol is
mutated in this condition (57). curative for the disorder. The gene for renal 25-hydroxyvi-
Some adolescents and young adults develop hypophos- tamin D–1α-hydroxylase has been cloned (64,65) and
phatemic rickets at an advanced age, compared with the localized to 12q13.3. In patients with this disorder, inacti-
classic X-linked disturbance. There do not appear to be any vating mutations have been described in the gene, which
specific differences in the pathophysiology or clinical con- lead to absence of enzyme activity in experimental systems
sequences of this disease, termed adolescent hypophos- (64,66) and explain the virtual absence of circulating
phatemic osteomalacia, and the X-linked disorder discussed 1,25(OH)2D.
earlier. The treatment is the same and includes both phos- Since the original description in 1978 by Brooks et al.
phorus supplementation and administration of calcitriol. (67), fewer than four dozen patients have been reported with
There is, in addition, an autosomal dominant variety of the classic clinical and radiographic findings of deprivational
hypophosphatemic rickets. In general, final height is not as rickets but significantly increased serum levels of 1,25(OH)2D
 11. Disorders of Phosphorus, Calcium, and Vitamin D 243

and an associated resistance to the healing effect of adults with primary hyperparathyroidism; infants and tod-
1,25(OH)2D. Some patients also have alopecia (areata or dlers with primary hyperparathyroidism show diffuse glan-
universalis) or other ectodermal defects (oligodontia, milia, dular hyperplasia.
or epidermal cysts). This disease, vitamin D–dependent rick- Severe neonatal hyperparathyroidism may result from
ets, type 2, represents a disorder in which some aspect of the homozygous inactivating mutations of the extracellular cal-
function of the vitamin D receptor is abnormal. The patho- cium receptor (85,86) or even from the presence of only
genesis of the disturbance has been shown to involve one of a one abnormal allele of the gene (87). In both cases, severe
number of identified point mutations in the vitamin D hypophosphatemia and phosphaturia are present. Total sur-
receptor gene (68). Treatment consists of maintaining gical parathyroidectomy is curative, and, interestingly,
suprapharmacologic levels of 1,25(OH)2D for prolonged patients are often normocalcemic thereafter. (See Familial
periods (6 to 12 months minimum), which often requires Hypocalciuric Hypocalcemia.)
parenteral therapy with the hormone and administration of
oral or parenteral calcium. The response of the rachitic disor-
Miscellaneous Disorders
der to therapy probably depends on the exact defect in vita-
min D receptor function (69). Many clinical situations have been associated with hypo-
phosphatemia and phosphaturia, although the exact relation-
ship between the agents implicated and the biochemical
Idiopathic (Genetic) Hypercalciuria
findings is less certain than in the disorders described earlier.
A minority of children with idiopathic (genetic) hypercalci- These situations include chronic corticosteroid usage, the
uria (see later) have a primary renal phosphorus leak that diuretic phase of acute tubular necrosis or a “postobstructive”
leads to hypophosphatemia and phosphaturia in association diuresis, chronic glycosuria (but not the entities of renal glyco-
with a frank elevation in serum 1,25(OH)2D levels. In such suria), hypokalemic disturbances with a normal GFR, and
children the response to phosphorus supplementation is chronic alcohol abuse.
reduced hypercalciuria, normalized serum phosphorus levels,
and further exaggeration of urine phosphorus excretion.
DISORDERS OF CALCIUM HOMEOSTASIS

Renal Transplantation Hypercalcemia Associated

with Hypocalciuria: Familial
Several disturbances in phosphorus homeostasis may result
Hypocalciuric Hypercalcemia
after renal allograft transplantation (70–83). Hypophos-
phatemia and phosphaturia occur in 70% of all patients The autosomal dominant condition familial hypocalciuric
with successful renal allografts within the first year after hypercalcemia (85,86,88,89), best characterized as an
transplantation, sometimes without distinct elevation in abnormality in the physiologic set point for serum calcium
serum PTH levels. A distinct phosphorus-losing tubulopa- concentration, may begin in infancy or childhood with the
thy is less common and leads to a more pronounced hypo- development of hypercalcemia. The infant or child is gen-
phosphatemia indistinguishable from that of Fanconi erally asymptomatic, and the elevated blood calcium level is
syndrome. It is not associated with an elevation of serum unexpected for the clinical disease being evaluated. The
1,25(OH)2D levels. Most often, the serum PTH level is in exception is the homozygous infant in whom life-threatening
the upper range of normal to frankly elevated levels in this hypercalcemia and hyperparathyroidism have been reported
situation. Phosphorus supplementation may exaggerate the (see reference 90 and earlier discussion).
mild hyperparathyroidism, if present; if hyperparathyroid- The disease results most often from an inherited abnor-
ism is absent, it is the treatment of choice. mality of the extracellular calcium receptor, an inactivating
mutation of one of the two alleles of the gene (see earlier
and reference 91). The gene defect changes the relationship
Primary Hyperparathyroidism
between PTH release and serum calcium level; the sigmoid
Primary hyperparathyroidism (see reference 84 and later in curve describing that relationship is shifted to the right.
this chapter) is uncommon in the first decade of life but Thus, the calcium set point (i.e., the serum calcium level
increases in incidence thereafter. It may occur as an isolated that suppresses maximal PTH release by 50%) is increased.
finding and most commonly presents with nephrolithiasis Apparently, a similar set point for renal tubular calcium
in the first several decades of life. Primary hyperparathy- reabsorption is increased as well, which accounts for the
roidism may be associated with polyglandular endocrinopa- relative hypocalciuria in the face of an elevated serum cal-
thy type II, which is characterized by medullary carcinoma cium level. However, there may be additional abnormali-
of the thyroid, hypercalcitoninemia, and hypercalcemia. ties in the disorder. PTH levels remain inappropriately
There is an adenomatous transformation of one of the four normal despite the elevated serum calcium levels, and his-
primary glands in more than 85% of children and young topathologic abnormalities in the gland itself have been
244 II. Homeostasis

seen in up to 20% of patients who have frank hyperpar- fiber atrophy; peptic ulcer disease; pancreatitis; anemia;
athyroidism (92). alteration in consciousness from lethargy through coma;
The treatment of the disorder is one of recognition and nausea and vomiting; constipation; and clinical depression.
no specific therapy. Subtotal parathyroidectomies produce Primary hyperparathyroidism may occur as part of the mul-
persistent hypercalcemia, and the production of hypopara- tiple endocrine syndromes. In type 1 disease (hyperfunction of
thyroidism by total parathyroidectomy is of no benefit to the parathyroid, pancreatic, and anterior hypophysis cells),
the patient who is largely asymptomatic. Diagnostic clues 95% of patients develop hyperparathyroidism, whereas fewer
include a family history, absence of the autoimmune than 30% develop the associated prolactinoma or gastrinoma.
polyglandular syndromes, a ratio of urinary calcium to cre- In type 2A disease (hyperfunction of the cells of the adrenal
atinine (both values in milligrams) of 0.03 or less with ele- glands, parathyroid glands, and C cells of the thyroid glands),
vated serum calcium levels and serum magnesium levels hyperparathyroidism occurs in only one-third of cases, whereas
elevated between 3 and 4 mg/dL. the development of C-cell cancer of the thyroid is more com-
mon. In the latter group of patients, the occurrence of a pheo-
chromocytoma is associated with hypercalcemia in 15 to 20%
Hypercalcemia Associated of patients, independent of parathyroid gland pathology.
with Hypercalciuria In addition to the isolated disease and the disease associ-
ated with multiple endocrine syndromes 1 and 2, another
Primary Hyperparathyroidism
variant of primary hyperparathyroidism has been described,
Although primary hyperparathyroidism (84) is common in termed cystic parathyroid adenomatosis, which also indicates
adult patients with de novo hypercalcemia, it is unusual in the its pathology (94). The disorder is one of recurrent adenoma-
first decade of life and occurs most commonly in neonates. In tous change over time, but usually only in one gland at the
neonates and infants in the first 6 months of life, primary time of hypercalcemia. To date, there have been no reports of
hyperparathyroidism has been reported approximately 100 children with the disorder. Associated fibrous tumors of the
times, always associated with extreme elevations in serum cal- cheekbones are present in the patients described.
cium levels (often greater than 14 mg/dL) and marked symp- Diagnostically, primary hyperparathyroidism is character-
toms related to that elevation (93). The pathology in neonatal ized by an elevation in the level of circulating intact PTH in
hyperparathyroidism is diffuse glandular hyperplasia in more almost all patients, and an inappropriately high level of PTH
than 90% of reported cases; adenomatous changes in one or for the degree of hypercalcemia in all patients. Corroborative
more glands are responsible for the additional 10% of cases. findings include elevated nephrogenous cAMP levels, mildly
When primary hyperparathyroidism occurs in children elevated 1,25(OH)2D levels, hypophosphatemia with phos-
younger than 10 years of age, a search for an associated endo- phaturia, and mild renal tubular acidosis.
crinopathy syndrome should be made (see later). In the second The treatment of symptomatic primary hyperparathy-
decade of life, the incidence of primary hyperparathyroidism roidism in children is surgical with exploration of all four
increases dramatically (compared with younger ages) and may neck glands strongly recommended. Although “asymptom-
approach 1 in 1000 population. There does not seem to be a atic” primary hyperparathyroidism exists as an entity in
gender predilection among children and adolescents with pri- adults, it is not clear that this occurs in children. Therefore,
mary hyperparathyroidism, which contrasts sharply with the medical treatment of that disorder (phosphorus supple-
female predominance of the disease in adults. mentation, the use of bisphosphonates, and provision of sex
In addition to hypercalcemia and hypophosphatemia, steroids) is probably inappropriate for most children with
the disease has other major features that result from the primary hyperparathyroidism.
effects of elevated PTH levels on the skeleton and the kid- A new class of drugs, called calcimimetics (95), has been
ney. The skeleton shows classically subperiosteal resorption designed to interact within the transmembrane-spanning
in the distal phalanges, a “salt-and-pepper” appearance of domains of the extracellular calcium receptor and act allosteri-
the cranium, resorption and tapering of the clavicles, cally to reduce the activation of the receptor by calcium levels
brown tumor formation in the long bones, and bone cysts in the ECF. Such agents may have great promise in the treat-
as a result of chronic stimulation from PTH excess. In ment of primary hyperparathyroidism, hyperparathyroidism
addition, severe osteopenia, especially in cortical bone, is that occurs in the course of chronic renal insufficiency and
demonstrated by determination of bone mineral density. end-stage renal disease or after renal transplantation, and, per-
In the kidney, primary hyperparathyroidism is associated haps, in severe neonatal hyperparathyroidism. Treatment with
with calcium nephrolithiasis in up to 25% of patients in calcimimetic agents is also without study in children.
several series, a reduction in GFR, polyuria from an antidi-
uretic hormone–resistant concentrating defect, nephrocal-
Syndromic Hypercalcemia
cinosis, and systemic hypertension, especially with an acute
onset of the disease. Other systemic manifestations may Two important syndromes in infants and children produce
include a myopathy, now shown to be due to type II muscle hypercalcemia: Williams syndrome and idiopathic infantile
 11. Disorders of Phosphorus, Calcium, and Vitamin D 245

hypercalcemia. Williams described infants with supravalvu- with Williams syndrome, increased thirst, and the associ-
lar aortic stenosis, peculiar facies (“elfinlike”), and hypercal- ated problems of symptomatic hypercalcemia. The distinc-
cemia during the first year of life (96,97). Currently, it is tion between this disorder and Williams syndrome remains
thought that the disorder represents a spectrum of abnor- a bit problematic because some infants have cardiovascular
malities from the facial dysmorphism alone through all of anomalies similar to those seen in the Williams disorder.
the described abnormalities. A scoring system has been Other clinical manifestations of idiopathic infantile hyper-
developed to categorize the suspected disease in infants as calcemia include hypertension, strabismus, inguinal her-
lying within or outside the syndrome itself (98). Two-thirds nias, disordered posture, kyphosis, radioulnar synostosis,
of infants with the disorder have been small for gestational and dislocated patellae. Hyperacusis is seen commonly,
age and many are born past their expected date of birth. may be persistent, and is problematic.
Facial abnormalities include structural asymmetry, tempo- We have identified (111) more than a dozen infants and
ral depression, flat malae with full cheeks, microcephaly, young children with this disorder in whom an elevated level
epicanthal folds, lacy or stellate irises, a short nose with a of N-terminal PTH-related protein (PTH-rp) was demon-
long philtrum, an arched upper lip with a fuller lower lip, strated at the time of hypercalcemia. Furthermore, in seven
and small, maloccluded teeth. The vocal tone is hoarse. The of those children who achieved normocalcemia, the levels
children are affable and have been described as “cocktail of PTH-rp were normal or unmeasurably low, and in two
party–like” with unusual friendliness to strangers. Addi- children with persistent hypercalcemia, the levels of PTH-
tional cardiovascular abnormalities include other congeni- rp remained elevated. No other nonmalignant, hypercalce-
tal heart defects and peripheral organ arterial stenoses of mic disorder that we have investigated, including Williams
the renal, mesenteric, and celiac vessels. Hypercalcemia, if syndrome, includes elevated levels of the peptide.
initially present, rarely remains by the end of the first year The therapy for this disorder is normalization of the
of life, but hypercalciuria persists throughout childhood extremely high serum calcium level. We have not seen a
and adolescence. recurrence of the disorder once the calcium level is normal-
Several reports (99,100) have emphasized the renal ized, although the systemic manifestations noted earlier are
abnormalities demonstrated by patients with Williams syn- not changed.
drome, including urinary frequency, daytime urinary
incontinence, and some structural abnormalities, including
Hypercalcemia with Malignancy
hypoplasia or dysplasia.
The genetic defect for Williams syndrome is a heterozy- There are three general mechanisms whereby patients with
gous microdeletion of 7q11.23 and encompasses the elastin malignancy develop hypercalcemia: lytic bone metastases,
gene (101). Rarely, it may involve a defect of chromosome the ectopic production of PTH-rp, and the ectopic produc-
11 [del(11)(q13.5q14.2)] or even chromosome 22 [r(22) tion of 1,25(OH)2D. Because most children with cancer
(p11→q13)] (102). develop hematologic malignancies, few of which metastasize
The pathogenesis of the disturbance in calcium is to bone with enough tumor burden to produce hypercalce-
unknown. Studies have largely focused on abnormalities of mia, and few children develop the solid organ malignancies
vitamin D metabolism (103–106). Previous studies of associated with tumoral production of PTH-rp, the ectopic
affected children have demonstrated increased circulating production of 1,25(OH)2D is most often responsible for the
levels of 25-hydroxyvitamin D after vitamin D administra- hypercalcemia seen with leukemias and lymphomas in
tion, increased levels of 1,25(OH)2D during periods of infants and children. Occasionally, primordial mesenchymal
hypercalcemia but not normocalcemia, and diminished tumors produce an osteoclast-activating factor that may lead
levels of calcitonin during calcium infusions (107). to local, osteolytic hypercalcemia. Such factors include
The treatment is directed toward the associated abnor- interleukin-1 and tumor necrosis factor-α, which are potent
malities, especially hypertension and cardiovascular anoma- promoters of bone resorption. Treatment of the malignan-
lies. There are no data on the clinical effect of normalizing cies eliminates the hypercalcemia from the ectopic produc-
mildly elevated serum calcium levels, but the author has tion of hormones and cytokines (112).
anecdotal evidence that normalization of persistent, mild
hypercalcemia may improve the final neurologic outcome
Miscellaneous Disorders
of children with Williams syndrome.
Idiopathic infantile hypercalcemia was reported by Subcutaneous Fat Necrosis
Lightwood (108) in the early 1950s in England and was Michael et al. (113) reported on the association of signifi-
ascribed to vitamin D intoxication. Other cases have been cant birth trauma with fat necrosis in two infants who were
described subsequently in which maternal exposure to vita- small for gestational age and who subsequently developed
min D was not an issue, which thus led to the designation severe, symptomatic hypercalcemia. Histologic examina-
of the disorder as “idiopathic” (109,110). Affected infants tions of the violaceous pressure sites that developed revealed
generally have much higher serum calcium levels than those an inflammatory, mononuclear cell infiltrate and calcium-
246 II. Homeostasis

containing crystals. The author has noted such hypercalce- PTH, PTH-related peptide, and calcitriol. However, reports
mia in patients with subcutaneous fat necrosis associated of patients with Jansen syndrome have demonstrated that,
with major trauma or disseminated varicella. The mecha- secondary to one of several point mutations in the structure of
nism of the disorder is unknown but may be related to the PTH/PTH-rp receptor, ligand-independent activation of
minimally elevated levels of 1,25(OH)2D or excessive pros- the receptor and consequent biologic action occur in such
taglandin production from the tissue trauma site. individuals (119–121).

Granulomatous Disorders Hypophosphatasia

Of children with sarcoidosis, an autoimmune disorder, 30 to Infantile hypophosphatasia, a disorder in which serum alka-
50% manifest hypercalcemia, and an additional 20 to 30% line phosphatase activity is absent or significantly dimin-
demonstrate isolated hypercalciuria (114). Some of the pre- ished, is associated with hypercalcemia, hypercalciuria, and
senting signs and symptoms of children with sarcoid are radiological signs of rickets. The diagnosis is confirmed with
related to hypercalcemia. Twins with cat-scratch disease were demonstration of an increase in urinary phosphoethanola-
reported to develop symptomatic hypercalcemia associated mine excretion. See Whyte (122) for a comprehensive review
with that granulomatous disorder (115). In such disorders, it of this disorder.
is believed that expression of 25-hydroxyvitamin D–1α-
hydroxylase, or another mixed-function microsomal oxidase Other Causes of Hypercalcemia
capable of transforming 25-hydroxyvitamin D, is expressed Additional causes of hypercalcemia are uncommon in
and leads to unregulated production of 1,25(OH)2D and infants and children. For the sake of completeness, one can
consequent hypercalcemia. Treatment of the underlying dis- list the following: thyrotoxicosis, adrenal insufficiency,
order abolishes the supraphysiologic levels of 1,25(OH)2D VIPoma, leprosy, use of thiazide diuretics, lithium therapy,
and the associated hypercalcemia. vitamin A toxicity, and milk-alkali syndrome. Aggressive
treatment with calcitonin, and later with thiazide diuretics,
Limb Fracture has been shown to ameliorate the hypercalcemia often seen
Isolated fracture of a weight-bearing limb that requires in the infantile forms (123).
immobilization for even a few days may be associated with
hypercalcemia and hypercalciuria in young children or ado-
lescents (116). Their occurrence probably reflects the more Normocalcemic Hypercalciuria
rapid skeletal turnover in this age group than in adults.
Normal Levels of Calcium Excretion
Vitamin D Metabolite Therapy Studies of several populations of children of diverse ethnicity
Children with renal osteodystrophy are commonly treated have revealed a rather uniform degree of calcium excretion.
with calcitriol and develop hypercalcemia as a result once On a daily basis, calcium excretion above 3.5 mg/kg is con-
every 12 to 15 treatment-months per patient. Newer thera- sidered excessive (hypercalciuria) (124–127), although some
pies of pulse oral or intravenous 1,25(OH)2D may produce normative data showing higher excretion rates may reflect
hypercalcemia every 6 to 9 treatment-months per patient. the effect of a high dietary intake of sodium on calcium
The use of calcitriol to treat other mineral disorders is asso- excretion dynamics (128). Data from multiple studies of
ciated with hypercalcemia only one-third as often as is the adults and children with hypercalciuria have demonstrated
use of calcitriol or any other vitamin D metabolite to treat that hypercalciuria is likely present when a randomly voided
renal osteodystrophy. Treatment with the parent com- urine sample shows a calcium to creatinine ratio (both values
pound, vitamin D, may produce long-lasting hypercalce- in milligrams) exceeding 0.2 (or exceeding 0.5 for values in
mia if used in excess, because of lipid storage (117). millimoles). Values of the ratio for randomly collected speci-
mens of 0.15 to 0.2 (for measurements in milligrams) are
Jansen Syndrome highly suspicious for frank hypercalciuria and, in the author’s
Jansen syndrome presents in neonates with rickets and experience, signal quantitative hypercalciuria in more than
hypercalcemia and is a form of metaphyseal dysplasia. After 65% of children. However, the clinician is encouraged to
infancy, the radiographic picture becomes one of mottled perform complete 24-hour urine collections to determine if
calcifications in the long bones, which represent partially hypercalciuria is indeed present, because there is a significant
calcified cartilage protruding into the diaphysis. The skull incidence of false-positive results when ratios are used (129).
and spine may be affected in the neonate as well (118). Urinary calcium to creatinine ratios in neonates and
The biochemical picture of the affected infant or child infants younger than 3 months of age should be interpreted
with this disorder resembles that of those with primary hyper- with caution, because such ratios have been shown to
parathyroidism, but with some important exceptions: hyper- depend on both gestational age and source of milk protein
calcemia, hypophosphatemia, and hypercalciuria are present (commercial formula vs. human milk) (130,131). In prac-
but in the virtual absence of calciotropic hormones, including tice, the author does not use ratios for screening for hyper-
 11. Disorders of Phosphorus, Calcium, and Vitamin D 247

TABLE 11.3. DIFFERENTIAL DIAGNOSIS OF

NORMOCALCEMIC HYPERCALCIURIA
Genetic (idiopathic) hypercalciuria
Furosemide therapy
Corticosteroid therapy
Sarcoidosis
Immobilization
Hereditary hypercalciuria with hypophosphatemic rickets
CLCN5 chloride-channel mutation
Bartter’s syndrome
Seyberth syndrome
Prostaglandin E infusion
Vitamin D toxicity (early)
Limb fracture
Thyrotoxicosis
Distal renal tubular acidosis

FIGURE 11.1. Pathophysiologic schemes for the production of

normocalcemic hypercalciuria. The postulated “primary” defects in
calciuria in this population but proceeds to a formal genetic hypercalciuria are depicted in the lightly shaded boxes. It
can be seen that the two defects merge in the production of hyper-
collection. On formal collection, daily calcium excretion of calciuria (solid box). Up-pointing arrows represent increases in the
more than 5 mg/kg is viewed as hypercalciuria. associated physiologic process, and down-pointing arrows repre-
sent decreases in the associated physiologic process. Ca2+, blood
ionized calcium level; Ca, calcium; PTH, parathyroid hormone.
Genetic Hypercalciurias
Hypercalciuria may result from an X-linked, recessively
inherited disorder associated with one of several mutational a primary renal calcium leak as a single pathophysiologic
defects in a renal chloride channel (132) or may be familial mechanism for the disorder. The current view is that both of
without a gene abnormality that has yet been described. The these disease mechanisms occur in a child with genetic
latter entity has been called idiopathic hypercalciuria. Chlo- hypercalciuria. The pathophysiologic mechanism that domi-
ride-channel defects in the kidney have a wide phenotypic nates the clinical picture is wholly dependent on the dietary
expression, ranging from complete Fanconi syndrome with intake of calcium during the time diagnostic studies are per-
rickets, termed Dent’s disease (133), to a disorder that is mild formed. On a calcium-rich or calcium-sufficient diet, the
in childhood and is characterized by nephrolithiasis or neph- gastrointestinal disorder manifests easily, whereas at very low
rocalcinosis but that in adulthood appears to be progressive levels of dietary calcium intake, “renal” hypercalciuria per-
to chronic renal failure (134,135). tains. Alternatively, gastrointestinal absorption of calcium
The differential diagnosis of normocalcemic hypercalci- may contribute little to the final urinary calcium levels (143).
uria is shown in Table 11.3. Normocalcemic hypercalciuria Habitual dietary calcium intake determines the level of
that has been termed idiopathic is more prevalent than the 1,25(OH)2D found: normal with elevated calcium intake
chloride-channel defect (for a general review, see references and elevated during dietary calcium restriction (144). Simi-
136–138). It may occur in 7 to 10% of all children. The larly, secondary hyperparathyroidism may be seen during
inheritance of this disorder appears to be autosomal domi- dietary calcium restriction when the renal excretion of cal-
nant with incomplete penetrance (139). The disorder is cium is manifest (145) but may be absent during dietary cal-
associated with nephrolithiasis, recurrent gross and micro- cium sufficiency (146). The two predominant mechanisms
scopic hematuria, or otherwise unexplained pyuria, or solely of genetic hypercalciuria are summarized Figure 11.1.
with recurrent abdominal pain, and has been implicated in Thus, it remains unknown whether the primary distur-
the frequency-dysuria syndrome (140–142). bance in genetic hypercalciuria is at the level of the intestine
Hypercalciuria may be seen in patients who ingest an exces- or the kidney. Experimentally, the intestine may have an
sive amount of dietary sodium, and the physician is encour- abnormal sensitivity to 1,25(OH)2D, perhaps because of the
aged to define the level of salt excretion in the assessment of presence of a vitamin D receptor with a higher maximum
urine calcium excretion. Especially in the small group of chil- transport velocity than that in normocalciuric experimental
dren with genetic hypercalciuria who form kidney stones (per- animals (147). Alternatively, the renal 1α-hydroxylase may
haps 2 to 3% of this group), other urine abnormalities may produce more of 1,25(OH)2D than is needed for systemic
occur, including hypocitraturia in 25%, dietary hyperoxaluria calcium homeostasis. Lastly, perhaps the abnormality in
in 30 to 40%, and hyperuricosuria in 20%. genetic hypercalciuria results from a disorder in the meta-
The mechanisms of genetic hypercalciuria have been bolic clearance of 1,25(OH)2D.
debated intensely in the literature, with evidence for primary Until recently, it was not thought that bone calcium
overabsorption of calcium from intestinal sources vying with contributed to the urinary excess of calcium in patients
248 II. Homeostasis

with genetic hypercalciuria. Studies in adults with the Hyperprostaglandin Syndromes

disease and in children with genetic hypercalciuria have Several disorders of prostaglandin metabolism in infants
now convincingly shown that excessive bone resorption and children are associated with hypercalciuria, although
occurs (148–153). The bone loss is from both cortical the urinary finding rarely dominates the clinical picture.
and trabecular bone and is associated with serum markers These include Seyberth (hyper–prostaglandin E) syndrome
that indicate increased bone resorption (C. B. Langman, (161,162) and the use of prostaglandin infusions in neo-
unpublished data, 2002). Frank osteoporosis with fractur- nates with congenital heart disease. Some data implicate
ing bone disease has been seen in adults and children, excess urinary prostaglandin excretion in genetic (idio-
especially when the latter group is placed on a calcium- pathic) hypercalciuria (163,164).
restricted diet.
Treatment of genetic hypercalciuria is directed toward Distal Renal Tubular Acidosis
normalization of calcium excretion. In view of the tendency An important feature of distal renal tubular acidosis is the
for bone disease to be part of the disorder, dietary calcium attendant hypercalciuria that arises in part from an
restriction seems contraindicated for most infants, children, acquired defect in citrate metabolism secondary to the aci-
and adolescents. Whether treatment of any sort need be dosis (165,166). Correction of the hypercalciuria with ade-
given is dictated by the clinical picture and local customs. quate alkali supplementation is critical to achieve normal
In the author’s view, indications for therapy include stone growth in children with the disorder (167).
formation, documentation of severe osteopenia without
other cause, and symptomatic urinary findings. Treatment Juvenile Rheumatoid Arthritis
for gross hematuria alone in the absence of stone formation Children with clinically active juvenile rheumatoid arthritis
does not seem warranted, although in follow-up studies, it have hypercalciuria and osteopenia (168,169). A reduction
appears that children with gross hematuria from hypercalci- in disease activity improves both the osteopenia and the
uria had associated nephrolithiasis more often than did a hypercalciuria (170).
control population of hypercalciuric children without gross
hematuria (154).
If dietary sodium restriction does not ameliorate the Normocalcemic Hypocalciuria
hypercalciuria, an increase in dietary potassium may be
Magnesium-Losing Tubulopathy
initiated (155) in the absence of stone disease. If stone
(Gitelman’s Syndrome)
disease is present, the next therapy to institute is admin-
istration of thiazide diuretics in dosages used to treat Gitelman’s syndrome may result in a frank hypokalemic
mild primary hypertension. Such agents directly inhibit metabolic alkalosis or have a more limited clinical expres-
tubular calcium secretion in the cortical thick ascending sion in which renal magnesium wasting with hypo-
limb (156,157) and promote whole-body calcium reten- magnesemia occurs as an isolated entity (171) (see Chapter
tion as well (158). Thiazides may have direct effects on 38). Urine calcium excretion is generally under 0.5 mg/kg
bone mineralization, and these effects are currently body mass per day. The lack of even normal levels of urine
being studied in children and adults in several centers. calcium excretion is unexpected, because severe hypo-
Some concern has been raised about long-term use magnesemia inhibits the release or action of PTH. The dis-
because thiazides alter lipid metabolism in a proathero- order has an autosomal dominant inheritance, although
genic manner (159). incomplete phenotypic expression occurs commonly. Chil-
Other treatments for hypercalciuria unresponsive to thia- dren manifest severe hypokalemia but grow and develop
zide therapy alone may include the administration of supple- normally, unlike children affected with Bartter’s syndrome
mental alkali, usually in the form of citrate salts, and/or the
provision of neutral phosphate salts to limit dietary calcium
absorption. Careful attention to all components of the urine TABLE 11.4. CLINICAL FEATURES
related to nephrolithiasis is mandatory when multiple thera- OF HYPOPHOSPHATEMIA
pies for genetic hypercalciuria are undertaken. Acute hypophosphatemia Chronic hypophosphatemia

Muscle weakness Musculoskeletal complaints

Bartter’s Syndrome Paresthesias Paresthesias
See Chapter 38 for a discussion of Bartter’s syndrome, a Hyporeflexia Depressed reflexes
metabolic disorder. The overwhelming majority of infants Cranial nerve palsies Tremor
and children with Bartter’s syndrome are hypercalciuric Tremor Seizures
(160), and occasionally hypercalciuria is the presenting sign Confusion → coma Cardiac failure
Respiratory failure
of the disease. Hypercalciuria may be associated with the Hemolytic anemia
development of nephrolithiasis, but it is commonly associ- Rhabdomyolysis
ated with nephrocalcinosis.
 11. Disorders of Phosphorus, Calcium, and Vitamin D 249

TABLE 11.5. CLINICAL FEATURES REFERENCES

OF HYPERCALCEMIA
Lethargy → confusion → coma Hyporeflexia 1. Langman CB. Vitamin D physiology: normal and abnor-
Depression, paranoia Abnormal electroencephalo- mal. In: Strauss J, ed. Pediatric nephrology. Coral Gables,
Muscle weakness graphic pattern FL: University of Miami Press, 1989:81–105.
Constipation Nausea with or without emesis 2. Whyte MP. Tumoral calcinosis. In: Favus MJ, ed. Primer on
Reduced QTc interval on elec- Bradycardia the metabolic bone diseases and disorders of mineral metabo-
trocardiogram Polyuria with concentrating and lism. New York: American Society for Bone and Mineral
Systemic hypertension diluting defects Research, 1990:264–265.
Nephrocalcinosis Nephrolithiasis
3. Whyte MP. Metabolic and dysplastic diseases. In: Favus MJ,
Extraosseous calcifications Malignant venous thrombosis
Coe FL, eds. Disorders of bone and mineral metabolism. New
Headache
York: Raven Press, 1992:1019–1021.
4. Lyles KW, Burkes EJ, Ellis GJ, et al. Genetic transmission of
tumoral calcinosis: autosomal dominant with variable clini-
cal expressivity. J Clin Endocrinol Metab 1985;60:1093–
(172–174). Nephrocalcinosis is uncommon, and nephroli- 1096.
thiasis is slightly more common than in the general popula- 5. Mozaffarian G, Lafferty FW, Pearson OH. Treatment of
tion; stones formed are composed of oxalate. When the tumoral calcinosis with phosphate deprivation. Ann Intern
disorder presents in infancy or early childhood, it may be Med 1972;77:741–745.
6. Prince MJ, Schaefer PC, Goldsmith RS, et al. Hyperphos-
associated with symptomatic hypocalcemia. In late adoles-
phatemic tumoral calcinosis: association with elevation of
cence and adulthood, chondrocalcinosis may develop serum 1,25-dihydroxyvitamin D concentrations. Ann Intern
whether the biochemical abnormalities have been effec- Med 1982;96:586–591.
tively treated or not. 7. Lyles KW, Halsey DL, Friedman NE, et al. Association of
1,25-dihydroxyvitamin D, phosphorus, and parathyroid hor-
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139. Coe FL, Parks JH, Moore ES. Familial idiopathic hypercal- treatment of children with hypercalciuria: effects and side
ciuria. N Engl J Med 1979;300:337–341. effects. Pediatr Nephrol 1993;76(6):699–702.
140. Langman CB, Moores ES. Hypercalciuria in clinical pediat- 160. Restreppo de Rovetto C, Welch TR, Hug G, et al. Hyper-
rics. Clin Pediatr 1984;23:135–140. calciuria with Bartter syndrome: evidence for an abnormal-
141. Stapleton FB, Noe HN, Roy S, et al. Hypercalciuria in chil- ity of vitamin D metabolism. J Pediatr 1989;115:397–404.
dren with urolithiasis. Am J Dis Child 1982;136:675–678. 161. Seyberth HW, Rascher W, Schweer H, et al. Congenital
142. Alon U, Warady B, Hellerstein S. Hypercalciuria in the fre- hypokalemia with hypercalciuria in preterm infants: a
quency-dysuria syndrome of childhood. J Pediatr 1990;116: hyperprostaglandinuric syndrome different from Bartter
103–105. syndrome. J Pediatr 1985;107:694–701.
143. Welch TR, Abrams SA, Shoemaker L, et al. Precise determina- 162. Leonhardt A, Timmermanns G, Roth B, et al. Calcium
tion of the absorptive component of urinary calcium excretion homeostasis and hypercalciuria in hyperprostaglandin E
using stable isotopes. Pediatr Nephrol 1995;9(3):295–297. syndrome. J Pediatr 1992;120:546–554.
144. Stapleton FB, Langman CB, Bittle J, et al. Increased serum 163. Henriquez-La Roche C, Rodriguez-Iturbe B, Herrera J,
concentrations of 1,25(OH)2 vitamin D in children with et al. Increased excretion of prostaglandin E in patients
fasting hypercalciuria. J Pediatr 1987;110:234–237. with idiopathic hypercalciuria. Clin Sci 1988;75:581–
145. Moore ES, Langman CB, Favus MJ, et al. Secondary hyper- 587.
parathyroidism in children with idiopathic hypercalciuria. J 164. House M, Zimmerman B, Smith C, et al. Idiopathic hyper-
Pediatr 1983;103:932–935. calciuria associated with hyperreninemia and high urinary
146. Stapleton FB, McKay CP, Noe HN. Urolithiasis in chil- prostaglandin E. Kidney Int 1984;26:176–182.
dren: the role of hypercalciuria. Pediatr Ann 1987;16:980– 165. Norman ME, Feldman NI, Cohn RM, et al. Urinary citrate
992. excretion in the diagnosis of distal renal tubular acidosis. J
147. Favus MJ, Coe FL. Evidence for spontaneous hypercalciuria Pediatr 1978;92:394–398.
in the rat. Miner Electrolyte Metab 1979;2:150–155. 166. Restaino I, Kaplan BS, Stanley C, et al. Nephrolithiasis,
254 II. Homeostasis

hypocitraturia, and a distal renal tubular acidification defect osteopenia in children with juvenile rheumatoid arthritis. J
in type I glycogen storage disease. J Pediatr 1993;122:392– Pediatr 1993;122:693–696.
396. 171. Gitelman HJ, Graham JB, Welt LG. A new familial disorder
167. McSherry E, Morris RC Jr. On the attainment and mainte- characterised by hypokalemia and hypomagnesemia. Trans
nance of normal stature with alkali therapy in infants and Assoc Am Phys 1966;79:221–224.
children with classical renal tubular acidosis. J Clin Invest 172. McCredie DA, Blair-West JR, Scoggins BA, et al. Potas-
1978;61:509–527. sium-losing nephropathy of childhood. Med J Aust 1971;
168. Stapleton FB, Hanissian AS, Miller LA. Hypercalciuria in 1:129–135.
children with juvenile rheumatic arthritis: association with 173. Booth BE, Johanson A. Hypomagnesemia due to a renal
hematuria. J Pediatr 1985;107:235–239. tubular defect in reabsorption of magnesium. J Pediatr
169. Reed AM, Haugen M, Pachman LM, et al. Abnormalities in 1974;84:350–355.
serum osteocalcin values in children with chronic rheumatic 174. Evans RA, Carter JN, George CRP, et al. The congenital
diseases. J Pediatr 1990;116:574–580. “magnesium-losing kidney.” Report of two patients. J Med
170. Reed AM, Haugen M, Pachman LM, et al. The repair of 1981;50:197–209.
 12

NUTRITION AND METABOLISM

CORINA NAILESCU
PHYLLIS J. KASKEL
FREDERICK J. KASKEL

NUTRITION AND METABOLISM IN CHRONIC resistance, there is abnormal insulin release attributable to
KIDNEY DISEASE reduced adenosine triphosphate content in the pancreatic
islets, induced partially by high intracellular calcium, sec-
Influences of Chronic Kidney Disease on ondary to augmented parathyroid hormone (PTH)–
Nutrition and Metabolism induced calcium entry into cells (4,5).
Regarding protein metabolism, abundant evidence from
Poor nutrition adversely affects weight gain, growth, and,
cross-sectional analyses indicates that low values of serum
likely, development of children with chronic kidney disease
proteins and loss of muscle mass are commonly found in
(CKD). The etiology of poor weight gain in CKD is thought
patients with CKD (7). In addition to an inadequate diet,
to be multifactorial. There is evidence that most infants and
CMA, inflammation, and resistance to anabolic hormones,
young children with CKD have subnormal caloric intake (1).
Foreman et al. found that mean caloric intake was 80 ± 23% especially to insulin, contribute to abnormalities in protein
[mean ± standard deviation (SD)], of the recommended turnover and ultimately result in low-serum proteins (8).
dietary allowance (RDA) for age in these children (1). There Each of these factors stimulates protein breakdown in muscle
was no correlation between caloric intake and height velocity. and activates a common proteolytic pathway, the ubiquitin-
Calcium, vitamin B6, zinc, and folate intakes were also low. proteasome pathway (9–12). CMA stimulates amino acid and
The mean protein intake in these children was 153 ± 53% of protein catabolism in children with CKD by either a primary
the RDA. The same study suggested that the amount of effect of acidification or secondary effects due to changes in
energy per unit body weight (kcal/kg/day) consumed by chil- hormonal responses induced by acidification (13). For exam-
dren with CKD is comparable to that of age-matched ple, CMA causes resistance to insulin and impaired function
healthy children, but because of their small size, children of growth hormone (GH), thyroid hormone, and the con-
with CKD consume fewer total calories and have a lower version of vitamin D to its most active form, 1,25(OH)2-
%RDA. Despite the general use of supplemental feedings in cholecalciferol (14–16). Inflammatory proteins, such as
children with CKD, it is still not known whether these sup- acute-phase reactant proteins, tumor necrosis factor, and
plements consistently improve height or weight gain (2). interleukins (IL), have been found in excess in serum from
This review suggests that additional mechanisms may be adult hemodialysis (HD) patients (17,18). These inflamma-
responsible for the growth deficits in CKD. tory factors could cause excessive protein catabolism and
CKD creates a state of glucose intolerance (3). The ana- suppression of hepatic synthesis of albumin. Insulin defi-
bolic effect of insulin is blunted, including the glucose ciency causes loss of muscle mass and activates the ubiquitin-
metabolism, as are amino acid uptake into cells and the proteasome pathway to degrade muscle protein (19). Further
lipoprotein lipase (LPL) activity. Clinically, this state usu- characterization of the biochemical mechanisms that regulate
ally consists of fasting euglycemia but abnormal glucose the ubiquitin-proteasome and other catabolic pathways will
tolerance with a delayed decrease in blood glucose in be necessary to identify new strategies for preventing protein
response to insulin and hyperinsulinemia. The mechanisms deficits in CKD.
responsible seem to be multiple, but the most prominent Dyslipidemia increases the risk of cardiovascular events
metabolic disturbance in uremic patients is insulin resis- among individuals with CKD and contributes to the pro-
tance, mainly due to a postreceptor defect (4,5). Chronic gression of renal disease itself (20). The characteristics of
metabolic acidosis (CMA), frequently associated with dyslipidemia are different in non-nephrotic patients com-
CKD, contributes to insulin resistance, and its treatment pared with the nephrotic ones. Hyperlipidemias can occur
increases insulin sensitivity (6). In addition to the insulin in as many as 70% of the non-nephrotic patients with
256 II. Homeostasis

CKD, as opposed to 100% in the nephrotics (21). The proliferation of the fetal growth plate chondrocytes, inhib-
characteristic abnormality in plasma lipids in non-nephrotic its the differentiation of these cells into hypertrophic chon-
patients with CKD includes elevated triglycerides (TG) drocytes, and stimulates accumulation of cartilage-specific
accompanied by an increased concentration of very-low- proteoglycans that are thought to act as inhibitors of miner-
density lipoproteins (VLDL), decreased high-density lipo- alization (31). However, despite the high serum PTH
proteins (HDL), and normal to below normal levels of levels, there is “PTH resistance” at the level of growth
low-density lipoproteins (LDL), which represents a type IV plates, owing to intertwined factors affecting the PTH/
hyperlipoproteinemic pattern. In the nephrotic patients, PTH-related peptide receptors. This receptor’s mRNA
serum lipoprotein patterns are nearly equally distributed expression was found to be downregulated in the kidney
among types IIa, IIb, and V. The finding of low total cho- and growth plate of uremic rats, in the osteoblasts of
lesterol (TC) level should not necessarily be reassuring, patients with ESRD (32), and as a result of high-dose inter-
because it could be a marker of malnutrition associated mittent calcitriol therapy (33,34). On the other hand,
with increased mortality risk in HD patients (22). The PTH/PTH-related peptide expression is upregulated by
mechanism of this low cholesterol involves a defective GH and physiologic doses of calcitriol (33).
catabolism of TG-rich lipoproteins and decreased activity CMA may affect growth independently of uremia,
of both LPL and hepatic TG-lipase (23). In addition, CMA because diseases such as renal tubular acidosis are associated
and hyperinsulinemia depress LPL activity (24). Disor- with growth defects, and catch-up growth can be achieved
dered lipoprotein metabolism results from complex interac- when patients are given alkali therapy. When the blood pH
tions among many factors, including the primary disease is less than 7.25, length gain is diminished earlier than
process, use of medications such as corticosteroids, the weight gain. A reduction of weight gain is observed only for
presence of malnutrition or obesity, and diet. more severe acidosis with pH less than 7.20 (35). This find-
The most recent data regarding growth in children with ing suggests that the longitudinal growth of bone is more
CKD is provided by the North American Pediatric Renal sensitive to acidosis than are such factors as protein synthe-
Transplant Cooperative Study (NAPRTCS, 2001). Patients sis, which influences body weight. The mechanisms appear
with CKD are 1.4 SD below age- and gender-adjusted to be a profound in vitro–negative effect of CMA on the
norms for height. Adolescents (older than 12 years) have GH/IGF-1 axis, mainly through a downregulation of the
less severe height deficits (–1.01 ± 0.04) at baseline, relative receptors for both hormones (36) plus a stimulation of
to infants (–1.81 ± 0.07; younger than 24 months) and osteoclastic and a suppression of osteoblastic activity (37).
toddlers (–1.77 ± 0.05; age, 2 to 5 years). The height deficit
for females is marginally worse than for males, and patients
Nutritional Support for
with baseline glomerular filtration rate (GFR) less than the
the Predialysis Child
median of 36.5mL/min/1.73 m2 have more severe growth
retardation than the patients with higher GFR. Children Nutritional guidelines for children with chronic renal
with end-stage renal disease (ESRD) are, overall, approxi- insufficiency (CRI) before dialysis are not as well estab-
mately 1.6 SD below the appropriate age- and gender- lished as those for children on dialysis. However, recent
adjusted height levels at the time of dialysis initiation, and studies show that disturbances in nutritional intakes, bone
younger patients, in particular, have more severe growth biochemistry, and growth occur early in CRI and suggest
retardation. Although the weight deficits of dialysis patients the need for joint medical and dietary intervention in chil-
are not as severe as height, patients are, on average, more dren even with mild and moderate CRI (38).
than 1 SD below normal for weight. Children who are not yet on dialysis need nutritional
The GH/insulin-like growth factor-1 (IGF-1) axis plays intake to provide calories for both maintenance and catch-
a fundamental role in the growth process during childhood. up growth. The protein requirements are slightly greater
Although serum GH and IGF-1 levels are usually normal than for the normal healthy controls (39) (Table 12.1).
or high in growth-retarded CKD children (25), it was rec- Vitamin and trace element supplementation may be
ognized long ago that uremic serum has a high IGF-1 bind- indicated during the predialysis period if their estimated
ing capacity, resulting in low IGF-1 bioactivity (26,27). intake is considerably less than the RDA. Iron supplemen-
The mechanisms for this increased IGF-1 binding capacity tation is usually required in the predialysis period when
seem to be related to decreased renal clearance of IGFBP depletion of iron stores is documented by laboratory stud-
(IGF binding proteins), because serum levels of IGFBP-1, ies. Patients receiving erythropoietin generally require iron
-2, -4, and -6 were shown to correlate inversely with GFR supplementation owing to increased turnover of red blood
(28,29). In addition, uremia raises hepatic mRNA levels for cells, and require regular checking of serum iron levels.
IGFBP-1 and -2 by an unknown mechanism (30). Zinc supplementation is not routinely administered to chil-
Secondary hyperparathyroidism has significant effects dren during the predialysis phase, but because foods rich in
on longitudinal growth. High PTH levels are associated zinc are often limited owing to poor appetite, supplementa-
with mobilization of calcium from bone. PTH also causes tion by pharmacologic means on the basis of RDA stan-
 12. Nutrition and Metabolism 257

TABLE 12.1. PREDIALYSIS: FLUID AND NUTRIENT RECOMMENDATIONS FOR CHILDREN

Infant (0–1 yr) Toddler (1–3 yr) Child (4–10 yr) Adolescent (11–18 yr)

Energy 0–0.5 yr: ≥108 kcal/kg 102 kcal/kg 4–6 yr: 90 kcal/kg Girls 11–14 yr: 47 kcal/kg
0.5–1 yr: ≥98 kcal/kg 7–10 yr: 70 kcal/kg Girls 15–18 yr: 40 kcal/kg
Boys 11–14 yr: 55 kcal/kg
Boys 15–18 yr: 45 kcal/kg
Protein 0–0.5 yr: 2.2 g/kg 1.2 g/kg 4–6 yr: 1.2 g/kg 11–14 yr: 1.0 g/kg
0.5–1.0 yr: 1.6 g/kg 7–10 yr: 1 g/kg 15–18 yr: 0.9 g/kg
Sodium Generally unrestricted; 1–3 Generally unrestricted; 1–3 Generally unrestricted; 1–3 Generally unrestricted; 1–3
mEq/kg if edema or HTN mEq/kg if edema or HTN mEq/kg if edema or HTN mEq/kg if edema or HTN
present present present present
Calcium 0–0.05 yr: 400 mg/d 800 mg/d (provided hyper- 800 mg/d (provided hyper- 1200 mg/d (provided hyper-
calcemia does not occur calcemia does not occur calcemia does not occur
and calcium-phosphorus and calcium-phosphorus and calcium-phosphorus
product does not exceed product does not exceed product does not exceed
70) 70) 70)
0.5–1.0 yr: 600 mg/day
(provided hypercalce-
mia does not occur and
calcium-phosphorus
product does not
exceed 70)
Potassium 1–3 mEq/kg, if needed 1–3 mEq/kg, if needed 1–3 mEq/kg, if needed 1–3 mEq/kg, if needed (usu-
(usually not until GFR is (usually not until GFR is (usually not until GFR is ally not until GFR is <10%
<10% normal) <10% normal) <10% normal) normal)
Phosphorus Use low-content formula if Usually 600–800 mg/d Usually 600–800 mg/d Usually 600–800 mg/d when
serum levels of phos- when serum levels are when serum levels are serum levels are elevated
phate are elevated; elevated elevated
restrict high-content
foods
Vitamins 1 mL multivitamin drops; Multivitamin, if needed; Multivitamin, if needed; Multivitamin if needed;
vitamin D metabolites if vitamin D metabolites, vitamin D metabolite, if vitamin D metabolite if
needed, based on if needed, based on needed, based on needed, based on serum
serum calcium, PTH, serum calcium, PTH, serum calcium, PTH, calcium, PTH, and alka-
and alkaline phospha- and alkaline phospha- and alkaline phospha- line phosphatase levels
tase levels tase levels tase levels
Trace minerals Supplement zinc, iron, or Supplement zinc, iron, or Supplement zinc, iron, or Supplement zinc, iron, or
copper, if needed copper, if needed copper, if needed copper, if needed
Fluid Unrestricted unless Unrestricted unless Unrestricted unless Unrestricted unless needed;
needed; then replace needed; then replace needed; then replace then replace insensible +
insensible + urinary out- insensible + urinary out- insensible + urinary out- urinary output
put put put

GFR, glomerular filtration rate; HTN, hypertension; PTH, parathyroid gland.

Adapted from Nelson P, Stover J. Nutrition recommendations for infants, children and adolescents with end-stage renal disease. In: Gillit D, Sto-
ver J, eds. A clinical guide to nutrition care in end-stage renal disease, 2nd ed. Chicago, IL: American Dietetic Association, 1994:79–97.

dards may be recommended if there is reason to suspect treatment strategies in these children (41). An initial
zinc deficiency. Fluoride supplementation is provided only evaluation and ongoing monitoring of nutritional and
when the water supply is not satisfactory (40). growth status is a major component of providing opti-
mal care to children with ESRD. For children on chronic
renal replacement therapy, it is essential that the evaluations
Monitoring Energy and Protein Intake for
are done by experienced pediatric renal nutritionists with
the Child on Dialysis
skills in age-appropriate data collection, interpretation,
The outcome measures chosen for adult studies, mainly and counseling.
mortality rates, are not appropriate for children, who A thorough nutritional assessment should include obser-
generally have low mortality rates while being treated for vation of physical signs and symptoms, dietary intake
ESRD. Alternatively, complications unique to younger assessment using the 24-hour or 3-day dietary history, bio-
patients, such as impaired growth, puberty, or school chemical values, anthropometric measurements, body com-
performance, are better end points for evaluation of position data, and assessments of protein catabolic rate if
258 II. Homeostasis

available. The physical exam may lead to clues suggestive of amino acids lost per HD session have been reported. This
deficiencies of proteins and vitamins (42). loss is further influenced by the choice of membrane: Newer
The protein-caloric status in children with CKD is usu- synthetic polyacrylonitrile membranes have decreased losses
ally indirectly assessed by measuring albumin and transferrin, relative to cellulose membranes (48).
both of which are affected by inflammation, infection, mal- In adults, multiple studies have reported that a good
nutrition, acidosis, hormonal influences, liver disease, dialy- nutritional status predicts a better outcome in terms of
sis, and blood loss. More sophisticated measurements of mortality, morbidity, and rehabilitation (49,50). In pediat-
protein stores involve protein turnover studies and biochemi- rics, similar studies have not been done owing to the small
cal analysis of skeletal muscle and the nitrogen balance (43). number of patients treated and the relatively short period
Growth parameters in a pediatric patient on dialysis must of treatment before transplantation. Recently, data from the
be measured according to standardized protocols with consis- United States Renal Data System (USRDS) were used to
tent equipment and preferably performed by the same per- identify all patients under the age of 18 years who initiated
son. The most used parameters are weight, height, head dialysis between 1995 and 1998. This multivariate analysis
circumference (in those less than 3 years of age), body mass performed on almost 2000 pediatric patients demonstrated
index (BMI), mid-arm circumference, and skinfold thick- that each decrease in serum albumin of ~1 g/dL was associ-
ness, for which nomograms are available depending on age ated with a 54% higher risk of death. This finding was
and gender. Anthropometric measurements in relation to independent of glomerular causes of their CKD and other
nutritional status can be thought of predominantly as meth- potential confounding variables (51).
ods of assessment of protein-calorie malnutrition (44). Good dialysis adequacy is a prerequisite for good nutri-
Body composition data are generally used for research tional status in adult dialysis patients. Observations made on
purposes only, owing to the difficulty of standardization pediatric patients undergoing dialysis in 13 units throughout
(45). For determination of fat-free mass, the following the six-state New England region over a period of 18 months
methods can be used: total body water, total body potas- showed that vigorous dialysis, evidenced by a high Kt/V (urea
sium, total body nitrogen and calcium, bioelectrical imped- amount cleared from plasma divided by distribution volume),
ance, and electromagnetic scanning. Determination of did not appear to improve serum albumin concentrations in
body fatness can be done by densitometry, inert gas absorp- younger HD patients (52). However, most of these patients
tion, or near-infrared interactance. Neutron activation or were well dialyzed, and thus this finding is likely consistent
dual-energy x-ray absorptiometry can be used to determine with the adult studies. Thus, there appears to be little addi-
the bone mineral content (46). tional benefit in nutritional status once a given level of clear-
Nutrition evaluation and counseling are recommended at ance is attained. In a recent report of a combination of
the initiation of dialysis, ideally within the first week, and after enhanced HD clearance and nutritional supplementation, this
that, on an ongoing basis because of the dynamic nature of the regimen was associated with improved growth in prepubertal
child’s medical condition and food preferences. The maximum to early-pubertal HD children monitored for more than 2
time between such updates should be 3 to 4 months. Condi- years while receiving an average of 90.6% and 155.9% of their
tions that could dictate a more frequent evaluation of the recommended energy and protein intake, respectively. Average
nutrition plan of care include decreasing dry weight, ongoing weekly treatment time was 14.8 hours, with a urea reduction
decrease in oral intake, change in gastrointestinal function, sig- ratio of 85% and Kt/V of 2.0 per treatment, all well above cur-
nificant change in SD scores (SDS), elevated or suboptimal rent guidelines (53). On this regimen, the patients gained, on
laboratory values related to nutrients, ongoing excess interdia- average, more than 0.3 SD in height without GH and normal
lytic weight gain, concern for appropriate compliance with pubertal development was also described (53).
recommendations, change in psychosocial situation, or when Children on maintenance HD require an adequate caloric
placement of a tube for feeding is under consideration (42). intake not only for weight gain and growth, but also to avoid
Compliance with the nutrition prescription and recom- the use of proteins as an energy source. Current recommen-
mendations from other team members are important at any dations are that children treated with maintenance HD
age, especially in adolescents. Integrating the treatment should receive caloric intake according to the RDA for chro-
goals of the dietitian, social worker, child development spe- nologic age at the time of the initial evaluation. Furthermore,
cialist, nurse, and physician helps to maximize patient and ongoing adjustments should be made, depending on the
family adherence to the overall plan of care. child’s clinical and biochemical response (42).
Current recommendations regarding the dietary protein
intake (DPI) in patients on maintenance HD are that, ini-
Nutritional Support for the Child on
tially, DPI should be based on the RDA for chronologic age
Maintenance Hemodialysis
plus an additional increment of 0.4 g/kg/day to achieve a
Chronic HD results in dialysate loss of amino acids and positive nitrogen balance (42) (Table 12.2).
water-soluble vitamins. In addition, the HD procedure itself Children on maintenance HD often have an insufficient
has catabolic effects (47). In adult studies, up to 8 g of free intake of oral nutrients because of anorexia and dietary
 12. Nutrition and Metabolism 259

TABLE 12.2. HEMODIALYSIS: FLUID AND NUTRIENT RECOMMENDATIONS FOR CHILDREN

Infant (0–1 yr) Toddler (1–3 yr) Child (4–10 yr) Adolescent (11–18 yr)

Energy 0–0.5 yr: ≥108 kcal/kg 102 kcal/kg 4–6 yr: 90 kcal/kg Girls 11–14 yr: 47 kcal/kg
0.5–1 yr: ≥98 kcal/kg 7–10 yr: 70 kcal/kg Girls 15–18 yr: 40 kcal/kg
Boys 11–14 yr: 55 kcal/kg
Boys 15–18 yr: 45 kcal/kg
Protein 0–0.5 yr: 2.6 g/kg 1.6 g/kg 4–6 yr: 1.6 g/kg 11–14 yr: 1.4 g/kg
0.6–1.0 yr: 2.0 g/kg 7–10 yr: 0.25 g/kg 15–18 yr: 1.3 g/kg (males)
1.2 g/kg (females)
Sodium 1–3 mEq/kg, if needed Same as for predialysis Same as for predialysis Same as for predialysis
Calcium Same as for predialysis Same as for predialysis Same as for predialysis Same as for predialysis
Potassium Same as for predialysis Same as for predialysis Same as for predialysis Same as for predialysis
Phosphorus Use low-content formula if 600–800 mg/d 600–800 mg/d 600–800 mg/d
serum levels of phosphate
are elevated; restrict high-
content foods
Vitamins 1 mL multivitamin drops, 1 Multivitamin, 1 mg folic acid, B-complex vitamin containing B-complex vitamin containing
mg folic acid, and vitamin and vitamin D metabolites, 1 mg folic acid, 10 mg pyri- 1 mg folic acid, 10 mg pyri-
D metabolites (in most as needed doxine, 60 mg ascorbic acid, doxine, 60 mg ascorbic
cases) 5 mg pantothenic acid, 1.0 acid, 10 mg pantothenic
mg thiamin, 1.2 mg ribofla- acid, 1.5 mg thiamin, 1.7
vin, 3 μg vitamin B12, 300 μg mg riboflavin, 6 μg vitamin
biotin; 15 mg niacin; active B12; 300 μg biotin; 20 mg
form of vitamin D, as niacin; active form of vita-
needed min D, as needed
Trace miner- Supplement zinc, iron, or cop- Supplement zinc, iron, or cop- Supplement zinc, iron, or cop- Supplement zinc, iron, or cop-
als per, if needed. Iron is usually per, if needed. Iron is usually per, if needed. Iron is usually per, if needed. Iron is usually
needed with recombinant needed with recombinant needed with recombinant needed with recombinant
erythropoietin. erythropoietin. erythropoietin. erythropoietin.
Fluid Provide insensible + urinary Provide insensible + urinary Provide insensible + urinary Provide insensible + urinary
output + ultrafiltration output output output
capacity (if possible)

Data from Nelson P, Stover J. Nutrition recommendations for infants, children and adolescents with end-stage renal disease. In: Gillit D, Stover J,
eds. A clinical guide to nutrition care in end-stage renal disease, 2nd ed. Chicago, IL: American Dietetic Association, 1994:79–97; and K/DOQI Nutri-
tion in chronic renal failure. Am J Kidney Dis 2000;35:S112–S115.

restrictions. The first step to provide nutritional support is by infusion of the hyperosmolar solution causing rapid fluid
oral supplementation (54) (Table 12.3). This issue is most shifts from muscle cell to interstitium. A long-term compli-
important during periods of very high-energy requirements cation of IDPN is the possible development of abnormal
during the first 2 years of life and the adolescent years (55). liver function tests due to fatty deposition in the liver and
When oral supplementation fails, enteral feeding through a cholestasis. Therefore, the patients on IDPN require close
nasogastric or gastrostomy tube is commonly used and is monitoring in terms of glucose control, hepatic function,
successful in opposing growth retardation and achieving and lipid profile (58).
catchup growth in infants and young children (56,57). Pediatric IDPN indications and appropriate composi-
Intradialytic parenteral nutrition (IDPN) can be initi- tions are not well defined but involve the infusion of 5 to 6
ated if enteral feeding has failed (58). Morbidity and mor- mg/kg/min of glucose, 1.2 to 1.4 g/kg/day of protein, and
tality were lower in adult HD patients who received IDPN possibly the addition of intralipids (63).
(59,60), and criteria exist for IDPN in this population IDPN provides minimal supplementation, usually
(61). However, one study failed to demonstrate a beneficial between 500 and 1500 kcal and is administered only three
effect of IDPN (62). One study evaluating the use of IDPN times per week. In addition, only approximately 70% of the
in the pediatric population demonstrated a dramatic infused amino acids are retained owing to rapid clearance by
increase in oral caloric intake and eventual weight gain after HD (58). Thus, in patients who are severely malnourished,
3 months, secondary to the improved dietary status (63). IDPN could not provide sufficient nutrients, and total par-
Possible side effects of IDPN are hyperglycemia associated enteral nutrition (TPN) is indicated. However, in patients
with glucose infusion or rebound hypoglycemia when the with moderate malnutrition who are intolerant to further
infusion is suddenly terminated. A frequent complaint in oral supplementation secondary to anorexia, a short course
patients receiving IDPN is painful cramps in the arm con- of IDPN may improve nutritional status sufficient to permit
taining the fistula, an effect thought to be due to the rapid more physiologic oral supplementation (63).
260 II. Homeostasis

TABLE 12.3. FORMULAS FOR CHILDREN WITH RENAL DISEASE

kcal/ Protein [g/L Carbohydrate Na/K Ca/P (mg/ mOsm/kg
Manufacturer mL (sources)] Fat [g/L (sources)] [g/L (sources)] (mEq/L) L) water

Similac PM Ross Products 0.67 16 (whey, casein- 38 (soy, coconut) 69 (lactose) 7/15 380/190 250
60/40 ate)
Amin-aid R & D Laborato- 2 19 (free amino 46 (partially hydro- 365 (maltodex- <15/<15 –/– 700
ries acids) genated soybean trin, sucrose)
oil, soy lecithin,
mono- and diglyc-
erides)
Suplena Ross Products 2 30 (caseinates) 96 (high oleic saf- 255 (maltodex- 34/29 1430/730 600
flower and soy oil, trin, sucrose,
soy lecithin) cornstarch)
Renalcal Nestle Clinical 2 34 (essential L- 82 (MCT oil, canola 290 (maltodex- –/– –/– 600
Diet Nutrition amino acids, oil, corn oil, soy trin, modi-
select nonessen- lecithin) fied
tial amino acids, cornstarch)
whey protein
concentrate)
Nepro Ross Products 2 70 (caseinates) 69 (high oleic saf- 213 (corn syrup, 37/27 1370/685 665
flower and soy sucrose, FOS)
oil)

–/–, none; FOS, fructo-oligosaccharides.

Adapted from Winter HS, Madden J. Nutritional support of the chronically ill child. In: Lifschitz CH, ed. Pediatric gastroenterology and nutrition in
clinical practice. New York: Marcel Dekker, 2002:405.

In conclusion, short-term IDPN is a promising safe and in HD patients (68). Adequate water treatment, including
effective nutritional intervention in children on HD who reverse osmosis, prevents the accumulation of the major-
are not receiving sufficient nutrition from enteral feedings. ity of trace elements in HD patients. Zinc supplementa-
However, IDPN is quite expensive, and generally restric- tion may be recommended for patients with proven zinc
tive criteria prevent reimbursement. As soon as the patient deficiency, but its use in all HD patients is questionable
can tolerate an increase in oral intake or becomes a candi- (69,70). Selenium deficiency is to be suspected in dialyzed
date for tube feedings, enteral supplementation should be patients, and supplementation may be beneficial by
undertaken. increasing glutathione peroxidase activity, cardioprotec-
The practice of prescribing water-soluble vitamins for tive effect, and immunostimulatory properties (71).
HD patients has not been rigorously tested but probably
does little harm. There are losses of water-soluble vita-
Nutritional Support for the Child on
mins in the dialysate fluid, particularly ascorbic acid
Chronic Peritoneal Dialysis
(64). Vitamin B6 and folate are important supplements,
especially because they are useful in reducing the homo- Malnutrition in children receiving chronic peritoneal dialy-
cysteine levels (65). In view of the reports of peripheral sis (PD) has specific etiologies and treatments (72). Benefits
neuropathy and hyperoxalemia with high-dose vitamin of this modality, such as a more constant control of uremia,
B6 and vitamin C supplementation, megavitamin ther- a liberalization of dietary restrictions, and an additional
apy with water-soluble vitamins should be avoided (66). source of calories from the dialysate glucose absorption, are
Fat-soluble vitamins should be given only when there is counter balanced by losses of proteins, amino acids, vita-
a clear indication because of the risk of toxicity. Vitamin mins and trace elements in the dialysate, anorexia possibly
A levels are invariably increased in the plasma of CRI related to the pressure effect of dialysate in the abdomen,
patients, because retinol-binding protein is increased in and to the hyperglycemia effects of absorption of glucose
uremia (67). from dialysate (73). Finally, there is a catabolic effect
Trace elements are indispensable components of many induced by episodes of peritonitis (74,75).
enzymes, and abnormalities are primarily the result of As with HD, good nutritional status in PD is associated
uremia and the dialysis procedure. Plasma trace element with a better clinical outcome. Studies done in adults showed
concentrations in adult HD patients are distinctly differ- that markers of nutritional status, mainly albumin, correlate
ent compared to those of healthy controls. Elements such well with morbidity and mortality in patients on PD (76). In
as cesium, magnesium, molybdenum, and rubidium are children, it was shown that better nutrition correlates with a
reduced, and cadmium, cobalt, and lead are accumulated reduced rate of infections, particularly peritonitis (77). Inter-
 12. Nutrition and Metabolism 261

TABLE 12.4. PERITONEAL DIALYSIS: FLUID AND NUTRIENT RECOMMENDATIONS FOR CHILDREN
Infant (0–1 yr) Toddler (1–3 yr) Child (4–10 yr) Adolescent (11–18 yr)

Energy 0–0.5 yr: ≥108 kcal/kg 102 kcal/kg 4–6 yr: 90 kcal/kg Girls 11–14 yr: 47 kcal/kg
0.5–1 yr: ≥98 kcal/kg 7–10 yr: 70 gm/kg Girls 15–18 yr: 40 kcal/kg
Boys 11–14 yr: 55 kcal/kg
Boys 15–18 yr: 45 kcal/kg
Protein 0–0.5 yr: 2.9–3.0 g/kg 1.9–2.0 g/kg 4–6 yr: 1.9–2.0 g/kg 11–14 yr: 1.7–1.8 g/kg
0.5–1.0 yr: 2.3–2.4 g/kg 7–10 yr: 1.7–1.8 g/kg 15–18 yr: 1.4–1.5 g/kg
Sodium Same as for predialysis Same as for predialysis Same as for predialysis Same as for predialysis
Calcium Same as for predialysis Same as for predialysis Same as for predialysis Same as for predialysis
Potassium 1–3 mEq/kg, if needed (usu- 1–3 mEq/kg, if needed 1–3 mEq/kg, if needed 1–3 mEq/kg, if needed (usu-
ally not until GFR is <10% (usually not until GFR is (usually not until GFR is ally not until GFR is <10%
normal) <10% normal) <10% normal) normal)
Phosphorus Use low-content formula if Usually 600–800 mg/d Usually 600–800 mg/d Usually 600–800 mg/d when
serum levels of phosphate when serum levels are when serum levels are serum levels are elevated
are elevated; restrict high- elevated elevated
content foods
Vitamins 1 mL multivitamin drops, 1 Multivitamin, 1 mg folic B-complex vitamin contain- B-complex vitamin contain-
mg folic acid, and vitamin acid, and vitamin D ing 1 mg folic acid, 10 mg ing 1 mg folic acid, 10 mg
D metabolites (in most metabolites as needed pyridoxine, 60 mg ascorbic pyridoxine, 60 mg ascorbic
cases) acid, 5 mg pantothenic acid, 10 mg pantothenic
acid, 1.0 mg thiamin, 1.2 acid, 1.5 mg thiamin, 1.7
mg riboflavin, 3 μg vita- mg riboflavin; 6 μg vita-
min B12, 300 μg biotin; 15 min B12, 300 μg biotin; 20
mg niacin; active form of mg niacin; active form of
vitamin D, as needed vitamin D, as needed
Trace minerals Supplement zinc, iron, or cop- Supplement zinc, iron, or Supplement zinc, iron, or Supplement zinc, iron, or
per, if needed. Iron is usually copper, if needed. Iron is copper, if needed. Iron is copper, if needed. Iron is
needed with recombinant usually needed with usually needed with usually needed with
ethropoietin. recombinant ethropoi- recombinant ethropoi- recombinant ethropoietin.
etin. etin.
Fluid Provide insensible + urinary Unrestricted unless needed Unrestricted unless needed Unrestricted unless needed
output + ultrafiltration
capacity (if possible)

Data from Nelson P, Stover J. Nutrition recommendations for infants, children and adolescents with end-stage renal disease. In: Gillit D, Stover J,
eds. A clinical guide to nutrition care in end-stage renal disease, 2nd ed. Chicago, IL: American Dietetic Association, 1994:79–97; and K/DOQI
Nutrition in chronic renal failure. Am J Kidney Dis 2000;35:S112–S115.

estingly, cardiovascular disease is also a surprisingly common ments occur, whereas protein losses are inversely related to
cause of death in children on PD, and low serum albumin the patient’s weight and peritoneal membrane total area (73).
levels in children on dialysis have recently been shown to be During peritonitis, the permeability of the peritoneum for
associated with an increased risk of patient death (51,78). proteins and amino acids increases significantly by 50 to
Adequate dialysis may be an important factor for obtaining 100%. Between 100 and 300 mg of proteins/kg/day are lost
and maintaining adequate nutritional status and growth in the drained peritoneal dialysate, which translates to up to
(79,80). The delta height velocity of children with a mean age 10% of the total protein intake (73,75). The main protein
at initiation of dialysis of 28.5 months was found to be signifi- lost is albumin, but there are also losses of immunoglobulins,
cantly correlated with total creatinine clearance, residual GFR, transferrin, opsonins (84), and water-soluble vitamins, such
and residual Kt/Vurea (81). On the other hand, on a cautionary as vitamin B6, vitamin C, and folic acid (84,85).
note, Schaefer et al. have also described an important inverse In addition to the nonspecific factors contributing to
correlation between growth rates and creatinine clearance in decreased caloric intake in CKD, there are specific factors
children on PD, perhaps attributable to dialytic losses of an related to PD, such as the abdominal fullness from the dialy-
essential factor (82). The change in height SDS over 18 sate and the absorption of the glucose from dialysate.
months in children on PD revealed that high-transporter state Whereas the glucose absorbed from the dialysate provides cal-
and total dialysate volume had a negative effect, whereas higher ories, it also contributes to the anorexia seen in PD patients
dialytic creatinine clearance had a positive effect (83). Alterna- (54,86). The number of calories provided by dialysate glucose
tively, Kt/V greater than 2.75 in PD patients had no effect on absorption may be predicted by an equation applied in adults
nutrition but resulted in increased albumin losses (52). (87). Pediatric studies reported dialysate glucose absorption
Malnutrition in PD patients is multifactorial. Increased ranging from 9 to 18 kcal/kg/day, representing 7 to 15% of
losses of amino acids, water-soluble vitamins and trace ele- the total daily caloric intake, respectively (39).
262 II. Homeostasis

Current recommendations for energy intake in children dialysate glucose load, such as hyperlipidemia, excessive
treated on PD should follow the RDA for chronologic age, weight gain, and glucose intolerance, could be ameliorated
including calories derived from the dialysate glucose, and (94,95). Usually, one exchange per day is replaced with the
adjusted accordingly (42). Malnourished children, how- amino acid solution consisting of both essential and nones-
ever, will require “catchup” energy supplementation sential amino acids with electrolyte composition similar to
detailed by the American Academy of Pediatrics (88). Sup- that of standard dialysis solutions (96) (Table 12.5).
plemental g-tube feeding facilitates weight gain in infants Between 50 and 90% of the amino acids are absorbed
and older children receiving PD and arrests the decline in without any changes in ultrafiltration compared with stan-
height SDS traditionally observed in infants with CRI total dard dialysate (97). Side effects include a rise in blood urea
protein and albumin (89,90). Gastrostomy feedings via a nitrogen, metabolic acidosis, anorexia, and nausea (98,99).
button in children on PD significantly improved BMI (91). This promising nutritional intervention is still rather
Current recommendations regarding DPI in patients on expensive and should be reserved for malnourished patients
PD are also based on the RDA for chronologic age, to who fail more conservative nutritional support.
which a supplement based on anticipated peritoneal losses Dietary intake of water-soluble vitamins is lower than
should be added (42,73) (Table 12.4). Dietary recommen- the RDA in the majority of children on PD and supple-
dations for children on PD could be further defined using a mentation results in intakes that exceed the RDA
series of nitrogen balance studies. The correlation between (44,100,101). Hyperhomocysteinemia, an independent risk
estimated DPI and nitrogen balance indicates that a DPI of factor for cardiovascular disease in adults (102), is associ-
more than 140% RDA and a total energy intake of more ated with deficiencies of folate, vitamin B6, and vitamin B12
than 85% RDA are required to obtain an estimated nitro- (103). Elevated plasma homocysteine levels in pediatric
gen balance of at least 50 mg/kg/day, which is considered patients on PD were significantly reduced after administra-
adequate for metabolic needs in children (92). High bio- tion of 2.5-mg folic acid daily for 4 weeks (104). Supple-
logic value protein (e.g., meat, milk, eggs) should consti- mentation of trace elements is reserved for specific
tute 60 to 70% of the DPI (39). deficiencies such as zinc (72,105).
The use of special amino acid–based dialysis solutions in
children on PD may compensate for losses into the dialysate
(93). Furthermore, the potential complications related to the NUTRITION AND METABOLISM OF THE
ACUTELY ILL CHILD
TABLE 12.5. COMPOSITION OF AMINO ACID 1.1%
DIALYSIS SOLUTION (NUTRINEAL, BAXTER)
There is no conclusive evidence that acute renal failure
(ARF) per se increases energy expenditure (EE). However,
Essential amino acids (g/L) ARF is often associated with other organ system failures
Valine 1.39
that increase energy demands (106). Metabolic changes
Leucine 1.02
Isoleucine 0.85 occurring in such situations are different from the mecha-
Methionine 0.85 nisms associated with starvation alone. The optimal intake
Lysine 0.76 of nutrients in children with ARF is therefore influenced
Threonine 0.65 more by the nature of the disease causing ARF, the extent of
Phenylalanine 0.57
catabolism, and the modality and frequency of renal
Tryptophan 0.27
Histidine 0.71 replacement therapy, rather than by renal dysfunction itself.
Nonessential amino acids (g/L) The stress response is characterized by increased catabo-
Arginine 1.07 lism with subsequent release of large amounts of glucose,
Alanine 0.95 amino acids, and fatty acids from the body’s stores. These
Proline 0.60
metabolic changes are mediated by proinflammatory cyto-
Glycine 0.51
Serine 0.51 kines such as tumor necrosis factor-α and the ILs IL-1α,
Tyrosine 0.30 IL-1β, and IL-6. These changes have consistently been
Electrolytes (mmol/L) viewed as adaptive or beneficial, as they may reduce and
Sodium 132 redirect energy consumption, postpone anabolism, and at
Calcium 1.25
the same time activate the immunologic reaction (107).
Magnesium 0.25
Chloride 105 However, a prolonged stress response can trigger a sus-
Lactate (mmol/L) 40 tained and irreversible catabolic state mediated by release of
Osmolarity (mOsm/L) 365 cytokines and by activation of the hypothalamic-pituitary
pH 6.7 axis (108,109).
The mortality rate remains very high in patients with
Adapted from Schroder CH. The choice of dialysis solutions in pediat-
ric chronic peritoneal dialysis: guidelines by an ad hoc European
ARF, reaching between 40 and 80% in adults (110,111)
committee. Perit Dial Int 2001;21(6):568–574. and between 30 and 80% in children (112,113). In adult
 12. Nutrition and Metabolism 263

populations, a correlation between energy balance and sur- TABLE 12.6. EQUATIONS FOR PREDICTING BASAL
vival has been established (114). Although studies suggest- METABOLIC RATE AND ENERGY REQUIREMENTS
ing that nutrition improves outcome of ARF in children Age range (yr) Basal metabolic rate (kcal/d)
have not been documented, it is reasonable to suggest that
the complication rate is reduced, and recovery is enhanced Males
0–3 60.9 • weight – 54
by adequate nutrition. 3–10 22.7 • weight + 495
Assessment of the general nutritional status before the 10–18 17.5 • weight + 651
acute episode becomes very important. Studies performed Females
in adults report that a poor prior nutritional status places 0–3 61.0 • weight – 51
the patient at risk in terms of morbidity and mortality: 3–10 22.5 • weight + 499
10–18 12.2 • weight + 746
Malnourished patients have longer hospitalizations and Level of activity Activity factor
increased morbidity and mortality (115). Unfortunately, Confined to bed 1.2
surveys of hospitalized pediatric patients indicate a 20 to Sedentary 1.5
40% prevalence of protein-energy malnutrition (116). Normal 1.7
There are four primary goals in managing the nutri- Athlete 2.0
tional needs of children in ARF: maintaining adequate
Note: Energy requirement = basal metabolic rate × activity factor.
caloric intake, avoiding excessive protein intake to control Adapted from WHO. Energy and protein requirements: report of a
the rise in blood urea nitrogen, minimizing potassium and joint FAO/WHO/UNU expert consultation. WHO Technical Report
Series No. 724. Geneva: World Health Organization, 1985.
phosphate intake, and reducing fluid intake (117). Enteral
nutrition should be considered first, whenever possible,
because it also supports maintenance of the gut barrier and to REE than predicted by the formulas (126). Therefore,
may prevent bacterial invasion (106). If the patient is not direct measurement of EE is the only means currently avail-
able to ingest adequate intake, tube feeding can be intro- able for accurate determination of caloric requirements.
duced. When this is not possible, TPN becomes necessary. For practical purposes, where indirect calorimetry is not
If the child is oligo-anuric and sufficient calories cannot be available, one must rely on one of numerous methods that
achieved while maintaining appropriate fluid balance, an attempt to predict EE and, therefore, nutritional require-
earlier initiation of dialysis should be instituted. ments, based on age, weight, and/or height. Caloric
A hypermetabolic state occurs during critical illness in requirements are a summation of basal metabolic needs,
adults, characterized by an EE significantly greater than activity, and growth (Table 12.6). In healthy children, REE
that in the normal resting state (118,119). Recent studies accounts for approximately 50% of total EE, whereas activ-
suggest the same EE state exists in pediatric patients with ity and growth account for the remaining 50% of total EE
critical illnesses (120). In an effort to estimate the EE in (127). To predict energy requirements for critically ill chil-
stress situations, including ARF, one needs to determine the dren, one must first determine basal energy requirements
resting EE (REE) and the 24-hour EE (24h EE) for healthy and adjust for hypermetabolism (128) (Table 12.7).
children first. There are traditionally several methods for Patients with ARF have high protein catabolic rates. Sev-
estimating REE in children, the most commonly used eral studies done in adult populations reported between 1.4
being the World Health Organization and the Schofield and 1.8 g/kg/day protein catabolic rates (129,114). Another
weight and weight-height equations (121,122). Recently, a parameter that is used to measure the protein catabolic
study using the enhanced metabolic testing activity cham- state, urea nitrogen appearance (UNA), has been found to
ber, a new indirect calorimetry technology, was able to pro- be significantly elevated in children with ARF, with levels
pose new equations to estimate the REE and 24h EE in more than 180 mg/kg/day (130). A recent study of criti-
infants 0 to 7 months of age (123). According to this study, cally ill children demonstrated that protein turnover but
both the World Health Organization and the Schofield not lipolysis correlated with the severity of the critically ill
equations underestimated REE by greater than 15 kcal/kg/ condition (131).
day. The new proposed equations were within 4% of the In addition to the catabolic effect of the illness, there are
REE obtained by using the enhanced metabolic testing amino acid losses through dialysate solutions. Although the
activity chamber. amino acid losses in conventional HD and PD were dis-
Extrapolating from adult studies, the energy require- cussed earlier, it is worthwhile discussing the case of contin-
ments in critically ill children might be estimated by adding uous renal replacement therapy–related amino acid losses.
a stress-related correction to the REE (124,125). A recent Amino acid clearances and calculated losses in adults on
prospective study using indirect calorimetry on children continuous venovenous hemofiltration have been reported
admitted to an intensive care unit (postcardiac surgery, sep- between 2 and 11% of the dietary intake, whereas studies
sis, systemic inflammatory response syndrome) showed done for continuous venovenous HD found 8 to 22%
that EE predicted by the specific formulas grossly overesti- dietary loss (132,133). The ultrafiltrate volumes, as well as
mates the actual EE, which means that 24h EE was closer blood flow rates, have varied widely between these individ-
264 II. Homeostasis

TABLE 12.7. METHOD TO PREDICT METABOLIC RATES DURING

CRITICAL ILLNESS
Average hospital energy requirements

Body weight (kg) kcal/kg/day Increases in energy with stress

0–10 100 Fever 12% per degree Celsius >37°C

10–20 1000 + 50/kg Cardiac failure 15–25%
>20 1500 + 20/kg Major surgery 20–30%
Burns Up to 100%
Severe sepsis 40–50%

Adapted from Holliday MA, Segar WE. The maintenance need for water in parenteral fluid therapy.
Pediatrics 1957;19:823–832; and Wesley JR, et al., eds. Parenteral and enteral nutrition manual. Chicago:
Abbot Laboratories, 1980:17.

ual studies, making direct comparison among the treat- NUTRITIONAL AND METABOLIC CHANGES
ments difficult. The only study performed in children AFTER RENAL TRANSPLANTATION
reported amino acid losses of 11 to 12% of dietary intake.
The patients had similar blood and dialysate/prefiltered Metabolic disorders post–renal transplantation result from
replacement fluid flow rates, and there were equivalent a complex interaction of factors such as genetic susceptibil-
amino acid losses and urea clearances with no significant ity; medications; and physiologic changes related to
difference in regard to modality, continuous venovenous ischemic, immunologic, vascular, and mechanical injury.
hemofiltration, or continuous venovenous HD (134). A Although other chapters discuss the electrolyte imbalances
negative nitrogen balance occurred in those children that frequently occur after renal transplantation, this sub-
despite the delivery of standard TPN containing 1.5 g/kg/ chapter focuses on common derangements pertinent to
day of proteins and caloric intake of 20 to 30% above REE. lipid and carbohydrate metabolism.
It is possible to estimate the protein requirements by
measuring the protein equivalent of total nitrogen appear-
Posttransplant Hyperlipidemia
ance (PNA), which is an estimate of the protein losses
(135). When the patient is in nitrogen balance, PNA Hyperlipidemia is a risk factor for cardiovascular disease in
should be equal to the protein intake. The PNA can be adult transplant patients (136–139) and may also cause
derived from the UNA by using the Bergström formula: chronic allograft nephropathy (140), whereas no data exist
in the pediatric transplant population.
PNA [g/day] = 20.1 + 7.5 UNA [g/day]
The prevalence of hyperlipidemia is highest immediately
The UNA results from measuring the urea nitrogen content posttransplant, declining to 30% after 5 years (139). A lon-
of all measurable outputs (urine, dialysate or ultrafiltrate, fis- gitudinal evaluation of pediatric renal transplant recipients
tula drainage) plus the change in body urea nitrogen: showed that the prevalence of elevated TC and TG declined
from 70 to 35% and 46 to 15%, respectively, between 1
UNA [g/day] = output urea nitrogen +
and 10 years posttransplant, most likely as the doses of
change in body urea nitrogen
immunosuppressive agents were decreased (141).
Where change in body urea nitrogen [g/day] is given by: Causes of post–renal transplant hyperlipidemia include pre-
existing hyperlipidemia, especially with a familial history; med-
(SUNf – SUNi)[g/L/day] × BWi[kg] × 0.6[L/kg] +
ications (immunosuppressives, diuretics, and beta blockers);
(BWf – BWi)[kg/day] × SUNf[g/L] × (1 L/kg)
graft dysfunction; older age; male gender; obesity; and hypoal-
where i and f are the pre- and postdialysis moments in buminemia secondary to nephrotic syndrome (142). Risk fac-
time, SUN is serum urea nitrogen, BW is the body weight, tors for high TC in transplanted children on cyclosporine A
0.6 is an estimate of the total body water and 1 L/kg is the (CsA) include preexisting hyperlipidemia plus a family history
volume of distribution of urea in the weight that is gained and, unlike the adult studies, worsen with time (143). In the
or lost. same study, elevated TG was associated with reduced GFR.
The PNA is normalized to body weight to obtain the The specific lipid profile abnormalities posttransplant
nPNA, which is expressed as g/kg/day. include increased TC, LDL, VLDL, and apolipoprotein B
In addition to the previously described particularities of levels. HDL levels can be low, normal, or slightly elevated.
ARF in regard to the energy intake and protein metabolism, The chronic use of immunosuppressive medications is
such children also need daily intake of lipids, minerals, and an important determinant of hyperlipidemia in posttrans-
vitamins, according to the normal daily requirements for age. plant children. The most commonly used medications, cor-
 12. Nutrition and Metabolism 265

ticosteroids and CsA, were clearly reported to produce posttransplant year and an increase in mortality in the sec-
hyperlipidemia. Tacrolimus seems to have a less prominent ond year (159). PTDM in adult post–renal transplant
effect, whereas sirolimus has a more pronounced effect. patients worsened mortality (160).
Corticosteroids were shown to be associated with The incidence of PTDM in adult kidney transplant
increased TC and TG levels (144). They enhance lipogene- recipients ranges from 4 to 41% (161,162). The very wide
sis by increasing the activity of acetyl–coenzyme A carboxy- range may be due to the lack of a standard definition of
lase and free fatty acid synthetase, thus increasing hepatic PTDM, as some patients have transient hyperglycemia
synthesis of VLDL, downregulating LDL receptor activity, after transplant, and other patients have persistent DM. In
and increasing the activity of 3-hydroxy-3-methylglutaryl pediatric populations, the prevalence of PTDM parallels
coenzyme A reductase. Concurrently, they induce insulin that of type 2 DM (163,164). A comprehensive study of
resistance resulting in decreased activity of LPL, thus rais- pediatric renal transplant recipients reported a 7% preva-
ing TG. Finally, the corticosteroid-induced decrease in lence of PTDM (serum glucose >200 mg/dL) (165).
adrenocorticotropic hormone release may contribute to the In adults with PTDM, risk factors include the type of
lipid abnormalities, because adrenocorticotropic hormone immunosuppressive regimen, ethnicity, age, BMI, family
upregulates LDL receptor activity (142). Withdrawal of history of DM, episodes of rejection, and HLA type
steroids posttransplantation significantly reduces TC and (166,167). PTDM in pediatric renal recipients occurred
LDL levels (145). However, there is a simultaneous within 1.2 years posttransplant (range, 1 day to 6.2 years),
decrease in HDL levels, which may decrease the HDL:cho- especially with a family history of type 2 DM, tacrolimus
lesterol ratio. Replacement of low-dose oral methylpred- use versus CsA (odds ratio, 9.1), and hyperglycemia in the
nisone with deflazacort resulted in decreased TC and LDL immediate posttransplant period (165).
and increased HDL in pediatric patients (146). Corticosteroids increase insulin resistance in peripheral
CsA is associated with increased TC and increased LDL, tissues by decreasing insulin receptor number and function,
whereas HDL and TGs are not strongly affected (147). CsA increasing hepatic glucose output, decreasing glucose
is highly lipophilic, and up to 80% is transported in the uptake in muscle, and increasing lipolysis and free fatty
blood mostly in the core of LDL particles (148). Its hyper- acid release from adipose tissue (161). In addition, cortico-
lipidemic effect is due to several known mechanisms. It steroids may inhibit insulin secretion by the pancreatic beta
binds to the LDL receptor and raises LDL levels (149). In cells (168).
addition, CsA increases hepatic lipase activity and decreases Tacrolimus has been clearly associated with PTDM in
LPL activity, resulting in impaired clearance of VLDL and children (169). It may affect beta-cell function and periph-
LDL (142). On the other hand, like the corticosteroids, eral insulin resistance and diminishes insulin secretion
part of the hyperlipidemic effect seems to be secondary to (170,171). Tacrolimus reversibly suppressed insulin gene
the insulin resistance. Tacrolimus has an effect on lipid transcription in normal rats (172). Furthermore, tacrolimus
metabolism similar to that of CsA, but it seems to be less damages beta cells directly and possibly irreversibly (173).
prominent (150,151). Sirolimus is associated mainly with Beta-cell antibodies were transiently found in the setting of
high TG and, to a lesser extent, elevated TC. The mecha- PTDM during tacrolimus use (174). Thus, in patients with
nisms of sirolimus-induced hyperlipidemia have not been a strong family history of type 2 DM or hyperglycemia in
completely elucidated, and the effect is dose dependent and the immediate posttransplantation period, tacrolimus
reversible (152). should be used cautiously (165). CsA also inhibits insulin
Lipid-lowering agents, such as fluvastatin, are effective secretion from beta cells and decreases glucose disposal by
in adult transplant recipients (153–155). A prospective 12- similar mechanisms.
week intensive dietary intervention study (American Heart
Association step 2 diet) was effective in reducing hyperlip-
idemia in postrenal transplant children (156). Among pedi-
INFLUENCES OF NUTRITION DISORDERS ON
atric cardiac transplant recipients, pravastatin was effective
RENAL FUNCTION
and well tolerated (157), whereas atorvastatin also effec-
tively lowered lipids but was associated with rhabdomyoly-
Influences of Obesity on Renal Function
sis and asymptomatic creatine kinase elevation (158).
Obesity was found to be an important risk factor for a cer-
tain number of diseases, including cardiovascular diseases,
Posttransplant Glucose Intolerance
hypertension, lipid and lipoprotein abnormalities, type 2
Hyperglycemia and posttransplant diabetes mellitus (PTDM) DM, nonalcoholic steato-hepatitis, pseudotumor cerebri,
are well-known complications of organ transplantation, obstructive sleep apnea, and orthopedic problems such as
which worsen graft outcome and increase patient mortality. Blount disease (175–177).
PTDM in adult liver transplant recipients results in an It is a problem of major concern that the prevalence
increase in the number of rejection episodes in the first and severity of obesity is increasing in the pediatric popu-
266 II. Homeostasis

lation. As a consequence, the prevalence of the sequelae of stress, leading to glomerular cell proliferation, matrix accu-
obesity is expected to be increasing as well. The last large mulation, and glomerular sclerosis.
investigation done by the National Health and Nutrition Hyperlipidemia is another potential contributing factor,
Examination Survey (NHANES III) reported a prevalence because the Zucker obese rat, a model in which FSGS
of childhood obesity of 25% in the United States, defined develops, is hyperlipidemic, and reduction in serum lipids
by a BMI of more than 95% for age and gender (178). appears to decrease or prevent development of FSGS (196).
This prevalence represented a 30% increase comparative A study of autopsies in obese adults noted that those with
to a previous similar investigation, NHANES II. The FSGS had higher lipid levels and showed lipid deposits in
same trend has been observed in Western Europe in the renal tubular epithelial cells (185).
past 30 years. Renal venous hypertension has been incriminated as
The association of obesity with proteinuria has been well another contributing factor to obesity-related FSGS. One
described in adults (179–181). Despite the prevalence of study demonstrated that adult obese patients had increased
childhood obesity, an association between obesity and renal plasma volume and increased right atrial pressure, which
abnormalities such as proteinuria has not been clearly decreased with weight reduction along with parallel decreases
reported until very recently (182). Obesity-associated pro- in urinary protein losses (179). Other conditions also associ-
teinuria in adults is associated with focal segmental glomer- ated with increased right atrial pressure and presumed renal
ulosclerosis (FSGS). However, the FSGS diagnosed in these venous hypertension, such as tricuspid atresia, constrictive
patients shares clinical features distinctive from those usu- pericarditis, and pulmonary hypertension, have been associ-
ally seen with primary FSGS: Albumin levels tend to be ated with proteinuria and nephrotic syndrome (197,198).
higher, often above 3 g/dL; TC levels are only mildly ele- The glomerular hypertrophy theory postulated a sequence
vated, often <300 mg/dL; proteinuria is moderate; blood of changes that may occur in the genesis of obesity-related
pressure tends to be normal or only mildly elevated; and FSGS from glomerular hypertrophy, stimulated in part by
edema may be minimal to absent (183). Patients who lost angiotensin II and other growth factors, leading to produc-
weight successfully showed marked decreases in pro- tion of excess amounts of extracellular matrix in mesangial
teinuria. Histologic features of obesity-associated pro- areas and the development of obesity-related FSGS (199).
teinuria in adults include focal segmental sclerotic changes Very recent studies showed the connection between adi-
often in a hilar location, mesangial proliferation and hyper- pose tissue and kidney at molecular level mediated by leptin,
trophy, foci of hyalinosis, and glomerulomegaly (184,185). a small peptide mainly produced in adipose tissue, therefore
In obesity-associated FSGS, on electron microscopy, the directly reflecting the amount of body fat. In glomerular
foot process fusion is often minimal and focal rather than endothelial cells, leptin stimulates cellular proliferation,
diffuse, as opposed to idiopathic FSGS. transforming growth factor β1 (TGF-β1) synthesis, and type
FSGS is the histologic expression of a variety of condi- IV collagen production. Conversely, in mesangial cells, leptin
tions that differ in cause, pathogenesis, clinical course, and upregulates synthesis of the TGFβ type II receptor but not
response to therapy (186–189). Although the pathophysi- TGF-β1 and stimulates glucose transport and type I collagen
ology of obesity-related FSGS is unclear, potential etiologic production through signal transduction pathways involving
factors seem to be hyperfiltration, hyperlipidemia, renal phosphatidylinositol-3-kinase. These data suggest that leptin
venous hypertension, and glomerular hypertrophy. triggers a paracrine interaction in which glomerular endothe-
Based on the above clinical and pathologic characteris- lial cells secrete TGF-β, to which sensitized mesangial cells
tics, the patients with obesity-related FSGS seem to share may respond. Both cell types increase their expression of
features with a very diverse group of patients whose initiat- extracellular matrix in response to leptin. Infusion of leptin
ing factor is a reduction in renal mass. The reduction in into normal rats produces the development of FSGS and
renal mass can be due to reflux nephropathy (190), nephrec- proteinuria (200).
tomy for Wilms’ tumor (191), unilateral renal agenesis An association between obesity and renal abnormalities,
(192), or unilateral nephrectomy (193). These reduced such as proteinuria, has not been reported until very
numbers of glomeruli may be subjected to hemodynamic recently. In one study, seven obese black adolescent patients
stress by taking on the filtration of a constant volume of had FSGS and significant proteinuria but no edema; serum
plasma. This sets up a cascade of proteinuria, mesangial cell albumin levels were slightly low and TC was normal or
gene expression, and focal scarring. Similarly, in case of mildly elevated. The histologic features included glomerular
obesity, there is increased plasma volume and cardiac out- hypertrophy, FSGS, increased mesangial matrix and cellular-
put that translates into a relative deficit in the number of ity, relative preservation of foot process morphology, and
glomeruli (194). In addition, obesity is associated with absence of evidence of inflammatory or immune-mediated
hypertension. Significant weight gain is accompanied by processes (182). These clinical and histologic characteristics
excess renal sodium resorption, leading to increased sys- resemble the descriptions of obesity-related FSGS found in
temic arterial pressure and glomerular hyperfiltration the adult studies. Obesity-related FSGS may lead to ESRD
(195). These changes result in glomerular capillary wall (182). Children with FSGS in hilar areas, as commonly
 12. Nutrition and Metabolism 267

seen in obesity-associated FSGS, have a much higher likeli- calorie diet and also a low-protein diet can independently
hood of developing CKD than patients with peripherally produce a fall in GFR or renal plasma flow (210). These
located focal segmental lesions (201). changes are usually reversible, not associated by renal struc-
There is clearly a predisposition of the black children to tural damage, and are not explained by hypoproteinemia or
develop obesity-related FSGS (182). Idiopathic primary edema, which can occur in these states (211).
FSGS is also much more common in adult black individu-
als (202). Epidemiologic data also suggest that there is a
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hemofiltration in critically ill anuric children. Child Nephrol ison of efficacy and safety profiles. Clin Transplant 1999;
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2263. cebo in primary renal allograft recipients. Transplant Proc
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ing continuous venovenous hemodiafiltration. Artif Organs 153. Akiyama T, Ishii T, Imanishi M, et al. Efficacy and safety of
1997;21(5):359–363. treatment with low-dose fluvastatin in hypercholesterolemic
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135. Rocco MV, Blumenkrantz MJ. Nutrition. In: Daugirdas JT, cal and safety experience. J Clin Pharm Ther 1999;24:397–
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140. Massy ZA, Guijarro C, Weiderkehr MR, et al. Chronic 159. Navasa M, Bustamante J, Marroni C, et al. Diabetes melli-
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144. Henkin Y, Como JA, Oberman A. Secondary dyslipidemia. Transplant 1992;7:346–349.
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1992;267:961–968. of type 2 diabetes in youth. Diabetes Care 1999;22:345–354.
145. Ingulli E, et al. Steroid withdrawal, rejection and the mixed 164. Pinhas-Hamiel O, Dolan L, Daniels SR, et al. Increased
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 13

FLUID AND ELECTROLYTE

THERAPY IN CHILDREN
MICHAEL J. G. SOMERS

The importance of fluid and electrolyte therapy in clinical 1957 (3), has subsequently been taught as the gold stan-
pediatrics cannot be underestimated. In ambulatory prac- dard to minimize complications and improve clinical out-
tice, given the frequency with which children develop comes. Recent reassessment of this traditional approach,
dehydrating illnesses, the clinician is often called on to for- again spawned in large measure by Holliday’s work, has
mulate approaches to rehydration. In hospitalized children, come to appreciate that such elaborate maneuvers are often
myriad clinical and practice parameters impact fluid and unnecessary and may be counter-therapeutic (4). Rather,
electrolyte therapy and frequently go beyond merely pro- the use of standard oral rehydration and maintenance
viding adequate water and electrolytes to replace losses. hydration fluids is a simple, safe, and efficacious alternative
Over the last several years, the role of fluid and electro- for most children in need of fluid and electrolyte therapy to
lyte therapy in clinical medicine has been subject to reinter- restore intravascular volume in the setting of a dehydrating
pretation as a better understanding of the physiologic gastroenteritis (5).
principles of fluid and electrolyte homeostasis has evolved Nonetheless, there continues to be a place for the more
and as common clinical practices have been critically precise traditional assessment and prescription of fluid and
reevaluated. Historically, it was two centuries ago that the electrolyte therapy, especially in children with nondiarrheal
first attempts to replace enteral losses from cholera led to an illness (6). In more complex disorders of fluid and electro-
initial understanding of the profound morbidity and mor- lyte pathophysiology (e.g., frequently seen postoperatively
tality that can accompany significant losses of salt and or in critically ill children with sepsis, burns, or trauma), an
water (1). The recognition that affected patients improved understanding of the distribution of body fluids, usual fluid
simply by providing repeated intravenous infusions of a and electrolyte requirements, and the effect of perturba-
saline solution served as an impetus to define and develop tions in normal homeostatic balance remains vital to the
parameters for fluid and electrolyte therapy. This under- correct prescription of initial therapy and the proper assess-
standing was later generalized to other illnesses in which ment of clinical response.
there was an element of dehydration and ultimately helped Such an understanding becomes more crucial when
to define the threshold for the minimum daily provision of approaching the care of a child with renal disease. Fre-
fluid and electrolytes—so-called maintenance require- quently, the presence of a preexisting renal condition or the
ments—as well as a threshold of maximal tolerance. development of an acute renal dysfunction can complicate
In the first decades of the twentieth century, the work of the fluid and electrolyte management of the child. Standard
Gamble and colleagues was especially significant to the devel- approaches to such therapy assume that normal renal
opment of fluid therapy in pediatrics (2). The concepts of homeostatic mechanisms come into play with the provision
extracellular and intracellular fluid compartments in the of adequate fluid and electrolytes. These approaches are ill
body helped to underscore the important role that renal advised for the child with renal disease in whom volume
mechanisms play in maintaining normal fluid and electrolyte and electrolyte regulation may be deranged. Similarly, these
balance. In turn, this enhanced knowledge of body fluid approaches may have limited value with the critically ill
physiology spawned a tradition of clinical therapy grounded child in whom normal fluid and electrolyte homeostasis
on rather elaborate guidelines for intravenous fluid and elec- may also be altered. In these circumstances, the clinician
trolyte replacement in the ill child based on continuing needs to approach fluid and electrolyte therapy systemati-
(maintenance) needs and past and current losses (deficits). cally and with attention to individual clinical circumstance.
This so-called deficit therapy approach to fluid therapy Otherwise, in the absence of such a customized fluid and
in children, cogently outlined by Holliday and Segar in electrolyte prescription, the possibility arises that a thera-
276 II. Homeostasis

peutic intervention may be deleterious, given preexisting pressure pushing water across the capillary from the lumen
reduced tolerance to alterations in body fluid volume, com- to the interstitium. Perturbations in the distribution of
position, or distribution. effective osmoles can result in redistribution of water
between the intracellular and extracellular spaces.
There is an ongoing dynamic equilibrium between the
intracellular and extracellular spaces. Diffusional gradients,
DISTRIBUTION OF BODY FLUIDS
osmotic forces, and the activity of cellular pumps or trans-
porters all combine to establish the differences in the com-
Total Body Water: Extracellular and
position of the body compartments. The intracellular space
Intracellular Fluid Compartments
cannot be directly accessed. As a result, access to the intra-
Water makes up a large proportion of body weight, varying cellular space is achieved via its communication with the
with age, body size, and body composition. In early gesta- extracellular space. Thus, any intake by ingestion or infu-
tion, 90% of the weight of the developing fetus may be sion into the extracellular space results in a new equilib-
water. In premature infants born early in the third trimes- rium being established with the intracellular space as solute
ter, total body water (TBW) approximates 80% of body and fluid come to be exchanged. Ultimately, the final equi-
weight. This falls to 70 to 75% in term infants, 65 to 70% librium is a result of complex biochemical, electrical, and
in toddlers and young children, and eventually to 60% in physical interactions.
older children and adolescents. Lean individuals have more The communication between the cellular spaces can be
body water than obese individuals, and pubertal adolescent bidirectional. In other words, there can be exchange from
boys with increasing muscle mass tend to have more TBW the intracellular space to the extracellular space allowing,
than adolescent girls. Although 60% is often the bench- for instance, for transfer or release of cell metabolites. In
mark for estimating TBW in older children and adults, the addition, because the extracellular space can communicate
actual percentage may be lower, especially in less well- with the external milieu, any output from the extracellular
conditioned individuals or the elderly (7). space to the external milieu results in effective excretion
Body water is distributed into intracellular and extracel- from the body. There is, however, no direct communication
lular compartments. The intracellular compartment con- between the intracellular space and the external milieu, and
sists of water found within the cells of the body and any output from the cells themselves is mediated via the
comprises approximately two-thirds of TBW or 40% of cell’s direct ability to interface with the interstitial fluid or
body weight. The extracellular compartment comprises the plasma water.
one-third of TBW or 20% of body weight and is divided For the clinician, it is always important to remember
into the interstitial fluid that bathes all cells and the plasma that any impairment in the patient’s normal homeostatic
water that is carried intravascularly. The increased TBW mechanisms regulating fluid and electrolyte balance has a
seen in young children is due to an increased extracellular striking impact on the patient’s TBW and its extracellular
compartment, the result of a relatively increased surface and intracellular constituents. Clinically, a common exam-
area as compared to body weight (8). ple of this disruption of normal balance is the hypertension
The boundary between the intracellular and extracellu- frequently exhibited by the renal patient with progressive
lar compartments is the cell membrane. Input or output renal dysfunction. As renal function declines, the ability of
from the body generally proceeds via some interface with the kidney to excrete free water (CH2O) also declines. Fre-
the extracellular compartment. For instance, provision of quently, this is in the setting of decreased effective nephron
intravenous fluid and electrolyte therapy is into the intra- number with concomitant impairment of overall tubular
vascular space, and subsequent delivery intracellularly solute excretion, most notably salt. Often superimposed on
depends on a host of factors. this baseline tendency for dysregulation of solute and water
Because most cell membranes are readily permeable to balance are clinical factors (e.g., circulatory failure and
water, the distribution of water between the intracellular decreased effective arterial volume) leading to further renal
and extracellular spaces largely reflects osmotic forces. Each salt and water retention. This salt and water overload can
body space has a solute that is primarily sequestered within lead to chronic expansion of the TBW and resulting sys-
that compartment and that maintains its osmotic gradient temic hypertension with expansion of the extracellular vol-
(9,10). For instance, activity of the sodium-potassium ume compartment. Appropriate therapy in this instance
pump found in cell membranes leads to an increased con- includes the judicious use of diuretics to reduce the total
centration of potassium intracellularly and an increased body burden of salt and water and to restore the TBW to a
concentration of sodium in the interstitium. Thus, sodium more physiologic state. In this instance, a clinician’s failure
serves as the effective osmole interstitially and potassium to appreciate the preexisting expansion of the TBW because
intracellularly. Similarly, plasma proteins, most notably of chronic salt and water overload could prove deleterious
albumin, exert an osmotic force to maintain water intravas- to the patient if management did not include some measure
cularly. Osmotic force is counterbalanced by hydrostatic to reduce the salt and water overload. This example also
 13. Fluid and Electrolyte Therapy in Children 277

underscores the concept that fluid and electrolyte therapy the tissues and accomplish effective cellular exchange. Thus,
may involve the removal of solute and water as well as the these children act as if they are volume depleted: They
more usual notion that it is solely concerned with the cor- excrete little sodium in their urine, and they vigorously con-
rection of deficits of electrolytes and volume. tinue to expand their already overexpanded extracellular
space by reabsorbing even more salt and water in response to
the effects of aldosterone and antidiuretic hormone (ADH).
Effective Circulating Volume
Similarly, this paradoxic state of sodium avidity and ADH-
The concept of effective circulating volume is somewhat mediated water reabsorption can be seen frequently in chil-
more abstract than the division of body water into intracell- dren with nephrotic syndrome or with cardiac failure despite
ular and extracellular fluid compartments. Through the cir- their preexisting expansion of the extracellular space.
culation of the vascular volume, oxygen and nutrients are In managing all aspects of a patient’s fluid and electro-
delivered to the intracellular space, and cellular metabolites lyte therapy, the clinician must accurately assess both the
are cleared from the intracellular space. The effective circu- patient’s current extracellular volume status and effective
lating volume refers to that portion of the extracellular vas- circulating volume and reconcile these with potential
cular space that is actually perfusing the tissues and causes of volume loss. At all times, it is most important to
accomplishing such an exchange. maintain an effective circulating volume and to make ther-
Any compromise in this exchange can prove deleterious apeutic decisions based on each patient’s unique clinical cir-
to usual cell homeostasis; as a result, the body constantly cumstances at that time. This may require rather disparate
senses and regulates effective perfusion of fluid through the therapeutic interventions. For instance, expansion of the
intravascular space. Homeostatic feedback mechanisms extracellular volume with vigorous rehydration therapy
include baroreceptors that respond to the stretch of special- may be called for in a child with poor perfusion secondary
ized areas of the carotid arteries and the atrium. Hypoper- to gastroenteritis-induced dehydration, whereas another
fusion of these areas results in decreased stimulation of child with equally poor perfusion due to cardiodynamic
these stretch receptors, triggering the secretion of vaso- compromise may be intravascularly replete and require the
pressin and eventually leading to increased water reabsorp- initiation of pressor therapy, and another child with edema
tion in the most distal nephron and expansion of the from relapsed nephrotic syndrome may actually require
vascular volume. Similarly, in response to glomerular fluid restriction. These examples underscore that loss of
hypoperfusion, there is not only decreased afferent arteri- effective circulating volume generally arises as a result of
olar stretch but also decreased glomerular filtration and one or more broad perturbations in the extracellular fluid
delivery of sodium to the macula densa. Both of these stim- compartment that impacts effective perfusion (Table 13.1).
uli can lead to the secretion of renin from the juxtaglomer- Clinical signs and symptoms of effective circulating vol-
ular cells of the afferent arteriole. Renin release initiates a ume loss may be subtle. At times, there may be preservation
cascade of events resulting ultimately in increased aldosterone- of effective circulating volume in the face of an overall
mediated sodium reabsorption from the kidney as well as depleted extracellular fluid compartment. Failure to initiate
increased angiotensin-mediated vasoconstriction and sodium appropriate fluid and electrolyte therapy in such a circum-
and water uptake. stance may result in eventual compromise of the effective
Thus, the effective circulating volume is a product of mul- circulating volume. Important initial clinical signs to assess
tiple factors, not the least of which includes the size of the in any patient being evaluated for fluid therapy include
vascular space and the influence of various regulatory hor- pulse rate and capillary refill. Tachycardia and sluggish refill
mones. As a component of the extracellular body water, the generally precede more obvious signs of ineffective circula-
size of the vascular space often parallels the size of the extra-
cellular space. However, the size of the vascular space and the
adequacy of the effective circulating volume do not necessar- TABLE 13.1. ALTERATIONS IN EFFECTIVE
ily vary coordinately. It is possible for the extracellular space CIRCULATING VOLUME
to be replete or expanded and the actual effective circulating Cause Mechanism
volume to be decreased. For instance, children with signifi-
cant liver disease are often edematous, a sign of sodium Contracted extracellular fluid Water or sodium chloride def-
space icit
retention and expansion of the interstitial component of the Massive vasodilatation Loss of vascular tone sustaining
extracellular space. The intravascular component of their perfusion pressure
extracellular space may also be expanded due to factors Loss of intravascular osmotic Osmotic fluid losses into inter-
resulting in avid salt and water reabsorption by the kidney. pressure stitium
However, because of portal hypertension, splanchnic vessel Overfill of the intravascular Hydrostatic fluid losses into
space interstitium
congestion, and multiple arteriovenous spider angiomas gen- Hemorrhage Direct loss of blood and
erally seen with this condition, much of the expanded intra- plasma water
vascular volume is ineffective—it does not serve to perfuse
278 II. Homeostasis

tion (e.g., hypotension and oliguria). Clinical symptoms a ventilator in the intensive care unit. Careful repeated assess-
may also be nonspecific and include fatigue and lethargy ment of the patient’s volume status and close attention to a
that are often attributed to an underlying illness rather than balance of overall daily input and output proves more useful at
to volume depletion. Proper restoration of effective circu- arriving at a correct estimate of daily fluid and electrolyte needs
lating volume or extracellular fluid compartment depletion than reliance on mathematic formulas alone. With these cave-
requires an understanding of baseline fluid and electrolyte ats in mind, it is nonetheless a common clinical practice to
needs as well as consideration of any extenuating clinical make certain empirical assumptions regarding daily needs for
circumstances unique to the patient in question. water and the major electrolytes.
Historically, daily maintenance water needs have been
estimated based on energy expenditure (Fig. 13.1) (3,11).
Thus, for every kilocalorie (kcal) of energy used per day, 1
FLUID AND ELECTROLYTE REQUIREMENTS
mL of water must be provided. Based on the computed
energy expenditure of the average hospitalized patient, for
Maintenance Water Therapy
the first 10 kg of body weight, 100 mL of water per kg is
The fluid and electrolytes required to replace daily losses provided daily. For the next 10 kg of body weight, 50 mL
and to maintain an overall net balance of zero fluid or elec- of water per kg is provided daily, and for every kilogram
trolytes gained or lost are often termed maintenance needs. of body weight in excess of 20 kg, 20 mL of water per kg
Such needs are a function of individual homeostatic and is provided daily. In addition, in the process of oxidation
environmental factors and, thus, vary from day to day and of carbohydrate and fat, approximately 15 mL of water is
from individual to individual. In the average child with generated for every 100 kcal of energy produced. This
adequate access to sources of fluid and nutrition, these water of oxidation contributes significantly to overall
maintenance needs are generally readily met (3,11). In the water balance.
ill or hospitalized child who requires therapeutic interven- Maintenance water losses occur from insensible sources
tion, these needs must be considered in the choice of fluid (almost exclusively evaporative and respiratory losses) and
therapy provided. from urine output. In the child with average metabolic
To assist in estimating these needs, fluid and electrolyte demands, for every 100 kcal of energy expended, 100 mL
requirements are generally calculated on the basis of weight or of water must be taken in. Of this 100 mL of water, 40 mL
surface area, but individual clinical circumstance must be con- is lost insensibly, and 75 mL is lost as urine output. There
sidered when making such calculations (12). For instance, the is, however, 15 mL of water generated from the water of
20-kg child who is well requires a far different maintenance oxidation rendering net water balance equilibrated.
quantity of fluid and electrolytes than the 20-kg child who is Clinical factors can have a striking impact on water
tachypneic and febrile or the 20-kg child who is anuric and on losses (Table 13.2). Fever increases insensible losses by

FIGURE 13.1. A comparison of the relation of

estimated energy expenditure to weights over
the range of 3 to 70 kg. (Courtesy of Malcolm
Holliday.)
 13. Fluid and Electrolyte Therapy in Children 279

TABLE 13.2. FACTORS AFFECTING INSENSIBLE WATER LOSSES

Increased losses Change (%) Decreased losses Change (%)

Prematurity 100–300 Enclosed incubator 25–50

Radiant warmer 50–100 Humidified air 15–30
Phototherapy 25–50 Sedation 5–25
Hyperventilation 20–30 Decreased activity 5–25
Increased activity 5–25 Hypothermia 5–15
Hyperthermia 12°C

nearly 10% per degree Celsius. Premature infants with rela- its concentration can be influenced profoundly by changes
tively increased surface areas for size can have insensible in water metabolism. An understanding of this link
losses two- to threefold higher than baseline, especially if between water regulation and serum sodium values is cru-
they are on open warmers or under phototherapy. On the cial when prescribing fluid and electrolyte therapy. Most
other hand, children on ventilators being provided humidi- important, the clinician must recognize that hypo- or
fied oxygen may have only half of the insensible losses of a hypernatremia is usually a manifestation of impaired water
nonventilated child. Similarly, urinary output can vary tre- regulation and that therapy must address regulation of
mendously. A child with a concentrating defect or ADH water balance rather than alterations in body sodium stores.
unresponsiveness may have urinary water losses of several
liters per day, whereas an anuric child has no urinary losses.
Factors Contributing to Altered Water
In any child with normal renal function, even in the setting
Homeostasis in Ill Children
of maximal ADH stimulation, there is a minimal volume of
urinary water losses that is obligatory to excrete the osmotic In children with acute illness, vasoactive hormone levels
load ingested by the diet and generated by basal metabo- appear to be increased, predisposing them to altered water
lism. Thus, even the child concentrating urine at 1200 to homeostasis and changes in measured serum sodium levels.
1400 mOsm/L loses approximately 25 mL of water as urine In one study of 103 acutely ill children, plasma vasopressin
for every 100 kcal of energy expended (3). and renin levels were significantly elevated when compared
to 31 control children awaiting elective surgery (16). These
elevations were independent of the child’s underlying ill-
Water Homeostasis and Serum Osmolality
ness and resulted in the ill children as a group having a
Water homeostasis is crucial to maintaining a stable serum lower measured serum osmolality than the control group.
osmolality. The major factors contributing to serum osmo- In ill children, there are multiple causes of both physio-
lality are the serum concentrations of sodium, glucose, and logic and aberrant vasopressin effect (Table 13.3). As a
blood urea nitrogen (BUN) (13). Serum osmolality is esti- result, in such at-risk children who receive hypotonic intra-
mated by the following equation: venous fluids for prolonged periods of time or in volumes
exceeding those generally recommended, there is the risk of
(2 × serum Na) + (serum glucose/18) + (BUN/2.8)
acute hyponatremia. After volume resuscitation with iso-
where the serum sodium is measured in mEq/L and the tonic fluids, most hospitalized children are provided hypo-
glucose and BUN in mg/dL. Normally, the contributions
of glucose and BUN to the effective osmolality are small,
TABLE 13.3. COMMON CAUSES OF VASOPRESSIN
and the serum osmolality can be estimated by doubling the EFFECT IN HOSPITALIZED CHILDREN
serum sodium concentration (14,15). Thus, most children
have a serum osmolality between 270 and 290 mOsm/L, Category Specific etiology
corresponding to serum sodium values of 135 to 145 mEq/ Physiologic Hyperosmolar state, hypovolemia
L. Chemoreceptors in the hypothalamus constantly sense Pulmonary Pneumonitis, pneumothorax,
serum osmolality and respond to even small variations asthma, bronchiolitis, cystic
toward either limit of normal by adjusting ADH release fibrosis
Drug effect Narcotics, barbiturates, carba-
from the posterior pituitary. Changes in osmolality in the mazepine, vincristine, cyclophos-
setting of hypovolemia augment ADH release further. phamide
ADH effect on water permeability of the collecting tubule Metabolic Hypothyroidism, hypoadrenalism,
is a principal influence on the regulation of water balance. porphyria
Alterations in water intake or excretion result in the Central nervous system Infection (meningitis or encepha-
litis), tumor, trauma, hypoxia,
development of hypo- or hyperosmolality as the usual ratio shunt malfunction, nausea,
of extracellular solute to water is perturbed. Because pain, anxiety
sodium is the largest component of extracellular osmolality,
280 II. Homeostasis

tonic fluids for their maintenance therapy. Given the levels often vary with these alterations in water balance.
tendency for ill children to have vasopressin effect indepen- Generally, serum sodium is kept regulated at levels between
dent of the usual osmotic and volume-related stimuli, some 135 and 145 mEq/L. Serum sodium below 130 mEq/L or
investigators have suggested that isotonic fluids may be above 150 mEq/L is out of the range of normal homeostasis
safer alternatives and should be continued as the source of and is often indicative of some problem with water balance.
maintenance fluid even after acute volume repletion (17). Because sodium is the major extracellular osmole, alter-
ations in serum sodium can result in water flux between the
intracellular and extracellular spaces. Because significant
Maintenance Electrolyte Therapy
water flux into or out of cells could prove deleterious to cell
Estimates for the maintenance requirements of the major function, cell volume is closely regulated to minimize such
electrolytes sodium, potassium, and chloride can also be shifts (19). For instance, with hyponatremia, there is
made based on metabolic demands or, by extension, on daily decreased effective osmolality in the extracellular space. As
water needs (3). For sodium and chloride, approximately 2 a result, water can shift from the plasma into the intracellu-
to 3 mEq/100 mL of water per day is required. For potas- lar space, and cell swelling occurs acutely. To counterbal-
sium, 1 to 2 mEq/100 mL of water per day is needed. Again, ance such swelling, the cell acutely attempts to regulate its
these estimates may require adjustment based on clinical cir- volume by transporting electrolytes, especially potassium,
cumstance, but the daily intake of most healthy individuals from the intracellular space into the extracellular space,
contains more than adequate electrolytes for maintenance thereby decreasing the osmotic gradient for water transfer.
needs. Although there can be significant electrolyte losses at Over several days, when faced with chronic hyponatremia
times through the skin or the gastrointestinal tract, most or chronic hypo-osmolality, the cells also achieve effective
electrolyte losses are urinary (12,18). Thus, in the setting of volume regulation by losing organic osmolytes (e.g., as tau-
anuric renal failure, in the absence of concomitant electrolyte rine and inositol), thereby further diminishing the osmotic
losses from other sources, a much lower level of maintenance gradient for water transfer into the cell (20).
electrolyte supplementation is needed, and often patients With hypernatremia, the osmotic gradient favors water
maintain adequate electrolyte balance with water supplemen- flux out of the cells into the extracellular space. Without
tation alone to provide insensible fluid losses. protective mechanisms, cell volume shrinks. Again, there
As with provision of water, it is vital in children who require are both acute and chronic mechanisms that attempt to
electrolyte therapy to tailor their electrolyte prescription to minimize changes in cell volume. Acutely, there is transport
their individual needs. For the provision of sodium and chlo- of electrolytes intracellularly. Over time, there is stimula-
ride, this requires careful assessment of the extracellular fluid tion of the production of organic idiogenic osmoles
space, especially the effective circulating volume. Providing too (20,21). Together, these act to ameliorate the loss of intra-
little sodium chloride results in volume contraction and circu- cellular volume that would otherwise occur.
latory compromise; providing too much causes volume over- Thus, alterations in serum sodium values that arise slowly
load and sequelae (e.g., hypertension and edema). Similarly, or are more chronic in nature tend to be better tolerated clin-
inappropriate potassium supplementation may have signifi- ically than acute alterations. Chronic or slow changes allow
cant clinical ramifications. In children with diminished renal for maximal counterregulation and fewer clinical sequelae.
function or who are at risk of hyperkalemia for other reasons, On the other hand, profound sudden alterations—serum
it is usually appropriate to forego any maintenance potassium sodium values falling to approximately 120 mEq or below or
supplementation. When supplementation is given acutely, it is 160 mEq or above—are often accompanied by dramatic
important to monitor serum potassium values closely. When neurologic complications directly related to the acute
chronic potassium supplementation is needed, there should changes in cell volume in the central nervous system (CNS).
continue to be periodic assessments. A course of supplemental These regulatory mechanisms must also be kept in mind
potassium administered orally is safer than a bolus intravenous when formulating specific therapeutic intervention. Acute
injection; intravenous potassium supplements rarely need to perturbations can be corrected more rapidly than chronic
exceed 0.5 mEq/kg/hr (13). conditions because the full gamut of responses to the
imbalance has yet to come into play.

PERTURBATIONS IN FLUID AND Hyponatremia: Initial Approach

ELECTROLYTE HOMEOSTASIS
Hyponatremia is usually defined as a serum sodium value
less than 130 mEq/L. As noted earlier, low serum sodium
Alterations in Water Balance, Serum
values are less likely to arise secondary to depleted salt
Sodium, and Cell Volume
stores and more likely to be the result of persistent ADH
As discussed earlier, the regulation of osmolality is achieved effect and a relative surfeit of water for solute in the extra-
by alterations in water intake and excretion. Serum sodium cellular space.
 13. Fluid and Electrolyte Therapy in Children 281

TABLE 13.4. ETIOLOGY OF HYPONATREMIA

Urinary Na (mEq/L)

Circulating volume <20 >20

Decreased Burns Adrenal insufficiency

Cystic fibrosis Diuretics—early
Diuretics—late Salt wasting
Gastroenteritis
Normal or increased Cardiac failure Renal failure
Hepatic cirrhosis Syndrome of inappropriate secretion of
Nephrotic syndrome antidiuretic hormone
Water intoxication

Infrequently, pseudohyponatremia may be seen. This is intravascular space of the extracellular fluid into the inter-
a result not of depletion of sodium stores but of an alter- stitial space (“third-spacing”) as can occur postoperatively
ation in the usual makeup of the extracellular space such in conditions of vascular leak, or with peritonitis. Such
that the relative concentration of sodium is now depressed. hyponatremia can also be seen after a period of diuretic
Common etiologies of pseudohyponatremia include hyper- therapy. In response to chronic diuretic-mediated volume
glycemia, hyperlipidemia, and hyperproteinemia. In these contraction, the mechanisms outlined above come into
clinical conditions, it is not uncommon for serum sodium play. Thiazide diuretics, especially in combination with a
values to be only modestly depressed. Because there is no loop diuretic (e.g., furosemide), are particularly prone to
underlying true anomaly in sodium or water stores in these inducing such hyponatremia.
conditions, the serum sodium need not be addressed with The appropriate therapeutic response to these condi-
any therapeutic maneuvers. With the introduction of ion- tions is the provision of sodium and water either by use of
sensitive electrodes for the measurement of plasma sodium intravenous saline solutions or oral electrolyte solutions.
concentration, pseudohyponatremia related to the presence This results in restoration of sodium balance and volume
of confounding factors in the laboratory assay is much less expansion.
commonly encountered.
In attempting to clarify the etiology of the hyponatre-
Hyponatremia: Decreased Volume Status and
mia, it is often useful initially to assess the patient’s extracel-
Urine Sodium Greater Than 20 mEq/L
lular volume status and determine if it is decreased, normal,
or increased. Then, by quantitating urine sodium excretion Decreased circulating volume and urinary sodium excretion
and determining the renal response to the hyponatremia, it greater than 20 mEq/L are indicative of renal salt wasting,
becomes easier to determine if the patient should receive either from an intrinsic tubulopathy or from early diuretic
sodium and water or if a sodium and water restriction is the effect. Less commonly, adrenal insufficiency can cause
appropriate therapy (Table 13.4). sodium wasting from the principal cell of the distal neph-
ron. Such cortisol deficiency can arise from an intrinsic
endocrine defect (e.g., congenital adrenal hyperplasia
Hyponatremia: Decreased Volume Status and
related to 21-hydroxylase deficiency); from some secondary
Urine Sodium Less Than 20 mEq/L
impairment of adrenal function caused by infection, bleed-
Decreased circulating volume is usually seen with states of ing, or malignancy; or from pharmacologic adrenal sup-
significant sodium loss. The most common site of this loss pression without adequate replacement therapy. In the
is the gastrointestinal tract as a result of vomiting, diarrhea, setting of adrenal insufficiency, replacement of appropriate
or tube drainage. The renal response to the decreased effec- adrenal hormones and provision of adequate sodium and
tive circulating volume includes increased activity of the water prove therapeutic. With renal salt wasting, supple-
renin-angiotensin axis and relatively high levels of aldoster- mentation with sodium and any other electrolytes exhibit-
one and angiotensin. As a result, urine sodium values are ing impaired renal reabsorption is useful.
generally low (less than 20 mEq/L), and water reabsorption
in the distal nephron is augmented by high levels of ADH.
Hyponatremia: Normal or Expanded Volume and
In the face of continuing sodium losses exceeding intake,
Urine Sodium Less Than 20 mEq/L
this state of vigorous ADH effect leads to a relative excess of
water and concomitant hyponatremia. Normal or increased circulating volume and urine sodium
Such findings can also be seen in cystic fibrosis in which excretion less than 20 mEq/L can be seen in conditions in
there are increased skin losses of sodium and chloride, with which there is an excess of both TBW and total body
bleeding, with burns, with certain losses of fluid from the sodium. The three major disorders that cause this type of
282 II. Homeostasis

hyponatremia are the nephrotic syndrome, hepatic failure is more commonly a reflection of a problem with water bal-
related to cirrhosis, and cardiac failure. In all these condi- ance rather than a sodium imbalance (23). In most
tions, there is a state of sodium and water avidity related to instances, the patient has a relative deficiency of water for
high levels of ADH and aldosterone. Most commonly, this normal extracellular solute content.
is in the setting of preexisting total body sodium overload Because sodium is the major determinant of plasma
as evidenced by edema. In all of these conditions, despite osmolality, as serum sodium levels rise, serum osmolality
the increased extracellular or circulating volume, the effec- concomitantly increases. Increases in serum osmolality are
tive circulating volume is often depressed. As a result of this sensed by hypothalamic osmoreceptors, triggering ADH
ineffective perfusion of the tissues, sodium and water avidity release from the posterior pituitary once serum osmolality
is only heightened by stimulation of the renin-aldosterone- begins to increase over 280 mOsm (24). Increased serum
angiotensin axis, further exacerbating the total body excess osmolality also causes a sensation of thirst. Thus, in the
of salt and water. Appropriate therapy includes striking a normal state, as serum osmolality increases, there is
balance between interventions promoting the maintenance increased fluid intake in the setting of high ADH levels.
of effective circulating volume and restricting the provision This results in increased reabsorption of CH2O from the
of excess water and sodium that only contributes to further cortical collecting and reequilibration of serum sodium
TBW and sodium overload. levels and serum osmolality before clinically significant
hypernatremia or hyperosmolality occurs.
Outside infancy, hypernatremia as a result of sodium
Hyponatremia: Normal or Expanded Volume and
excess or salt poisoning is infrequently seen in pediatrics.
Urine Sodium Greater Than 20 mEq/L
Its major cause is improper preparation of powdered or
Hyponatremia in the setting of normal or increased effec- liquid concentrated formula resulting in a hypertonic,
tive circulating volume is always related to persistent ADH hypernatremic solution. Because infants do not have free
effect (10). If the urine sodium value is greater than 20 access to water, they cannot respond to their increasing
mEq/L, the most common clinical scenario is the syndrome sense of thirst as they develop hypernatremia. Exacerbat-
of inappropriate secretion of ADH (SIADH). SIADH can ing the whole situation are the facts that, most typically,
arise from disparate clinical conditions, including in the caregivers continue to provide the same incorrectly pre-
postoperative child, the child with significant pain, or the pared formula for further feedings and that young infants
child with pulmonary disease; as alluded to earlier, ill chil- are unable to excrete sodium loads as efficiently as older
dren are at risk for both SIADH and inappropriate ADH children. Iatrogenic sodium loading can also be seen in
effect (22). In SIADH, despite a state of hypo-osmolality, children who have received large doses of sodium bicar-
the urine is inappropriately concentrated as a result of an bonate because of persistent acidosis or during a resuscita-
inability to suppress ADH secretion and block ADH-medi- tion or in children who have been given inappropriate
ated water reabsorption from the distal nephron. Appropri- amounts of sodium in peripheral nutrition. Iatrogenic
ate therapy for SIADH includes restricting water intake sodium loading can also be seen in the child who has
and attending to any underlying clinical factors predispos- received repeated large volumes of blood products, gener-
ing to this syndrome. ally isotonic or sodium-rich solutions.
Normal or increased extracellular volume and high urine Children who have hypernatremia from sodium excess
sodium concentration can also be seen in the setting of should exhibit physical signs and symptoms compatible
renal failure as glomerular filtration and water clearance fall with extracellular space expansion. Thus, they frequently
and the fractional excretion of sodium rises. A more have peripheral edema and may have hypertension or
unusual cause of this form of hyponatremia is polydipsia, symptoms of pulmonary edema. These children can
usually psychogenic in nature. Such water intoxication is respond to therapy aimed at augmenting the elimination of
rare in children but can occasionally be seen in emotionally excess sodium. The use of diuretics and the provision of
disturbed older children or infants inappropriately pro- adequate CH2O serve to decrease the total body sodium
vided large volumes of water or very hypotonic fluid. In burden. Rarely, dialysis may need to be considered in cases
both of these circumstances, restriction of the volume of in which it is clinically indicated to ameliorate the hyper-
CH2O ingested on a daily basis may be beneficial. natremia rapidly (25,26).
More commonly, hypernatremia is the result of a CH2O
deficit or a combined water and sodium deficit in which
Hypernatremia
the water losses exceed the sodium losses. Hypernatremia
Hypernatremia is defined as a serum sodium value greater secondary to a water deficit arises in the setting of inade-
than 150 mEq/L. Generally, higher serum sodium values quate access to water or an impairment in ADH release or
can be tolerated, and significant clinical and neurologic response. It is uncommon to see hypernatremia secondary
effects of hypernatremia do not occur until sodium values to poor water intake except in infants or young children
exceed 160 mEq/L. As with hyponatremia, hypernatremia who cannot get water for themselves in response to their
 13. Fluid and Electrolyte Therapy in Children 283

sense of thirst (27). As for ADH-related anomalies, there tion, manifesting with less than 5% weight loss, usually
are many causes of central or nephrogenic diabetes insipi- respond to 30 to 50 mL/kg of fluid. In the setting of more
dus (28). Again, given normal access to water, it is rare for significant dehydration or loss of vascular tone, more than
the older child to develop hypernatremia even with an 200 mL/kg may be needed. The clinician can approach
impairment in the ADH axis because of the strong drive to such fluid resuscitation with either intravenous or oral
drink in response to thirst (29). In very young children rehydration therapy.
with diabetes insipidus, however, the issue of access to
water arises, and hypernatremia may be a concern.
Clinical and Laboratory Assessment of
The most common etiology of hypernatremia in chil-
Volume Depletion
dren is the loss of hypotonic fluid (i.e., fluid with a relative
excess of water for its sodium content). In these situations, In estimating the severity of dehydration, the most objec-
the TBW is decreased more than the total body sodium. tive measure is usually the change in weight from baseline
The usual clinical scenario leading to such a condition is (30). As rehydration proceeds, following weights on a serial
viral diarrheal illness in the setting of poor water intake or basis becomes an important adjunct in assessing the efficacy
persistent vomiting. In this condition, there is loss of stool of fluid repletion. Frequently, the clinician is faced with the
with a sodium content generally less than 60 mEq/L. These situation in which no baseline weight is known, and various
children tend to excrete small volumes of concentrated parameters based on history and physical examination need
urine with urine sodium content less than 20 mEq/L, to be used to judge the severity of dehydration (Table
underscoring the fact that they are conserving both water 13.5). Children with mild dehydration have minimal clini-
and sodium. Their hypernatremia is not a manifestation of cal signs and only a modest decline in urine output. As
a total body excess of sodium but a depletion of sodium dehydration becomes more significant, more classical find-
that is overshadowed by a larger relative depletion of body ings (e.g., dry mucous membranes, tenting skin, sunken
water. Therapy is aimed at restoring water and sodium bal- eyes, and lethargy) become prominent. With profound
ance by providing back the hypotonic fluid that was ini- dehydration, there is anuria, marked alterations in con-
tially lost either by the use of intravenous saline solutions or sciousness, and hemodynamic instability.
with oral electrolyte therapy. A capillary refill time greater than 2 seconds is a useful
physical finding in effective volume depletion (31). However,
delayed capillary refill is neither a sensitive nor specific
FLUID REPLACEMENT THERAPY marker of dehydration (32,33) and may be most useful if
normal, as this does seem to exclude reliably severe dehydra-
Most commonly, the importance of fluid replacement ther- tion. In a prospective cohort study of dehydrated Egyptian
apy lies with restoration of an adequate effective circulating children between 3 and 18 months of age, the best correla-
volume. In its absence, significant metabolic derangements tion between clinical assessment of degree of dehydration
can occur, which only serve to exacerbate perturbations in and actual volume depletion came in children who had such
fluid and electrolyte homeostasis. The volume of fluid clinical parameters of significant dehydration as prolonged
replacement required varies with the extent and etiology of skinfold tenting, a dry mouth, sunken eyes, and altered sen-
the compromised circulation. Children with mild dehydra- sorium—findings generally associated with more substantial

TABLE 13.5. CLINICAL ASSESSMENT OF DEHYDRATION

Degree of dehydration

Physical sign Mild Moderate Severe

Vital sounds
Pulse Normal Rapid Rapid and weak
Blood pressure Normal Normal to slightly low Shock
Weight loss
Infant <5% 10% >15%
Older child <3% 6% >9%
Mucous membranes Tacky Dry Parched
Skin turgor Slightly Decreased Tenting
decreased
Eye appearance Normal tears Decreased tears ± No tears + very sunken
sunken
Capillary refill Normal Delayed (>3 s) Very delayed (>5 s)
Urine output Decreased Minimal Anuric
284 II. Homeostasis

dehydration (34). Similarly, in a review of preschool children child is not showing progressive improvement. Clinical
with dehydration, the best clinical indicators of volume parameters to follow include monitoring the child’s gen-
depletion—decreased skin turgor, poor peripheral perfusion, eral appearance and sensorium, the change in weight
and Kussmaul breathing—accompanied more significant from initiation of rehydration, as well as urine output
dehydration (35), underscoring the difficulty with which and urine osmolality. In children with some types of
more subtle degrees of dehydration may be estimated by the underlying renal dysfunction, there may often be a
clinician without access to prior weights. chronic urinary concentrating defect. In these children,
A study of 97 American children who needed intrave- relatively dilute urine flow may be maintained even in
nous fluids in an emergency department for rehydration the face of clinical dehydration. In these children, mark-
underscored the difficulty in assessing accurately even ers other than urine output and osmolality should be
severe dehydration by standard clinical estimates (36). Phy- followed.
sicians’ initial estimate of dehydration compared to the
actual percent loss of body weight varied dramatically, with
Oral Rehydration Therapy
a sensitivity of 70% for severe dehydration (greater than
10% loss) but only 33% for moderate dehydration (6 to Parenteral fluid and electrolyte therapy have been the main-
10% loss). This study suggested that adding a serum bicar- stays of medical treatment for most children presenting
bonate level to the assessment may be useful, increasing the with fluid and electrolyte imbalances even though oral
sensitivity of the clinical scales to 100% in severe dehydra- rehydration with electrolyte solutions is actually a safe, effi-
tion and 90% in moderate dehydration if standard clinical cacious, and convenient alternative that can treat mild to
features and a serum bicarbonate less than 17 mEq/L were severe volume depletion (44–47). Although this approach
found. has been especially underused in North America, oral ther-
Other studies have found that laboratory studies by apy has proved successful in clinical settings worldwide in
themselves are poor indicators of the degree of dehydration. resuscitating children of all ages with profound fluid and
In 40 children receiving intravenous fluids for dehydration, electrolyte anomalies; short of significant circulatory com-
prehydration assessment of serum BUN, creatinine, uric promise, it can be used as first-line therapy in all fluid and
acid, anion gap, venous pH, and venous base deficit were electrolyte aberrations (48,49). An example of a situation in
made, as well as assessment of urinary specific gravity, uri- which oral hydration therapy is indicated is presented in
nary anion gap, and fractional excretion of sodium. Only the following case study:
the serum BUN/creatinine ratio and serum uric acid signif-
A healthy 5-year-old boy with no significant medical
icantly correlated with increasing levels of dehydration, but
history presents to his pediatrician’s office after 2 days
both lacked sensitivity or specificity for detecting more
of a febrile illness. For most of this time, his appetite
than 5% dehydration (37). Similarly, in a retrospective
has been severely depressed, and his parents estimate
review of 168 dehydrated children, elevated serum urea
that he has only had a few cups of fluid in the last 8 to
levels and depressed serum bicarbonate levels were found to
12 hours. Today, he vomited once, prompting the
be useful adjuncts to clinical evaluation in accurately assess-
office visit. He has continued to urinate, although
ing the degree of dehydration but were not by themselves
urine volume is less than usual. On physical examina-
predictive (38).
tion, the boy is nontoxic and alert. Initially, his pulse is
In fact, with the most common clinical scenario (i.e.,
120 beats per minute; however, after acclimation to the
viral gastroenteritis) leading to dehydration in children,
examination, it has decreased to 100 beats per minute.
there rarely is a significant laboratory anomaly despite clini-
His sitting blood pressure is 90/50 mm Hg, and he is
cal volume depletion. In a cohort of children from the
not compliant with attempts at orthostatic vital signs.
United Kingdom admitted for rehydration due to viral gas-
His mucous membranes are dry, and his weight today
troenteritis, only 1% of admitted children had an electro-
is 20 kg, exactly the same as his weight at a well-child
lyte derangement (39–41).
examination 6 months previously. His parents are con-
In children with volume depletion accompanying
cerned that he is becoming dehydrated.
trauma, sepsis, surgery, or underlying renal dysfunction, it
is more likely to find perturbations in electrolyte and acid- Pediatricians, family practitioners, and emergency
base status. Thus, in the absence of a straightforward case of room physicians face such clinical scenarios regularly. The
mild to moderate diarrheal dehydration, the general con- otherwise well child with an apparent viral illness causing
sensus is that blood should be obtained for assessments of mild to moderate dehydration is frequently treated by
electrolytes, bicarbonate, and renal function to help guide intravenous therapy under the oftentimes misguided
fluid and electrolyte therapy (42,43). assumption that oral rehydration rarely succeeds, is too
As the child is volume resuscitated, it is important to labor intensive, or takes too much time. In fact, such chil-
assess the child’s response. Initial estimates as to the dren are excellent candidates for oral rehydration. In most
degree of dehydration may need to be adjusted if the developed countries where a viral disease is believed to be
 13. Fluid and Electrolyte Therapy in Children 285

TABLE 13.6. ORAL REHYDRATION THERAPY (ORT) FOR PREVIOUSLY HEALTHY,

WELL-NOURISHED CHILDREN
Type of dehydration Rehydration prescription Rehydration duration

Mild (<5%) 30–50 mL/kg ORT 3–4 h

Moderate (5–10%) 50–100 mL/kg ORT 3–4 h
Severe (>10%) 100–150 mL/kg ORT 3–4 h
Evidence of shock 20 mL/kg 0.9% NaCl IV Repetitive infusions until perfu-
sion restored then transition
to ORTa
Accompanying hypernatre- Per type of dehydration ≥12 h for ORT
mia Monitor fall in serum Na

aWith fluid containing 45 to 90 mmol/L Na, 90 mmol/L glucose, 20 mmol/L K, and 10 to 30 mmol/L citrate.

the etiology of the dehydration and there is little concern osmotic diarrhea in some children; it was a common prac-
about a cholera-like enteritis, oral rehydration solutions tice at that time for parents to use high-solute fluids (e.g.,
with sodium contents from 30 to 90 mEq/L boiled skim milk) as an adjunctive home remedy. Taken
(54,55,57,59) can be chosen as oral therapy with expecta- together, these early experiences contributed to a reluctance
tion of success. by many clinicians to use oral rehydration solutions.
With this child, given his history and physical examina- Over time, there came to be a better understanding of
tion, it is unlikely that any electrolyte perturbations would the physiology of water and solute absorption from the
be found; hence, there is little indication for assaying elec- gut. Of prime importance was the recognition that many
trolytes or renal function before starting oral rehydration substances actively transported across intestinal epithe-
(39–41). The family is given a commercially available oral lium had an absolute or partial dependence on sodium for
rehydration solution containing 75 mEq/L sodium, 20 absorption and that sodium itself was actually better
mEq/L potassium, 30 mEq/L citrate, and 2.5% glucose. absorbed in their presence (51–53). This led to the rou-
They are asked to provide 1 L of fluid (50 mL/kg) to the tine introduction of glucose into oral rehydration solu-
child over the next 4 hours. The child should be offered tions in a fixed molar ratio of no more than 2:1 with
small aliquots of fluid very often—5 mL every 1 to 2 min- sodium. Moreover, it became clear that the sodium/
utes at initiation. If this regimen is tolerated with no vomit- glucose cotransporter remained intact not only in the face
ing, the aliquots may be gradually increased in volume and of enterotoxic gastroenteritis (e.g., as seen with cholera or
the frequency reduced, aiming to deliver at least the pre- Escherichia coli) but also in more common viral and bacte-
scribed total volume over approximately 4 hours. rial enteritides (48,49).
A common cause of failure with oral rehydration is not The World Health Organization (WHO) and the
recognizing that the initial provision of fluid given “little United Nations Children’s Fund have championed the use
and often” is far more likely to be well tolerated than larger of a rehydrating solution that includes Na 90, Cl 80, K 20,
aliquots. If families are unwilling to provide the fluid in this base 30, and glucose 111 (2%) mmol/L. This WHO solu-
manner, a nasogastric tube may be placed for continuous tion has proved useful in many clinical trials in children
infusion. with dehydration from various causes, including secretory
Although some children may not respond to this and nonsecretory diarrhea, and has been shown to reduce
approach and require intravenous rehydration, most chil- the morbidity and mortality associated with diarrheal ill-
dren with mild to moderate rehydration can be rehydrated ness regardless of its etiology (54,55). Most commercially
orally without a great deal of trouble. A guide for the vol- available oral rehydration solutions differ from WHO solu-
umes of fluid to provide and the duration of rehydration tion in that they have a somewhat higher carbohydrate con-
can be found in Table 13.6. tent, a lower sodium content, and a higher carbohydrate to
The first oral rehydration solutions were developed in sodium ratio (Table 13.7).
the 1940s at academic medical centers. Within 10 years, a These formulation changes arose from concerns that
commercial preparation was available, but its use was asso- using an oral rehydrating solution with a sodium content
ciated with an increased incidence of hypernatremia (50). greater than 60 mmol/L may prove problematic in devel-
Several factors contributed to the development of this oped countries where most gastroenteritis is viral in nature
problem: The preparation was a powder meant to be recon- and has a lower sodium content than the secretory diar-
stituted with water and was sometimes incorrectly adminis- rheas commonly seen in less developed areas. It was feared
tered as the powder itself or improperly diluted with too that if minimally dehydrated children losing small amounts
little water. When correctly reconstituted, the solution had of sodium in their stools were exclusively provided WHO
a final carbohydrate concentration of 8% leading to an solution without provision of some excess CH2O, hyper-
286 II. Homeostasis

TABLE 13.7. ORAL REHYDRATION SOLUTIONS sodium or potassium supplementation. Moreover, the base
Concentration (mmol/L)
composition and the carbohydrate source are often subop-
timal for the dehydrated child, especially in the setting of
Product Na Glucose K Cl Citrate diarrheal illness (Table 13.8). Similarly, most beverages
WHO ORSa 90 111 20 80 30 marketed as sports drinks for “rehydration” after exercise
Rehydralyte (Ross) 75 140 20 65 30 are also deplete of sufficient electrolytes given that the elec-
Pedialyte (Ross) 45 140 20 35 30 trolyte composition of sweat is many times lower than the
Ricelyte (Mead 50 170b 25 45 34 composition of gastrointestinal fluid. In prescribing oral
Johnson)
rehydration to children in an ambulatory setting, the clini-
aOral
cian should clarify for the family the appropriate fluid and
rehydrating solution provided as powder. Needs to be reconsti-
tuted with water. volume for the child to ingest, emphasizing the need to use
bRice-syrup solids substituted for glucose.
a fluid with appropriate electrolyte content if there is con-
cern about evolving imbalances in sodium, potassium, or
natremia might ensue. A few studies did indeed document bicarbonate homeostasis.
iatrogenic hypernatremia related to such rehydration tech-
niques (56). In cases of mild dehydration stemming from
Oral Rehydration and Significant
causes other than secretory diarrhea, solutions with lower
Sodium Alterations
sodium contents may be useful; in fact, solutions with sodium
content ranging from 30 to 90 mmol have proved quite effec- Although oral rehydration is often associated with the child
tive in this setting (57–59). A more recent metaanalysis of with modest dehydration and no presumed electrolyte
studies focused on the safety and efficacy of oral rehydration anomalies, it has also been used in cases of dehydration
solution in well-nourished children living in developed coun- accompanied by hyponatremia or hypernatremia (60,61).
tries documented little evidence that WHO solution was It has met with overall success when a WHO-like solution
more likely to cause aberrations in serum sodium than oral has been used. In children with severe hypernatremia
rehydration solutions with lower sodium content. (greater than 160 mEq/L), although most can be success-
fully rehydrated orally, there have been reports of seizures,
generally as a result of too rapid correction of serum
Oral Rehydration with Fluids Other Than Oral
sodium stemming from the provision of supplemental
Rehydration Solution
water in addition to the glucose-electrolyte solution (60–
Despite the proven efficacy and fairly widespread availabil- 62). In those cases, the average serum sodium fell by 10 to
ity of commercial oral rehydration solutions and the ease 15 mEq/L over 6 hours rather than over 24 hours as
with which other electrolyte solutions can be mixed at advised. In follow-up studies, no seizure activity was seen in
home with recipes requiring few ingredients other than a similar cohort of hypernatremic children who received 90
water, sugar, and salt, there are, nonetheless, many children mmol Na rehydration solution alone at a rate calculated to
who are given common household beverages in attempts at replace the infant’s deficit over 24 hours (60). It is impor-
oral rehydration. In children with dehydration accompa- tant for the practitioner to remember that once peripheral
nied by electrolyte losses from vomiting or diarrhea, most perfusion has been stabilized with initial volume expansion,
common household beverages do not contain adequate there is no benefit to correcting any deficit rapidly, and tak-

TABLE 13.8. COMPOSITION OF COMMON ORAL FLUIDS

K Carbohydrate
Fluid Na (mEq/L) (mEq/L) Source of base (g/100 mL)

Apple juice <1 25 Citrate 12

Orange juice <1 55 Citrate 12
Milk 20 40 Lactate 5
Cola 2 <1 Bicarbonate 10
Ginger ale 4 <1 Bicarbonate 8
Kool-Aid <1 <1 Citrate 10
Gatorade 20 2.5 Citrate 4
Jell-O 25 <1 Citrate 14
Coffee <1 15 Citrate <0.5
Tea 2 5 Citrate 10

Adapted from Feld LG, Kaskel FJ, Schoeneman MJ. The approach to fluid and electrolyte therapy in
pediatrics. Adv Pediatr 1988;35:497–536.
 13. Fluid and Electrolyte Therapy in Children 287

ing 24 to 48 hours may be a more prudent course in the group of American academic pediatricians, private practi-
face of significant electrolyte anomalies. tioners, and pediatric house staff actually acknowledged
using such therapy (57). Oral rehydration schemes have
been shown to be used significantly more frequently by
Oral Rehydration Schemes
emergency room physicians who were familiar with the
Several oral rehydration schemes have been shown to be quite American Academy of Pediatrics’ oral rehydration recom-
effective and well tolerated. In one approach used extensively in mendations (65). Yet, even in these more knowledgeable
developing countries, the patient’s volume deficit is calculated emergency physicians, oral therapy was underused in chil-
on the basis of weight loss and clinical appearance (63). The dren with all degrees of dehydration. Worldwide, WHO
volume deficit is doubled; this is the target rehydration volume estimates that less than 25% of patients who could benefit
to be given over 6 to 12 hours. Two-thirds of this volume is from oral rehydration are actually treated with an appropri-
given as a glucose-electrolyte solution containing 90 mmol Na ate oral therapy (66). Moreover, even in areas of the world,
over 4 to 8 hours; once this has been ingested, the remaining such as Bangladesh, where oral rehydration has been cham-
volume is provided as water alone over 2 to 4 hours. In cases of pioned by both local and international medical agencies for
suspected or confirmed hypernatremia with serum sodium decades, its use is still suboptimal (67,68).
exceeding 160 mEq/L, the volume deficit is not doubled and is When used, oral rehydration has been demonstrated to be
administered as 90 mmol Na glucose-electrolyte solution alone quite successful, with more than 95% of children in a meta-
over 12 to 24 hours. Patients who refuse to take fluids by analysis of oral rehydration studies achieving some degree of
mouth have nasogastric tubes placed. With this approach, suc- volume repletion (69). Other advantages to oral therapy
cessful oral rehydration is the rule; 95% of children are fully include its ability to be administered readily anywhere by the
rehydrated without the need for intravenous therapy. child’s usual caretaker, the safety and stability of the product
An alternative approach has been to have the child begin despite its lengthy shelf-life, and avoidance of the discomfort
by taking 15 mL/kg/hr of a 60- to 90-mmol Na rehydra- and potential complications associated with intravenous
tion solution by mouth or nasogastric tube (64). The solu- catheter placement (70). In developed countries, there has
tion is given in small, frequent quantities and increased up been concern that the WHO solution may not be looked on
to 25 mL/kg/hr until hydration has improved, at which by parents as a convenient hydration solution because it
point solid feedings are reintroduced and volumes of 5 to involves preparation from a powdered packet. In fact, in a
15 mL/kg of rehydration solution offered after feeds until randomized controlled trial of an urban pediatric clinic and a
the volume deficit has been delivered. suburban medical practice in the United States, parents were
The American Academy of Pediatrics has issued guidelines equally as satisfied with the ease of administration and effec-
for the treatment of fluid and electrolyte deficits with oral tiveness of the WHO solution as compared to a commer-
rehydration solutions (48). Children with acute dehydration cially prepared oral rehydration solution (71).
and extracellular volume contraction should be provided 40 to Oral rehydration is somewhat less successful in hospital-
50 mL/kg of a glucose-electrolyte solution containing 75 to 90 ized children than in children treated in an ambulatory set-
mmol/L Na, 110 to 140 mmol/L glucose (2.0 to 2.5%), 20 ting (69). This difference may be directly related to the
mmol/L potassium, and 20 to 30 mmol/L base. This volume degree of dehydration or other complicating clinical issues
should be administered over 3 to 4 hours; once there has been leading to hospital admission. Moreover, the relatively
amelioration of the extracellular volume contraction, the labor-intensive, slower approach to oral rehydration may be
patient should be changed to a maintenance solution with 40 problematic in medical facilities with time constraints or
to 60 mmol Na at half the rate. If the child is still thirsty on space limitations (70,72).
this regimen, there should be free access to supplemental water There are recent reports that frozen flavored oral rehydra-
or low-solute fluid (e.g., breast milk). tion solution may be more readily accepted than conven-
In a recent evidence-based guideline for treating dehydra- tional oral solution. Its use resulted in higher rates of
tion in children from industrialized European countries, oral successful rehydration in children with mild to moderate
rehydrating solution containing 60 mmol/L of sodium, 90 dehydration, even if these children initially did not respond
mmol/L of glucose, 20 mmol/L of potassium, and 10 mmol/ to conventional oral rehydration (70). Frozen flavored rehy-
L of citrate was recommended, with rehydration occurring dration solution is now commercially available in some parts
over 3 to 12 hours using from 30 to 150 mL/kg of fluid of the world, as is a variety of flavored rehydration solutions.
depending on the degree and type of dehydration (30). This Another potential issue with oral rehydration is that its
rehydration scheme is summarized in Table 13.6. use does not alter the natural course of the child’s illness.
Thus, in gastroenteritis with dehydration, by far the most
common illness requiring rehydration in children, oral
Limited Use of Oral Rehydration Solutions
rehydration does not lower stool output or change the
Despite the promulgation of guidelines for oral rehydration duration of diarrheal illness (73). As a result, caretakers may
in a variety of clinical situations, less than 30% of a mixed abandon oral rehydration because the child is not getting
288 II. Homeostasis

TABLE 13.9. COMPOSITION OF COMMON INTRAVENOUS FLUIDS

Osmolarity Na K Buffer (source) Mg (mEq/ Ca (mEq/ Dextrose

Fluid (mOsm/L) (mEq/L) (mEq/L) Cl (mEq/L) (mEq/L) L) L) (g/L)

Crystalloids
0.9% Saline 308 154 0 154 0 0 0 0
Lactated Ringer’s 275 130 4 109 28 (lactate) 0 3 0
solution
5% Dextrose water 252 0 0 0 0 0 0 50
Normosol 295 140 5 98 27 (acetate) 3 0 0
23 (gluconate)
Plasma-Lyte 294 140 5 98 27 (acetate) 3 0 0
23 (gluconate)
Colloids
5% Albumin 309 130–160 <1 130–160 0 0 0 0
25% Albumin 312 130–160 <1 130–160 0 0 0 0
Fresh frozen plasma 300 140 4 110 25 (bicarbonate) 0 0 0
3.5% Haemaccel 301 145 5 145 0 0 6 0
6% Hetastarch 310 154 0 154 0 0 0 0
Dextran 40 or 70 310 154 0 154 0 0 0 0

better and is not appreciating the benefits of ongoing there are occasional children who are truly unable to sustain
hydration. Oral rehydrating solutions have been formu- an adequate rate of oral fluid intake despite concerted
lated with lower electrolyte composition and different car- efforts or have such persistent losses that parenteral therapy
bohydrate moieties with the goal to reduce the osmolarity comes to be necessary. The mainstays of fluid therapy in
of solutions and theoretically augment fluid absorption children are saline or buffered saline crystalloid solutions.
from the small intestine (74). The rice-based oral solutions Isotonic versions of these crystalloids are used for volume
have been studied most extensively. In these solutions, glu- resuscitation, and hypotonic saline solutions may be used
cose is substituted with 50 to 80 g/L of rice powder. In a in addition to provide supplemental maintenance hydra-
metaanalysis of 22 randomized clinical trials comparing tion. In addition to crystalloid solutions, there are several
rice-based solutions to conventional glucose-containing colloid fluids that are used by many clinicians. Table 13.9
solutions, stool output dramatically decreased in children lists the electrolyte content of some of the more common
with cholera given rice-based hydration but did not change intravenous solutions used for pediatric fluid therapy.
in children with other bacterial or viral enteritides (75).
There are some reports that suggest that providing chil-
Choice and Volume of Parenteral Fluid
dren with noncholera enteritis with reduced osmolarity rehy-
dration solution may be beneficial. In one study, 447 boys Children with significant extracellular volume contrac-
younger than 2 years of age admitted for oral rehydration tion (greater than 10% acute weight loss in an infant or
were assigned to receive either the WHO solution (osmolar- 6% weight loss in an older child) should receive an iso-
ity, 311 mmol/L) or a solution containing less sodium and tonic crystalloid solution [e.g., 0.9% saline (154 mEq/L
chloride (osmolarity, 224 mmol/L). Children who received NaCl) or Ringer’s lactate (130 mEq/L NaCl)] at a rate of
the lower-osmolarity solution had reduced stool output, 20 mL/kg over 30 to 60 minutes. Children with a more
reduced duration of diarrhea, reduced rehydration needs, moderate degree of dehydration may not exhibit signs or
and reduced risk of requiring intravenous fluid infusion after symptoms of volume contraction. In certain situations,
completion of oral hydration (76). A metaanalysis of nine however, it may be clinically warranted to provide them
trials comparing WHO solution to reduced-osmolarity rehy- with an initial rapid intravenous bolus to initiate rehydra-
dration solution concluded that children admitted for dehy- tion therapy.
dration had reduced needs for intravenous fluid infusion, Concomitant with the placement of intravenous access,
lower stool volumes, and less vomiting when receiving the blood should be obtained for determination of serum elec-
reduced-osmolarity solution (77). trolytes, osmolality, and renal function. Given that dehy-
drated children often have high levels of vasoactive
hormones and high vasopressin levels, it is most circum-
Intravenous Therapy
spect to establish baseline electrolyte levels, as it is possible
Although absolute indications for parenteral intravenous to alter electrolyte balance rapidly with intravenous ther-
therapy are limited, they do include any condition of apy. In the face of inadequate tissue perfusion, a parenteral
impaired peripheral circulation or overt shock. In addition, fluid infusion should begin immediately before the return
 13. Fluid and Electrolyte Therapy in Children 289

of any pertinent laboratory results. If hemorrhagic shock is tion of these results from ill adults may not be germane to
suspected, resuscitation with packed red blood cells is opti- all critically ill, volume-depleted children. For instance, a
mal. In cases of severe volume depletion, if the child does report of 410 children with meningococcal disease suggests
not improve with the initial 20-mL/kg crystalloid bolus, that albumin infusion in this population may not have
this should be repeated up to two additional times. In chil- been harmful, as case fatality rates were lower than pre-
dren who have not improved despite administration of 60 dicted (86). Overall, however, there seem to be no substan-
mL/kg of total volume over 1 hour or in children in whom tive data to support the routine use of colloid to
underlying cardiac, pulmonary, or renal disease may make complement or replace crystalloid in fluid resuscitation.
empiric aggressive rehydration more problematic, consider- Rather, repetitive infusions of large volumes of crystalloid
ation should be given to placement of a central monitoring seem to be well tolerated in volume-depleted children; do
catheter to more accurately assess intravascular volume and not seem to predispose to excessive rates of acute respira-
cardiac dynamics (78). In some instances of profound inef- tory distress syndrome or cerebral edema; and in some con-
fective circulating volume (e.g., those that might accom- ditions (e.g., sepsis), play an important role in improved
pany certain cases of sepsis), initial volume resuscitation survival (87). A recent survey of pediatric anesthesiologists
may require sequential infusions of fluid ultimately exceed- in western Europe reported that colloid solutions are being
ing 100 mL/kg. used less frequently in infants and older children and sug-
Within minutes of infusion of a crystalloid fluid, it gested that familiarity with some of the issues raised in
becomes distributed throughout the extracellular space. these systematic reviews is affecting practice patterns (88).
Because this involves equilibration of the fluid between the Repetitive infusions of crystalloid may also prove prob-
two components of the extracellular space—the intravascu- lematic in some children. Most notably, if very large vol-
lar and interstitial spaces—actually only one-third to one- umes of 0.9% saline are used acutely for volume
fourth of infused crystalloid stays in the blood vessels (78). resuscitation, it is not unusual for children to develop a
This accounts for the need to give large volumes of crystal- hyperchloremic metabolic acidosis. This occurs as acidotic
loid in the setting of circulatory collapse and leads some to peripheral tissues begin to reperfuse and already depleted
suggest that colloid solutions (e.g., 5 or 10% albumin) extracellular bicarbonate stores are diluted by a solution
should play a role in resuscitation (79,80). with an isotonic concentration of chloride (78,89). This aci-
dosis can be ameliorated by supplemental doses of bicarbon-
ate as well as the addition of supplemental potassium as
Colloid Solutions for Volume Resuscitation
needed. There is sometimes a tendency for clinicians to react
The use of colloid solutions for volume resuscitation is con- to the hyperchloremic metabolic acidosis with further saline
troversial. In the 1990s, colloids were included in a number bolus infusions. In the face of corrected hypoxia or hypo-
of widely promulgated guidelines for the care of patients in volemia, however, such maneuvers may only exacerbate the
emergency facilities and intensive care units both for hem- chloride-driven acidosis (90). This hyperchloremic acidosis
orrhagic shock before the availability of blood and for non- is seen less frequently when Ringer’s lactate solution is used
hemorrhagic shock as an adjunct to crystalloid use (81). as the resuscitation fluid because of the metabolic conver-
Types of colloids used included 5% albumin, fresh frozen sion of lactate to bicarbonate. In the setting of significant
plasma, modified starches, dextrans, and gelatins. These preexisting acidosis or underlying hepatic dysfunction pre-
guidelines, generally aimed toward the fluid resuscitation of venting the metabolism of lactate, infusion of Ringer’s lac-
adults, were composed despite the prior publication of a tate solution may, however, exacerbate an acidosis.
systematic review of randomized controlled trials that dem- Large-volume infusion of blood may predispose to elec-
onstrated no effect on mortality rates when colloids were trolyte anomalies as well as manifestations of citrate toxic-
used in preference to crystalloids (82). Moreover, there is a ity. If aged whole blood is infused, there is the possibility
distinct cost disadvantage to using colloid solutions. that a large potassium load is delivered to the patient, as
Two subsequent systematic reviews have looked at this potassium, over time, migrates down its concentration gra-
issue anew. In one meta-analysis of 38 trials comparing col- dient from less viable erythrocytes into plasma. Because
loid to crystalloid for volume expansion, there was no most patients receive packed red blood cells instead of
decrease in the risk of death for patients receiving colloid whole blood, this potential problem is minimized, as little
(81). In the other review, albumin administration was actu- plasma is infused; thus, the relatively small amount of
ally shown to increase mortality by 6% compared to crys- infused potassium can be accommodated by movement
talloid (83). Proposed mechanisms contributing to this intracellularly.
worse outcome include anticoagulant properties of albumin Citrate is used as the anticoagulant in stored blood.
(84) and accelerated capillary leak (85). Because citrate complexes with calcium, there can be a fall in
A drawback of all these systematic reviews, however, has ionized calcium levels if large volumes of blood are infused
been the limited number of studies that included children rapidly or if there are concomitant perturbations in calcium
other than ill premature neonates. As a result, generaliza- homeostasis. Similarly, citrate may complex with magne-
290 II. Homeostasis

sium, and magnesium depletion may occur. The liver usually Symptomatic Hyponatremia
metabolizes infused citrate into bicarbonate. Alkalosis may,
In the setting of symptomatic hyponatremia, especially if
therefore, occur if large volumes of citrate are metabolized. In
the child has seizures, it is important to raise the serum
the setting of hepatic dysfunction, however, citrate is not
sodium approximately 5 mEq/L acutely. Generally, this
metabolized, serves as an acid load, and helps to create an
results in stabilization of the clinical situation and allows
acidosis or exacerbate any underlying acidosis.
for further evaluation and treatment of the child to proceed
Regardless of the initial infusion with either colloid or
in a nonurgent fashion. This is one of the few situations in
crystalloid, once sufficient volume to restore circulatory
which hypertonic saline (3% saline) should be used.
integrity has been infused, less rapid volume expansion is
To calculate the proper volume of 3% saline to infuse, the
necessary. During this phase, the rapidity of fluid repletion
child’s TBW must be multiplied by the 5-mEq/L desired
is most probably not a concern unless there are severe
increase in serum sodium to determine the amount of
underlying aberrations in the serum sodium or serum
sodium (in milliequivalents) to infuse. Because every millili-
osmolality. In the absence of these derangements or pro-
ter of 3% saline contains 0.5 mEq of sodium, doubling the
found volume deficit, if the child has improved signifi-
number of milliequivalents of sodium needed results in the
cantly with the initial parenteral volume expansion,
proper volume in milliliters of 3% saline to infuse. Thus, in
attempts should be made to reinstate oral rehydration. Pro-
the 20-kg child, the TBW is approximately 12 L (0.6 L/kg ×
longed intravenous therapy should rarely be needed.
20 kg), and the desired sodium dose is 60 mEq (12 L × 5
mEq/L). If 120 mL of 3% saline were infused, the serum
Rapid Rehydration sodium would be expected to rise by approximately 5 mEq/
Over the last decade, a scheme of rapid intravenous resusci- L. The infusion should be given at a rate to increase the
tation and follow-up oral rehydration has been adopted by serum sodium by no more than 3 mEq/L/hr and is often
many pediatric emergency departments to successfully treat given more slowly over the course of 3 to 4 hours (93). If the
children with up to 10% dehydration secondary to vomit- child continues to be symptomatic from hyponatremia after
ing and gastroenteritis (91). After infusion of 20 to 30 mL/ this infusion, additional 3% saline may be given until the
kg on intravenous crystalloid, the child is allowed to take symptoms improve or the serum sodium is in the 120 to 125
up to several ounces of a standard oral rehydration fluid; if mEq/L range. At that point, further correction of the hypo-
this intake is tolerated without vomiting for 30 to 60 min- natremia should consist of a slower infusion of more dilute
utes, then the child is discharged home to continue rehy- saline to cover the sodium deficit, the sodium maintenance
dration, initially with a prescribed volume of standard needs, and any volume deficit.
rehydration solution.
If the child does not tolerate oral rehydration or if there
Asymptomatic Hyponatremia
are such significant electrolyte anomalies that there are con-
cerns regarding potential adverse CNS sequelae of too rapid If a child has severe hyponatremia but is not symptomatic,
rehydration, then intravenous rehydration may be the best there is no need to administer hypertonic saline based
route for continued hydration. It is rare for children to solely on a laboratory anomaly. With or without symp-
become symptomatic from serum sodium aberrations until toms, in cases of severe hyponatremia, the child should be
levels less than 120 mEq/L or greater than 160 mEq/L are carefully evaluated as to the etiology of the hyponatremia,
reached. Children who have had very sudden fluxes in elec- keeping in mind that hyponatremia may be the result of an
trolytes may become symptomatic earlier. On the other imbalance of water regulation. If this is the case, CH2O
hand, children whose severe sodium abnormalities are should be restricted and appropriate supplementation with
believed to be more chronic in nature must be treated in a intravenous saline solutions begun to provide maintenance
more controlled fashion, as they are at higher risk for devel- sodium requirements of approximately 2 to 3 mEq/kg/day
oping CNS symptoms during treatment. and any ongoing losses of sodium.
The vast majority of children treated in emergency Besides these maintenance sodium needs, if the child has
facilities for volume repletion do well with such rapid an element of dehydration, every kilogram of body weight
rehydration. These children are generally healthy with nor- lost from baseline represents a 1-L deficit of normal saline
mal cardiac and renal function and have developed extra- from the TBW as well. These losses are often referred to as
cellular volume depletion relatively rapidly. As a result, isotonic losses. These account for a sodium deficit of 154
they experience no ill effects from rapid rehydration. In mEq/L that also must be included in the calculations for
fact, the clinical success of this aggressive restoration of sodium replacement.
extracellular volume underlies the calls to reexamine the In the setting of hyponatremic dehydration, there have
traditional deficit therapy approach to rehydration with its been additional sodium losses as well. Generally, this
tedious calculations of fluid and electrolytes losses and sodium deficit occurs because stool losses are replaced with
requirements (4,92). fluids with a sodium content less than 60 mEq/L. To esti-
 13. Fluid and Electrolyte Therapy in Children 291

mate these sodium losses, the difference between the child’s The child’s water deficit is 1.2 L, reflecting the 1.2-
desired serum sodium and current serum sodium is multi- kg weight loss. She has normal maintenance water
plied by the child’s estimated TBW. This product represents needs of an additional 1 L/day based on her normal
the hyponatremic sodium losses that must be added to the weight of 10 kg. She is having no other ongoing water
maintenance sodium needs, any ongoing losses, and the losses and has already received nearly 250 mL in intra-
sodium losses that accompanied weight loss. An example of venous fluid in the form of 0.9% NaCl and 3% NaCl.
the calculations and therapeutic maneuvers that need to be Thus, her current water needs are 1950 mL.
considered with significant hyponatremia is presented in The child’s normal maintenance sodium needs are
the following case study: 30 mEq/day (3 mEq/kg/day). She has lost 1.2 kg in
body weight that represents 185 mEq of sodium. In
A girl who normally weighs 10 kg presents having gener- addition, she has hyponatremic sodium losses that
alized seizures. She has had 1 week of gastroenteritis have arisen as her diarrheal stool was replaced with
with intermittent fever and has been drinking water water alone. To calculate these needs, her normal TBW
only, refusing any other feeds for several days. Emer- needs to be multiplied by the difference in her serum
gency intravenous access is obtained. Lorazepam is sodium from a normal value of 135 mEq/L. Her
administered, and the seizure activity stops. Blood work TBW is 6 L (TBW = 0.6 L/kg × 10 kg), and the dif-
is obtained, and the child is weighed and found to be ference in serum sodium is 23 mEq/L (135 mEq/L to
8.8 kg. A bolus infusion of 200 mL of 0.9% NaCl is 112 mEq/L); her hyponatremic losses are, therefore,
administered, after which the girl appears adequately 138 mEq (6 L × 23 mEq/L). Thus, total sodium needs
perfused but still lethargic. She no longer has diarrhea are 30 mEq of maintenance, 185 mEq of isotonic
but vomits any small volumes of oral rehydration solu- losses, and 138 mEq of hyponatremic losses, or a total
tion offered. The serum sodium is then reported to be of 353 mEq. She has already received 52 mEq of
112 mEq/L. While further evaluation of the child’s over- sodium from the 400 mL of 0.9% NaCl given in the
all status is ongoing, it is important to begin correcting emergency department. Her current sodium needs are,
the apparent symptomatic hyponatremia. thus, approximately 300 mEq.
The child has actually already received approximately
To choose the proper solution for this child, the deficit
30 mEq of sodium in the 0.9% NaCl bolus given
of 1950 mL of water should contain 300 mEq of sodium.
because of her dehydration and poor perfusion. Given
This is best approximated by 0.9% NaCl with its NaCl
her TBW of roughly 5.4 L (weight in kg × 0.6 L/kg),
content of 154 mEq/L NaCl. In the past, it has been sug-
this should result in her serum sodium having already
gested that half of the fluid and sodium deficit be replaced
increased by approximately 5 mEq/L. Because the child
over 8 hours and the remainder over the ensuing 16 hours.
has had hyponatremic seizures and is still exhibiting
Although such a plan can be followed, there is little evi-
some CNS effect with her lethargy, it is probably pru-
dence that more rapid correction of the hyponatremia is
dent to raise the serum sodium by approximately 5
harmful except if the patient has been symptomatic with
mEq/L so that it is in the 120- to 125-mEq/L range.
hyponatremia or has profound asymptomatic hyponatre-
Because she is hemodynamically stable, it is probably
mia of chronic duration. In these cases, it is safest to plan to
best not to provide an excess of further volume until the
correct the serum sodium by no more than 12 to 15 mEq/L
child’s brain is imaged to assess for cerebral edema, espe-
over 24 hours. More rapid correction has resulted in
cially given the history of seizures, lethargy, and hypona-
osmotic demyelination injury to the brain with devastating
tremia. By using a small volume of hypertonic saline, the
long-term neurologic outcomes (93,96,97).
serum sodium could begin to be raised in a controlled
manner while further evaluation of the child continues.
It would take approximately 22 mEq of sodium (TBW
Severe Hypernatremia
× desired increase in serum sodium = 5.4 L × 5 mEq/L)
to accomplish the desired elevation. Because each millili- With hypernatremia, intravenous therapy is again guided by
ter of 3% saline contains approximately 0.5 mEq of the clinical situation and is usually reserved for those children
sodium, a total of 44 mL of 3% saline could be infused with very elevated serum sodium values. In cases of hyper-
over approximately 3 to 4 hours. natremia due to salt poisoning, there should be signs of over-
In addition to this acute management to restore cir- hydration and volume expansion. Excretion of sodium should
culation and perfusion and to raise the serum sodium be enhanced by using a loop diuretic to augment urine sodium
to a safer level, plans must be formulated to attend to losses and by replacing urine output with CH2O. If the patient
the patient’s overall volume and sodium deficit. To pre- has significant renal or cardiac compromise because of the elec-
scribe the proper follow-up intravenous fluid, the trolyte imbalance, dialysis and ultrafiltration may be necessary
patient’s initial water and electrolyte deficits must be (25,26). With hypernatremia and volume expansion, however,
reconciled with her therapy thus far. it is detrimental to provide further intravenous saline.
292 II. Homeostasis

In hypernatremia accompanied by volume loss, any sig- The baby has maintenance sodium needs of 15
nificant alterations in effective circulation should be mEq/day (3 mEq/kg/day). Her sodium deficit reflects
addressed with 20-mL/kg bolus infusions of an isotonic crys- only her isotonic fluid losses that have been estimated
talloid solution until effective peripheral perfusion is above at 240 mL of normal saline or 37 mEq of
restored. Then, further provision of water and sodium sodium. Thus, over the next 2 days, her sodium needs
should be provided based on calculated water and sodium are 67 mEq, of which she has already received the vast
needs. In the majority of cases, however, with mild elevations majority in the emergency department due to her need
in serum sodium and minimal degrees of dehydration, the for initial volume expansion.
actual calculation of deficits is probably unnecessary, as the Initiating an infusion of 30 mL/hr of CH2O should
child is hemodynamically stable initially and a candidate for result in the slow and steady correction of the hyper-
exclusive oral rehydration. In situations in which there is pro- natremia over 2 days. The serum sodium should be
found hypernatremia or circulatory compromise, it remains monitored every 4 hours initially; if it is falling faster
necessary, however, to be able to calculate a CH2O deficit to than desired (approximately 0.5 mEq/hr), then sodium
tailor intravenous rehydration therapy. An example of such a should be added to the rehydration fluid.
situation is outlined in the following case study:
After 2 days of refusing any fluid intake, a 5-kg infant
with a viral syndrome presents in shock and is 15% FLUID AND ELECTROLYTE THERAPY WITH
dehydrated with a weight of 4.25 kg and a serum RENAL DYSFUNCTION
sodium of 170 mEq/L. She receives 300 mL of 0.9%
NaCl in the emergency department and is now admit- Impact of Renal Disease on Fluid and
ted for further therapy. Electrolyte Therapy
The child has lost 750 g of weight. Because this is Compromised renal function reduces the tolerance for
hypernatremic dehydration, there has been loss of changes in TBW as well as changes in the composition or dis-
water in excess to salt. Thus, part of the weight loss tribution of volume between the intracellular and extracellu-
represents isotonic losses, but a larger proportion rep- lar body spaces. Similarly, alterations in electrolyte balance are
resents CH2O loss. The child’s CH2O deficit can be less readily tolerated because the normal homeostatic mecha-
calculated by the following equation: nisms are frequently perturbed. Thus, in the child with cer-
[(serum Na actual)/(serum Na desired) × TBW] – TBW tain renal disorders (e.g., marked nephrosis or significant
Substituting the appropriate data for this baby: impairment in renal clearance), it becomes vital to approach
the provision of fluids and electrolytes with great care.
[(170/145) × (0.6 × 4.25)] – (0.6 × 4.25) =
As far as fluid therapy is concerned, it is important to
(1.2 × 2.55) – 2.55 = 0.51 L
recognize that any scheme to provide maintenance fluids or
Therefore, of this baby’s 750-mL fluid deficit due to electrolytes presupposes normal renal function. Roughly
dehydration, 510 mL is CH2O and 240 mL is nor- two-thirds of any daily maintenance fluid prescription is to
mal saline. replace urinary water losses. Similarly, urinary electrolyte
Too rapid correction of the child’s serum sodium losses figure prominently in daily electrolyte balance. In the
with CH2O could result in cerebral edema as the water setting of oliguria or anuria, provision of maintenance flu-
infused into the extracellular space follows osmotic ids could contribute to and potentially exacerbate volume
forces and moves into the intracellular space. In cases overload, and maintenance electrolyte therapy could result
of hypernatremia in which the serum sodium exceeds in electrolyte anomalies.
160 mEq/L, it is considered safest to correct the serum It is better to approach the fluid and electrolyte needs of the
sodium by no more than 15 mEq/day. In this baby’s child with renal dysfunction in the context of his or her cur-
case, this means that correction to a serum sodium in rent volume status and electrolyte needs. The patient who is
the normal range would take approximately 2 days. symptomatic due to volume depletion benefits from volume
If the fluid and electrolyte therapy must be given expansion regardless of urine output. Once volume is replete,
intravenously, the appropriate prescription again the child’s needs can be reassessed along with his or her current
depends on calculation of water and sodium require- renal function. The child who is volume overloaded is best
ments and deficits. Her original fluid deficit was 750 managed by volume restriction and provision of only insensi-
mL, and her maintenance water needs are estimated at ble fluid losses of approximately 300 mL/m2. Insensible fluid
500 mL/day. Thus, over the next 2 days, the fluid losses should be considered essentially CH2O. The child who
needs to replace her deficit and to provide maintenance is volume replete should be kept volume replete. This is most
are 1750 mL. Of this, she has already received 300 mL readily accomplished by providing a combination of insensible
of fluid in the emergency department, so she has a net losses as CH2O and any other volume losses (urine output,
deficit of 1450 mL. diarrheal stool, surgical drain output, vomitus) on an addi-
 13. Fluid and Electrolyte Therapy in Children 293

tional milliliter-for-milliliter basis. If there are significant ongo- efficacy of tissue perfusion, current ventilatory requirements,
ing losses from a single source, the electrolyte composition of and current renal function. Whenever there is concern about
this fluid can be assayed so that the replacement fluid may incipient or exacerbating volume overload, it is important to
more accurately reflect the electrolyte losses. Otherwise, a solu- review the volume and types of fluids being provided.
tion of 0.45% NaCl can be used initially and altered as the Increasing the concentration of continuous medication drips
clinical situation continues to develop and further electrolyte and assessing medication compatibility for simultaneous
determinations are made. infusion are important steps in limiting total daily fluid
If the child’s volume status or the adequacy of renal input. Initially, it is often appropriate in these critically ill
function is difficult to discern initially, it is probably best to children to ascertain that their intravascular space is replete
provide the child with replacement of both insensible and to help maintain hemodynamic stability. Once the patient is
ongoing losses. This should maintain the child’s current believed to be intravascularly replete, maintaining euvolemia
volume status and allow for further determination of the by providing insensible water losses as well as replacing any
appropriateness of more vigorous hydration or, conversely, ongoing fluid and electrolyte losses should maintain the
fluid restriction as the clinical situation clarifies. Monitor- patient in fluid and electrolyte balance.
ing the child’s weight on at least a daily basis and docu- Oftentimes, despite a desire to limit fluids in the critically
menting the child’s total fluid intake and output also assists ill child, medication requirements, nutritional needs, and
in arriving at a proper hydration regimen. hemodynamic insufficiency may result in the development of
Assessing the child’s current electrolyte status and moni- significant volume overload. There may also be situations in
toring the loss of electrolytes in the urine or in any other which due to the “vascular leak” phenomenon, the critically ill
source of significant electrolyte losses assist in tailoring the child becomes massively volume overloaded but has a
daily electrolyte prescription. An understanding of the decreased effective circulating volume. In other words, renal
pathophysiology underlying the child’s renal dysfunction is and tissue perfusion may be a sluggish because fluid has leaked
also useful. The child who has profound tubular electrolyte from the intravascular space into the interstitial space. In this
losses requires more sodium on a daily basis than the child setting, there may be a need to continue to administer large
who is edematous and has total body salt overloaded from volumes of fluid to maintain circulatory integrity with the
his or her nephrotic syndrome. The child with chronic knowledge that such infusions only exacerbate the total body
renal insufficiency and hypertension mediated by long- fluid overload. Aggressive diuretic therapy may prove useful
standing salt and water overload may actually benefit from especially if renal function is not compromised. Combination
diuretic therapy to remove salt and water rather than any diuretic therapy using agents that work at separate sites along
further volume expansion with saline. the renal tubule may be necessary. Ultimately, the use of either
Certainly, the provision of supplemental potassium to periodic or continuous ultrafiltration may be beneficial to
the child with renal dysfunction must be done judiciously. these patients by allowing ongoing fluid administration but
The oliguric or anuric child should receive no potassium limiting the daily imbalance between fluid intake and output.
until it is well documented that serum potassium levels are Ultrafiltration may be accomplished via peritoneal dialysis, by
low or that there are extrarenal potassium losses (e.g., gut intermittent hemodialysis with ultrafiltration, or by using one
losses from diarrheal stool). The child with marginal renal of the slow continuous ultrafiltration techniques now known
function should receive small amounts of potassium as continuous renal replacement therapy.
(approximately 1 mEq/kg/day) with at least daily assess- If ultrafiltration is initiated, extreme vigilance is neces-
ment of electrolyte balance to reassess the adequacy and sary to prevent exacerbation of intravascular depletion and
appropriateness of continued potassium supplementation. the development of prerenal azotemia or frank renal failure.
Special care must be taken with the continuous modalities
to insure that ultrafiltration rates are periodically reassessed
Fluid and Electrolyte Therapy in the
and readjusted. Furthermore, because the electrolyte losses
Intensive Care Unit
that accompany the ultrafiltration of fluid are isotonic, the
Many children who are critically ill present a challenge to the electrolyte content of infused fluids must be adjusted to
clinician attempting to prescribe appropriate fluid and elec- match the composition of the ultrafiltrate. Serum electro-
trolyte therapy. Oftentimes, there may be renal dysfunction, lyte values need to be followed in a serial fashion with peri-
and frequently there may be multiorgan failure complicating odic review and readjustment of the composition of
management decisions. What frequently creates significant supplemental intravenous fluids.
fluid and electrolyte anomalies is rather rote reliance on stan-
dard formulas to prescribe fluid and electrolyte therapy.
Alterations in Serum Sodium Complicated
Rather than relying exclusively on a set maintenance require-
by Renal Failure
ment of fluid or electrolytes, it is almost always best to assess
the patient’s individual fluid and electrolyte needs in the con- Because of the important contribution of serum sodium to
text of the underlying pathophysiology, volume status, the serum osmolality, alterations in serum sodium, especially
294 II. Homeostasis

coupled with alterations in BUN related to renal failure, essary for urea clearance. By performing controlled
can complicate the usual approach to a child with fluid and ultrafiltration on the patient and replacing back with
electrolyte anomalies. Generally, there are greater concerns CH2O the volume ultrafiltered, the serum sodium
with hypernatremia and renal failure, as the need to correct could be corrected without exacerbating the volume
the sodium in a slow fashion can be problematic when status.
renal replacement therapy needs to be initiated for clear- With a serum sodium of 165 mEq/L and an esti-
ance of urea. Balancing the correction of sodium and the mated TBW of 42 L (70 kg × 0.6 L/kg), this patient
hyperosmolar state with the clearance of urea requires a has CH2O needs of 7.5 L to lower his serum sodium to
carefully considered plan that is grounded in a firm under- the 140-mEq/L range [(165/140 × 42) – 42]. Because
standing of fluid and electrolyte homeostasis. the patient is now significantly hypernatremic and has
In most cases of hypernatremia related to severe dehydra- been subject to various fluid and electrolyte shifts as his
tion, some degree of acute renal insufficiency is present. This diabetic ketoacidosis has been treated, it is prudent to
renal failure is usually prerenal in nature, a result of a correct his serum sodium by no more than 10 to 12
decreased effective circulating volume rather than an intrin- mEq/day over the course of 3 days. Thus, if the patient
sic glomerular or tubular disorder. Most often, in the course is ultrafiltered for 2.5 L/day, and the ultrafiltration vol-
of rapid restoration of perfusion and early rehydration, the ume each day is replaced back as CH2O, the serum
urine output increases, and the azotemia begins to resolve. sodium should be in the normal range in 3 days’ time.
Alternatively, there are occasional cases in which, due to The ultrafiltration goal could be achieved over the
intrinsic renal dysfunction or acute tubular necrosis, the renal course of a few hours each day if the patient were
insufficiency does not respond to volume infusion; in fact, the hemodynamically stable or over a more prolonged
provision of excess volume may contribute to significant vol- period of time each day if there were concerns regard-
ume overload. In these cases, there may be need to consider ing hypotension. Thus, a conventional hemodialysis
some form of renal replacement therapy to assist in the con- setup could be used for relatively rapid ultrafiltration
trolled correction of fluid and electrolyte derangements, espe- only or a continuous filtration circuit for either rapid
cially if the renal failure is oliguric or anuric in nature. Such an or slow filtration.
example is detailed in the following case study: Because the fluid removed in ultrafiltration is
isonatremic to the serum sodium, the sodium concen-
A 15-year-old boy presents with a several-week history tration of each liter of ultrafiltrate should mirror the
of polyuria, severe weight loss, fatigue, and poor oral serum sodium concentration at the time of ultrafiltra-
intake. He is diagnosed by his pediatrician as having tion. Thus, on the initial day of ultrafiltration, each
diabetes mellitus with ketoacidosis and is referred to an liter of ultrafiltrate should contain a sodium content of
emergency department for management. At this point, 165 mEq/L. By providing back the volume ultrafil-
his serum sodium is 154 mEq/L, his creatinine is 3.0 tered each day as CH2O, the serum sodium content is
mg/dL, and his BUN is 30 mg/dL. In the emergency expected to fall, in this case, by approximately 8 to 10
department, the child receives several bolus infusions mEq/L/day.
of normal saline supplemented with sodium bicarbon- It is important to recognize that CH2O must be pro-
ate and is started on an insulin drip. He is admitted vided back to the patient to make up for the ultrafiltra-
and continues to receive brisk intravenous hydration tion losses. Otherwise, because the ultrafiltrate is
with normal saline with bicarbonate supplementation. isotonic, there is no change in the serum sodium con-
He is noted to be oliguric, and this does not improve centration, and the ultrafiltration may potentially exac-
with several more hours of hydration with normal erbate the renal failure by depleting the intravascular
saline. The next morning, laboratory values reveal a space and the effective circulating volume.
serum sodium of 165 mEq/L, a creatinine of 4.5 mg/ Moreover, it is also important to recognize that the
dL, and a BUN of 50 mg/dL. He has made only 75 patient’s overall daily fluid needs are greater than the
mL of urine in the last 8 hours and is developing some daily ultrafiltration volume alone, as maintenance fluid
mild peripheral edema. requirements and any ongoing fluid losses must also be
In this case, the renal insufficiency and poor urine considered. Because the patient is in renal failure, his
output have complicated the usual management of dia- maintenance fluid needs are scaled back to insensible
betic ketoacidosis and have exacerbated an underlying losses of 300 mL/M2/day; in this case, there are no ongo-
hypernatremia. Given the patient’s evolving renal fail- ing losses. Thus, each day for the next 3 days, this 70-kg
ure, it is not feasible to provide the necessary volume of patient needs to receive approximately 500 mL/day of
CH2O to correct the hypernatremia without contrib- insensible losses and 2500 mL/day of ultrafiltration
uting to further volume overload. Because of the replacement, or a total of 3000 mL/day. His mainte-
apparent progressive renal failure, it is also useful to nance sodium requirements are 3 mEq/kg/day. Although
correct the hypernatremia in case dialysis becomes nec- it may seem counterintuitive to provide a hypernatremic
 13. Fluid and Electrolyte Therapy in Children 295

patient with maintenance sodium, disregarding these it is easier to use renal replacement therapy in the setting of
requirements results in a more rapid correction of the severe hyponatremia and concomitant renal insufficiency.
hypernatremia than desired. If the child were to receive a Again, the focus needs to be on the rapidity of the correc-
saline infusion of 0.45% NaCl at a rate of 125 mL/hr, tion of the serum sodium. In conditions of chronic severe
this would provide just over 3 mEq/kg/day of sodium in but asymptomatic hyponatremia, the rate of correction of
a total volume of 3 L/day. serum sodium should parallel the rate of correction recom-
mended in hypernatremia—approximately 10 to 12 mEq/
If the child with hypernatremia has profound renal fail- L/day. Correction of chronic hyponatremia at a more rapid
ure and requires dialysis for urea clearance, the dialysis pre- rate has been associated with the development of central
scription must take into account the need to slowly correct pontine demyelinosis.
the serum sodium. Normally, regardless of the modality of All of the manipulations described above for hypernatre-
renal replacement therapy, most dialysate contains sodium mia and renal failure can be used with hyponatremia and
isotonic to the normal serum sodium range. It may prove renal failure with the understanding that the dialysate
detrimental, however, to dialyze a patient who is very sodium concentration should now not exceed the serum
hypernatremic against a dialysate with a sodium concentra- sodium value by 10 to 12 mEq/L. Conventional hemodial-
tion at least 30 mEq/L less than the patient’s serum sodium ysis machines can be adjusted to produce dialysate with a
concentration. The diffusional gradient during dialysis sodium concentration as low as the mid-120s. In the very
leads to more rapid correction of the serum sodium than rare situation in which a child with profound hypernatre-
the desired drop of approximately 1 mEq every 2 hours. mia (less than 110 mEq/L) was being hemodialyzed, brief
Although most hemodialysis machines can be readjusted hemodialysis runs may be necessary initially to prevent too
so that the dialysate produced has a sodium content as high rapid correction of the serum sodium level and the atten-
as the low to mid-150s, this still may not reduce the gradi- dant risk of central pontine demyelinosis. If dialysate is
ent sufficiently in cases of severe hypernatremia. In those being custom prepared for peritoneal dialysis or hemodia-
situations, by maximizing the sodium concentration of the filtration, precise alterations in the electrolyte content can
dialysate and by performing dialysis for limited amounts of be made more readily to reduce the sodium gradient.
time, one can minimize the drop in serum sodium. Still, The local resources, the training of ancillary staff, the
there need to be frequent assessments of the serum sodium unique circumstances of each patient, and the comfort of
concentration, and overall clearance may need to be sacri- the clinician with different modalities of renal replacement
ficed to prevent too rapid correction of the serum sodium therapy guide the choice of therapy when faced with renal
and a rapid concomitant decrease in the serum urea that failure and significant serum sodium anomalies. The actual
may increase the chances for dialysis dysequilibrium. modality of renal replacement therapy used is less impor-
Alternatively, a continuous hemodiafiltration technique tant than careful attention to the rate of correction of the
such as continuous venovenous hemodiafiltration can be electrolyte anomaly, to the rate of urea clearance being
performed. By asking the hospital pharmacy to increase the achieved, and to the clinical response of the patient to
sodium content of the dialysate fluid to within 10 to 12 ongoing therapy.
mEq/L of the serum sodium concentration, the diffusional
gradient for sodium clearance can be minimized. Then, by
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apy be improved? WHO Drug Information 2000;14:88. CD002847.
56. Nalin DR, Harland E, Ramlal A, et al. Comparison of low 75. Fontaine O, Gore SM, Pierce NF. Rice-based oral rehydra-
and high sodium and potassium content in oral rehydration tion solution for treating diarrhoea. Cochrane Database Syst
solutions. J Pediatr 1980;97:848–853. Rev 2000;2:CD000567.
57. Snyder JD. Use and misuse of oral therapy for diarrhea: 76. International Study Group on reduced-osmolarity ORS
comparisons of US practices with American Academy of solutions. Multicentre evaluation of reduced-osmolarity oral
Pediatrics recommendations. Pediatrics 1991;87:28–33. rehydration salts solution. Lancet 1995;345:282–285.
58. Leung AKC, Taylor PG, Geoffroy L, et al. Efficacy and 77. Hahn S, Kim Y, Garner P. Reduced osmolarity oral rehydra-
safety of two oral solutions as maintenance therapy for acute tion solution for treating dehydration due to diarrhoea in
diarrhea. Clin Pediatr 1988;27:359–364. children: systematic review. BMJ 2001;323:81–85.
59. Santosham M, Daum RS, Dillman L, et al. Oral rehydra- 78. DeBruin WJ, Greenwald BM, Notterman DA. Fluid resus-
tion therapy of infantile diarrhea: a controlled study of well citation in pediatrics. Crit Care Clin 1992;8:423–438.
nourished children hospitalized in the United States and 79. Awazu M, Devarajan P, Stewart CL, et al. “Maintenance”
Panama. N Engl J Med 1986;306:1070–1076. therapy and treatment of dehydration and overhydration.
60. Pizarro D, Posada G, Levine MM. Hypernatremic diarrheal In: Ichikawa I, ed. Pediatric textbook of fluids and electrolytes.
dehydration treated with “slow” (12-hour) oral rehydration Baltimore: Williams & Wilkins, 1990:417–428.
therapy: a preliminary report. J Pediatr 1984;104:316–319. 80. Tullis JL. Albumin. 2. Guidelines for clinical uses. JAMA
61. Pizarro D, Posada G, Villavicencio N, et al. Oral rehydra- 1977;237:460–463.
tion in hypernatremic and hyponatremic diarrheal dehydra- 81. Alderson P, Schierhout G, Roberts I, et al. Colloids versus
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J Dis Child 1983;137:730–734. crystalloids in fluid resuscitation: an analysis of randomized
63. Pizarro D, Posada G, Levine MM, et al. Oral rehydration of controlled trials. J Fam Pract 1991;32:387–390.
infants with acute diarrhoeal dehydration: a practical 83. The Cochrane Injuries Group Albumin Reviewers. Human
method. J Trop Med Hyg 1980;83:241–245. albumin administration in critically ill patients. Cochrane
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therapy revisited. S Afr Med J 1995;85:655–658. 84. Soni N. Wonderful albumin? BMJ 1995;310:887–888.
65. Ozuah PO, Avner JR, Stein RE. Oral rehydration, emer- 85. Fleck A, Raines G, Hawker F, et al. Increased vascular per-
gency physicians, and practice parameters: a national survey. meability: a major cause of hypoalbuminaemia in disease
Pediatrics 2002;109:259–261. and injury. Lancet 1985;1:781–784.
66. Avery ME, Snyder JD. Oral therapy for acute diarrhea: the 86. Nichani S. Albumin: saint or sinner? Arch Dis Child 1999;
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894. 87. Carcillo JA, Fields AI. Clinical practice parameters for
67. Tanzi VL. An econometric study of the adoption of oral hemodynamic support of pediatric and neonatal patients in
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68. Chowdhury AM, Karim F, Sarkar SK, et al. The status of blood transfusion and intraoperative glucose by APA and
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is it used? Health Policy Plan 1997;12:58–66. 11:685–689.
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 S E C T I O N

III

RESEARCH METHODS
 14

MOLECULAR BIOLOGY
FRANCESCO EMMA
GIAN MARCO GHIGGERI

OVERVIEW OF MOLECULAR CELL BIOLOGY in the completion of the human genome project (8). It has
provided new insight into the complexity of living organ-
The discipline of molecular biology began in the mid-1940s isms that can only be partially studied with commonly
through an effort by scientists to discover the universal laws available techniques.
that govern the process of self-replication in living organisms The complexity of biologic systems presents new chal-
(1). Earlier efforts by Mendel, Wilson, Morgan, and Garrod lenges for molecular biology in the post-genomic era.
to study genetic inheritance used organisms with complex Recent progress in microengineering, computer technology,
genomes. These studies were based entirely on the natural and bioinformatics has led to the development of nano-
breeding systems of the organisms that were analyzed. As a technologies and to the emergence of new fields of investi-
result, a factor that determined a phenotypic future (i.e., a gation, including functional genomics and proteomics.
gene) could be shown to reside at a specific location, or locus.
Until the advent of molecular biology, the concept of genes
and loci remained intangible. Chromosomal loci were merely Basic Concepts
the sites determining differences, usually between “normal”
Central Dogma
and “mutant,” and genes remained detectable only if
mutated (2). The research efforts described led to the formulation of the
From the 1940s through the 1960s, these concepts were so-called central dogma of molecular biology soon after the
revolutionized. After Beadle and Tatum had established for discovery of DNA structure in 1953. According to the cen-
the first time a link between genes and proteins, Griffith tral dogma, DNA sequences are transcribed into RNA mol-
and Avery proposed that DNA was the molecule contain- ecules that carry the flow of genetic information out of the
ing genetic information. Soon after, work by Wilkins, Fran- nucleus to be translated into proteins (1,3). These processes
klin, Watson, and Crick described the structure of DNA as have been well defined and are detailed in most introduc-
a double helix. The discovery of the genetic code and the tory cell biology texts (3,9). The simple concept of a one-
establishment of the role of RNA in the synthesis of pro- way flow of information from DNA to RNA to protein has
teins laid the foundations for the development of modern- been thoroughly revised. Genetic information is conveyed
day molecular biology (3). in both directions through a complex series of feedback
Efforts by many scientists have provided key technologi- loops.
cal innovations since the 1960s, allowing recombinant
DNA technology to be applied to a wide variety of biologic
Proteins and Nucleic Acids: Different Molecules
problems. Major milestones include the discovery of
with Common Features Linked by a Genetic Code
restriction nucleases and DNA ligases; the development of
DNA libraries (4), DNA cloning procedures, nucleic acid Both DNA and proteins are composed of a limited number
hybridization techniques, rapid sequencing techniques (5), of units that are assembled sequentially in a step-by-step
and the polymerase chain reaction (PCR) (6); and the pro- process that always proceeds in one unique direction (9).
duction of transgenic animals (7). These advances permit The link between nucleic acids and proteins is provided by
detection, amplification, and engineering of DNA sequences the genetic code. To translate four nucleotides into 20 dif-
and delineation of the role of genes in cellular physiology ferent amino acids, groups of at least three nucleotides
and pathophysiology (2). (codons) yielding 64 possibilities are required. Of these, 61
The molecular revolution has affected all of the sciences, correspond to specific amino acids, whereas three corre-
including medical and clinical research, and has culminated spond to termination codons. The code is said to be degen-
302 III. Research Methods

erate, as it contains redundancies. Codons corresponding to

the same amino acid generally differ by the nucleotide in
the third position (3,9).
As a consequence of the degeneracy of the genetic code,
nucleotide substitutions (point mutations) in the third
nucleotide of a given codon may not change the primary
sequence of a protein and are frequently found in nature, as
they are not subject to natural selection. Point mutations in
the first and second position result in an amino acid substi-
tution or in a termination codon, either of which can dra-
matically alter protein structure and function.
In principle, each messenger RNA (mRNA) can be trans-
lated in any of the three possible reading frames determined
by overlapping triplet codes. With few exceptions, only one
reading frame produces a functional protein because stop
codons encountered in the other two reading frames termi-
nate translation. As the only punctuation signal is located at
the start codon (ATG), the reading frame is set at the begin-
ning of the translation and proceeds until a termination
codon is reached. Thus, finding the correct reading frame and
locating the start codon are essential steps in the process of
cloning genes and defining their protein products. Mutations
causing the deletion or insertion of one or two nucleotides
result in a shift of the reading frame (frame-shift mutations)
and cause the production of aberrant protein products.
FIGURE 14.1. General organization and processing of a eukary-
otic gene. A gene consisting of four exons and three introns is
shown. The promoter region (black box) is located near the 5'
Gene Structure end of the gene. Transcription of the gene yields a primary mes-
senger RNA (mRNA) transcript that contains both exon and
The complementary strands of chromosomal DNA are intron sequences. Differential splicing of this mRNA transcript
arranged in an antiparallel fashion as dictated by hydrogen yields two mature mRNA species by inclusion or exclusion of
bond pairing of nucleotide bases. It is estimated that the exon 3 (hashed box). To be exported into the cytosol and trans-
lated, mRNAs need to be modified by polyadenylation of their
human genome is composed of approximately 3 billion 3' end and capping of their 5' end with methylated guanosines.
base pairs of DNA containing 30,000 to 100,000 genes Differential splicing results in two different protein isoforms
arrayed on 23 pairs of chromosomes (10). Overall, the that are encoded by a single gene.
amount of DNA containing genes comprises a minority of
nuclear DNA.
The physical gap between the enhancer and the promoter
Traditionally, a gene is depicted as a “transcriptional
explains the need for accessory factors that convey tran-
unit” as illustrated in Figure 14.1. The DNA double helix is
scriptional signals to the RNA-polymerase through pro-
represented as a line interrupted by rectangular boxes corre-
tein-protein interactions (Fig. 14.2). All molecules,
sponding to exons, with its 5' end on the left and its 3' end
generally proteins, which are physically involved in the
on the right. Each gene is divided into two major regions,
regulation of transcription are referred to as trans-acting
namely the promoter and the coding regions.
elements, because their DNA sequence resides in a differ-
Typically, the promoter region is located upstream near
ent location of the genome (3,9).
the 5' boundary of the coding region and contains clusters
The coding region contains the information for protein
of short sequences (fewer than ten base pairs) spread over
synthesis. In this region, most genes are composed of a suc-
a few hundred bases that bind transcription factors. These
cession of exons and introns. Exons are coding sequences
regulatory proteins mediate the attachment and activation
that ultimately transfer into mature mRNA, whereas
of type II RNA polymerase, which mediates the transcrip-
introns are edited out of the newly synthesized transcript by
tion along the DNA while unwinding the duplex and
a process called splicing.
adding nucleotides to the growing RNA molecule. DNA
sequences in the promoter region that bind to transcription
factors are referred as cis-acting elements. In some cases,
Control of Gene Expression
transcription factors also interact with other cis-acting
elements (termed enhancers) that are located at a greater As only a fraction of the available genes are expressed in a
distance (up to a few thousand bases) from the promoter. given cell at a given developmental stage, differential trans-
 14. Molecular Biology 303

and a factor σ that recognizes the promoter region located 10

to 35 bases upstream to a transcription starting point (11) (Fig.
14.2). Thus, σ factors are the simplest examples of trans-acting
elements. Once transcription begins, σ factors separate from
the core enzyme. Sequential expression of different σ factors
promotes the activation of different genes in a cascade of
events that modifies the phenotype. During sporulation, for
example, σ factor 43 promotes the transcription of specific
genes involved in sporulation and the transcription of σ factors
E and K that regulate and terminate this process (12). Con-
versely, intrinsic palindromic regions forming DNA hairpins
or other trans-acting elements termed p factors that bind to the
core enzyme can terminate transcription by the RNA poly-
merase. In addition, natural σ factor competitors, such as pN,
NusH, or NuH6, can maintain RNA polymerase in an inac-
tive state (13).
Therefore, gene expression in prokaryotes is controlled by
balanced initiation and termination of transcriptional signals
and by the specificity of different σ factors. However, this sim-
ple organization cannot provide for highly coordinated profiles
of gene expression. This is achieved by physical alignment of
clusters of genes termed operons behind the same promoter
that enables sequential synthesis of proteins that are function-
ally related (14). The lacZYA, for example, is a classic operon
FIGURE 14.2. Transcriptional regulation in prokaryotes and
eukaryotes. A: In prokaryotes, genes are organized in a cluster of that codes for proteins involved in the β-galactose metabolism
related genes under the control of a single promoter, forming an (breaking, transport, and acetylation of galactose) and is under
operon. The RNA polymerase complex is constituted by a core the negative control of the lacI gene that encodes for the lac
enzyme that is activated on binding of a σ factor. B: This modular
system is much more complex and sophisticated in eukaryotes. Dif- repressor (15). The repressor maintains the operon in the inac-
ferent transcription factors bind to the promoter and enhancer tive state until it is inactivated by end-products of galactose
regions and position type II RNA polymerase at the starting point metabolism (16). In this way, transcription of enzymes respon-
of transcription. General transcription factors shown in black inter-
act with the TATA box and hold together the enhancer-promoter- sible for galactose metabolism is activated only in the presence
RNA polymerase complex. When not activated, eukaryotic genes of galactose in a highly efficient and ergometric manner. The
are hidden in nucleosomes that are composed of a central core lacZ gene is frequently inserted in recombinant bacterial plas-
formed by histones. Activation of eukaryotic genes requires remod-
eling of the chromatin to allow the transcriptional apparatus to mids, allowing colorimetric detection of colonies containing
interact with the regulatory sequences. exogenous DNA material (Fig. 14.3).

cription and processing of genes provides for enormous Transcriptional Regulation in Eukaryotes
diversity between cells within the same organism. Although Three general principles govern the control of gene trans-
gene expression can be controlled at different levels, tran- cription in eukaryotes:
scriptional regulation generally predominates.
The information that governs transcription is located in 1. Unlike prokaryotes, genes operating within the same
DNA sequences that correspond to cis-acting elements or that metabolic pathway are not usually physically aligned
encode for transcription factors. These sequences occupy a along genomic DNA and are often located on different
minimal portion of the entire genome but are the key determi- chromosomes.
nants of cell organization by ensuring a balanced expression of 2. Recurrent structural motifs in DNA-binding domains
different genes that preserves phenotypic stability. They also (enhancers and promoters) indicate that the expression
provide for differences among cell types within the same of functionally related genes is achieved by the activa-
organisms and are at the core of the evolutionary process. tion of shared classes of transcription factors rather
then by unique, gene-specific regulatory proteins.
Gene Expression in Prokaryotes: A Simple Model 3. Transcription factors are often involved in develop-
of Regulation mental regulation.

The prokaryotic RNA polymerase has a simple structure The eukaryotic type II RNA polymerase differs from its
formed by a core enzyme that mediates RNA transcription prokaryotic ancestor by the complexity of transcription fac-
304 III. Research Methods

that holds together the enhancer-promoter-RNA polymer-

ase complex and positions the enzyme at the starting point
of transcription (18). Although enhancers are not essential,
they increase promoter efficiency.
A second major characteristic of transcription in eukary-
otic cells is related to the association of nuclear DNA with
histones, forming nucleosomes. This particular organiza-
tion prevents interaction between regulatory sequences and
transcription factors unless conformational changes of the
chromatin permit gene activation (Fig. 14.2).
It is suggested that chromatin remodeling is achieved by
specific energy-dependent reactions that displace nucleo-
somes along the DNA strand. Alternatively, histone acety-
lation is thought to inactivate the natural binding of
histones to DNA, allowing nucleosome remodeling, bind-
ing of transcription factors, and functional interactions
between enhancers and promoters (18,19). This view is
supported by the fact that histone acetylation correlates
with the state of activation of gene expression.
Finally, gene transcription can also be chemically
blocked in eukaryotic cells by methylation of specific DNA
regions located near the promoter. Experimentally, the state
of DNA methylation can be determined by inhibition of
the nuclease activity of restriction enzymes, such as HpaII,
that recognize non-methylated GC doublets.

Transcription Factors
FIGURE 14.3. Basic enzymes and techniques in recombinant DNA
technology. The figure summarizes basic procedures used in rou- The major characteristic of transcription factors is to con-
tine laboratory experiments. A: Messenger RNA (mRNA) molecules
are reverse-transcribed into complementary DNA (cDNA) using the tain specialized domains that allow for DNA interaction.
enzyme reverse transcriptase to synthesize the first cDNA strand, Zinc-fingers, helix-turn-helix (HTH) domains, helix-loop-
which is then used to generate double-stranded cDNA using a DNA helix domains, and leucine zippers are examples of DNA-
polymerase. Both enzymes require priming with complementary
oligonucleotides. B: cDNA and other DNA molecules can be ampli- binding domains that are encountered in more than 80%
fied in bacteria after insertion into plasmids using restriction of transcriptional factors (20).
enzymes and DNA ligase. Recombinant plasmids generally contain Zinc-finger motifs are composed of a Zn ion holding
a polylinker that offers different restriction sites to facilitate the
insertion of exogenous DNA. In addition, they are engineered to together a peptide loop (the finger) through interaction
contain antibiotic resistance genes (Ampr) for selection of trans- with two histidines and two or four cysteines. The func-
formed bacteria and often contain other useful genes, such as the tional DNA binding domain is located in the amino acid
lacZ in this example, which allows color detection of colonies trans-
formed with “empty” plasmids. C: Alternatively, DNA can be ampli- residues at the base of the finger and recognizes specific
fied by polymerase chain reaction (PCR) with the Taq polymerase. consensus sequences that are generally located in the
Repeated cycles of DNA denaturation (95°C), primer annealing enhancer region. Steroid receptors are the most popular
(58°C in this example), and DNA synthesis (72°C) allow for exponen-
tial replication of DNA strands encompassed by the two primers. examples of this class of transcription factor, including glu-
When reverse-transcription and PCR are combined, the procedure cocorticoid, mineralocorticoid, androgen, progesterone,
is referred to as RT-PCR. thyroid hormone, and vitamin D receptors (21). Increasing
evidence implicates the involvement of steroid receptors,
such as the proto-oncogene bcl-6 or WT1, in cancer (22).
tors that are required for its activation (17) (Fig. 14.2). This Similar DNA binding activity characterizes other classes
elaborate modular system allows for flexible and highly of transcription factors. HTH domains are often found in
coordinated regulation of gene transcription. proteins that have key roles during morphogenesis such as
On transcription, DNA rearranges to allow interaction the homeobox group of transcription factors (23). Members
between transcription factors located at the promoter and of the helix-loop-helix class include proteins that control
at the enhancer site. The TATA box, which contains con- myogenesis, such as MyoD, that have been implicated in the
sensus sequences recognized by general transcription factors trans-differentiation of myofibroblasts, which promote renal
(TFIID, TBP, and TAF), is usually located approximately fibrosis (24). Leucine-zipper motifs are used by the Jun and
24 bases upstream from the promoter and acts as a bridge Fos proteins. Both are members of the AP-1 heteromeric
 14. Molecular Biology 305

transcription complex that is involved in cell proliferation Before RNA is exported into the cytosol, splicing of
and regulation of cell matrix during fibrogenesis (25). An intron segments should be performed. The exact function of
important transcription factor that is often implicated in introns remains unclear. Because they facilitate recombina-
renal diseases is nuclear factor-κB, which regulates many tion events, they have played a major role in the evolution of
proinflammatory pathways and is itself under the control of species and are often targeted in the process of generating
other proteins, such as angiotensin II, known to promote knockout animals. Their physical structure may be more
inflammatory reactions in the renal parenchyma (26). important than their actual nucleotide sequence, as these
diverge more rapidly between species than exons (30).
Introns can range in size from 80 to several thousand
Transcriptional Control during Development
nucleotides. They contain specific sequences at their
As discussed above, transcriptional regulation plays a cen- extremities referred to as the 5' splice site (always ending
tral role during development by governing complex with 5'-GU-3') and the 3' splice site (always ending with 5'-
sequences of events that transform undifferentiated embry- AG-3') that come together in the process of splicing. Dur-
onic cells into highly specialized mature cells (3,9). ing splicing, a large catalytic heteromeric complex, termed
Three major classes of developmental regulatory genes spliceosome, is formed by the assembly of different ribonu-
have been identified. These include maternal genes, segmen- cleoproteins (31). After RNA binding, spliceosomes bridge
tation genes, and homeotic (Hox) genes. Each is expressed at together two exons and excise their intron segment.
different stages of cell maturation following complex In the majority of genes, each 5' splice site pairs with the
sequences of activation in which gene products that are closest 3' splice site in the spliceosome, producing only one
expressed at a given stage activate genes at a following stage. form of mRNA. In some cases, however, splicing of RNA
This highly sophisticated sequence of events creates a hierar- enables a single gene to produce different mRNA trans-
chy according to which maternal, segmentation, and cripts by jumping from a given 5' splice site to a more dis-
homeotic genes are expressed sequentially. tant 3' splice site (Fig. 14.1). Each of these mRNAs yields
Four maternal systems activate the expression of specific different isoforms of the same protein that can be alterna-
transcription factors, termed morphogens, that are responsi- tively produced in different types of cells.
ble for the initial patterning of the embryo. Segmentation Recent genome-wide analysis of alternative splicing
genes are zinc-finger proteins that control the boundaries of indicates that 40 to 60% of human genes have alternative
compartments, whereas Hox genes control the differentia- splice isoforms (32), including molecules involved in kid-
tion steps of each segment. Hox genes are characterized by ney diseases such as angiotensin-converting enzyme and
a common HTH-type DNA-binding motif at their car- nephrin. Splicing of mRNA is an important regulatory step
boxy-terminus termed homeobox and are organized in the in the production of cell proteins that requires a high
human genome in four different clusters (A,B,C,D) con- degree of accuracy. One pressing and still inadequately
taining up to ten genes each. answered question is the functional meaning of these alter-
As the number of identified developmental transcription native transcripts. In general, DNA mutations that involve
factors is rapidly increasing, their impact on renal develop- splice sites (splice site mutations) do not prevent splicing
ment and involvement in congenital renal anomalies is a but instead cause the normal partner to seek alternative
field of active research, as detailed in Chapter 1. Moreover, splice sites, producing the synthesis of various abnormal
during the recovery phase of renal cell injury, cells reacti- proteins lacking one or more exons, as, for example, in
vate several developmental genes in repair and restoration Frasier syndrome (33).
of function (27). Strategies to stimulate these patterns of
expression are being investigated to promote early recovery
Translation of Messenger RNA into Proteins
from acute injury (28,29).
Mammalian ribosomes are the site of mRNA translation
and are composed of two asymmetric subunits, the 40S
Messenger RNA Modification and Splicing
that binds mRNAs and the 60s that interacts with transfer
During cleavage and processing of primary RNA transcripts, RNAs. The genetic code is housed in the transfer RNA
the RNA molecules are capped at their 5' origin by the addi- molecules that associate specific codons with the corre-
tion of methylated G nucleotides, and a segment of poly-A sponding amino acid.
residues is attached near their 3' end (Fig. 14.1). These mod- Eukaryotic cells can decrease their rate of protein syn-
ifications are necessary to allow the export of the transcript thesis in various conditions such as infections or heat
from the nucleus, to stabilize mRNA molecules, and to allow shock. One important mechanism that mediates these
interaction with ribosomes. The presence of a poly-A tail is a types of nonselective responses involves phosphorylation of
major characteristic of mRNAs and constitutes a key tool to a repressor protein termed elF-2 that interacts with target
isolate mRNAs from other RNA molecules, mostly riboso- regions containing the AUG start codon preventing riboso-
mal, using complementary oligo-dT primers. mal binding.
306 III. Research Methods

A second gene-specific mechanism of translational regula- the human genome project, focus is gradually shifting from
tion is termed attenuation and involves the formation of the first to the second goal and the more complex task of
mRNA hairpins that block the translation. Similar mechanisms delineating complex patterns of gene expression. In the fol-
also regulate mRNA stability and degradation by RNases. lowing sections, several recombinant DNA technologies are
Given the fact that each molecule of mRNA can serve as a tem- described to illustrate common experimental procedures that
plate for multiple copies of proteins, the rate of mRNA degra- are routinely used in molecular diagnostics as well as basic
dation is a major determinant of protein abundance and is research. A few examples of new, promising approaches are
often the site of complex regulatory processes. In general, these also provided.
processes operate primarily on unstable mRNAs (such as cyto-
kines) that are stabilized under specific conditions by their
interaction with trans-acting elements (34). The stability of Basic Recombinant DNA Technology
transferrin mRNA, for example, increases during iron depriva-
Hybridization and Detection of Nucleic Acids
tion, triggering the synthesis of more transferrin molecules (35).
The pairing of nucleotide bases in DNA and RNA allows a
wide variety of specific recognition processes both in vivo and
Protein Sorting and Degradation
in vitro. These not only form the basis of many critical cellular
Newly synthesized polypeptides are processed by a complex functions but also provide the molecular biologist with tools to
network of cellular enzymes and other binding proteins that detect and study single genes. Based on specific hydrogen
are arranged in a highly organized fashion in various organelles bonding arrangements, G pairs with C and A with T or U.
in the cell. Information resident in the primary amino acid The extraordinary specificity of nucleic acid-base recogni-
sequence as well as the folded structure of the proteins allow tion has been exploited in the process of hybridization of com-
each to be recognized and targeted to its ultimate destination plementary DNA or RNA in vitro. Under appropriate
(3,9). Proteins that are synthesized in free cytosolic ribosomes conditions, a unique nucleotide sequence present within a
are normally directed to the nucleus or to the mitochondria, complex mixture of nucleic acids can be identified with a reso-
whereas membranous ribosomes are the site of synthesis for lution of greater than one part per million (37). Using stan-
proteins that enter the reticuloendothelial system (cotransla- dard techniques, DNA or RNA is isolated from cells or tissues,
tional transport) to be redirected to their final destination after stripped of proteins, and denatured into single strands. When
being processed in the Golgi apparatus. An excellent example incubated under conditions favoring renaturation, comple-
of this process is highly polarized epithelial cells in renal mentary sequences again reassociate. The experimental condi-
tubules. In these cells, transport proteins are located specifically tions (commonly temperature and salt concentration) can be
on the apical or basolateral plasma membranes. This arrange- altered to allow for only perfect or nearly perfect sequence
ment enables epithelial cells to perform net transport of solutes matches. This is called stringency. Lowering stringency condi-
and water to either secrete or reabsorb fluid. tions is sometimes desirable to identify close relatives of partic-
Some proteins are able to self-assemble by spontaneous ular nucleotide sequences. Thus, one can search for a gene or
interaction among reactive amino acid groups, whereas other mRNA in kidney that is a close relative of a transcript
proteins require the assistance of chaperon molecules such as expressed in other tissues as well as species (38,39).
the Hsp70 system and chaperonins. These molecules control Nucleic acids are commonly fractionated by agarose gel
the accessibility of reactive groups and maintain the peptide in electrophoresis. Under these conditions, the agarose acts as a
a relatively flexible state until it reaches its final conformation. molecular sieve, retarding larger strands while allowing smaller
The final fate of most cell proteins is degradation into strands to migrate in the electric field placed across the gel.
proteosomes. A process called ubiquitination that involves Fractionated DNA or RNA is then eluted from the gel or
covalent linkage of small peptides called ubiquitins to target transferred to a filter and exposed to a labeled DNA probe.
proteins precedes this step. Ubiquitination is also involved When the filter contains DNA, this process is called a South-
in important signal-transduction pathways, such as the ern blot (40), whereas it is called Northern blot if it contains
nuclear factor-κB pathway, in which inhibitory subunits RNA (41).
are degraded after stimulatory signals and activate various This same procedure has also been adapted to tissue sec-
signaling cascades (36). tions to localize the expression of mRNA transcripts by spe-
cific cell types within a complex organ such as the kidney.
This technique is called in situ hybridization (42).
RECOMBINANT DNA TECHNOLOGIES AND Binding of specific DNA sequences to DNA or RNA spe-
PROTEIN ANALYSIS cies can also occur in solution. One example is protection of
specific mRNA transcripts from digestion by S1 nuclease via
When reduced to its basics, molecular biology has until hybridization (43). This technique, referred to as S1 nuclease
recently addressed two major objectives; namely, to identify protection assay, is more sensitive than standard Northern
genes and to analyze their function. With the completion of blotting.
 14. Molecular Biology 307

Restriction Endonucleases riophage λ, are more advantageously used. The λ phages are
composed of a head that contains the viral genome and a tail
In eukaryotic chromosomes, strings of DNA are several
that infects bacterial cells with high efficiency. Phages can be
million bases long. The discovery by Arber in 1962 of bac-
packaged in vitro after insertion of exogenous DNA and are
terial nucleases that cut DNA molecules at specific loca-
replicated in bacteria.
tions constitutes one of the cornerstone steps in the
Both λ phages and plasmids can amplify DNA fragments
development of recombinant DNA techniques (44).
up to 20 kilobases (kb). They are ideal for cDNA and other
Most restriction enzymes recognize palindromic DNA
relatively small DNA molecules, but are insufficient for large
sequences, meaning that the 5' to 3' sequence in the upper
strings of genomic DNA. In these cases, other vectors can be
strand is identical to the 5' to 3' sequence in the lower strand.
used. These include cosmid vectors, containing elements of
To date, nearly 1000 different restriction enzymes have been
both plasmids and λ phages that can accommodate up to 45-
purified. Each enzyme can produce defined DNA restriction
kb fragments or bacteriophage P1 housing up to 100 kb of
fragments possessing specific nucleotide sequences at each end
exogenous DNA. If even larger fragments need to be repli-
from any given DNA sample.
cated, bacterial artificial chromosomes or yeast artificial chro-
With these enzymes, strings of DNA can be isolated,
mosomes can incorporate up to 300 or 1000 kb of DNA,
ligated into plasmid or phage genomes, and amplified (Fig.
respectively (9).
14.3). Restriction nucleases have also allowed construction
of the first detailed maps of various genomes (restriction
maps) and are used for allelic discrimination by restriction DNA Amplification with Polymerase
fragment length polymorphism. In this technique, DNA Chain Reaction
mutations that modify the recognition sequence for specific
The second breakthrough in DNA amplification was achieved
restriction enzymes can be identified by the length of the
in 1985 by Mullis and coworkers who developed PCR (6).
restriction reaction product.
PCR relies on the binding of two priming oligonucleotides
that flank a region of DNA to amplify the region located in
between (Fig. 14.3). These two oligonucleotides are comple-
DNA Amplification Using Prokaryotic Systems
mentary to the opposite DNA strands. Addition of DNA
The possibility of replicating specific strings of DNA to polymerase results in the synthesis of new DNA. Repeated
obtain quantities sufficient for analysis and further manipu- cycles of denaturation, annealing, and DNA synthesis are per-
lation is central to all recombinant DNA technologies. In formed in a chain reaction such that the newly synthesized
the early 1970s, work by Boyer and by Cohen provided the strands become templates for further DNA synthesis. This
first fundamental tools for DNA cloning with the discovery process exponentially increases the number of DNA copies
of DNA ligases and the characterization of bacterial plas- containing the sequence of interest. Modern PCR uses ther-
mids. Plasmids are circular molecules of DNA that replicate mostable DNA polymerase species derived from the thermo-
in the cytoplasm of bacterial cells (Fig. 14.3). philic bacterium Thermus aquaticus (Taq polymerase), which
Specific regions of plasmids not vital for vegetative growth retain activity after being heated to 95°C and obviate the need
under laboratory conditions can be engineered using restric- for addition of fresh enzyme after each round of DNA synthe-
tion nucleases for the insertion of exogenous DNA fragments. sis and denaturation (45). Several modified enzymes that guar-
When inserted into Escherichia coli cells, plasmid genes encod- antee more reliable DNA duplication or allow for the
ing for antibiotic resistance are expressed, allowing selection of amplification of longer strings are also available.
bacteria that have been transformed. During this process, the PCR permits selective amplification of minute quantities of
inserted DNA is replicated along with the rest of the bacterial DNA, facilitating every aspect of molecular biology research
genome. This technology enables the amplification and char- and diagnostics (including forensic pathology), including site-
acterization of virtually any DNA string of appropriate size. directed mutagenesis, labeling, and sequencing of DNA. Even
Because bacteria have a mean generation time of approxi- fixed tissues on slides or small tissue fragments, such as renal
mately 20 minutes during exponential growth and contain biopsy specimens, can provide sufficient material for PCR
often as many as 500 copies of plasmids per bacterium, virtu- amplification.
ally limitless amounts of DNA can be grown and harvested
routinely. Most available plasmids are engineered to incorpo-
Amplification of Messenger RNA with
rate a polylinker corresponding to a portion of DNA that con-
Reverse Transcription
tains multiple restriction sites that facilitate cloning of DNA
fragments generated by various restriction enzymes. Many experimental circumstances require direct amplifica-
Because of their simplicity, DNA is generally cloned into tion of mRNAs. Indeed, mRNA transcripts reflect the
plasmids. The relatively low efficiency of bacteria transforma- actual genes that are activated in a cell system and contain
tion with plasmids limits their use, however, when generating nucleotide sequences that can be directly translated into
DNA libraries. In this case, bacterial viruses, such as the bacte- proteins, obviating the tedious task of sorting exon seg-
308 III. Research Methods

ments from introns when working with genomic DNA. Analysis of Gene Expression
This process is achieved with a reverse transcriptase derived
A critical issue in normal physiology and renal pathophysi-
from retroviruses, which are one major exception to the
ology is the determination of the expression of given genes
central dogma, as they harbor their genetic information in
under different cellular and environmental conditions.
RNA molecules that are copied into DNA on infection of
Classically, gene expression analysis is performed by protein
host cells. The DNA obtained by reverse-transcription is
detection with specific antibodies in Western blotting or,
called cDNA, as it reflects the nucleotide sequences of
when antibodies are not available, by measuring the
mRNAs (Fig. 14.3). Similar to DNA polymerases, reverse
amount of mRNA transcripts by Northern blotting or with
transcriptase requires complementary oligonucleotide
the more sensitive S1 nuclease assay. Densitometric meth-
priming to begin transcription. Oligo-dT hybridizing to
ods have been developed to compare the amount of
poly-A tails can be used to reverse-transcribe in a nonselec-
expressed protein or mRNA with respect to a control prep-
tive manner mRNA molecules (3,9). In other circum-
aration. These semiquantitative techniques, however, have
stances, gene-specific primers are designed to amplify
severe limitations. They require relatively large amounts of
selected mRNA molecules. Once converted into cDNA,
starting material, can only study a limited numbers of genes
nucleic acids are often ligated into plasmids or phages or
simultaneously, and require development of specific probes
directly amplified by the PCR reaction, a process that is
such as antisera. To overcome some of these limitations,
referred as reverse transcription polymerase chain reaction
other techniques have recently been developed and are
(RT-PCR) (Fig. 14.3).
briefly reviewed.

Sequencing Nucleic Acids

Quantitative Reverse Transcription Polymerase
The highly specific binding of small oligonucleotides to
Chain Reaction
DNA also lies at the heart of the dideoxy chain termination
sequencing of DNA. DNA sequences are obtained from a RT-PCR is more sensitive than traditional Northern blot
uniform population of DNA, generally synthesized in large analysis and requires less RNA. The major difficulty in
amounts by expansion in a bacterial plasmid or by PCR. quantifying mRNA by RT-PCR is related to the exponen-
After DNA is denatured, a complementary primer is added. tial nature of the method.
In the traditional manual sequencing, synthesis of comple- In competitive RT-PCR, an internal standard sharing
mentary radioactive DNA strands is initiated by DNA the same priming sequences as the transcript of interest is
polymerase after addition of all four deoxynucleotides (dATP added to the mixture and acts as a competitor during the
labeled with 35S, dGTP, dCTP, and dTTP) in four different reaction (47,48). The abundance of mRNA is determined
reaction tubes containing one of the four dideoxynucleotide by comparing the signal obtained from the target signal
analogs of G, A, T, or C. In each reaction, chain termination with the internal standard. As this method is time consum-
can occur whenever a dideoxy analog is inserted in the newly ing and often difficult to set up, it is increasingly replaced
formed DNA strand, preventing further extension. After res- by real-time RT-PCR.
olution of each mixture of nested DNA fragments by gel Real-time RT-PCR allows detection of products of the
electrophoresis, the nucleotide sequence is determined by PCR reaction as they are being formed (Fig. 14.4). In the
reading the order of bands in the four lanes of the corre- modern version of this technique, quenched fluorescent
sponding autoradiogram. Practically, 300 to 400 nucleotides dyes linked to the 5' end of one primer are released by the
can be determined with a single manual gel run. This 5' nuclease activity of the Taq DNA polymerase and
sequence can be extended by construction of other oligonu- become fluorescent (49). The emitted light is measured in
cleotides complementary to a short portion of the nucleotide real time during the PCR reaction and is proportional to
sequence determined previously (46). the amount of PCR product. The number of cycles
These procedures have been improved and automated. required to cross a given fluorescence threshold is inversely
The basic principles behind automated sequencing remain proportional to the amount of mRNA present in the origi-
similar to the manual technique. The major difference nal reaction mixture.
resides in the fluorescence labeling of one deoxynucleotide Multiplex real-time RT-PCR represents an extension of
in each reaction tube using different dyes. The reaction this technique and is based on differential fluorescent label-
mixtures are then pooled and separated in a single lane on a ing of primers that amplify for different genes. This permits
sequencing gel or electrophoresis capillary. As the bands comparison within the same PCR reaction of the relative
advance along the electric gradient, they pass under a laser amount of different transcripts (50). By this method,
beam that excites their fluorescence, which is read and ana- housekeeping genes that are presumed to be stably
lyzed by a computer. Reliable DNA sequences can often be expressed serve as internal controls, allowing correction of
obtained for more than 500 nucleotides per run, and mul- the results for the amount of RNA that was loaded in the
tiple lanes can be read simultaneously by the machine (46). initial sample reaction. Multiplex real-time RT-PCR allows
 14. Molecular Biology 309

FIGURE 14.4. Real-time reverse transcription polymerase chain

reaction (PCR). The figure illustrates an actin calibration
curve. Total RNA was extracted from HK2 cells and loaded in
increasing concentrations in the sample reaction. Fluores-
cence was measured in real time as primers were incorpo-
rated in newly synthesized PCR fragments with an ABI Prism
7700. The number of cycles required to cross a given fluores-
cence threshold (A) is proportional to the initial amount of
loaded messenger RNA (B). (See Color Plate 14.4.)

determination of gene expression even from extremely

small samples, such as renal biopsies, and has been devel-
oped, for example, to detect gene expression in human FIGURE 14.5. Differential display on DNA microarrays. Messen-
renal specimens (51). ger RNA (mRNA) obtained from two different samples are
labeled with two fluorescent cyanine dyes with one round of
reverse transcription. The fluorescent targets are then pooled
and hybridized under stringent conditions to the clones on the
Differential Messenger RNA Display Using microarray chip. The emission light is measured with a scanning
DNA Microarrays confocal laser microscope at two different wavelengths that are
specific for each dye. Monochrome images are then pseudo-
With the identification of increasing numbers of genes, colored, combined, and analyzed with suitable computer soft-
DNA microarray technology has rapidly developed as a valu- ware. (See Color Plate 14.5.)
able technique for comparative gene expression analysis.
DNA chips are composed of thousands of known
expressed sequence tags (see Gene Cloning and Analysis of For differential display, two populations of mRNA are
Cloned Sequences) or synthetic oligonucleotides that are labeled with different fluorophores, hybridized to the chip
deposited in gridded arrays by a robotic spotting device on and analyzed with a laser scanning device (Fig. 14.5). The
a solid support such as a glass microscope slide or a mem- intensity of fluorescence emitted by each dye is propor-
brane matrix. Oligonucleotide sequences are usually tional to the amount of RNA that has hybridized at a given
directly selected from databases such as GenBank, dbEST, location, reflecting the level of a gene expression repre-
or UniGene (52). As many as 10,000 genes can be spotted sented by that spot. The enormous quantity of information
on a single microscope slide. generated by expression data from thousands of genes
310 III. Research Methods

requires sophisticated computer analysis to generate mean- interest. Routinely, 50,000 phages (each containing a unique
ingful results (53). inserted DNA sequence) are plated on a lawn of bacteria in
Currently, DNA microarray technology is rapidly evolv- an agar plate. Phage DNA can be transferred in replicate
ing. Despite increasing miniaturization and the development fashion to a nylon filter and probed with a labeled DNA of
of more flexible techniques to generate “custom made” chips, interest to identify clones that hybridize with the probe. Indi-
it still has technical limitations, particularly in terms of chip vidual plaques are picked from the plate, and the cloned
reproducibility and variability in the efficiency of labeling DNA insert is characterized. When partial transcripts are
and hybridization. New protocols and technical improve- obtained, these can be used to further screen libraries in
ments are solving many of these limitations (52). search of overlapping sequences. Alternative procedures,
The major challenge of this technology is the development termed rapid amplification of cDNA ends, allow the amplifica-
of computer-based inferential analysis strategies that recognize tion of the missing 5' or 3' ends by RT-PCR (57).
patterns of gene expression within complex genetic networks This approach has tremendous flexibility and power.
such as the human genome. The so-called cluster analysis is Knowledge of only a partial mRNA or protein sequence
emerging as a powerful statistical tool, permitting the grouping allows synthesis of oligonucleotides that can be used as
of genes in hierarchical clusters that follow similar patterns of probes. Because of the degeneracy of the genetic code (3,9),
expression. This information can then be used as a molecular it is impossible to specify the exact nucleotide sequence cor-
fingerprint in diagnosis or in monitoring response to therapy. responding to a specific peptide fragment. In these cases,
mixtures of degenerate oligonucleotides containing several
possible sequence combinations are used (58). Cloning
Gene Cloning and Analysis of
strategies can also aim at identifying genes based on their
Cloned Sequences
homology with other proteins expressed in other tissues or
Cloning of genes responsible for genetic disorders can be species. By computer alignment of related sequences, con-
achieved without prior knowledge of the molecular nature stant regions that have been preserved throughout evolu-
of the disease by a strategy termed positional cloning, which tion within the same family of proteins can be identified
is based on the genomic localization of the locus of interest and used to design degenerate oligonucleotides. By a simi-
using genetic markers present at a known chromosomal lar approach, cDNA clones from related genes can serve
location (54). (See Chapter 19.) directly as probes. By lowering the stringency of the hybrid-
Alternatively, gene cloning is performed by screening ization conditions, homologous clones that contain high
tissue-specific collections of recombinant vectors contain- percentages of sequence identity with the probing DNA
ing sequences of foreign cDNA, called cDNA libraries (4). can be identified.
This basic approach can also be refined. Subtractive librar-
ies, for example, are obtained by subtracting unwanted
Library Screening with Antibodies,
mRNA species from the library before screening (55).
with Functional Assays, or by
Altogether, the success of any cDNA cloning strategy is
Protein-Protein Interaction
related to the quality of the library to be screened and to
the efficiency of the probing system that is used. In addition to the use of DNA fragments or oligonucle-
Since the 1990s, the process of cloning genes has been otides as probes, phage plaques can be screened with anti-
revolutionized by the creation of electronic databases. More sera raised against purified proteins. Using expression
than 53,000 partial sequences of cDNA called expressed plasmids, peptides encoded by exogenous cDNAs can be
sequence tags that cover most human genes have been col- directly translated in the bacterial lawn and identified with
lected (56). In addition, computer programs have been specific antisera (59).
developed to identify putative genes within genomic data- With a similar approach, phage epitope libraries express-
bases by sorting out and mending together potential exon ing randomly generated oligopeptides can be screened to
regions. All of these sequences and sequences from already find domains recognized by antibodies or by other proteins
cloned genes have been collected in databases that can be (60). The recognized epitope sequence generally corre-
accessed online (www.ensembl.org and www.celera.com). sponds to a partial amino acid sequence contained in the
Taken together, these databases constitute virtual DNA natural antigen or binding protein. From this sequence, the
libraries that can be screened electronically using partial protein can be identified or cloned. This technique is par-
DNA or protein sequences. By this method, entire genes ticularly powerful for identifying autoantibodies or to clone
can sometimes be identified. proteins by their reciprocal interactions such as in receptor-
ligand association. The two-hybrid system is an alternative
strategy that permits investigators to fish for clones that
Screening Libraries by Nucleic Acid Hybridization
code for peptide that interact with other proteins offered as
The object of screening a DNA library is to identify single bait in the screening process (see Protein-Protein Interac-
plasmids or phages containing the nucleotide sequences of tion) (48).
 14. Molecular Biology 311

Another powerful approach relies on screening cDNA ous cDNAs using retroviral vectors. In these systems, viral
clones that produce functionally active proteins when promoters initiate and produce high levels of expression of
expressed in a suitable system. Xenopus oocytes have emerged the desired protein. However, such studies are often ham-
as a favorite system to clone membrane transport proteins. pered by the transient nature of the expression of transfected
Assays using oocytes have used electrode impalements, patch genes. Using the same system, regulatory regions of genes
clamping, and optical or isotope flux techniques. Large may be transfected into cells and their effects on a neighbor-
quantities of RNA (cRNA) can be synthesized in vitro from ing gene determined using a gene product that is easily
cDNA libraries, fractionated, and used for injection into assayed, such as chloramphenicol acetyltransferase (67).
oocytes. When the oocyte demonstrates the expected physio- Sorting and trafficking of proteins within highly polar-
logic response, smaller fractions of the original cRNA pool ized epithelial cells of the kidney has been addressed using
are injected until the process is narrowed down to a single recombinant DNA technology and cultured epithelial cells
clone (61). such as the MDCK cell line. These cells maintain a polar-
ized morphology and possess distinct apical and basolateral
plasma membrane domains. Selective budding of viruses,
Initial Analysis of Cloned Complementary DNAs
such as vesicular stomatitis virus exclusively from the apical
DNA sequences are generally read and entered into a suit- or basolateral membranes of MDCK cells, has permitted
able computer program for analysis and storage. Computer detailed study of the fundamental determinants of epithe-
programs provide important clues as to the nature of newly lial cell polarity (68).
cloned proteins, such as structural aspects, including mem- Xenopus oocytes are a popular system to express mem-
brane-spanning domains, antigenicity, and the presence of brane transport proteins. The cystic fibrosis gene was
specialized amino acid sequences coding for phosphory- among the first gene to be isolated without knowledge of its
lation or glycosylation. Sequence alignment in databases actual function. Oocytes were used to demonstrate its func-
helps define the relationships with other genes and identify tion as an epithelial cell chloride channel (69). Other fun-
functional motifs, such as DNA-binding and protein-bind- damental membrane transport proteins cloned using Xenopus
ing domains, that can give important clues to the biologic oocytes include Na-H exchangers (70), bumetanide-sensi-
function of the newly cloned sequence (62). tive Na-K-2Cl and thiazide-sensitive NaCl cotransporters
(71), the renal outer medullary adenosine triphosphate–
regulated potassium channel (72), multiple aquaphorin
Expression of Specific Genes in Various
water channels (73), and the amiloride-inhibitable epithe-
Experimental Systems
lial Na+ channel, or ENaC (74). These data provide molec-
An important aspect of recombinant DNA technology is ular links between epithelial cell transport data and the
the demonstration that a clone selected from a library codes expression of specific genes within individual kidney epi-
for a gene of interest. In cases in which structural analysis of thelial cells. In turn, detailed knowledge of these trans-
the clone demonstrates a high degree of conservation with porter proteins has allowed the identification of specific
another homologous gene or cDNA, expression studies can gene abnormalities in humans that cause inherited disor-
compare these structural and functional differences. Site- ders of renal tubular function, including nephrogenic dia-
directed mutagenesis studies can further refine knowledge betes insipidus (75) and Bartter’s (76,77), Gitelman’s (78),
of the function of various portions of a single protein (63). and Liddle’s (79) syndromes.
Bacteria infected with recombinant phage can express
polypeptides coded by cDNAs as fusion proteins. These
Expression and Suppression of Specific Genes in
fusion proteins can be purified in large quantities and used
Animal Models
for functional studies or as immunogens to raise antisera.
Mammalian proteins expressed in bacteria, however, are Single cell expression systems cannot be used to study
not posttranslationally modified. This limitation has been cir- expression in multicellular organisms in which a gene’s
cumvented by use of the baculovirus expression system, in expression or lack thereof has complex effects on an ani-
which recombinant proteins are produced by viral infection of mal’s development and physiology. For these studies, the
cultured insect cells (64). These proteins are then properly pro- introduction and disruption of specific genes into amphib-
cessed, glycosylated, and phosphorylated. Yeast has also been ian, insect, and mammalian embryos have permitted stud-
used extensively as an expression system (65). Detailed knowl- ies of gene expression in the resultant offspring.
edge of yeast physiology and genetics coupled with the ability Xenopus and Drosophila embryos have been used to char-
to mutagenize this organism has enabled detailed dissection of acterize various developmentally specific transcripts govern-
genomic control mechanisms as well as identification of pro- ing tissue-specific differentiation (3,9). This research is
teins that regulate endocytosis and membrane fusion (66). greatly facilitated by the ability to manipulate various cells
A variety of mammalian cells in culture, including green of the earliest embryo stages, then transplant them to other
monkey kidney or COS cells, have been used to express vari- embryos and allow them to develop. This research has pro-
312 III. Research Methods

duced a fundamental understanding of pattern formation The yeast two-hybrid system is also an attractive method
in these animals. Homologs of these pattern genes have based on the modular structure of gene activation, which
been characterized in mammals. complements or is used as an alternative to pull-down
In a similar fashion, transgenic mice have been used experiments. In the yeast two-hybrid system, the GAL4
extensively to produce a stable transfer of new or altered transcriptional activator is often exploited (59). This trans-
genes into the germ line of rodents (7). Both the homozy- cription factor has a DNA-binding domain and an activa-
gous and heterozygous offspring of transgenic animals can tion domain, both of which must be in close association to
then be studied extensively. Production of transgenic ani- activate transcription. By DNA recombinant techniques,
mals involves injection of a new gene (transgene) into the two protein sequences acting as bait and target are fused
pronucleus of a one-cell stage embryo so that it can inte- with sequences encoding with one of the GAL4 domains.
grate into the genome. This embryo is then implanted into When expressed in yeast cells, the activation domain and
a foster mother and allowed to develop. Transgenes are usu- the DNA-binding domain are bridged together if the two
ally passed from parent to offspring. This permits propaga- proteins interact, thus promoting transcription of a
tion of the new trait. Inclusion of the appropriate reporter gene. These systems can be used to study interac-
regulatory elements of a gene allows study of gene expres- tions between known molecules or to clone new proteins
sion in its various target tissues at precise times during the using cDNAs libraries, as mentioned before.
life span of the transgenic animal. This technology has been Similar prokaryotic systems have been developed based
used on a large scale to determine not only gene functions on the modular structure of bacterial RNA polymerases, in
that are critical to embryo development and survival but which target and bait cDNAs are fused to either the core
also genes that produce specific phenotypes that mimic enzyme or a σ factor.
human abnormalities (80).
Moreover, this technology has been carried one step fur-
Protein-DNA Interaction
ther through development of gene targeting. Instead of
introducing genes into cells, special vectors are designed to Techniques for protein separation also offer an important
disrupt or knock out specific genes in mice. The offspring tool to study protein-DNA interactions. This permits anal-
of these mice possess various copy numbers of the dis- ysis of regulation of DNA expression by transcriptional fac-
rupted gene and can be examined using a wide array of ana- tors. Such experiments are essential to identify DNA
tomic, physiologic, and cellular methods (81). binding sites and have been successfully used to purify and
identify a number of regulatory proteins.
The basic principles behind these studies rely on the fact
Protein Analysis and Proteomics
that protein-DNA interaction forms high molecular weight
Protein analysis involves protein detection, protein separa- complexes that are retarded during polyacrylamide gel elec-
tion and purification, characterization of peptide sequences, trophoresis or protected from digestion with DNAse (82).
and identification of interactive partners. Direct recognition In the example shown in Figure 14.6, a nuclear protein
of proteins is usually achieved with specific antibodies after extract from cells stimulated with angiotensin II was coin-
protein separation in polyacrylamide gels (Western blotting) cubated with DNA strings containing the promotor
or can be directly performed in situ on biologic samples by sequences of collagen III. As shown, angiotensin II pro-
immunocytochemistry. Antibodies can also be used to pull motes the synthesis of a protein that binds to the 32P-
down or blot specific proteins from solutions, which are then labeled DNA target, forming a macromolecular complex
used for further manipulations or are quantified (i.e., by that is retarded in the gel (83).
enzyme-linked immunoassays).
Protein Separation and Purification
Protein-Protein Interaction
Separation of proteins is another important aspect of pro-
One important question in protein analysis is to identify tein analysis required as a preliminary step for peptide
interactions between proteins, which regulate most intra- sequencing or to perform other tasks such as functional
cellular signaling and are critical to the assembly of func- assays. These complex sets of experimental procedures need
tional peptides. Protein-protein interaction assays have high levels of expertise in protein biochemistry and are gen-
been developed in which labeled proteins are extracted erally based on prior knowledge of the physical characteris-
from a crude mixture as they bind to target proteins linked tics of the protein of interest, including size, isoelectric
to solid supports. These approaches can be further refined point, and hydrophobicity. The availability of antibodies or
with recombinant DNA techniques, allowing for bacterial specific ligands greatly simplifies the experimental proce-
synthesis of partial protein fragments that are used as tar- dures by allowing detection of the final products or the use
gets to identify specific domains that mediate protein- of alternative strategies such as the above-mentioned pull-
protein interaction. down assay.
 14. Molecular Biology 313

FIGURE 14.7. Two-dimensional electrophoresis analysis. The fig-

ure shows platelet proteins resolved in a low polyacrylamide–
concentration, macroporous, two-dimensional gel. The initial pro-
tein mixture was solubilized with a tri-n-butyl phosphate:ace-
tone:methanol solution according to an optimization protocol
for high molecular weight proteins (85). Proteins were sepa-
rated according to their isoelectric points using an immobilized
pH gradient (IPG) in the first dimension (horizontal) and by their
molecular weight (M.W.) in the second dimension (vertical). Sin-
gle spots on the gel represent individual protein species.

vents and macroporous gels also allow for the detection of

poorly soluble high molecular weight peptides (more than
150,000 kDa) that cannot be resolved by traditional gels
(Fig. 14.7) (84). Moreover, new staining procedures based
on silver stains or negative dyes offer increased sensitivity,
FIGURE 14.6. Gel retardation assay. The figure demonstrates with detection limits in the nanograms range (85).
binding of regulatory nuclear proteins to cis-elements in the
COL3A1 promoter after angiotensin II (Ang II) stimulation.
Nuclear extracts were obtained from Ang II–stimulated cells Proteomics
(Ang II+) and incubated with a 32P-labelled oligonucleotide (414)
that contains sequences +3 to +20 of the COL3A1 promoter. Ang One of the challenges of the post-genomic area is the char-
II stimulates the synthesis of a peptide that binds to the target
DNA and retards its migration in the gel (lane 2). This reaction acterization of global patterns of gene expression directly at
can be competed with increasing amounts of nonlabeled, wild- the protein level. This new field of investigation has come
type oligonucleotide (lanes 3 to 5) but not with a cold mutated to light in recent years with the combined developments of
analogue sequence (414m) (lanes 6 to 8). In the absence of Ang
II stimulation (Ang II–), no DNA-protein complex generating gel mass spectrometry techniques, nanotechnologies, and bio-
retardation is observed (lane 1). informatics, and is generally referred to as proteomics (86).
With a similar approach to differential transcription analy-
sis, techniques permitting analysis of patterns of protein
In general, protein separation involves prepurification expression have been developed.
chromatographic steps that are performed with ion Although antibodies can only measure the expression of
exchange, size exclusion, hydrophobic interaction, or affin- few proteins simultaneously, two-dimensional gels com-
ity chromatography, followed by gel electrophoresis. Since bined with chromatography techniques considerably
the 1990s, two-dimensional electrophoresis has gained con- increase the number of gene products that can be studied
siderable favor. This powerful technique has been the simultaneously. The complexity of peptide expression pat-
object of major developments and permits fine resolution terns represents, however, a major obstacle in proteomics.
of proteins. Recent advances based on the use of new sol- In general, the abundance of proteins in biologic samples is
314 III. Research Methods

in the range of 106 whereas no more than 103 different pro- 3. Alberts B, Bray D, Lewis J, et al. Molecular biology of the cell,
teins can be visualized even with high-quality two-dimen- 3rd ed. New York: Garland Publishing Inc, 1994.
sional gels. For these reasons, the initial step of any 4. Huynh TV, Young RA, Davis RW. Constructing and screen-
proteomic approach requires reduction of sample complex- ing cDNA libraries in lgt10 and lgt11. In: Glover DM, ed.
ity by preliminary removal of uninteresting components DNA cloning techniques: a practical approach. Oxford, UK:
IRL Press, 1984.
using various affinity strategies.
5. Sanger F. Determination of nucleotide sequences in DNA.
In two-dimension differential protein display, proteins Science 1981;214:1205–1210.
from different sources are labeled with different fluorescent 6. Mullis KB, Faloona FA. Specific synthesis of DNA in vitro via a
cyanine dyes and run contemporaneously in the same gel. polymerase chain reaction. Methods Enzymol 1987;155:335–
Similarly to differential display with DNA microarray tech- 350.
niques, the gel is analyzed with a laser beam scanner that 7. Hanahan D. Transgenic mice as probes into complex sys-
measures the emitted fluorescence from each dye after exci- tems. Science 1989;246:1265–1274.
tation at different wavelengths. Computer analysis deter- 8. Lander ES, Linton LM, Birren B, et al. Initial sequencing and
mines the relative contribution of the original protein analysis of the human genome. Nature 2001;409:860–921.
mixtures to every spot on the gel, which correspond to 9. Lodish H, Berk A, Zipursky SL, et al. Molecular cell biology,
unique protein species. These can then be recognized by 4th ed. New York: W H Freeman & Co, 1999.
10. Risch NJ. Searching for genetic determinants in the new
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 15

IN VITRO METHODS IN RENAL RESEARCH

PATRICIA D. WILSON

Significant advances in the understanding of renal cell func- pretreatment is now considered undesirable because of the
tion, epithelial cell biology, and morphogenesis have been resultant damage to cell membrane proteins, such as recep-
made in recent years by the application of in vitro tech- tors, and the deleterious effects on cell adhesion properties,
niques. The researcher today is presented with a wide choice which limit viability. For these reasons, physical microdis-
of in vitro models, and the aim of this chapter is to provide section of tubules without enzyme pretreatment is often the
not only an overview of techniques available but also suffi- preferred choice. Many segments of the nephron can be
cient information to allow insight into the advantages and isolated with precision by microdissection, and the ease of
limitations of each system. This provides the experimental dissection is increased by using pathogen-free animals or
pediatric nephrologist with an appreciation of the range of salt solution–perfused human kidneys. Although physical
renal in vitro methods currently available and allows selection microdissection of tubules provides the most accuracy, only
of the most appropriate in vitro system to adequately answer relatively small numbers of tubules can be obtained from a
the questions posed. For a more complete methodologic single isolate because of the limited period of tissue viabil-
review of isolation and culture techniques, the reader is ity. This limitation can be alleviated to some extent by
referred to standard tissue culture texts (1–3). freezing tissue slices in 5% dimethyl sulfoxide–containing
medium for subsequent use, but the technique remains
time consuming and labor intensive.
RENAL CELL TYPES AND If purity is less important than large quantities of cells,
ISOLATION TECHNIQUES the isolation technique of choice is gentle enzymatic diges-
tion and/or calcium chelation (by ethylenediaminetetraace-
The kidneys are extremely heterogeneous, predominantly tic acid) of thin tissue slices followed by differential sieving
epithelial organs, each containing 1 million nephrons that or Percoll centrifugation. These techniques are widely used
are highly segmented and composed of many different cell for the derivation of highly enriched proximal tubule prep-
types (Fig. 15.1). Detailed studies of the properties of these arations from cortical slices, but no discrimination between
individual cell types are needed for the fundamental under- S1, S2, and S3 segments is possible in these types of iso-
standing of normal function and development of the kid- lates. Similar techniques have been applied to isolate thick
ney, and progress toward achievement of this goal has ascending limbs of Henle and collecting tubules using cor-
accelerated dramatically since 1990. tical, outer, or inner medullary slices. The limitation of
The initial requirement for the establishment of renal compromised cell type purity of this technique can be
cell culture was the development of reproducible and reli- addressed by the application of a subsequent immunoab-
able isolation techniques for individual cell types. The most sorption step using cell type–specific antibodies. In this
successful of these are summarized in Table 15.1 and case, the quality of the preparation is a function of the spec-
include physical microdissection, size discrimination by ificity of the antibodies used. In addition to tubule epithe-
graded sieving or centrifugation over a Percoll or other sim- lial cells, it is also possible to isolate glomerular epithelia by
ilar gradients, and removal of specific components by trypsinization and two rounds of sieving to produce pure
immunoabsorption (4–11). Certain fundamental differ- podocytes (12–14).
ences between these starting isolates should be stressed. There are many obvious advantages to long-term study
Most of these techniques use an initial period of tissue dis- of defined cell populations in vitro. The ability to adapt
aggregation by incubation in collagenase, pronase, or other conditions to allow the proliferation of isolated renal cells
dissociative enzyme solutions. The length of time and con- in vitro has led to a dramatic increase in our understanding
ditions of this incubation determine whether tubule frag- of renal cell biology and provides a powerful tool for study
ments or single cells are obtained. However, enzyme of renal tubule function, development, and disease. Most
318 III. Research Methods

TABLE 15.1. ISOLATION TECHNIQUES

Technique Tubule Species Collagenase References

Microdis- PCT S1,Human, – 19,21

section S2 rabbit
PST Rabbit, – 20,21
human
mTAL, Rat, rab- + —
cTAL bit,
human
Tdl, DCT, Mouse + 19–21
CNT
CCT Human, – 19–21
rabbit
OMCT, Human, – —
IMCT rabbit
Sieve Glomer- Human, + 12–14
uli rat
Bovine + —
FIGURE 15.1. Schematic representation of distribution of cell PT, IMCT Rabbit, + 4,7
types in the mammalian nephron. All tubule cell types can be mouse
isolated by microdissection techniques, and those shaded can be Percoll PT, CT Dog, rab- + 8,9
grown as primary culture monolayers in serum-free defined bit
media. Glomeruli can be isolated by sieving and individual cell
Immunoab- PT, CCT Dog + 6
types grown in culture. Additional cells (not shown) include
juxtaglomerular, vascular endothelia and smooth muscle cells, sorption cTAL, Rabbit, + 10,11
and interstitial fibroblasts of the cortical and medullary zones. mTAL rat
CAL, cortical thick ascending limb of Henle’s loop; CCT, cortical
collecting tubule; CNT, connecting piece; DCT, distal convoluted CCT, cortical collecting tubule; CNT, connecting piece; cTAL, cortical
tubule; En, endothelial cells; G, glomerulus; IMCT, inner medul- thick ascending limb of Henle; DCT, distal convoluted tubule; IMCT,
lary collecting tubule; M, mesangial cells; MAL, medullary thick inner medullary collecting tubule; mTAL, medullary thick ascending
ascending limb of Henle’s loop; OMCT, outer medullary collect- limb of Henle; OMCT, outer medullary collecting tubule; PCT, proximal
ing tubule; PCT, proximal convoluted tubule; PE, parietal epithe- convoluted tubule; PST, proximal straight tubule; PT, proximal tubule;
lial cells; PST, proximal straight tubule; ThDL, thin descending Tdl, thin descending limb of Henle.
limb of Henle’s loop; ThAL, thin ascending limb of Henle’s loop;
VE, visceral epithelial cells.

these pure or highly enriched cell populations to defined cul-

tubule isolation techniques have used adult kidneys as start- ture media conditions. This has allowed the production of
ing material, but it has been easy to adapt these basic tech- primary cultures of homogeneous populations of several renal
niques to the isolation and proliferation of fetal tubules (2). cell types (Table 15.2).
Glomeruli can be readily isolated from mammalian kidney
cortex by graded sieving (14), and the differential supplemen-
CELL AND TISSUE CULTURE tation of culture media and extracellular matrix to the collage-
nase digests favors the primary outgrowth of epithelial,
Cell and tissue culture techniques have been developed for endothelial, or mesangial cells. Mesangial cells are the easiest to
the in vitro proliferation of many of the defined cell types in obtain because they grow on uncoated plastic in Roswell Park
the kidney. These are summarized in Table 15.2, but for a Medical Institute (RPMI) medium containing high levels
more detailed survey, the reader is referred to several (20%) of serum (16). Endothelial cells require gelatin for
reviews (3,13,15). attachment and endothelial cell growth factor supplementa-
tion in the medium (17), whereas epithelial cells require col-
lagen as matrix and supplementation of the medium with
Primary Cell Cultures
transferrin, insulin, dexamethasone, triiodothyronine, and
Traditional explant techniques, in which small pieces of the prostaglandin E2 (18). These cells undergo few passages, and
whole kidney (separated into cortex or medulla) are chopped cloning is recommended for purity. Epithelial cells are usually
and placed into tissue culture media containing serum and considered to be visceral (podocyte) in origin because Bow-
cell monolayer outgrowths studied in primary culture, are of man’s capsules containing parietal epithelia are removed by the
little use in kidney research because of the extreme heteroge- collagenase treatment. However, definitive marker analysis is
neity of cell types and the nonselective nature of serum stimu- required to substantiate this assertion (Table 15.3).
lation of growth. These problems stimulated the development Primary cultures of defined renal tubule epithelia have
of cell type–specific isolation techniques. Significant advances been derived from several species, using a wide variety of
in renal cell biology have been made by the application of techniques. As discussed earlier, microdissected tubules pro-
 15. In Vitro Methods in Renal Research 319

TABLE 15.2. MAMMALIAN RENAL CELL AND TISSUE CULTURES

Cell type Species References

Primary cultures
Glomerular epithelia Human, rat 13,18
Glomerular endothelia Human, bovine, rat 15,17,23
Glomerular mesangial cells Human, bovine, rat 16
Proximal convoluted tubule S1 Human, rabbit, mouse 3,19–21
Proximal convoluted tubule S2 Human, rabbit, mouse 3,19–21
Proximal straight tubule S3 Human (adult, fetal), rabbit, mouse 3,19–21
Mixed proximal tubules Dog, rabbit, rat, mouse 7,8,10
Medullary thick ascending limb Human, rabbit 19,20,24,25
Cortical thick ascending limb Human, rabbit, mouse 19,20,24,26
Distal convoluted tubule Rabbit, rat, mouse 26–28
Cortical collecting tubule Human (adult, fetal), bovine, dog, rabbit 11,19,20,29
Intercalated cells Rabbit 30
Principal cells Rabbit 31
Outer medullary collecting tubule Human, rabbit 19,20
Inner medullary collecting tubule Human, rabbit, rat 19,20,32,33
Juxtaglomerular cells Rat 34
Cortical interstitial fibroblasts Human, rabbit, rat 22,35,36
Medullary interstitial fibroblasts Rabbit, rat 36–38
Renal vascular smooth muscle cells Rabbit 39
Spontaneous permanent cell lines
Proximal-like: JTC-12, BSC-1, OK, LLC-PK1, PT Monkey, opossum, pig, mouse 40–43
Distal-like: MDBK, MDCK, GRB-MAL, MmTAL-1C Bovine, dog, rabbit, mouse 44–46
Fetal kidney Human 44
Immortalized/transfected cell lines
Glomerular epithelia Human, mouse 47,48
Proximal tubule Human, rabbit, rat 49–52
Distal convoluted tubule Mouse 52,53
Cortical collecting tubule Rabbit, human 54,55
Inner medullary collecting tubule Mouse 56
Fetal kidney Human 44

LLC-PK1, pig kidney; MDBK, Madin-Darby bovine kidney; MDCK, Madin-Darby canine kidney; MmTAL-1C, mouse medullary thick ascending limb;
OK, opossum kidney; PT, proximal tubule.

vide maximum purity and allow discrimination between the To assess the validity of any primary culture technique,
S1, S2, and S3 portions of the proximal tubule (19–21). marker analysis (Table 15.3) is essential because each cell
Commonly used and reliable techniques are available for the type contains some specific protein(s) in vivo. This allows
culture of proximal tubules, thick ascending limbs of Henle’s the analysis of both the purity of preparation and the main-
loop, and collecting tubules of cortical and medullary origin, tenance of the differentiated state in vitro. If these criteria
and these are used for the primary monolayer culture of cells are fulfilled, primary cultures are particularly useful for the
of adult and fetal origin. In addition to epithelial cells, renal study of normal cell function. Most cell cultures derived
interstitial fibroblasts can also be cultured with ease from the from isolated glomerular or tubule preparations survive a
cortical or medullary explants (22). These are fairly easy to few (three to five) passages but then die out. This is consis-
generate because fibroblasts proliferate rapidly in response to tent with normal cell properties and is thought to represent
10% serum stimulation, whereas renal epithelial prolifera- the maximal repair capacity of renal cells in vivo. This con-
tion is inhibited under these conditions. This means that the trasts with neoplastically transformed cells, which possess
renal fibroblasts initially present in mixed cell populations unlimited proliferative potential and produce tumors in
from explants eventually overgrow the cultures because of vivo and immortal cell lines in vitro.
their selective growth in response to 10% serum in simple
Dulbecco modified Eagle medium. Subculture with trypsin
Permanent Cell Lines of Renal Origin
results in the acquisition of pure preparations of fibroblasts
by the third passage, because renal epithelia are more sensi- Several permanently growing cell lines of renal epithelia are
tive to the destructive effects of trypsin than fibroblasts (22). in current use and can provide a convenient supply of large
Additional cell types, including juxtaglomerular and renal numbers of cells for the study of polarity, ion transport, and
vascular smooth muscle cells, have also been grown in pri- hormone receptor interactions. However, their limitations
mary culture (34,39). should be appreciated. Although LLC-PK1, OK, and JTC-
320 III. Research Methods

TABLE 15.3. MARKERS OF DIFFERENTIATED CELL TYPES: ADULT

Glomerulus
Visceral epithelium Podocalyxin, synaptopodin, podocin, laminin α5, integrin α3, C3B receptor, angiopoietin, VEGF
Parietal epithelium Cytokeratin, VCAM1
Endothelial cells Factor VIII, angiotensin-converting enzyme, Weibel-Palade bodies, acetylated low-density lipo-
protein uptake, PDGFB, Tie 2
Mesangial cells Actomyosin, contractility, angiotensin receptors, kinin receptors, protein kinase C, desmin, PDGFRβ
Tubules
Proximal Abundant brush border, alkaline phosphatase, γ-glutamyl transpeptidase, isomaltase, leucine
aminopeptidase, meprin, aminopeptidase N, dipeptidylpeptidase Na-glucose transporter, Na-
amino acid transporter, Na-amino acid transporter, parathyroid hormone receptors, aqua-
porin-1, midkine, NHE3; ClC5, rBAT, PEX, NBC, hUAT, GLUT-2, villin, 1,25-hydroxy 2D 3
Thin descending limb of Henle Aquaporin 1
Thick ascending limb of Henle No brush border, abundant mitochondria, highest Na-K-ATPase activity, preproepidermal growth
factor, ROMK channel, CaSR, Tamm-Horsfall protein, glucocorticoid receptor, osteopontin,
NKCC2 (BSC1)
Juxtaglomerular apparatus Renin
Distal convoluted tubule Epidermal growth factor, osteopontin, Ca-ATPase, NCCT, claudin 16
Connecting tubule Kallikrein, calbindin
Collecting tubule:
Principal cells Light cytoplasm, few organelles, calcium-binding protein, vasopressin, receptor V2 aquaporin 2,
ENaC 11-hydroxysteroid dehydrogenase, mineralocorticoid receptor, AE1, aquaporin 4
Intercalated cells Dense cytoplasm, many organelles, band 3, carbonic anhydrase II, H +ATPase, mineralocorticoid
receptor
Fibroblasts
Cortical Vimentin I; CAM-1; α1, α4, α5β, integrins
Medullary Vimentin, tenascin

CaSR, calcium sensing receptor; ClC5, chloride channel; ENaC, epithelial sodium channel; GLUT-2, Na+/glucose cotransporter; hUAT, urate trans-
porter; PDGF, platelet-derived growth factor; PEX, Na/phosphate transporter; NBC, Na+/HCO3– transporter; NCCT, Na+/Cl– transporter; NHE3, Na+/
H+ exchanger; NKCC2 (BSC1), Na+K+Cl– transporter; rBAT, cystine and dibasic amino acid transporter; ROMK, renal outer medullary potassium
clearance; VCAM1, V-cell adhesion molecule 1; VEGF, vascular endothelial growth factor.

12 cells have properties suggesting proximal tubule origin, these types of cells also suffer from the limitation that they
and Madin-Darby canine kidney (MDCK) cells have some are permanently transformed with an abnormal prolifera-
properties of distal tubules, the precise cells of origin of tive phenotype conferred by the expression of the intro-
these spontaneously immortal lines is unknown. In addi- duced oncogene.
tion, like all immortal cell lines, they have undergone
genetic drift because of numerous passages, which has led
Conditionally Immortalized Cell Lines
to the loss of some properties and acquisition of other
anomalous properties. Therefore, spontaneously immortal By far the most successful advance made toward the goal of
renal cell lines (40–46) may not be the system of choice for production of large numbers of permanently proliferating
the study of normal epithelial function, although they have but normally differentiated renal cell cultures has been the
proved invaluable for the study of certain specific proteins, introduction of a temperature-ensitive (tsA58) allele-
notably transporters, and for analysis of mechanisms con- containing Simian virus (SV-40) T antigen into defined
trolling epithelial cell polarity. However, their value for renal cell types (50,55,57–60). This has allowed for the
studies of renal development is limited. immortalization by virtue of expression of the integrated
SV-40 T antigen at the permissive temperature of 33ºC but
the differentiation and loss of T antigen expression after 7
Immortalized Cell Lines
to 10 days at nonpermissive, normal temperature (37ºC).
An important advance in the generation of cell lines for This technique has been successfully applied either by mak-
renal research has been the successful application of immor- ing cultures from renal tissues from the SV-40tsA58 trans-
talization techniques using recombinant viral vectors intro- genic mouse (58,61) or by transfection of human renal
duced into cells by infection or transfection. Initial studies epithelial cells in culture (50). The latter technique has
used oncogenic viruses such as adenovirus or papilloma proved to have a wide use and has been used to condition-
virus to generate cell lines (47–57) (Table 15.2) such as ally immortalize renal epithelial cells of normal and dis-
HEK 293, HKC-8, mIMCD, and HK-2, which have eased origin, including human normal fetal and normal
proved to be very useful for several types of studies, not adult proximal tubules, thick ascending limbs and collect-
least because they are permissive to transfection. However, ing tubules, and epithelia from patients with cystinosis and
 15. In Vitro Methods in Renal Research 321

TABLE 15.4. CHARACTERIZATION OF CONDITIONALLY IMMORTALIZED CLONAL EPITHELIAL CELL LINES

HFPT HFCT NHPT NHTAL NHCT ADPKD ARPKD

Keratin + + + + + + +
Vimentin –/+ – – – – + +
Alkaline phosphatase + – + – – +(–) –
Leucine aminopeptidase – – + – – +(–) –
Multidrug resistance P- + – + – – + –
glycoprotein
Aquaporin 1 + – + – – +(–) –
NHE3 – – + – – + ±
Epidermal growth factor +/– +(A) +(B) – – +(A) +(A)
receptor
Erb-B2 – +(A) – – – +(A) +(A)
α2 Integrin +/– + + +/– +/– + +
α6 Integrin – –/+ + + + + +
β1 Integrin + + + + + + +
Tamm-Horsfall protein – – – + – –(+) –
Aquaporin 2 – +(–) – – +(–) +(–) +
H+ATPase – –(+) + – +(–) –(+) –
Carbonic anhydrase II – – – – +(–) –(+) –
ENaC – + – – + + +
NaK-ATPase α1 – +(A) +(B) +(B) +(B) +(A) +(A)
NaK-ATPase β1 – – +(B) +(B) +(B) +(C) –
NaK-ATPase β2 +(A) +(A) – – – +(A) +(A)
Polycystin 1 – + – – + +(C) +
Protein kinase – + – – – + +
Sprouty-1 – + + – – + –(+)
WT1 – – – – – + –
Oct-1 – + – – – + +
Pax-2 – + – – – + +
Midkine – – + – – + +/–

–/+, weak signal; +/–, most clones positive, few clones negative; (A), apical; ADPKD, autosomal dominant polycystic kidney disease; ARPKD, autoso-
mal recessive polycystic kidney disease; (B), basal; (C), cytoplasmic; HFCT, human fetal collecting tubule; HFPT, human fetal proximal tubule; NHCT,
normal human adult collecting tubule; NHPT, normal adult human proximal tubule; NHTAL, normal human adult thick ascending limb.

autosomal dominant and autosomal recessive polycystic (matrigel), or amnion. This not only provides adhesion but
kidney disease. By using actively proliferating microdis- also produces epithelial cultures with more differentiated
sected renal tubule segments as starting material—6 weeks characteristics. In addition, cultures of microdissected
of neomycin selection and dilution analysis—clones have tubules and glomerular epithelia have an absolute require-
been isolated with the marker characteristics of their nor- ment for extracellular matrix. By contrast, cells of mesen-
mal renal epithelia of origin (Table 15.4). chymal origin in the adult kidney (i.e., mesangial and
interstitial cells) have no matrix requirement and grow on
uncoated plastic.
Role of Matrix
Studies in several in vitro systems have elucidated a major
Role of Growth Factors
role for the extracellular matrix in the regulation of epithe-
lial proliferation, differentiation, and gene expression (62). Soluble growth factors have long been known to play a
In vivo, the renal extracellular matrix is made up of the major role in the control of cell proliferation. In vitro,
mesenchymal layer of interstitial fibroblasts that secrete serum was first used to induce cell growth. However, serum
types I and III collagen and the thin basement membrane is a complex mixture of proteins, and detailed composition
on which epithelial cells rest, composed of type IV col- has not been fully elucidated. The value of serum in renal
lagen, laminin, fibronectin, entactin, and heparan sulfate cell culture is that it favors the growth of fibroblasts at the
proteoglycans. Confluent renal tubule epithelial cells in expense of epithelia. In mixed epithelial and fibroblast cell
vitro also secrete a basement membrane with normal struc- populations, this enables the differential survival of fibro-
tural and biochemical characteristics (19,63,64). Several of blasts because in vitro, after approximately three passages,
the techniques for primary cultures of renal tubule epithelia the epithelial cells have disappeared and fibroblasts con-
include precoating tissue culture dishes with type I (rat-tail) tinue to proliferate. A significant advance for renal epithe-
collagen or growth on collagen gels, EHS sarcoma matrix lial cell culture was the definition of serum-free growth
322 III. Research Methods

factor–supplemented culture media that support the prolif- MORPHOGENESIS AND DIFFERENTIATION:
eration of renal epithelial cells (65). Modifications of the APPLICATION OF IN VITRO TECHNIQUES
original formulation have led to the ability to culture most TO RENAL DEVELOPMENT
renal tubule epithelia in serum-free media. All epithelia
have an absolute requirement for transferrin and proximal Mammalian renal development is a complex process that
tubules are stimulated to proliferate by combinations of involves the induction of undifferentiated mesoderm to form
dexamethasone (or hydrocortisone) and insulin, although epithelia (73,74). As described in detail in Chapter 1, the
this requirement can be substituted by epidermal growth ureteric bud (a branch of the mesonephric duct) invades and
factor (EGF). Optimal proliferation of thick ascending is induced to branch by the mesenchyme of the metanephric
limb epithelia requires additional supplementation with tri- blastema and in return the ureteric bud induces the
iodothyronine, whereas collecting tubules require only undifferentiated blastemal cells to proliferate, condense, and
dexamethasone and transferrin (19–21). Although it is not differentiate into primitive renal vesicles, comma bodies, and
surprising that different renal epithelial cell segments with S-shaped bodies. The exquisite regulation of gene expression
their individual complements of membrane proteins, necessary to complete this elaborate set of reciprocal interac-
including receptors, should require distinct hormone sup- tions is beginning to be understood (75).
plementation, the precise mechanisms regulating renal cell–
type specific proliferation are not fully understood. Because
Transfilter Techniques
normal renal epithelial cell division is complete before birth
in humans, it is thought that the limited ability to prolifer- For studies of renal development and nephrogenesis, the
ate in vitro represents a repair response. most important modification of organ culture, the transfil-
ter technique, was first introduced by Grobstein (67) and
continues to be used extensively. Metanephric blastemas are
ORGAN CULTURE TECHNIQUES attached to a filter suspended above a tissue culture dish or
well. To induce nephrogenesis, tissue fragments of ureter or
It is often desirable to study tissue and cell interactions, and spinal cord can be applied to the undersurface of the filter.
although this can be accomplished by co-cultures of pri- Alternatively, the natural inducer, the ureteric bud is left in
mary monolayers or cell lines, an alternative is to use an the blastema of a day 11 to 12 embryonic kidney, and cul-
organ culture technique. Such techniques maintain the ture on the membrane is carried out in optimized serum-
three-dimensional organization of the tissue of origin, free, supplemented media that can support the normal
including interactions with endogenous matrix proteins three-dimensional development of organogenesis in culture
and intercalated growth factors. The emphasis in such sys- for up to 2 weeks.
tems is to retain viability of tissues rather than to encourage Progressively refined analysis of the transfilter organ cul-
monolayer outgrowth of new cells. ture system has provided the basis for much of our present
These techniques have proved particularly useful for understanding of the cellular and molecular events associ-
the study of renal development, because the embryonic ated with nephrogenesis and has led to an understanding of
organ can be removed and placed in organ culture in the complex temporally and spatially coordinated interac-
which the influence of a variety of inductive factors can be tions involved. These include cell-cell contact and cell-
studied in detail. Most studies of this type have used matrix attachment, allowing regulated migration and cell
mouse metanephric blastema (66–68), although rat and shape changes as well as appropriate growth factor–receptor
human renal organ culture have been reported (69,70). interactions regulating proliferation and apoptosis as well as
Small pieces of tissue or whole embryonic kidneys are dis- epithelial polarization events associated with renal tubule
sected and placed on a stainless steel raft or membrane fil- maturation.
ter apparatus that has been placed in a tissue culture dish.
Sufficient tissue culture medium is added to the dish to
Proliferation and Differentiation
bathe the tissue from below and form a thin film of media
of Primary Cultures
above but not to submerge it. Organ culture techniques
have also been used to study mechanisms of cyst forma- Cell culture techniques have been used to study renal devel-
tion and regression in normal mouse kidneys and in those opment and epithelial differentiation and have included the
cultured from mice with congenital recessive polycystic isolation and culture of S-shaped bodies from newborn rab-
kidney disease (71,72). This technique is also now being bit kidneys that give rise to the outgrowth of collecting duct
used to microinject recombinant viral reporter-DNA con- principal cell monolayers (31). More recently, several pri-
structs into either the blastema or the ureteric bud to ana- mary and conditionally immortalized cell lines of normal
lyze lineage tracing of progenitor cells or the effects of human fetal proximal and collecting tubule epithelia have
specific gene expression in the developing ureteric bud, been generated from microdissected nephron segments pro-
respectively. viding a readily manipulatable system for the analysis of epi-
 15. In Vitro Methods in Renal Research 323

FIGURE 15.2. Phase-contrast light micrographs of confluent primary cultures. A: Epithelial

monolayer grown from individually microdissected human fetal proximal tubule explanted onto
type I collagen-coated plastic plates in Click–Roswell Park Medical Institute medium supple-
mented with human transferrin, dexamethasone, insulin, and fetal bovine serum (1%). Note
polygonal morphology. B: Epithelial monolayer grown from individually microdissected human
fetal collecting tubule explanted onto type I collagen-coated plastic plates in Click–Roswell Park
Medical Institute medium supplemented with transferrin, dexamethasone, and fetal bovine
serum (1%). Note polygonal morphology. C: Renal fibroblasts derived from cortical explants
plated onto uncoated tissue culture plastic in Dulbecco modified Eagle medium supplemented
with 10% fetal bovine serum. Note elongated spindle morphology.

thelial differentiation, polarization, and transport events (Fig. of induction and differentiation. Major advances have
15.2). These processes can be particularly well studied when been made in the development of renal progenitor and
cells are grown on filters suspended in a tissue culture well ureteric bud cell culture systems. The isolation of nor-
because there is individual access to the separate culture mal human renal progenitor cells from 12- to 16-week
medium compartments bathing the apical and basal surfaces human fetal kidneys and their dependence on condi-
of the cell monolayer. For instance, the polarized fate of a tioned media from a Wilms’ tumor cell line (WT-CM)
radioactive tracer added to one compartment can easily be has led to the identification of a new soluble growth fac-
traced by scintillation techniques (Fig. 15.3). These types of tor activity (midkine) involved in the control of renal
studies have been widely applied in functional analysis of progenitor proliferation (79–81). Human renal progeni-
normal fetal, adult, and diseased (e.g., polycystic kidney dis- tor cells in vitro not only retain the distinctive morphol-
ease) epithelia (76–78). An additional advantage of these ogy of renal cortical progenitors but also retain the
membrane-grown cultures is that the differentiated and capability to convert into epithelial phenotypes with
polarized characteristics of the cells are maximized under appropriate switches in gene transcription and protein
these conditions of nutrient access to both basal and apical expression after withdrawal of WT-CM and addition of
cell surfaces. serum and/or collagen matrix (82) (Fig. 15.4). Most
Although much has been learned about induction recently, similar studies have resulted in the isolation
and differentiation associated with nephrogenesis using and characterization of renal progenitor cells from
the transfilter organ culture system, recent important mouse kidneys and progress is being made in isolating
technical advances provide the potential for more subpopulations of epithelial, and stromal/fibroblast pro-
detailed molecular analyses of the underlying processes genitors (D. Hyink, personal communication, 2002).

FIGURE 15.3. Schematic representation of in vitro sys-

tem for study of transport and differentiation properties
of epithelial monolayers. Cells (C) are seeded at high
density on permeable, translucent membranes (M)
mounted on a plastic ring and suspended in a tissue cul-
ture well. In this way, the medium overlying the apical
surface of the cell monolayer (AM) is in a separate com-
partment from the medium bathing the basal surface of
the cell monolayer (BM), which allows addition of radio-
active tracer to either the basal or the apical compart-
ment. Loss from that compartment or appearance in the
contralateral compartment can be measured by scintilla-
tion counting of media aliquots.
324 III. Research Methods

FIGURE 15.4. A: Outer cortical region of human fetal kidney of 14 weeks’ gestation showing
small, round undifferentiated mesenchymal blastemal cells. B: Phase-contrast light micrograph of
human fetal nephroblasts in vitro, plated on gelatin and grown in serum-free medium containing
Midkine from G401 conditioned media (CM). Cells show small round morphology, small overall
diameter (4 μm), large nuclei, and thin rim of cytoplasm with few organelles. C: Light micrograph
of differentiated focus of cultured renal epithelial progenitor cells after removal of G401 CM and
addition of 10% fetal bovine serum. Note polygonal cells with large overall diameter and exten-
sive cytoplasm. D: Electron micrograph of outer cortical region of kidney in A. E: Electron micro-
graph of cultured nephroblasts in B. F: Electron micrograph of epithelial focus in C.

TABLE 15.5. MARKERS OF RENAL DEVELOPMENT

Cell type or structure Markers

Metanephric blastema (undifferentiated mes- Vimentin; syndecan; integrins α1, α9; collagen I, III; laminin β1, γ1; fibronectin; N-CAM;
enchyme) IGF-II; GDNF HGF; c-Met; NGFR; midkine; CRABP; BF2; tenascin; WT1 cadherin-11;
Hox AII; Hox DII; Six-2; Sca-1; P75NGFR; FGF2; BMP4; PNA+; apoptosis
Condensates Vimentin, syndecan, α8 integrin, N-CAM, N-myc, WT1, IGF-II, HGF, c-Met, NGFR, mid-
kine, pax-2, wnt4, cadherin 6, E-cadherin
S-shaped bodies Laminin α1 integrin, E-cadherin, syndecan, tenascin, N-myc, NGFR, wnt4, Notch-1
Glomerular pole Vimentin, WT1, α3 integrin, podocalyxin, C3b receptor, IGF-II, nephrin B 2
Tubular pole γGT, MDR, α6 integrin, α1-dystroglycan
Fetal tubule epithelia Cytokeratin, laminin α1, collagen IV, α6 integrin, E-cadherin, desmoplakin, HGF, c-Met,
LIM-1, α1-dystroglycan
Proximal Alkaline phosphatase, leucine aminopeptidase, renal peptidase, meprin, MDR, mid-
kine, aquaporin 1, CFTR, cadherin 6
Thin descending limb Aquaporin 1
Thick ascending limb Na-K-ATPase α1β2, Tamm-Horsfall protein, osteopontin, L-myc, integrin β4, integrin α2,
calcium-binding protein, E-cadherin, Brn 4/1
Collecting tubule/ureteric bud Midkine, Wnt7b, PKD-1, polycystin-1, CFTR, Pax-2, carbonic anhydrase, H-ATPase, aqua-
porin 2, Hox B7, c-ret, calbindin, Pax-2, E-cadherin and DBA+
Fetal glomeruli Vimentin; integrin α3, β1; podocalyxin; C3bR; WT1; ACE
Fetal interstitium/stroma Vimentin, tenascin, GD-3, ganglioside, IGF-II, FGFa, NGFR, RARα, RARβ, BF-2

ACE, angiotensin-converting enzyme; ATPase, adenosine triphosphatase; BMP, bone morphogenetic protein; CAM, cell adhesion molecule; CFTR,
cystic fibrosis transmembrane regulator; FGF, fibroblast growth factor; GDNF, glial derived factor; γGT, γ-glutamyl transpeptidase; HGF, hepato-
cyte growth factor; IGF, insulin-like growth factor; MDR, multidrug resistance; NGFR, nerve growth factor receptor; RAR, retinoic acid receptor;
WT1, Wilms’ tumor 1.
 15. In Vitro Methods in Renal Research 325

Differentiation of Immortalized Cell Lines in fetal cells. An absolute requirement for collagen has been
demonstrated for the attachment and growth of human
Mouse metanephric renal progenitor cells have also been
fetal proximal and collecting tubules, whereas undifferenti-
transfected with SV-40 T antigen (83,84) and provide a
ated renal progenitor cells not only grow in suspension in
useful system for the analysis of growth factor interactions.
the absence of matrix but also require its absence to retain
Together with immortalized cells derived from the ureteric
their undifferentiated state.
bud (85), these cellular approaches promise to increase our
The action of soluble factors via specific receptors plays an
molecular understanding of proliferation, induction, and
important role in proliferation and nephrogenesis, but stud-
differentiation in nephrogenesis.
ies in transfilter organ cultures were unable to identify a role
for many candidate growth factors (90). More recently, how-
Marker Analysis ever, the combination of embryonic kidney organ culture
and cell culture studies has begun to identify important roles
The differentiation process involved in conversion of undif-
for a variety of growth factor ligands, including midkine,
ferentiated, nonadhesive, nonpolarized mesenchymal cells
neuregulin, fibroblast growth factor, and leukemia inhibitory
into polarized, adhesive, highly specialized renal tubule
factor (81,85,91,92).
(and glomerular) epithelia involves several stages of modifi-
cation under strict temporal control. These include specific
matrix (laminin) and receptor (integrin) expression, inter- Establishment of Epithelial Cell Polarity
cellular tight-junction formation and E-cadherin expres-
Reabsorption and secretion in the kidney are the byproducts of
sion, and segregation of membrane proteins to apical and
vectorial transport—the physiologic hallmark of renal epithe-
basolateral domains. This is followed by elaboration of spe-
lia. This is possible because renal tubule epithelial cells are
cialized membrane and cytoplasmic proteins, including
polarized with asymmetric distributions of membrane proteins
enzymes, transporters, receptors, and structural compo-
on their apical and basolateral membranes. This polar distribu-
nents of the brush border and cytoskeleton (86–89). To
tion of transporters, channels, and other proteins is a charac-
study such a complex array of events in cell or organ cul-
teristic of fully differentiated functionally competent adult
tures, a series of developmental cell- and stage-specific
renal epithelia. Polarity is not only a functional characteristic
markers are necessary. Known markers derived from tissue
but also reflects structural differences of apical and basolateral
localization studies are summarized in Table 15.5. These
membranes. Typically, the apical (luminal) membrane is elabo-
markers can be used to characterize a culture system and
rated into a brush border, although the degree of specialization
evaluate its relevance for further study. For instance, in cul-
of microvilli depends on the tubule cell type. The basal mem-
tures of undifferentiated human and mouse renal progeni-
brane is characterized by its close proximity to the secreted
tors, vimentin and syndecan expression is strong. After
basement membrane, which is composed of a thin electron-
differentiation into cells with epithelial characteristics,
dense layer and separated from the epithelial cell by a thin
however, the former are lost, and E-cadherin, alkaline phos-
electron-lucent zone. Proteins of the apical and basolateral
phatase, and cytokeratin are highly expressed (79,82),
domains are prevented from intermixing by the occluding
whereas Pax-2 and N-CAM are appropriately, transiently
tight junctional complexes that form a continuous belt
expressed. The use of the renal progenitor cell system for
between epithelial cells at the apical pole of the cell and serve
study rests on the fidelity of expression of these markers
to prevent intercellular leakage. When renal tubule epithelia
before and after induction in vitro.
are grown in culture, they also establish polarity characterized
by formation of tight junctions and segregation of membrane
proteins to appropriate apical or basolateral domains. This
Regulation of Differentiation by Matrix and
results in the vectorial transport of ions characteristic of those
Soluble Factors
epithelia in vivo. The degree of polarization achieved in vitro is
The interactions of extracellular matrix (e.g., collagens I, dependent on the cell type and the culture conditions. Polar-
III, IV, and V; laminin; and proteoglycans) with specific ization begins when cells attach to a substrate, which can be
matrix receptors such as syndecan and integrins are criti- glass or plastic but is induced by collagen. Maximal polariza-
cally important in nephrogenesis (74,86,87,90). These tion of epithelia is seen when cells are grown to confluence on
interactions appear to be of particular importance in estab- collagen-coated filter membrane (transwell) inserts in which
lishing cell polarity, a prerequisite for epithelial differentia- feeding is from the basal as well as the apical surface.
tion, in which laminin α1-chain expression and interaction During renal development, a key feature of nephrogenesis
with an epithelial integrin α6 subunit is required (86,87). is the conversion of undifferentiated, nonpolarized mesen-
An important role for the adhesion protein E-cadherin chymal cells of the metanephric blastema through several
(uvomorulin) has also been proposed (88). In vitro tech- stages into polarized, highly specialized tubule and glomeru-
niques allow the analysis of the influence of matrix proteins lar epithelia. After induction by reciprocal interaction with
on proliferation (64), but few studies have been carried out the ureteric bud, mesenchymal cells first adhere to one
326 III. Research Methods

TABLE 15.6. MARKERS OF POLARITY: NORMAL ADULT genesis can be studied by embryonic organ culture tech-
Apical Basal and lateral
niques. Co-culture techniques of epithelial and fibroblast
components have also shown the importance of these inter-
Influenza hemagglutinin VSV G actions in the adult kidney (97).
Alkaline phosphatase Na-K-ATPase
γ-Glutamyl transpeptidase Ankyrin
5'-Nucleotidase Fodrin Morphogenesis Assays
Meprin E-cadherin
Diaminopeptidase IV, meprin V2 receptors Three-Dimensional Gels
Aminopeptidases M, N, P Epidermal growth factor
receptors Experimental confirmation of the role of mesenchymal
Maltase Insulin-like growth factor-1 and extracellular matrix induction on normal renal epi-
receptors thelia has also been shown by the ability of collagen or
H+-ATPase Laminin receptors
reconstituted basement membrane gels to promote the
Trehalase Type IV collagen receptors
Carcinoembryonic antigen HSPG receptors development of three-dimensional tubule-like structures
Na-glucose transporter GLUT2 Integrin α6 from mouse (inner medullary collecting duct) and
Na-amino acid transporter Integrin α2 bovine (MDCK) collecting tubules (98,99) as well as
Na-H exchanger NHE3 Integrin β1 human, dog, and rabbit proximal tubules (3). When ini-
Aquaporin 2 Integrin β4
tially seeded into type I collagen gels, MDCK and LLC-
Na+K+2Cl– symporter Cl/HCO3 transporter, AEI
Urate transporter AQP 4 PK1 cells form cysts (100,101), the specific polarity of
which is a reflection of the responsiveness of the epithe-
AQP, aquaporin; ATPase, adenosine triphosphatase; HSPF, heparin lium to inductive signals from the collagen matrix
sulfate proteoglycan. (95,96). Landmark studies showed that co-cultures of
MDCK cysts in type I collagen gels with fibroblasts or
the addition of fibroblast-conditioned media elicits a
another in the condensate stage and then begin to establish
tubulogenic effect on MDCK cells in collagen gels that
polarity in the vesicle and S-body stages. An important role
is mediated by hepatocyte growth factor (102,103).
for the adhesion protein E-cadherin has been suggested (88),
These studies have led to the wide use of this technique
and inhibitory antibody studies suggest a critical role for
to further dissect the mechanisms of hepatocyte growth
laminin α1 chain and its receptor α6β1 integrin because these
factor regulation (104,105) as well as to elucidate the
are expressed concomitantly with onset of epithelial polariza-
role of cyclic adenosine monophosphate, protein kinase
tion (86,87). The formation of occluding tight junctions
A, and protein kinase X (106,107). The use of this sys-
between cells has also been correlated with the expression of
tem is that it provides a robust three-dimensional assay
adherens junction and desmosomal plaque proteins, includ-
system to analyze the roles of cell-cell, cell-matrix, and
ing ZO-1, catenins, and desmoglein (93, 94). Subsequent
cell-soluble factor interactions. The concentration on
differentiation into tubule cell types is a result of further
MDCK cells will most likely in the future give way to
insertion of polarized tubule cell–specific membrane pro-
the use of more normal cell lines derived from condi-
teins. This is particularly marked on the apical surface, which
tionally immortalized mouse and human nephron seg-
is elaborated into a brush border to provide increased surface
ments from adult and fetal kidneys.
area to accommodate an array of enzymes, transporters, and
receptors. Table 15.6 shows some typical markers of apical
and basolateral membranes in renal tubular epithelia, which Embryonic Organ Culture
might be used to analyze differentiation processes. The analy-
An increased level of three-dimensional organization and
sis of cell polarization events in monolayer primary cultures
normal cell-cell, cell-matrix, and cell-soluble factor
derived from differentiated renal progenitors will in the
interactions can be achieved by organ culture. Because
future provide a temporal and spatial map of sequential
fetal mouse kidneys can now be encouraged to grow and
events involved in tubule maturation during nephrogenesis.
differentiate relatively normally for periods up to and
exceeding 14 days, this system can be used very success-
EPITHELIAL-MESENCHYMAL INTERACTIONS fully for analysis by adding growth factors, antibodies,
or even antisense to the media or by introducing genes
The regulation of ordered growth in the developing kidney into the organ rudiments. Studies of this type have been
and the subsequent maintenance of differentiation is a func- instrumental in identifying important matrix and
tion of reciprocal interactions between the epithelial and growth factors in the regulation of ureteric bud branch-
mesenchymal components of the organ (95,96). In the ing and progenitor cell production including laminin,
embryonic kidney, the mesenchyme provides a pool of pro- α6β1-integrin, sulfated proteoglycans, galectin-3, gangli-
genitor cells that differentiate along epithelial (glomerular oside D3, retinoic acid, midkine, EGF, and neuregulin
and tubule), vascular, and fibroblast lineages, and morpho- (75,91,108–113).
 15. In Vitro Methods in Renal Research 327

TABLE 15.7. IN VITRO TECHNIQUES FOR THE STUDY OF CELL

INJURY AND DISEASES
Insult or abnormality Cell type Species References

Ischemia (anoxia) PT, PCT, PST Human, rat 117

cTAL, mTAL, CCT Rabbit 118
Gentamicin LLC-PK1, PT PCT, PST, CCT Human, dog, rabbit 119–122
Cyclosporine LLC-PK1, PCT, PST, TAL, CCT, Human, dog, rabbit 123–126
mesangial, endothelia, vas-
cular smooth muscle
Nephropathic cystinosis T Human 50
ADPKD Primary monolayer: epithelia Human 22,63
fibroblasts
Conditionally immortalized: Human 50,55
epithelia
ARPKD Primary monolayer epithelia Human, mouse 127–130
Conditionally immortalized Human, mouse 50,55,131
epithelia
Organ culture Mouse 72
Nephronophthisis Primary monolayer Human 132
Wilms’ tumor G401, SK-NEP, WIT 13, GOS-4 Human 44,133
Renal adenocarcinoma A704, AHCN RAG Human, mouse 44
Renal carcinoma CAKI-1, 2: A-498; RC-1; RCC; Human 44,123,
CCF-RC1, -2 134–136

ADPKD, autosomal dominant polycystic kidney disease; ARPKD, autosomal recessive polycystic kidney
disease; CCT, cortical collecting tubule; cTAL, cortical thick ascending limb of Henle; mTAL, medullary
thick ascending limb of Henle; PCT, proximal convoluted tubule; PST, proximal straight tubule; PT, prox-
imal tubule.

IN VITRO TECHNIQUES FOR ANALYSIS OF from diabetic kidneys and/or by addition of glucose, trans-
RENAL CELL INJURY AND DISEASE forming growth factor β, interleukin-1β, or galectin-3 to
their tissue culture media in vitro (110,116).
Renal tubule cell injury in vivo can be induced by
ischemia, nephrotoxins, genetic abnormalities, or cancer.
Ischemic Injury
An understanding of some of the cellular and molecular
mechanisms involved is beginning to emerge, in large Isolated tubules were the first preparations used to study
part because of the use of in vitro techniques, as summa- anoxic and ischemic injury in vitro. These studies identified
rized in Table 15.7. a role for increased calcium in postischemic cell death
(117). Subsequent studies also showed protective effects of
extracellular acidosis and glycine. Molecular mechanisms of
Glomerular Disease
cell injury can be studied easily in long-term cultures, but
The availability of pure glomerular cell types in vitro has led permanent cell lines including MDCK and LLC-PK1 are
to their use in elucidating cellular mechanisms of disease. not well suited for the study of ischemic damage because
For example, glomerular epithelial cells in culture have they have adapted to the relatively hypoxic conditions of
been exposed to Heymann nephritis antigen and provided tissue culture and are abnormally resistant to ischemic
experimental evidence for binding in coated pits, patching, damage. However, primary cultures of rat, rabbit, and
capping, and final shedding from the cell surface (114). human renal tubule epithelial cells retain their sensitivity to
More recently, the advent of well-differentiated, condition- hypoxic and postischemic reperfusion injury. They have
ally immortalized podocyte cell systems from normal mice been used successfully to demonstrate differential tubule
and their genetically manipulated diseased counterparts has cell sensitivity and attenuation of cell death by polyethylene
significantly contributed to the understanding of the roles glycol, extracellular calcium restriction, calcium channel
of collagen 4α4 in Alport’s syndrome, nephrin in Finnish- blockers, and inhibitors of calmodulin and cysteine pro-
type nephropathy, and podocin in immunoglobulin A tease activities (117,118).
nephropathies (61,115). Glomerular epithelial cell cultures
have also been used to demonstrate a role for loss of nega-
Nephrotoxic Injury
tive charge and adhesive properties in puromycin aminonu-
cleoside nephrosis. Mesangial cell cultures have also been LLC-PK1 and primary cultures of rabbit and human proxi-
used as a model system to study diabetes by their isolation mal tubules and conditionally immortalized human proxi-
328 III. Research Methods

mal, thick ascending limb, and collecting tubules have been ADPKD epithelia. Similar abnormalities in Na-K-ATPase
used to reproduce the characteristics of gentamicin and ami- and EGF receptor proteins have been found in human
noglycoside antibiotic toxicity in vivo. They have demon- ARPKD, in cpk/cpk, and PKD1 and PKD2 knock-out
strated a major role for lipid changes, including decreased mice, which is consistent with their fundamental roles in
turnover and degradation caused by reductions in sphingo- cyst formation in genetically determined polycystic kidney
myelinase activity in cellular injury primarily in the proxi- disease. However, ADPKD differs from ARPKD in that
mal tubule (50,119–122). cysts are derived from all segments of the nephron, and
Cyclosporine toxicity can also be studied in LLC-PK1 there are additional interstitial abnormalities, including
rabbit proximal tubule and human proximal, thick ascend- increased extracellular matrix (heparan sulfate proteogly-
ing limb, and collecting tubule cultures and has been shown can) turnover and overproliferation of fibroblasts (22,64).
to exert differential toxic effects on proximal tubule cells. The identification of the PKD-1–encoded protein polycys-
These toxic effects can be attenuated by extracellular calcium tin-1 and its localization to cell membranes of normal fetal
restriction, calcium channel blockers, and cysteine protease collecting duct and ADPKD epithelia in vivo and in vitro
inhibition. The mechanism of action may be through intra- correlates with demonstrated abnormal fetal patterns of
cellular binding to cyclophilin and increased intracellular gene transcription in adult epithelia from ADPKD kidneys.
calcium-dependent protease activity (123,124). Renal vascu- Primary and conditionally immortalized cell lines derived
lar effects have also been demonstrated by observation of from these cell types provide an essential tool to analyze the
increased proliferation of endothelial and vascular smooth precise function of polycystin-1 and to determine the pri-
muscle cells in culture as well as increases in intracellular free mary effects of PKD-1 mutations (Table 15.4).
calcium in mesangial cells (125,126) that are inhibited by In addition to monolayer cultures, organ cultures of
cyclosporine. embryonic and postembryonic kidneys from cystic and
other disease states have provided invaluable information
(72). As more mouse models of genetic disease become
Genetic Renal Tubular and Cystic Diseases
available and developmental defects suspected, these types
The first cell culture system devised for the study of genetic of techniques are likely to be used increasingly. Not only
cystic disease was for human autosomal dominant polycys- will the results from the mutant phenotype be analyzed but
tic kidney disease (ADPKD) in which individual cysts were also the effects of addition of exogenous compounds or
microdissected and grown in primary culture (63). Subse- introduction of DNA constructs will allow for in vitro test-
quently, several primary, immortalized, and conditionally ing of potential therapeutic agents.
immortalized monolayer systems for human ADPKD, Culture techniques are also being used increasingly to
human and mouse autosomal recessive polycystic kidney study mechanisms of disease from genetically engineered
diseases (ARPKD), and nephronophthisis (132) have been mice such as the orpk model of ARPKD and the gy model
established (Table 15.7). Monolayer primary cultures were of X-linked hypophosphatemia (139). It is to be antici-
particularly instrumental in delineating the excessive tubule pated that further crosses with the immortomouse (58) will
epithelial cell proliferation, matrix adhesion, and polarity lead to isolation of cells from a variety of mouse models of
and fluid secretion defects in cystic epithelia (137) (Chap- disease. To advance the study of human disease, the same
ter 35). The advantages of these in vitro monolayer culture temperature-sensitive conditionally immortalizing con-
techniques are the capacity to carry out strict comparisons struct was first used to generate a unique model for analysis
of normal and abnormal cells, which have allowed struc- of nephropathic cystinosis (50), as well as human ADPKD
tural and functional characterization of the modified phe- and ARPKD (55). The success of generating immortal cell
notype in ADPKD epithelia, compared with similarly lines from these human disease states that retain their
cultured normal tubule epithelia of proximal, thick ascend- unique characteristics suggests the general use of this
ing limb or collecting tubule origin. Use of these tech- approach for the future. In addition, the ability to generate
niques in combination with in vivo studies has identified identical clonal cells will facilitate the detailed analysis of
several abnormalities and determined their functional con- the functional effects of mutations.
sequences. Mislocalization of Na-K-ATPase to the apical
membranes of ADPKD epithelia is associated with simi-
Cancer: Wilms’ Tumor
larly mislocated ankyrin, fodrin, and E-cadherin and a
basal-to-apical vectorial transport of sodium, suggesting a Although it has been surprisingly difficult to culture cells
mechanism for the secretion of ions and fluids into cysts from primary Wilms’ tumors, some permanently growing
(138). A mechanism for increased proliferation in ADPKD cell lines are available for study, the most widely used of
cyst epithelia is suggested by detection of hypersensitivity which are G401 and SK-NEP. The Wilms’ tumor suppres-
of ADPKD epithelia in vitro to the mitogenic effects of sor gene, WT1, localized on chromosome 11p13, is a zinc-
EGF, apical secretion of EGF into cyst fluids, and mislocal- finger DNA-binding protein involved in normal renal
ization of EGF receptors to apical cell membranes of development, and the deletion of this gene in vivo results in
 15. In Vitro Methods in Renal Research 329

Wilms’ tumor formation, and, therefore, cell lines that fail ACKNOWLEDGMENTS
to express WT1 should provide a suitable model system for
the study of Wilms’ tumor. Four candidates have been I am indebted to Drs. Christopher Burrow, Deborah
described: WIT 13, GOS-4, G401, and SKNEP (Table Hyink, Katalin Polgar, and Jill Norman for continued stim-
15.7). Each has limitations, however. WIT 13 has two dif- ulating discussion concerning the application of renal in
ferent 11p13 deletions (both lacking the WT1 locus), vitro techniques for studies of normal and abnormal renal
grows slowly, and has fibroblast morphology because it was development.
derived from the stromal component of a Wilms’ tumor.
GOS-4 and G401 have intact 11p13 regions but fail to
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 16

ANIMAL MODELS
JORDAN A. KREIDBERG

The use of animal models has been an essential aspect of nearly physiology, and, thus, they receive appropriate emphasis in this
all areas of nephrologic research since its earliest days. Research chapter. Several genetic approaches are available for use with
on kidney formation and malformation, physiology and patho- animal model systems. There are reverse genetic systems, in
physiology, immunologic injury, and tolerance or transplant which a gene of interest is mutated using gene targeting or
rejection all depend on the use of animal experimentation. This expressed in transgenic mice in such a way to interfere with its
chapter emphasizes genetic approaches that use animals, as this normal function. Using forward genetic approaches, one starts
area has shown the most progress in the development of novel with a phenotype of interest, which could either be obtained as
technologies since the previous edition of this book. a spontaneous mutation or from mice treated with a mutagen,
and an effort is made to identify the mutated gene responsible
for the phenotype. Reverse genetic approaches using gene-
INSTITUTIONAL OVERSIGHT targeted or transgenic mice are useful for a wide variety of
developmental and physiologic studies in which there is a need
There is increasing public awareness of the use of animals in to study the function of a known gene. Forward genetic
research and with this comes increasing concern about the approaches, on the other hand, are mainly used in projects in
appropriateness of the use of animals and whether much of the which the goal is the identification of novel genes.
research that does involve animal models could be accom-
plished using nonanimal models. Therefore, it is important to
Gene Targeting
note that all animal research in the United States must be eval-
uated by institutional committees before any experimentation Gene targeting was originally used to introduce a deletion or
may commence. Furthermore, the U. S. Department of Agri- interruption into a gene of interest, using the scheme shown in
culture provides constant oversight through the use of frequent Figure 16.1, such that it could be determined whether mice
and usually unannounced visits to animal facilities of research would be able to develop in the absence of that gene’s func-
institutions. These regulatory committees and agencies are tion. In cases in which a gene was shown not to be essential for
charged with evaluating animal protocols to make certain that development, the homozygous mutant mouse might serve as a
animals are used in an ethical manner, with proper use of anes- useful model in which to study the role of a specific gene in a
thetics or analgesics to minimize or eliminate any source of physiologic or disease process. For example, targeted deletions
pain during experimentation. They are also charged with veri- of the Wt1 (1), Pax2 (2), GDNF (3–5), Wnt4 (6), and BMP7
fying that animals are indeed required for the specific research (7,8), among others, showed these genes to be essential for var-
in question, that large animals are not used when smaller ones ious aspects of early kidney development. On the other hand,
would suffice, and that the investigators are trained and knowl- the absence of many immunology-related genes does not result
edgeable about proper use of animals. Despite these several lay- in any developmental impairment, but these mice have served
ers of oversight, in the end, it is up to the principal investigator as useful models to study the role of the immune system in
to be thoughtful about whether the intended experimental transplant rejection.
approach will yield sufficiently important and worthwhile The advent of gene targeting was made possible through
results to justify the use of laboratory animals. the use of two technologies developed mainly in the 1980s.
The first was the development of tissue culture conditions that
allowed embryonic stem (ES) cell lines to be grown indefi-
GENETIC MODELS nitely in culture while retaining their totipotency (9). ES cells
grown in culture could then be introduced into mouse preim-
The early 1990s to the present have witnessed an explosion in plantation embryos or blastocysts and become fully integrated
the use of genetic approaches to understand development and into those embryos such that their descendant cells would give
336 III. Research Methods

FIGURE 16.1. Gene targeting in mice. A: The scheme

for targeting a deletion of an exon in embryonic stem
(ES) cells. Exons are shown as black boxes along a chro-
mosome. Restriction sites for restriction enzyme
BamH1A are shown. The replacement vector is con-
structed such that the neomycin resistance gene (neo)
is shown as an open box, in place of one of the exons.
An external probe specifically does not overlap with
the replacement vector. A double homologous recom-
bination results in the integration of the vector into
the chromosome, thus replacing the exon with the
Neo gene. The BamH1 site within the neo gene results
in a shorter BamH1 restriction fragment detected by
probe after homologous recombination. B: ES cells can
be injected through a micropipette into a blastocyst, in
which they become part of the inner cell mass. The
injected blastocyst is introduced into the uterus of a
hormonally primed mouse and gives rise to a chimeric
mouse, partially derived from the ES cells and partially
from the original inner cell mass (ICM) cells. If the ES
cells and blastocysts are derived from strains with dif-
ferent coat colors, then the chimeric mouse will have a
variegated coat color pattern on its fur, providing an
indication of its overall extent of chimerism. In the
best cases, the resultant mouse is nearly entirely
derived from ES cells. C: Shows a possible pattern
obtained in a Southern blot, based on the scheme
shown in A, using the external probe. A wild-type
mouse shows only the longer band. A heterozygous
mouse shows both the wild-type and gene-targeted
band, and the homozygous mutant shows only the
shorter band, due to the presence of the BamH1 site in
the neo gene. D: The mating involved in obtaining
germ-line transmission of the mutation and subse-
quently obtaining homozygous mutant mice.

rise to all developmental lineages that are found in adult mice would in essence be heterozygous for a mutation in the tar-
(10). The second technology involved is the use of homolo- geted gene, and heterozygous ES cells can be isolated and
gous recombination to introduce mutations into mammalian expanded to provide a population for injection into blasto-
genes (11–13). As shown in Figure 16.1, when long stretches cysts. Therefore, by combining the ES cell technology and
of genomic DNA in recombinant DNA constructs are intro- homologous recombination, it became possible to target
duced into cells in culture, this DNA will, at variable and often mutations into genes in ES cells and then introduce ES cells
quite low frequency, recombine into the locus from which the carrying these mutations into blastocysts, finally obtaining a
genomic DNA was originally derived. Therefore, homologous mutant adult mouse.
recombination of the correctly designed genomic fragment can In a typical experiment, gene-targeted ES cells would con-
be used to introduce a deletion or insertion into a genomic tain one mutated allele and one normal or wild-type allele for
locus that renders the gene unable to be expressed. This ES cell the gene under study. The targeted ES cells would be injected
 16. Animal Models 337

into preimplantation blastocysts, and groups of these blasto- embryogenesis. In this situation, an embryo will become
cysts would be introduced into female mice that were previ- nonviable at the first point at which expression of the inac-
ously hormonally primed to allow implantation of the injected tivated gene becomes essential for survival. However, it
blastocysts into their uteri to begin a pregnancy. The resultant may be highly desirable to study the function of a gene
mice from these injections are termed chimeras, because any product in many later events during development or adult
specific cell is either derived from an ES cell or the original life. Conditional gene targeting allows the inactivation of a
injected embryo (i.e., the chimeric mouse essentially has four gene in particular tissues or at particular times during
parents, the male and female that provided the blastocyst and development or adult life (14–16). This technology has
the male and female that provided the embryo from which the been developed more recently, and has proved more diffi-
ES cell line in use was originally derived). In the best cases, a cult to use on a widespread basis thus far, for reasons that
chimera might be nearly entirely derived from the ES cells. are discussed next.
Among the tissues that ES cells contribute to are the germ The general approach to conditional gene targeting is
cells: spermatocytes or oocytes. When ES cells heterozygous shown in Figure 16.2. This is a variation on traditional gene
for a mutation are used to make a chimera, germ cells derived targeting, in that it also relies on homologous recombination to
from the ES cells have a 50% chance of carrying the mutant introduce a segment of recombinant DNA into the locus of a
rather than the wild-type allele. Therefore, mating a chimeric gene in ES cells. However, whereas traditional gene targeting
and wild-type mice can result in some of the offspring being inactivates the gene, conditional gene targeting must modify
true heterozygotes for the mutated gene. After obtaining both the gene such that it can be expressed until such time as its
male and female heterozygotes, they can be mated to obtain inactivation is desired. The most commonly used approach
homozygous mutant embryos or mice, depending on whether involved the insertion of LOX sites, which are 34 base pair sites
or not the gene is essential for development. involved in site-specific recombination by Cre recombinase, an
enzyme originally derived from a bacteriophage (15). Because
LOX sites are rather small, it is usually possible to insert them
Conditional Gene Targeting
in introns in which they have no effect on gene expression. By
The process described in the preceding section results in placing two LOX sites in a gene to flank an exon, Cre can be
the inactivation of a target gene from the beginning of used to inactivate a gene by recombining out the DNA seg-

FIGURE 16.2. Conditional gene targeting. The target-

ing vector is different from the previous figure in that
LOX sites flank the exon that will eventually be deleted,
and the Neo gene is flanked by FRT sites. The vector is
incorporated into the chromosome through homolo-
gous recombination, and embryonic stem (ES) cells with
this knock-in are used to make chimeric mice, and
germline transmission is obtained. Although the LOX
sites should not interfere with expression of the gene,
the Neo gene is likely to interfere with normal gene
expression. However, in most cases, mice will tolerate
one inactive gene, as long as the other allele is func-
tional. After obtaining heterozygous mice, they are
mated with Flp –deleter mice, that express Flp recombi-
nase in germ cells. Flp will recombine the FRT sites and
eliminate the neo gene. Mice without the neo gene,
but still containing the exon flanked by LOX sites, are
mated with mice expressing Cre in a particular tissue or
cell type, or expressing an inducible Cre, to obtain the
conditional knock-out. The breeding scheme shown in
the figure is oversimplified. In the actual experiment, a
more complicated breeding scheme is required to
obtain a mouse that is homozygous for alleles with LOX
sites and that also has the Cre-expressing transgene. An
alternative is to breed mice with the conditional allele
with mice carrying a traditional knock-out. This has the
advantage that to obtain the conditional knock-out,
Cre must only recombine one, and not two, pairs of
LOX sites in each cell.
338 III. Research Methods

ment containing the exon that was situated between the two
LOX sites, thus inactivating the gene. There are experimental
approaches for expressing Cre in temporally or spatially specific
manners or both. Spatial- or lineage-specific expression of Cre
is most often obtained by placing the Cre cDNA downstream
of a known tissue-specific promoter. Sometimes this is
achieved by using homologous recombination to insert the Cre
gene into the genomic locus of a gene with known tissue-
specific expression, such that Cre replaces the first exon of that
gene. Temporally, specific expression of Cre has proved more
difficult to obtain. One approach is to regulate Cre using the
tetracycline system for inducible gene expression (17). The
other approach makes use of a fusion protein consisting of Cre
and a portion of the estrogen receptor that confers steroid-
mediated nuclear localization (18,19). The latter is modified to
bind tamoxifen or tamoxifen derivatives instead of estrogen.
The Cre-modified estrogen receptor fusion protein will remain
in the cytoplasm and therefore not be able to mediate site-
specific recombination of LOX sites until tamoxifen is admin-
istered to the mouse to induce nuclear translocation of the Cre
fusion protein. This system can be used to induce recombina-
tion in embryos when tamoxifen is administered to pregnant
mice. The major obstacle to using conditional gene targeting
on a widespread basis is the availability of promoter/enhancer
elements that are able to confer robust tissue or cell-lineage–
specific expression of Cre recombinase. For example, in the
kidney, there are not promoters available that are able to direct
expression of a transgene in every distinct cell lineage or groups
of lineages. However, this situation is gradually improving. For
example, the nephrin and podocin promoters have recently
FIGURE 16.3. Gene targeting using bacterial artificial chromo-
been shown to confer podocyte-specific expression (20–22), some (BAC) clones. Homologous recombination is done in Escheri-
and the upstream region of the aquaporin-2 promoter confers chia coli instead of in embryonic stem (ES) cells. In the first step, a
expression in collecting ducts (23). DNA fragment is prepared that contains the kanamycin resistance–
positive selectable marker, and the SacB-negative selectable marker,
As more tissue-promoter elements become available, condi- which also contains homologous ends (a and b, each approximately
tional gene targeting promises to have a large impact on 50–60 bp), is introduced into E. coli. This fragment can usually be
genetic approaches to kidney disease. As noted earlier, there are prepared by polymerase chain reaction, using primers that contain
the homologies to the genomic region and also to a vector contain-
many genes expressed both in the developing and adult kid- ing the selectable markers. Usually, a strain of E. coli is used that
ney, in which the knock-out of the gene results in embryonic allows transient activation of the enzymes required for homolo-
lethality. This precludes study of how the product of that gene gous recombination. Selection for kanamycin resistance obtains
BAC clones where the selectable markers have recombined into the
might function in postnatal kidneys or why a mutation in that BAC. In a second round of homologous recombination, a DNA frag-
gene leads to kidney disease in humans. It also raises the ques- ment with the same homologous ends but containing a mutated
tion of why humans carrying such mutations are able survive, exon 2, denoted by the “X,” is introduced into the E. coli containing
the BAC. Selection against SacB obtains BACs in which the mutated
albeit with a genetic disease, when mice carrying mutations in exon 2 has replaced the selectable markers. A third round of
the same gene do not survive embryogenesis. Sometimes this is homologous recombination is used to insert the Neo gene, flanked
simply because mice and humans differ in their respective by FRT sites, so that the BAC can be used for homologous recombi-
nation in ES cells. As shown, this scheme is used to introduce point
requirements for specific genes, but more often, it is because mutations or small deletions into a gene. It can also be used to con-
humans with genetic disease often have point mutations that struct conditional knock-out vectors, similar to those shown in Fig-
lead to partial loss of function, whereas mouse knock-outs ure 16.2. An additional use is to knock-in a green fluorescent
protein or β-galactosidase (LacZ) reporter gene into a locus to
often involve complete loss of function mutations. Condi- obtain information about patterns of gene expression.
tional gene targeting can sometimes offer a solution to this
problem by allowing normal gene expression during embryo-
genesis and then inactivating a gene in adult mice. Alterna- by recent advancements that facilitate homologous recombina-
tively, there are variations on the Cre-LOX approach that allow tion into BACs (bacterial artificial chromosomes) in Escheri-
the introduction of point mutations into mice. The introduc- chia coli (Fig. 16.3). BACs are used because they contain large
tion of point mutations into mice has been greatly facilitated amounts of genomic DNA and, thus, are ideal for use as gene-
 16. Animal Models 339

targeting vectors. The longer length of BACs compared with reverse genetic approaches start with a known gene and
shorter genomic clones should improve the frequency of attempt to characterize the phenotype resulting from muta-
homologous recombination in ES cells. tion of that gene. Knock-out mice described earlier are one
type of reverse genetic approach; ENU mutagenesis
described later is an example of a forward genetic approach.
TRANSGENIC MICE This approach would also be used with spontaneous muta-
tions that affect renal function. In either case, whether or
As mentioned earlier, many mutations that result in human not a mutagen is applied, these projects are designed to lead
disease are point mutations that result in hypomorphic, or to the identification of the gene whose mutation is causing
partial, loss of function alleles of a gene. In this case, a dis- the phenotype. Now that the human and mouse genomes
ease state may result from decreased activity of a gene prod- have been sequenced, forward genetic approaches may tend
uct. In other cases, a point mutation or deletion mutation more often to assign diseases to previously identified genes
may produce a protein that interferes with the function of than identify the gene outright.
the normal gene; this is referred to as a dominant negative Animal models of disease that have a genetic basis may
effect. This could occur in instances in which a protein either result from spontaneous or induced mutations.
requires homodimerization for activity, and dimerization of Spontaneous mutations or phenotypes are those noticed
a wild-type and a mutant form of a protein leads to an inac- either by chance or through the directed observation of
tive complex. Dominant-negative effects can also be found large numbers of mice that were not otherwise treated to
in cases in which two proteins heterodimerize, and an inac- induce a mutation. In contrast, induced mutations are
tive mutant protein is able to complex with its partner pro- those resulting from the treatment of mice with irradiation
tein, but the complex is inactive. Dominant-negative effects or mutagenic agents known to introduce point mutations
can be studied in animal models using transgenic mice. or deletions into the genome. At present, several major
Although gene-targeted mice discussed in the previous sec- efforts in several countries involve the use of N-ethyl-N-
tions can also be considered to be transgenic because for- nitrosourea (ENU) to introduce small mutations through-
eign DNA is used to disrupt the endogenous gene, here the out the mouse genome (24–31). These large genome-scale
term transgenic is reserved for those mice in which foreign approaches, which can involve very large mouse colonies,
DNA has been inserted into the murine genome through are justified by the following arguments:
pronuclear injection. In contrast to gene-targeting schemes
in which genes are modified in ES cells and ES cells are 1. Most disease-related human mutations are caused by
then used to derive chimeric mice, in transgenic strategies, point mutations; therefore, an ENU-mutagenic approach
DNA is directly microinjected into the pronucleus of a fer- may have a greater chance of producing a phenotype
tilized egg or zygote, and the injected zygotes are then reim- resembling a human disease than will gene-targeted muta-
planted into the oviduct of a hormonally primed female tions that usually completely inactivate a gene.
mouse. The injected DNA is able to recombine by nonho- 2. An ENU-based approach does not rely on previous
mologous or illegitimate recombination into random loca- identification or cloning of the gene (i.e., any gene is a
tions within the genome and in variable amounts from theoretical target and can be studied) to the extent that
zygote to zygote. Once mice are derived from the injected some degree of compromise in the gene product’s
zygotes, they are tested to determine whether they carry the activity will result in an observable phenotype. The
injected DNA within their genomes as a transgene, and if obvious disadvantage in comparison with gene target-
they do, whether the transgene is expressed. By injecting ing is that a large amount of work lies between the
DNA constructs that contain a tissue-specific promoter and observation of a phenotype and the final identification
a mutated gene of interest, it is possible to study whether of the mutated gene.
expression of the mutant gene leads to an observable phe- 3. Given a large enough effort, it should be possible to
notype. In other instances, the gene to be expressed is not eventually “saturate” the genome with mutations—
mutated, and the experiment is designed to determine that is, examination of several hundred thousand
whether overexpression or de novo expression of the gene mutagen-treated mice is likely to provide the opportu-
results in an observable phenotype or disease model. nity to observe the effects of placing a mutation in
every gene capable of causing an observable phenotype.

FORWARD AND REVERSE GENETICS However, one important point remains to be mentioned
that dramatically increases the labor and expense of an
Forward genetic approaches begin with a phenotype and ENU-based effort. Most observable phenotypes tend to be
attempt to identify the gene. These phenotypes may be genetically recessive instead of dominant, meaning that
obtained through mutagenesis screens or from spontaneous they are not apparent in the first-generation offspring of
mutations identified within a population. In contrast, mutagen-treated mice. Instead, it is necessary to breed a
340 III. Research Methods

sequences that will amplify the (CA)n sequence between the

two primers. Within a genetically inbred strain of mice,
each individual mouse will contain the same number of CA
dinucleotides at each repeat. However, similar to the varia-
tion observed between human individuals, different inbred
strains may differ in the number of CA dinucleotides at any
particular repeat. In addition, there are species of mice
closely related to Mus musculus, such as Mus spretus, that
provide even greater differences in the number of CA dinu-
cleotides at many repeats than are found between the inbred
strains of M. musculus. As depicted in Figure 16.5, genetic
mapping using microsatellite markers takes the following
approach: A mouse (or mice) with a phenotype produced by
induced or spontaneous mutagenesis is mated with a mouse
from a different inbred strain or from a different species,
such as M. spretus, to produce F1 mice that are now hetero-
zygous at all loci, containing one allele from each of the two
parental strains and one mutant allele. In the case of a reces-
sive phenotype, these F1 mouse are now either backcrossed
to the original mutant strain or intercrossed among them-
selves to produce approximately 100 progeny mice, approxi-
mately 25% of which can be expected to show the mutant
phenotype. For dominant phenotypes, the backcross can be
to a wild-type mouse of either parental strain, and 50% will
display the phenotype. Importantly, during this back- or
intercross, there is independent segregation of chromo-
FIGURE 16.4. N-ethyl-N-nitrosourea (ENU) mutagenesis. A
scheme is depicted for finding recessive phenotypes through somes, such that each individual of the 100 mice is geneti-
ENU mutagenesis. Dominant phenotypes require a less compli- cally unique, in that at any given locus it may be
cated approach, as phenotypes will be apparent in the first homozygous for alleles from a parental strain or hetero-
generation derived from crossing founders with wild-type
mice. In this scheme, a mutagenized male founder that proba- zygous, containing an allele from each strain. A set of
bly carries many mutations after mutagenesis is mated with a polymerase chain reaction primers corresponding to
wild-type mouse to produce heterozygote offspring that carry approximately 40 to 50 microsatellite repeats (about two to
a subset of these mutations. These heterozygotes are mated
with wild-type mice to produce a second generation, which three per mouse chromosome) are used in the first round of
will carry a smaller subset of the original mutations. These are analysis. These are chosen such that the two parental strains
then mated to the original heterozygous offspring of the are known to differ in the length of the CA repeat between
founders, and 25% of the offspring of this cross will be homo-
zygous for any particular mutation that was present in the sec- each primer pair. DNA samples are now obtained from all
ond heterozygous generation. the progeny and are tested for the length of the CA sequence
at each of the microsatellites, and these results are correlated
with the observed phenotypes (in practice, a computational
second generation and then backcross it to the first-genera- result, called a lod score is produced). Most of the microsat-
tion mice, resulting in a third generation (Figure 16.4). ellites will not be genetically linked to the locus contain-
Doing this on a large scale will result in many third-genera- ing the mutation, and there will be no observable
tion mice that are now homozygous for mutations resulting correlation between the strain genotype at a particular mic-
from the original mutagenic treatment, and some will have rosatellite and the presence or absence of the phenotype. In
observable phenotypes that can be studied for biologic contrast, if a microsatellite marker is sufficiently closely
interest and to map the responsible gene. linked to the site of a mutation causing a recessive pheno-
Mapping sites of induced or spontaneous mutations in type, both alleles of the microsatellite marker are more likely
mice has been greatly aided by the development of sets of to be derived from the parental strain originally containing
microsatellite repeat markers. Microsatellite repeats used in the mutation. Thus, the goal of the first round of screening
mapping are stretches of CA dinucleotide repeats that are is to identify at least one marker that is linked to the muta-
found interspersed throughout mammalian genomes tion. Thereafter, subsequent rounds will use sets of markers
(32,33). Typically, these CA repeats contain 10 to 20 CA linked to the original positive marker, with the expectation
dinucleotides. These CA repeats are flanked by unique that it will be possible to identify a marker or pair of mark-
sequences, and thus it is possible to design pairs of polymer- ers very closely linked to the mutation that will delimit the
ase chain reaction primers that correspond to these flanking region of a single chromosome on which the mutation is
 16. Animal Models 341

FIGURE 16.5. A scheme for mapping a mutation to a

genetic locus. A: Depicts the use of microsatellite CA
repeats. Strains A and B are two inbred strains of mice
that differ in the length of many CA repeats, including
the one shown here. Strain A has 23 CA dinucleotide
repeats, whereas strain B has 25. They have the same
unique 5' and 3' sequences flanking the CA repeats;
thus, the same polymerase chain reaction primers can
be used for both strains, but amplification will yield a
longer product from strain B than A. B: The mating
scheme to begin mapping the mutation. Only chromo-
somes 1 and 2 are shown. Strain A has a homozygous
mutation on chromosome 1, marked as an “X” that is
linked to the CA repeat, here designated by the black
inverted triangle. This recessive mutation yields an
observable phenotype. Strains A and B are mated to
produce an F1 progeny, which will be heterozygous at
all loci, including the one mutated in strain A. They will
also be heterozygous for all CA repeats, including those
on chromosome 2. Thus, any CA repeats that differ
between the two strains will yield two bands on a poly-
merase chain reaction using the flanking unique prim-
ers for that CA repeat. F1 mice are backcrossed to strain
A homozygotes, and many offspring are examined.
Fifty percent of these offspring should be homozygous
for X and have the observable phenotype. When these
mice are analyzed for the CA repeat close to the locus
for X, most mice with the phenotype will show only the
strain A amplification product, whereas most of those
without the phenotype will show both the strain A and
strain B bands. In contrast, amplification of any CA
repeats from chromosome 2 or any other chromosome
will not show any correlation of strain A homozygosity
with the observed phenotype.

located. This can then be used to initiate either a candidate inexpensively maintained mammal. However, it should be
gene approach or a chromosome walking approach to even- mentioned that other animals may provide advantages that
tually identify the mutated gene. Newer sets of markers suggest them as an alternative to mice. Zebrafish are much
include single nucleotide repeats and improved radiation less expensive to maintain than mice, and genetic tools to
hybrid maps that will also aid in the mapping and identifi- map zebrafish mutations will soon equal those available for
cation of mutated genes (34,35). mice (36–38). The zebrafish excretory system involves a
These forward genetic approaches are not only suited to pronephric duct and glomus that bears important similarity
study developmental anomalies. Some of the large-scale to mammalian nephrons and has already been the subject
efforts on mouse mutagenesis ongoing around the world of many research studies (39–46). Alternatively, there is
involve performing basic blood work, urinalyses, and renal often a desire to use genetic approaches in animals that
structural studies on each mouse from the group being allow more complex physiologic studies than are usually
screened for new phenotypes. Thus, this approach has the done with mice. Rats provide an alternative, although the
potential to identify genes involved in disease progression, availability of genetic markers has not progressed as far as
as well as those responsible for morphogenetic processes. for murine systems (33,47), and they are more expensive
than mice to maintain.

GENETIC MODELS IN OTHER SPECIES

ANIMAL MODELS OF KIDNEY DISEASE
The previous discussion focuses on mice as the obvious
model for genetic approaches to kidney development or The selection of an animal model for some aspect or type of
disease. Mice are the most popular model system for kidney disease takes several factors into consideration. Most
genetic approaches, given their long history of study, the important, the similarity to human disease that can be
availability of many inbred lines, and that they are the most observed in a particular model is taken into account. Other
342 III. Research Methods

important factors include the cost of the animals involved: TRANSPLANT MODELS
The cost of maintaining animals larger than rodents increases
dramatically with size, and the numbers of animals that can Animal models have been used extensively to study transplant
be studied consequently decreases. For this reason, some stud- rejection and in efforts to understand how tolerance to trans-
ies may begin with a rodent model, and then progress to a planted tissue may be improved. Over the past 20 years, there
larger model once the rodent model establishes the feasibility has been an extraordinary advancement in our mechanistic
of the hypothesis under study. The size of an animal may be understanding of the immune function, and this has been
important to the extent that it affects the ability to perform brought to bear on the study of transplant rejection and toler-
surgical manipulations or physiologic measurements. How- ance (99–105). Important models under study include skin
ever, because it has become increasingly desirable to obtain and heart transplants in mice and kidney transplants in rats
physiologic measurements on various strains of knock-out (106–113). In addition, it is possible to produce “humanized”
mice, the equipment available to perform these measurements mice by transplanting human tissue into immunodeficient or
has improved and become commercially available. irradiated mice whose immune system has been reconstituted
with human lymphocytes, thus allowing the study of human
immune function in an animal model (114–117). One area of
MODELS OF RENAL FAILURE research that remains controversial is involving xenografts
(118,119). Because the supply of human kidneys and other
Approaches to the study of renal failure include acute and organs for transplants continues to fall far short of the demand,
chronic models. Acute renal failure has been induced using there is a desirability of determining whether nonhuman ani-
pharmacologic agents, antisera against kidney tissue or other mals provide an alternative source of organs for transplanta-
antigens in which immune complex formation leads to glo- tion. The major concerns here include the strong immunologic
merular disease (48–54). Ischemia-reperfusion models of rejection to a xenograft that must be overcome and the danger
acute renal failure, achieved by temporarily ligating a renal that xenografts might serve as vectors for the introduction of
artery, allow study of the pathologic processes involved in novel infectious agents into the human population.
tubular damage, as well as the effect of various pharmaco-
logic treatments on the pathologic process (55–75).
There is often considerable variability in the response to a ANIMAL MODELS FOR THE STUDY
treatment among animals of a particular species. For example, OF RENAL PHYSIOLOGY
different species of rats or mice may respond differently to dif-
ferent treatments. This difference in response is presumably Historically, several animal models have been used to study
due in a large part to genetic differences between different renal physiology, including swine, sheep, guinea pigs, rab-
strains of rats or mice. The identification of so-called modifier bits, rats, and mice. Fetal lambs have been a particularly
genes that are responsible for these interstrain differences may important model in which developmental aspects of physi-
contribute importantly to the understanding of human disease ology have been studied, particularly relating to obstructive
(76–82). Differences among humans in the sensitivity to uropathy (120–132). As alluded to earlier, when genes
nephrotoxic agents or differences in the severity of a disease encoding proteins involved in the regulation of physiologic
process among different individuals may be due to polymor- processes are knocked out, there is a desirability to perform
phisms in these same modifier genes. physiologic studies in murine systems to determine how
physiologic responses are altered in the absence of a partic-
ular protein (133–140). Although gene knock-outs are not
MODELS OF IMMUNOLOGIC INJURY yet done in rats, the availability of genetic models and gene
mapping of physiologic traits has yielded important infor-
There are many models of autoimmune injury to the kidney. mation (141–145). The ability to perform sophisticated
The traditional model for a lupus-like autoimmune disease is physiologic experiments in mice has improved dramatically
the New Zealand black mouse, which has been studied for over the past several years, as new equipment has become
many years (83–87). These mice develop autoantibodies available (139). As in other situations, there is a constant
similar to those observed in humans with systemic lupus need to balance the advantages of a large animal model
erythematosus and other related autoimmune disorders. with the lower costs of smaller animal models.
More recently, many strains of mice carrying mutations in
genes involved in the regulation of the immune response
have been used to increase our understanding of the role the SUMMARY
immune system plays in the onset and progression of kidney
disease (88–98). These knock-out strains have allowed inves- Animal models are of increasing importance in the study of
tigators to begin a genetic dissection of genes involved in kidney disease. An important shift since the early 1990s is
autoimmune and other disorders. the use of rodent models and the use of genetic models.
 16. Animal Models 343

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studies. The use of large versus small animals must take into specific expression in transgenic mice. J Am Soc Nephrol
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21. Wong MA, Cui S, Quaggin SE. Identification and character-
ization of a glomerular-specific promoter from the human
nephrin gene. Am J Physiol Renal Physiol 2000;279:F1027–
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renin-containing cells from the ovine fetal kidney. J Soc genetic susceptibility to renal impairment after unilateral
Gynecol Investig 1997;4(4):191–196. nephrectomy. J Am Soc Nephrol 2000;11(11):2068–2078.
129. Berry LM, et al. Preterm newborn lamb renal and cardiovas- 142. Kwitek-Black AE, Jacob HJ. The use of designer rats in the
cular responses after fetal or maternal antenatal betametha- genetic dissection of hypertension. Curr Hypertens Rep 2001;3
sone. Am J Physiol 1997;272(6 Pt 2):R1972–R1979. (1):12–18.
130. Matsell DG, Bennett T, Bocking AD. Characterization of 143. Stoll M, Jacob HJ. Genetic rat models of hypertension: rela-
fetal ovine renal dysplasia after mid-gestation ureteral tionship to human hypertension. Curr Hypertens Rep 2001;
obstruction. Clin Invest Med 1996;19(6):444–452. 3(2):157–164.
131. Peters CA, et al. Fetal compensatory renal growth due to uni- 144. Stoll M, et al. A genomic-systems biology map for cardio-
lateral ureteral obstruction. J Urol 1993;150(2 Pt 2): 597–600. vascular function. Science 2001;294(5547):1723–1726.
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 17

CLINICAL INVESTIGATION
SUSAN L. FURTH
JEFFREY FADROWSKI

CLINICAL RESEARCH QUESTION study, technical expertise of the research staff, and the time
and money that can be devoted to the project. Once a ques-
How can we best evaluate, treat, and assess long-term risks for tion is framed, the researcher needs to outline the study pro-
children with kidney disease? Who is at risk of developing end- tocol or methods, which include specifying the recruitment
stage renal disease (ESRD) in childhood or young adulthood? method, number of subjects and how they will be recruited,
Clinicians are often faced with questions such as these with how each variable will be operationally defined, and the plan
uncertain answers in the practice of pediatric nephrology. Par- for data analysis. A poorly designed study is worse than no
ents ask, “Why did my child get this disease?” “What is the study at all because, like imprecise measurements and an
most effective method to treat this condition?” “What’s the improper analytic plan, it can also lead to false conclusions.
prognosis of this condition in my child?” Frequently, these
answers are not known, and these questions are the inspiration
Steps in Refining a Good Research Question
for high-quality clinical research. The first step in developing a
valuable clinical study is determining whether the initial query A good research question usually begins with a broadly
can be translated into a good research question. stated concept. The initial question is then made more spe-
cific by identifying independent and dependent variables.
Often, research questions are concerned with causal rela-
Hallmarks of a Good Research Question
tionships. Independent variables are those conceptualized to
A good research question gives useful information, is interest- be causes; dependent variables are those conceptualized to be
ing to the researcher, builds on what is known, and can be effects. The research question can be modified to ask about
answered with available resources. Research is a labor of love, the role of multiple potential causes in leading to the spe-
demanding attention to detail, perseverance, honesty, and cific outcome. A simple research question asks whether x
imagination. Developing a good research question is an itera- (independent variable) causes y (dependent variable). More
tive process. One needs input from knowledgeable colleagues complex research questions could assess the relative impor-
and collaborators. The researcher must become thoroughly tance of x and other variables (e.g., a and b) as causes of y. A
familiar with what is already known about the topic by review- different research question might ask how strongly x pre-
ing the literature and consulting with experts in the area. dicts y in one population versus another.
Investigating what is already known has several benefits. First, The next step is to translate a research question into a
it can reveal that the candidate research question has already hypothesis. In our simple example, the researcher may
been answered adequately. Second, learning what is already hypothesize that x causes y. In the actual research project, the
known provides insight into potentially useful methods for information collected is examined to determine whether it is
addressing a research question. For example, previous studies reasonable to conclude that x does cause y. In examining the
may demonstrate good ways to measure a variable of interest data, the investigator tests the null hypothesis that x does not
or provide background information for determining sample cause y versus the alternative hypothesis that it does.
size. Third, a literature review may suggest ways to frame the
research question at hand. For example, a literature review may
reveal that particular modifiable risk factors are consistently SCIENTIFIC METHOD
associated with a disease process, and an intervention to mod-
ify these risk factors may form a sound basis for a clinical trial. A study’s potential value is determined by the relevance of
Finally, a good research question needs to be answerable the research question. Its ultimate worth is determined by
with available resources. These include subjects available for the study methods. Methodologic issues concern the study
348 III. Research Methods

design, subject selection, data collection techniques, and not manipulate the risk factor but merely selects children
the analytic plan. Subsequent sections in this chapter dis- with and without disease and compares them in terms of
cuss each of these aspects. As a foundation, this section the risk factor(s). Study designs are experimental when the
describes the concepts of inference, generalizability, and investigator not only observes but actually manipulates the
validity. relationship between two variables. Observational designs
provide somewhat weaker evidence of causation, because
they fail to rule out explanations other than association
Inference
between the variables studied. Experimental designs can
As Figure 17.1 shows, scientific research begins with the provide much stronger evidence for causation. In an experi-
research question. It then moves (clockwise in the figure) to mental study, the investigator controls the independent
the controlled arena of the study design and then through variable, which is the factor hypothesized to produce
the implementation of the actual study and findings. Infer- change in a dependent variable. In an experimental study,
ences from the findings in the study approximate the “truth subjects are randomized to receive or not receive the inde-
in the study.” From these “truths” we attempt to infer the pendent variable. The goal of the process of randomization
applicability of the findings to general clinical practice. is to produce study groups that are “balanced” in terms of
Researchers describe and explain reality by sampling a por- other factors that could influence the dependent variable.
tion of it, measuring characteristics of the sample, analyzing Unfortunately, experimental designs often are not feasible,
the measurements, and interpreting the results. Researchers ethical, or desirable. Epidemiologic studies of disease pre-
make inferences from the sample measures of operationally clude manipulation of risk factors in humans. Health ser-
defined variables to the hypothesized relationships of the vices researchers studying the public health impact of
theoretical constructs in the larger population of interest. changes in health policy rarely can control these changes.
Errors in the design or implementation of the study can Within the broad categories of observational and experi-
lead to false conclusions. The strength of inference depends mental designs, there are many variations. These variations,
largely on the research methods used in the recruitment of distinguishing characteristics, primary uses, strengths, and
study subjects (sampling) and in the choice and integrity of weaknesses are discussed in the section Study Design. Study
the study design. design also influences the validity of study results.
Statistical inference depends on the methods used to
define and sample the population. The researcher uses
Validity
inferential statistics to extrapolate the sample findings to the
larger population of children from which the sample was Validity is the extent to which study findings correctly
drawn. Inferential statistics assume that the studied sample reflect and explain reality. The concepts of internal and
is drawn by probability methods and can be used to make external validity are illustrated in Figure 17.2. As a research
inferences about the larger population. The size of a proba- question’s relevance increases, so does the need for validity.
bility sample determines the certainty of inferences from it. To some extent, every research question worth answering is
All other things being equal, the larger the sample size, the controversial because of its implications for policy and
greater the certainty of inferences to the population. practice and because it addresses areas in which existing evi-
The researcher’s ability to make a causal inference from dence is scant or contradictory. If a question is not contro-
study results depends largely on issues of study design.
Study designs are observational when the investigator does

FIGURE 17.2. External and internal validity in experimental

designs. (From Fletcher RH, Fletcher SW, Wagner EH. Clinical epi-
FIGURE 17.1. The role of inference in drawing conclusions demiology: the essentials, 3rd ed. Baltimore: Williams & Wilkins,
from clinical research studies. 1996:12, with permission.)
 17. Clinical Investigation 349

versial, there is little need for a scientifically rigorous search drugs or toxins. An example of a case series is the report by
for its answer. The clarity and rigor of study design as well Furth et al. on diabetes associated with the use of tacrolimus
as the careful implementation of the research plan increase in pediatric renal transplant recipients (2). The authors iden-
the likelihood that inferences from the study are valid. tified a number of pediatric transplant recipients treated with
Cook and Campbell (1) define several aspects of validity. tacrolimus who developed diabetes. The authors summarized
Statistical validity is the correctness of study conclusions the case histories and reviewed existing literature regarding
regarding the existence of a relationship between two vari- diabetes associated with immunosuppressive therapy in adult
ables. A study lacks statistical validity when it concludes transplant recipients. A case series such as this can provide
there is no relationship between variables when in fact there useful information for the clinician. However, as a method to
is one or when it concludes there is a relationship when in determine the risk associated with a particular factor, this
fact there is none. Statistical validity is jeopardized most design is extremely weak because there is no means of com-
often by inadequate sample size and by improper use of sta- parison. Even if a risk factor is highly prevalent among the
tistical tests. cases (in this example, all the cases of diabetes posttransplant
If there is a relationship between two variables, internal had been treated with tacrolimus), there is no way of know-
validity (Fig. 17.2) is the correctness of conclusions about ing whether the risk of the disease is greater with exposure to
whether the relationship between the operationally defined tacrolimus than with exposure to other immunosuppressive
independent and dependent variables is causal. Internal medications—for example, cyclosporine or steroids. The case
validity is jeopardized when a study design fails to control series design cannot provide an estimate of risk.
for factors that could confound the hypothesized causal
relationship.
Cross-Sectional Design
Finally, given a causal relationship between the indepen-
dent and dependent variables, external validity (Fig. 17.2) is A cross-sectional study is one in which the disease and risk
the correctness of generalizing to other persons, settings, factors are measured at the same time in a sample of sub-
and times. Poor choice of a study population and inade- jects. Subjects can be categorized as either having or not
quate research procedures are common challenges to exter- having the risk factor. Within each group, the presence of
nal validity. the disease can be determined. Analytically, the association
In summary, the scientific method involves extrapolating between a particular risk factor and the disease is measured
inferences from a study situation to the larger world. The as the relative prevalence of the disease among those with,
value of research depends on the validity of such inferences, versus those without, the risk factor.
which in turn is determined by the researcher’s choice of The cross-sectional study design is superior to the case
methods. The following sections explain the strengths and series in that it provides a means for comparison. Cross-
weaknesses of the methodologic choices available. sectional studies are relatively economic, easy to conduct, and
allow simultaneous examination of multiple risk factors. An
example of a cross-sectional study is the analysis of demo-
STUDY DESIGN
graphic factors associated with rates of hospital admissions for
infection in pediatric ESRD patients in the U.S. Renal Data
Observational Studies
System (USRDS). In this analysis, presented in Figure 17.3,
There are four major types of observational studies: case admissions for infection from 1997–1999 for incident and
series, cross-sectional, case-control, and cohort. Observa- prevalent pediatric ESRD patients were described according to
tional designs are weaker than interventional designs in age, race, gender, and ESRD treatment modality (3). In this
establishing causation, but they are useful when it is not analysis, the youngest patients were at highest risk for hospital
feasible to manipulate the independent variable. Studies of admission because of infection, and dialysis patients were at
disease etiology usually are observational. In these types of greater risk than transplant patients.
observational studies, risk factors or exposures are the inde- Cross-sectional studies have a number of limitations. For
pendent variables, and disease is the dependent variable. example, in the analysis of risk factors for hospitalization for
infection in the USRDS, patients studied are those with hospi-
talization data in the USRDS database. These are patients that
Case Series
have survived ESRD long enough to be counted in the data-
In a case series study, a sample of cases is chosen, and the pres- base. If they died early after developing ESRD, they may not
ence of the risk factor is measured. A case series study is easy be included. In addition, because of varying Medicare eligibil-
to conduct and is useful as a preliminary study to reinforce ity criteria (which influences the data available in this particu-
anecdotal evidence, to generate hypotheses, or to establish lar database), patients who have gone on to have long-term
variable distributions in planning future research. Case series successful kidney transplants may not be included. Therefore,
can sometimes identify previously unrecognized constella- the characteristics increasing risk for hospitalizations for infec-
tions of symptoms or morbidities attributed to exposures to tion in this study may not be generalized to all pediatric
350 III. Research Methods

FIGURE 17.4. Design of a case-control study. (From Fletcher RH,

Fletcher SW, Wagner EH. Clinical epidemiology: the essentials, 3rd
ed. Baltimore: Williams & Wilkins, 1996:213, with permission.)

control study starts with the identification of persons with

the disease or other outcome variables of interest in the
population at risk (Fig. 17.4). A suitable control group of
persons without the disease or outcome is also selected
from the population at risk. This is pictured on the right
side of Figure 17.4. To examine the possible relation of one
or more exposures to the given disease or outcome, the
researcher then looks back in time to compare the propor-
tions of the cases and controls exposed and not exposed to
the risk factor in question.
The case-control design has several advantages. It pro-
vides stronger evidence of causation than the cross-sectional
design. In a cross-sectional study, outcomes and exposures
are assessed simultaneously, and the investigator must infer
cause and effect relationships because the temporal
sequence cannot be established. In a case-control design, an
attempt to establish a temporal relationship between the
outcome and exposure is made by starting with a popula-
tion of persons with and without the outcome and then
working backwards to examine suspected exposures. Thus,
compared to the cross-sectional design, the investigator is
more confident that the exposure of interest came before
FIGURE 17.3. Cross-sectional study of risk factors for infection
in pediatric end-stage renal disease (ESRD). HD, hemodialysis; the outcome, not as a result of the outcome. As compared
PD, peritoneal dialysis. (From U.S. Renal Data System. USRDS to other study designs, case-control studies are efficient in
2001 Annual Data Report: atlas of end-stage renal disease in the the study of rare diseases or those with long latent periods
United States. Bethesda, MD: National Institute of Health,
National Institute of Diabetes and Digestive and Kidney Dis- between exposure and outcome. Whereas cross-sectional or
eases, 2001:114, with permission.) cohort designs would require a large number of subjects
and time to identify risk factors for a rare disease, a well-
designed case-control study can identify similar risk factors
patients with ESRD. Studying prevalent patients runs the risk with comparatively fewer subjects and much less time and
of missing those patients who were “cured” or who died soon expense. Also adding to the efficiency of the case-control
after developing the disease. Also, because in cross-sectional design, several potential risk factors for a disease or out-
analyses the presence or absence of two factors is assessed at the come can be examined simultaneously.
same time, it is not possible to attribute causality. A recent case-control study by Fored et al. confirmed the
association of acetaminophen and aspirin with chronic
renal failure (4). Adult Swedish patients with early-stage
Case-Control Studies
chronic renal failure were identified as cases (N = 918) from
Case-control studies are also known as case-referent, com- monthly reports of serum creatinine measurements from
peer, retrospective, case history, and cohort studies. A case- medical laboratories. Controls were randomly selected
 17. Clinical Investigation 351

throughout the ascertainment period from the Swedish spect in their recall of such exposures. This introduces recall
Population Register (N = 980). Aspirin and acetaminophen bias, a form of information bias because of better, and
were used regularly by 37 and 25%, respectively, of the sometimes exaggerated, recollection of exposures by cases as
patients with renal failure and by 19 and 12%, respectively, compared to controls.
of the controls. Regular use of either drug in the absence of It can be difficult for an investigator to remain objective
the other was associated with an increase by a factor of 2.5 in collecting exposure information. In interviewing subjects
(odds ratio via logistic regression) in the risk of chronic and in reviewing records, there may be a tendency to look
renal failure from any cause. more carefully or evaluate evidence differently for cases
Case-control studies yield an odds ratio as an estimate of than for controls. Strategies for dealing with this problem
relative risk. This measure is calculated by dividing the include the use of objective measures and to ensure that the
odds that a patient was exposed to a given risk factor by the individuals collecting data are unaware of the subject’s
odds that a control was exposed to the risk factor. It can group status (blinding/masking). The more subjective the
also be obtained from logistic regression analysis. Logistic method for measuring the exposure, the more important it
regression allows the investigator to obtain the odds ratio is to mask the observer. Blinding as to the specific exposure
for a given risk factor independent of other potential risk being studied or study hypothesis is useful and also can be
factors or confounders using the technique of adjustment. used to attempt to control recall bias.
Odds ratios are generally a good approximation of relative Nested case-control studies and nested case-cohort studies
risk if the outcome is rare. are alternative case-based hybrid designs that have many
As with any study design, the case-control method has advantages. A nested case-control study involves selecting all
limitations. Case-control studies allow for the study of only cases and control subjects from a known cohort. In this
one disease at a time, as opposed to cross-sectional or design, the controls are free of the outcome or disease.
cohort studies. This design does not allow for the measure- Nested case-control studies eliminate the problem of recall
ment of incidence, prevalence, or excess risk. Case-control bias, because the exposure information is obtained before the
studies are also subject to error, or bias, which can threaten outcome has developed (cohort design). Also, the temporal
the validity of the study. Selection and information biases, sequence between exposure and outcome is defined. This
the two major categories of bias, are possible in the case- design is also much more economical and efficient; the entire
control design. Selection bias arises if the manner in which cohort need not be analyzed for a given exposure (e.g., via a
cases and controls were selected yields an apparent associa- laboratory specimen). Nested case-cohort studies also use the
tion when, in reality, exposure and disease are not associ- selection of cases and controls from a known cohort. How-
ated. For example, cases, by definition, include only ever, in this design, controls are randomly selected from the
individuals who have been identified as having the disease initial cohort irrespective of outcome. This design permits
and who are available for study. Those who have not been the delineation of relative risk for an exposure.
diagnosed, have been misdiagnosed, or have died are
excluded. If diagnosis or availability is related to the expo-
Cohort Design
sure being studied, the sample of cases will be biased.
Avoiding selection bias can be even more challenging in Various names, including prospective, follow-up, and lon-
the selection of controls. The control group must be compa- gitudinal, have been used to label cohort studies in the
rable to the cases. They should not be chosen in such a way past, reflecting the temporal sequence of exposure and
that important differences between cases and controls exist disease in this category of observational studies (Fig.
that might influence exposure history and thus limit the infer- 17.5). The word cohort originated from the Latin word
ences derived from the study. A number of strategies exist to cohors, describing a group of warriors that marched
select a control group that is at risk for the disease and other- together. Clinical investigators have adapted this term to a
wise representative of the same population as the cases. These
include sampling cases and controls in the same way (e.g.,
from the same clinical setting), matching controls to cases on
key variables related to the disease (e.g., age), using multiple
control groups, and using population-based samples of both
cases and controls (e.g., using disease registries).
Information bias occurs when the case and control
groups differ in terms of the quality of the data collected to
measure risk factors. The retrospective approach to measur-
ing an exposure in the case-control design introduces the
possibility of differential recall between the cases and con-
FIGURE 17.5. Design of a cohort study. (From Fletcher RH,
trols. Cases may have been asked more often about the Fletcher SW, Wagner EH. Clinical epidemiology: the essentials, 3rd
presence of a given exposure and/or may be more circum- ed. Baltimore: Williams & Wilkins, 1996:102, with permission.)
352 III. Research Methods

specific type of research study: a group of individuals free them in the analysis. Multivariable analyses are examples of
of the disease(s) of interest is assembled, their risk status is statistical tools used to adjust for confounders. However,
determined, and the group is followed over time to mea- unsuspected confounders might still jeopardize the validity
sure the incidence of disease. Comparison of the inci- of conclusions.
dence of disease (or rate of death from disease) between For example, Wong et al. (5) used a multivariate logistic-
those with and without the exposure of interest permits regression analysis to account for potential confounders in
measurement of the association between the risk factor the association of antibiotic use for E. coli O:157–associ-
and the disease. ated diarrhea and HUS. Adjustments were made for the
The significance of the cohort design has been empha- initial white blood cell count and the day of illness on
sized by the wealth of scientific data obtained from famous which the initial stool culture was obtained for analysis.
cohorts such as the Framingham Study or the Physicians These factors had been previously associated with increased
Health Study. Pediatric nephrology has also benefited from risk of HUS. A higher initial white blood cell count could
studies using the cohort design. For example, Wong et al. be a potential confounder, for example, because it is associ-
used the cohort design to demonstrate a 17-fold increase ated with an increased risk of HUS (the outcome) and
in the risk of hemolytic uremic syndrome (HUS) associ- might make the physician more likely to prescribe antibi-
ated with antibiotic use in children with Escherichia coli otics (the exposure), thus potentially falsely linking antibi-
O:157 diarrheal infections (5). In this study, children with otic usage with HUS. After adjustment for these factors,
E. coli O:157 were followed to assess risk factors for the the multivariate analysis revealed a persistent association,
development of HUS. reassuring the discerning reader.
The cohort design has an obvious niche in clinical Cohort studies have several advantages. Because risk
research. Ethical and practical considerations often do factors are measured before disease, the temporal
not allow for randomization of individuals to an expo- sequence of risk and disease is established, and the
sure of interest. Cohort designs allow for the examina- potential for biased risk measurement is avoided. Several
tion of exposure and disease associations under such diseases or outcomes can be measured, and disease
circumstances. occurrence can be measured in terms of incidence, not
Cohort studies can be classified as concurrent or non- just prevalence. Cohort studies often require large sam-
concurrent. In a concurrent cohort study (also referred ple sizes and are unsuitable for studying rare diseases.
to as a prospective or longitudinal study), the clinical Large sample size and long follow-up periods can make
investigator identifies the population and collects extant cohort studies costly. A nonconcurrent cohort design
exposure information and then follows the cohort to a can reduce cost, but it decreases the investigator’s con-
designated point in the future. Nonconcurrent cohort trol over subject selection and risk factor measurement.
studies (i.e., retrospective, historical, and nonconcurrent
prospective) require the investigator to identify a cohort
Experimental Design
that has been delineated in the past, along with informa-
tion regarding the exposure(s) of interest. This popula- In an experimental design, the investigator controls the
tion can then be followed for the development of a given independent variable or intervention and uses random-
disease in the more recent past, the present, or into the ization to determine which subjects will receive the
future. intervention (the intervention, study, or experimental
Traditionally, the outcome of interest in cohort studies group) and which subjects will not (the control or pla-
is the ratio of the incidence of disease in those with the cebo group) (Fig. 17.6).
exposure divided by the incidence of disease in those
without the exposure. This can be interpreted as the rela-
tive risk for disease in many cases. When calculating and
interpreting risks in the cohort design, the absence of ran-
domization must be taken into account. Because the
investigator is merely observing the exposure and not con-
trolling for it via randomization, subjects with and with-
out the risk factor might differ in terms of other
characteristics that are related to the disease. If the charac-
teristic is related to both the exposure being evaluated and
the disease, it can lead to a misleading association between
the exposure and the disease. Such a characteristic would
be a confounder.
FIGURE 17.6. Design of a randomized trial. (From Fletcher RH,
To avoid misinterpreting such an association, the inves- Fletcher SW, Wagner EH. Clinical epidemiology: the essentials, 3rd
tigator must measure potential confounders and adjust for ed. Baltimore: Williams & Wilkins, 1996:139, with permission.)
 17. Clinical Investigation 353

The North American IgA Nephropathy Study is an tion to assign subjects to study groups. Such designs are
example of a randomized controlled clinical trial. In this useful in evaluating programs that are targeted to groups
study, eligible patients younger than 40 years with immuno- of individuals (e.g., a community-based health educa-
globulin A (IgA) nephropathy on kidney biopsy, glomerular tion program). In this case, the investigator might con-
filtration rate >50 mL/min/1.73 m2, and evidence of pro- trol the program’s design but cannot randomly assign
teinuria are randomly assigned to receive alternate day pred- community residents to be exposed to it.
nisone, fish oil, or placebo (6). The goal of this study is to Quasi-experimental designs fall into two broad catego-
determine the relative benefits of fish oil or alternate day ries: nonequivalent control group designs and time series
prednisone on the progression of IgA nephropathy. In this designs. In the former, two or more study groups are com-
and other controlled clinical trials, randomization is the key pared. The investigator tries to assemble groups that are
feature of its experimental nature. Through randomization, comparable but cannot be assured of their comparability.
all potential confounders, both those recognized by the In time series designs, the investigator measures the out-
investigator and those that are not suspected, are likely to be come of interest at several points in time before and after
balanced between the study groups. In other words, the the intervention. Agreement between hypothesized and
three groups in this study are considered to be the same observed patterns of outcome measures supports a causal
except for the treatment they receive. Differences in rates of relationship between intervention and outcome. There are
progression of kidney disease in the three groups are attrib- many variations on these two broad types of quasi-experi-
utable to the intervention, because the effect of confounders mental designs. Some variations incorporate both types.
has been ruled out by the balance achieved by randomiza- For example, studies of community health programs often
tion. Therefore, experimental designs offer stronger evi- involve time series analysis of two or more communities,
dence of causality than do observational designs. some with the health program and some without.
In an experimental study, it is important to ensure that In summary, many research designs are available to the
subject assignment is truly random. This can be achieved investigator. No single design is best for all research ques-
by using random numbers, either through computer- tions. Although experimental designs are superior to obser-
assisted assignment or manually, with a table of random vational designs in addressing threats to internal validity,
numbers. Sometimes blocking is used in conjunction with they are not always feasible or ethical. The most appropri-
random assignment. A block of subjects is simply a set ate design for a given research question is the design that
number of consecutive study enrollees. Within each block, maximizes internal validity within the constraints of the
a predetermined number of subjects is randomly assigned research environment.
to each study group. For example, if the block size is set at
six and two study groups of equal size are desired, then
three subjects in each block of six are randomly assigned to
IMPORTANT ISSUES IN CARRYING OUT A
one group and three to the other. Blocking is useful when
RESEARCH PLAN
study enrollment is expected to be prolonged. Over
extended periods, both study procedures and outside con-
Selection of Subjects
ditions can change. Blocking ensures that the study groups
will be balanced with regard to such changes. In any research study, one would like to extrapolate the
Experimental studies, like observational studies, are sub- findings to all patients with the condition of interest (Fig.
ject to measurement bias. Research staff should be masked 17.2). The study population is the group that is meant to
or blinded to the subject’s group assignment during data represent the target population from which a sample is
collection, especially if any outcome measures are not drawn. Sampling decisions involve defining the study pop-
strictly objective. In the IgA study, for example, both the ulation and sample.
research staff and the study subjects are unaware of which
treatment they receive. In one arm of the study, placebo
Defining the Target Population
capsules are made to look exactly like fish oil capsules. In
another arm, placebo tablets are distributed that are identi- Although there is no one single ideal target population,
cal to prednisone. If outcomes are measured without sub- the investigator needs to consider the ramifications of
ject or staff knowledge of the subject’s group status, they are one definition versus another. If the investigator were
less likely to be influenced by expectations about potential interested in studying risk factors for a specific disease,
differences between treatment and control group outcomes. the target population could be defined as all children
with this disease or a subset of them (e.g., children of a
certain age). The broader the target population, the
Quasi-Experimental Designs
greater the generalizability of the study findings. On the
In a quasi-experimental study, the investigator maintains other hand, the increased heterogeneity of a broadly
control over the intervention but cannot use randomiza- defined target population could introduce variability
354 III. Research Methods

among subgroups in terms of the importance of risk fac- Probability theory is the rationale for extrapolating
tors. For example, a particular characteristic could be a inferences from a study sample to the reference population.
major risk factor in some population subgroups but not A probability sample is one in which every subject, or ele-
in others. Assessing the importance of risk factors within ment, in the study population has a known probability of
subgroups requires a larger study sample and perhaps a being selected. A nonprobability sample is one in which the
more complex sampling design. probabilities of selection are unknown. It is legitimate to
extrapolate from a sample to its population only if proba-
bility sampling has been used.
Defining the Study Population
There are several types of probability sampling. In
A practical consideration in defining the target popula- simple random sampling, each element has an equal
tion is availability of the population for study. The chance of being selected. In systematic sampling, each ele-
investigator could have all children available seen in a ment in the population is assigned a consecutive num-
particular clinical setting. Insofar as children seen in this ber, and every nth element is sampled. Systematic
setting are representative of the target population, the sampling is easy to use, but it will generate a biased sam-
clinical site would be a good choice for study; the experi- ple if the sampling fraction (e.g., every tenth case) is the
ence of its enrollees could be considered generalizable to same as some periodicity in the ordering of cases in the
the target population. If children enrolled in the clinical population. For example, if every tenth patient is sam-
setting differ systematically from the target population, pled in a clinic where ten patients are seen each session
sampling bias is introduced. For example, tertiary care and the most complex cases are scheduled first, then the
pediatric nephrology centers might be more likely to sample will contain either all complex cases or no com-
serve children with advanced stages of the kidney disease plex cases, depending on the first element drawn. Strati-
or more severe or complicated cases. Studying only these fied random sampling is useful when one believes that
cases may introduce bias toward only studying the most population subgroups differ in important ways. The
complex forms of a particular disease. Sampling bias population is divided into the subgroups, or strata, of
impairs the generalizability of study findings. Represen- interest. Simple or systematic random samples are then
tativeness, therefore, is a prime consideration in defining drawn from each stratum. Cluster sampling is useful
a study population. Investigators should evaluate the when it is difficult or costly to sample elements in a pop-
representativeness of candidate settings and the likely ulation individually. Instead of elements, groups of ele-
implications of potential biases. One possible approach ments are sampled. For example, in a study of school
to this in studies of patients with kidney disease is to children, the investigator could take a probability sam-
compare the characteristics of participants in a study to ple of classrooms and then study all the students within
known characteristics of the larger population to whom each selected classroom. The selected classrooms, in
one would like to generalize the results. combination, must be representative of the overall popu-
lation. As with stratified sampling, formulas for calculat-
ing variance must be modified, and consultation with a
Defining the Sampling Scheme
statistician is recommended.
Just as we generalize from the study population to the tar- Nonprobability sampling techniques include convenience
get population, we generalize from the study sample to the sampling, quota sampling, and purposive sampling. A conve-
study population. Sample statistics are measures that pertain nience sample is one that is most readily obtained without the
to the samples that are studied. A sample mean, for exam- use of random sampling. A quota sample is a convenience
ple, is the sample’s average score on a particular measure, sample drawn to assure specified numbers of subjects in speci-
and a sample standard deviation expresses the variability of fied strata, without the use of random sampling. A purposive
the sample scores. The sample statistics are the investigator’s
best estimates of the population parameters. The sample
mean is the best estimate of the population mean; the sam- TABLE 17.1. EXAMPLE ILLUSTRATING HOW α, β, AND
ple standard deviation is the best estimate of the population EFFECT SIZE AFFECT SAMPLE SIZE
standard deviation.
Confidence Power
Extending beyond inference to hypothesis testing, sam-
1 – α (%) 1 – β (%) Effect size (%) Total sample No.
ple statistics of the association between variables are the
best estimates of these associations in the target population. 95 80 10 → 40 76
The association between a hypothesized risk factor and the 95 90 10 → 40 96
occurrence of disease in the study sample (perhaps mea- 95 90 10 → 20 572
95 99 10 → 40 156
sured by odds ratios or relative risks) is the investigator’s
best estimate of the association between the risk factor and Adapted from Sample size calculator (Statcalc) in Epi-info Stat Calc.
disease in the target population. Available at: http://www.cdc.gov/epiinfo/. Accessed April 11, 2003.
 17. Clinical Investigation 355

sample is one in which subjects are selected because they are Table 17.1 illustrates how varying the acceptable levels
judged to be representative of the population of interest. of α, β, and effect size influences sample size. For example,
Probability sampling is preferred but not always possi- if we designed a study to determine whether a new drug
ble. In clinical research, the investigator is often limited to could “cure” 40% of patients compared to an old drug that
a particular clinic population. If a clinic population is “cured” 10% of patients, we would have 90% power to see
believed to be representative and if it is larger than the such an effect with 95% confidence in a total sample of 96
number of subjects needed for study, the investigator patients (see row 2 in Table 17.1). In contrast, to obtain a
should use a probability sampling technique to draw the significant result documenting a smaller effect size from the
study sample. old drug cure rate of 10% to a new drug cure rate of 20%
An example of probability sampling using stratified sam- with the same α = 0.05 and 90% power, we would need to
pling techniques can be seen in a recent survey study of adult study 572 patients.
and pediatric nephrologists (7). The authors created a survey
containing ten case vignettes to assess whether increased
Attrition of Study Subjects
experience with pediatric patients influenced nephrologists’
recommendations for peritoneal or hemodialysis in other- Sample size calculations determine the number of subjects
wise identical patients with ESRD described in the vignettes. needed at the study’s conclusion. In determining the num-
Because the authors wanted the survey respondents to repre- ber of subjects to enroll, the investigator must estimate
sent the population of U.S. adult and pediatric nephrolo- attrition rates and enroll a sufficiently large sample to com-
gists, they randomly selected a representative sample of pensate for study dropout.
nephrologists in five geographic regions of the United States. Even if probability sampling is used to define the study,
Each randomly selected nephrologist was mailed a survey subject attrition could produce a biased sample at the
containing ten case vignettes to assess what factors affected study’s conclusion. An attrition rate of more than 25% is
the nephrologists’ dialysis recommendations. cause for concern. In data analysis, subjects completing
the study should be compared with those who drop out to
determine whether the two groups differ in a clinically
Determining Sample Size
significant way. Such differences must be considered in
In any study, several factors determine the required sample interpreting the study findings.
size. This section describes those that come into play in sev-
eral common types of investigations. Detailed sample-size
formulas and tables are beyond the scope of this chapter, DATA COLLECTION: MEASUREMENT
but several excellent references are listed in Suggested Read-
ing. Briefly, to estimate sample size, the researcher needs to Decisions on what data to collect and how to do so begin
set the acceptable level of α (probability of type I error), β with specifying the variables that need to be measured and
(probability of type II error), and determine the effect size operationally defining each. The investigator will need to
that one is likely to see. In determining sample size, the evaluate the suitability of existing measures and determine
probability of making a type I error, α, is usually set at whether to use an existing measure or develop a new one.
0.05. This is the probability of concluding that an associa- The data sources for each variable must also be identified.
tion between two variables exists when it does not. β error Finally, the investigator should specify the level of measure-
is the probability of concluding that no association exists, ment of each variable. An efficient way to document the
when in fact it does. The reader will be more familiar with data collection plan is to make a table with columns listing
β error in terms of its relationship to “power.” The power of the variables to be measured, their operational definitions,
a study is equal to 1-β. In many studies, β is customarily set the data source(s) for each variable, and level of measure-
at 0.2 for a power of 80%. If β is set to 0.1, the power of ment for each. This section describes issues pertaining to
the study is 90%. each of these tasks.
In addition to specifying α and β, the researcher must
also determine an estimate of the response to treatment in
Identifying the Variables to Be Measured
one of the groups (in a clinical trial) or the rate of occur-
rence of disease (in a cohort study). The effect size is an Researchers are often tempted to collect as much informa-
estimate of how much better than the comparison group tion as possible. This can be costly, in terms of time, money,
you expect a treatment group to be in a clinical trial or how and data quality. The investigator should be able to justify
increased the risk of a particular disease is in the setting of a each variable to be measured. Most important are the
particular risk factor (in a cohort study). An illustration of hypothesized independent and dependent variables. In addi-
estimated sample sizes for given α, β, and effect sizes is tion, identified potential confounders should be measured.
shown in Table 17.1 for a study comparing differences in Finally, data characterizing the study population and sample
proportions in two groups (8). will be needed to describe the study’s generalizability.
356 III. Research Methods

Sources of Data A pilot test is a dress rehearsal of the activities for selecting
study subjects, contacting them, securing informed con-
Study data can be collected from existing sources or can be
sent, and collecting and processing data. Activities that do
generated specifically for the specific research hypothesis
not work as planned should be modified and the pilot test-
being tested, using surveys, interviews, or observations.
ing continued until the fieldwork procedures run smoothly.
Most studies combine both strategies.
Data quality should be monitored during the main
An enormous variety of existing data sources is available,
study. Interviews and questionnaires should be reviewed as
including medical records, vital records, national and local
they are completed to allow recontacting subjects to correct
health surveys, and census data. Health programs often keep
errors. The reliability of subjective measures and those
records of services provided, and billing records can be espe-
requiring special technical skill should be assessed. For
cially helpful. Examples of existing data sources in pediatric
studies with unsupervised interviewing of subjects, it is wise
kidney disease include the USRDS (3) and data collected by
to validate a portion of the completed interviews handed in
the North American Pediatric Renal Transplant Case Study
by fieldworkers. This can be done by recontacting a ran-
(NAPRTCS) (9). Existing sources can provide data for time
dom sample of subjects and then asking them to verify
periods and individuals otherwise unavailable to the investiga-
their responses to a sub-sample of the interview questions.
tor. The number of studies published by the NAPRTCS and
their tremendous contribution to our understanding of clinical
outcomes in pediatric kidney transplantation and dialysis illus- ANALYTIC PLAN
trate this point. The chief disadvantage of existing sources
gleaned from registry data is that the data are often not col- Data Analysis
lected as systematically as in a prospective research study.
Investigators often defer considering data analysis until the
Important data elements are sometimes missing. Incomplete
data have been collected. This is a serious mistake. Study
data and inaccuracies are also possible with registry data.
planning should include an analytic plan of the steps
Primary data collection is expensive and is limited to sub-
needed to answer the study questions once the fieldwork is
jects available to the investigator. On the other hand, the
completed.
investigator’s control over data collection makes data quality
more certain. Many primary data collection strategies are
available, including mail surveys, mass-administered ques- Use of Statistical Tests
tionnaires, telephone and in-person structured and unstruc-
As noted earlier, statistical validity is the correctness of
tured interviews, direct observation, and videotaping and
study conclusions regarding group differences and variable
audiotaping. Choosing a strategy should be based on the
relationships. A key threat to statistical validity is the use of
research question, the sensitivity of the data to be collected,
inappropriate statistical tests. Parametric statistical tests are
the literacy of the population to be studied, and the resources
based on assumptions about parameters of the population
available for the study. The key factor should be data qual-
and are the most powerful tests available in situations in
ity—that is, which method will provide the most complete
which these assumptions are met. Nonparametric statistical
and accurate information within budgetary constraints.
tests are based on fewer assumptions about the population,
so they are appropriate in situations in which the assump-
tions underlying parametric statistics are not met.
Assessing Data Quality
Assumptions vary by statistical test. If a test is used in a
One strategy to enhance data quality is to train research staff situation that violates its assumptions, it will be inaccurate,
thoroughly. Often, data-collection staff work independently. leading to a misleading measure of statistical significance.
To ensure that they follow study procedures, the protocol for This, in turn, will lead to an incorrect estimate of the likeli-
data collection should be detailed in a study manual. Train- hood of a type I error.
ing sessions should be held to explain the study’s aim to staff, In developing the analytic plan, the investigator should
as well as how each one’s role fits into the big picture. Staff consider the assumptions of candidate tests in determining
should be given ample time to practice their data-collection which ones to use. A discussion of specific statistical tests is
skills. Once the fieldwork of the main study begins, staff beyond the scope of this chapter, but a framework for decid-
should be encouraged to bring problems to the attention of ing which tests to use can be given. In this framework, three
supervisory staff. Such problems should be resolved in a factors determine the type of test to use: the major analytic
timely fashion, and the resolution should be documented question to be answered, the levels of measurement used, and
and added to the study manual. In this way, research staff are the number and independence of comparison groups.
kept apprised of changes in the study protocol and are In preparing the analytic plan, the investigator needs to
impressed with the importance of adhering to it. translate the research question into analytic terms. Three
After the instrument pretesting and staff training, the major analytic approaches are to describe characteristics of
investigator should pilot test the data-collection activities. the sampled population, to compare groups of subjects,
 17. Clinical Investigation 357

TABLE 17.2. BIVARIATE STATISTICAL TESTS

Two groups Three or more groups

Independent Independent
Level of measurement (unpaired groups) Paired groups (unmatched groups) Matched groups

Nominal dichotomy Chi-square or Fisher’s McNemar’s test Chi-square Cochran’s test

exact test
More than two categories Chi-square McNemar’s test Chi-square Cochran’s test
Ordinal Mann-Whitney test Sign test Kruskal-Wallis one-way Friedman two-way
analysis of variance ANOVA
(ANOVA)
Wilcoxon matched-pairs
signed-ranks test
Interval t Test for groups t Test for pairs One-way ANOVA ANOVA for repeated
measures

and to measure associations among variables. In addition, of their association, the appropriate test of the statistical sig-
the appropriate analytic test is determined by the level of nificance of the association, and the certainty of estimates of
measurement of the variables of interest. Measurements can its strength. For dichotomous variables, the odds ratio and
be normal, ordinal, or interval. relative risk are measures of the degree of association. For
Where group comparisons are to be made, the appropri- continuous data, Pearson’s correlation coefficient is used to
ate statistical test is also determined by the number of groups measure association.
to be compared (two vs. three or more) and by whether com- Studies of the combined and relative impacts of multiple
parison groups are independent or matched. Thus, in a study independent variables, or the effect of an independent variable
of cases matched with sibling controls, it would be inappro- after controlling for other factors, will require multivariable
priate to use a statistical test for independent groups. analytic tests. The appropriateness of a multivariable technique
As decisions about level of measurement and the selection is determined by the levels of measurement of the independent
of study groups are part of study planning, it is easy to see and dependent variables. The references cited at the end of this
how these decisions are better informed if their ramifications chapter describe the applicability and interpretation of the
for data analysis are considered. Level of measurement, study most commonly used multivariable statistical tests.
group formation, and data analysis are all interrelated, and all
should be considered part of study planning.
Statistical Significance and Confidence Intervals
When the study’s purpose is to describe a population,
the investigator makes inferences from sample statistics to Most readers of the medical literature will be familiar with the
population parameters. Sample proportions and measures term statistical significance, which is most often referred to in
of central tendency (mean, median, and mode) and disper- clinical reports as a “p value <.05.” This highly sought after
sion (standard deviation, range) are used to estimate these result of a statistical test refers to the probability of α, or a
parameters in the population. Confidence intervals can be type I error. A p value of .05 in a study means that there is a
constructed around proportions and means to express the 5% chance that the results seen in the study could have
certainty of the sample-based population estimates. When occurred by chance. However, the authors have concluded
the study’s objective is to compare two or more groups, that this probability is low enough for them to accept the
sample group differences in proportions and means are alternative hypothesis (that there is a real difference between
used to estimate such differences in the population. Statisti- groups) and to reject the null hypothesis (there is no differ-
cal tests of significance can be used to assess the certainty of ence between groups). It is important to note that the p value
sample-based inferences about group differences in the in a study result depends on the size of the observed difference
population. The appropriate statistical test depends on the between the groups in question and the size of the sample of
number of groups compared, whether subjects in the groups patients studied. Standing alone, the p value does not convey
are matched, and the level of measurement of the variable any sense of the magnitude of the treatment effect seen in the
on which the groups are being compared. Table 17.2 dis- study or the precision of the estimate of the size of the treat-
plays bivariate statistical tests commonly used in assessing ment effect. Confidence intervals, in contrast to p values, can
the significance of group differences. convey this information in a more meaningful way.
When the research aim is to measure the association For any estimated value, it is useful to have an idea of the
between variables, sample statistics again are used to estimate uncertainty of the estimate in relation to the true value it is try-
population parameters. The variables’ levels of measurement ing to approximate. For example, if we designed a study to esti-
determine the appropriate statistical measure of the strength mate the beneficial effect of a new lipid-lowering medication in
358 III. Research Methods

chronic renal failure in adolescents, we would try to recruit a cal decision or a national organization is developing clinical
large representative sample of hyperlipidemic adolescents and practice guidelines, efforts should be made to systematically
randomize them to treatment with a new lipid-lowering medi- assess the strengths of scientific evidence related to a particular
cation. From our study, we might want to estimate the magni- clinical diagnostic or treatment plan. Guidelines first devel-
tude of lipid level reduction associated with the new medicine. oped more than 20 years ago at the Department of Clinical
We might also want to use this estimate as an approximation Epidemiology and Biostatistics at McMaster University first
for the “true” reduction in lipid levels that would be seen in the introduced tools to allow clinicians to critically review original
“universe” of pediatric patients with hyperlipidemia and articles on etiology, diagnosis, prognosis, and therapy (10). In
chronic renal failure. To estimate the “true” reduction in lipid the following decade, the series was widely read and cited, was
levels (which can never be directly measured), we can generate modified for use by the general public, and was published in
a confidence interval around our estimate. clinical epidemiology texts (11). At the same time, clinicians at
In any study, construction of a confidence interval around McMaster University and across North America continued to
the point estimate gives us a range of values in which we can expand and improve the guidelines. Their focus has expanded
be confident that the true value resides. A confidence interval to include clinicians’ ability to access, summarize, and apply
gives a sense of the estimate’s precision; it extends evenly on information from the literature to everyday clinical problems,
either side of the estimate by a multiple of the standard error transforming the Readers’ Guides to Users’ Guides (12–35).
(SE) of the estimate. In our example, our study might yield Such systematic approaches have also been adapted to
an estimate of the drop in serum low-density lipoprotein assess entire bodies of research on particular subjects. In
cholesterol levels of the treatment group of 30% with a stan- 1999, the U.S. Congress directed the Agency for Health
dard deviation of that estimate of 20%. One could then use Care Policy Research and Quality to identify methods to
this estimate to generate a confidence interval around this assess health care research results. The results of that effort
estimate. In the medical literature, one will most often see were published in a report entitled “Systems to rate the
references to 95% confidence intervals. The general equation strength of scientific evidence” (36). Key features of this
for a 95% confidence interval is equal to the estimate ±1.96 report and the users’ guides are summarized in Randomized
times the SE of the estimate. The factor 1.96 comes from the Controlled Trials.
standard normal distribution, in which 95% of estimates
would fall within ±1.96 SEs of the mean. If one wanted to
Randomized Controlled Trials
increase the probability of including the true estimate in the
confidence interval, one could generate a 99% confidence If a clinician or researcher is interested in evaluating the evi-
interval, which would equal the estimate ±2.56 times the SE dence that a cause-effect relationship exists between a treat-
of the estimate. Because the SE of an estimate is equal to the ment and a result, a randomized experiment is the design of
standard deviation of the population divided by the square choice. In evaluating the quality of the randomized clinical
root of the sample size, N, one can see that a larger sample trial, one must assess whether a number of important ele-
size is needed in a study to generate a precise estimate of a ments are addressed in the clinical trial. These are outlined
treatment’s effect. Given the same standard deviation, the SE in Table 17.3. The study population should be clearly
in our study would be smaller if we studied 100 children defined. Randomization should have resulted in study
compared to 20 children. The larger study would generate a groups that are balanced at the start of the study in terms of
narrower 95% confidence interval. The strict interpretation potential confounders. The reader should be wary of two
of a 95% confidence interval is that this is the range of values threats to this balance. The first is differential group attri-
for the true population estimate that is consistent with the tion during the course of the study, resulting in groups that
data observed in the study. In our hypothetical example, the are no longer balanced with respect to confounders by the
smaller study might give us the opportunity to conclude that study’s end. To guard against this, the investigator should
the new lipid lowering is associated with a 30% reduction in provide evidence of low attrition rates or should analyze
low-density lipoprotein cholesterol with a relatively broad treatment effectiveness on the basis of “intent to treat,”
95% confidence interval of 21 to 39%, whereas the larger including in the analysis of all study subjects both those
study yields a more precise estimate. The 95% confidence who completed the study and those dropping out. An
interval around the same point estimate of a 30% reduction intent-to-treat analysis maintains individuals and their out-
is 26 to 34% in the study with the larger sample size. comes in the treatment group to which they were assigned.
This maintains the balance achieved by randomization and
is the most conservative analysis plan. The second threat to
EVALUATING THE LITERATURE: RATING THE validity is biased outcome measurement. The reader should
STRENGTH OF SCIENTIFIC EVIDENCE look for evidence that outcome measurement was objective
and not biased by observer knowledge of subject group sta-
Health care decisions should be based on research-based evi- tus. Finally, in determining treatment impact, the investiga-
dence. Whether the individual nephrologist is making a clini- tor should provide evidence that both statistical and clinical
 17. Clinical Investigation 359

TABLE 17.3. IMPORTANT DOMAINS AND ELEMENTS TABLE 17.4. IMPORTANT DOMAINS AND ELEMENTS
FOR ASSESSING STRENGTH OF EVIDENCE IN FOR ASSESSING STRENGTH OF EVIDENCE IN
RANDOMIZED CONTROLLED TRIALS OBSERVATIONAL STUDIES
Domain Element Domain Element

Study question Clearly focused and appropriate question Study question Clearly focused and appropriate question
Study population Description of study population Study population Description of study populations
Specific inclusion and exclusion criteria Sample size justification
Sample size justification For all observational studies
Randomization Adequate approach to sequence genera- Specific inclusion/exclusion criteria for
tion all groups
Adequate concealment method used Criteria applied equally to all groups
Similarity of groups at baseline Comparability of groups at baseline
Blinding Double-blinding (e.g., of investigators, with regard to disease status and
caregivers, subjects, assessors, and prognostic factors
other key study personnel as appropri- Study groups comparable to nonpartic-
ate) to treatment allocation ipants with regard to confounding
Interventions Intervention(s) clearly detailed for all factors
study groups (e.g., dose, route, and Use of concurrent controls
timing for drugs and details sufficient Comparability of follow-up among
for assessment and reproducibility for groups at each assessment
other types of interventions) Additional criteria for case-control studies
Compliance with intervention Explicit case definition
Equal treatment of groups except for Case ascertainment not influenced by
intervention exposure status
Outcomes Primary and secondary outcome measures Controls similar to cases except without
specified condition of interest and with equal
Assessment method standard, valid, and opportunity for exposure
reliable Exposure or inter- Clear definition of exposure
Statistical analysis Appropriate analytic techniques that vention Measurement method standard valid and
address study withdrawals, loss to fol- reliable
low-up, missing data, and intention to Exposure measured equally in all study
treat groups
Power calculation Outcome measure- Primary/secondary outcomes clearly
Assessment of confounding ment defined
Assessment of heterogeneity, if applicable Outcomes assess blind exposure or inter-
Results Measure of effect for outcomes and vention status
appropriate measure of precision Method of outcome assessment standard
Proportion of eligible subjects recruited valid and reliable
into study and followed up at each Length of follow-up adequate for ques-
assessment tion
Discussion Conclusions supported by results with Statistical analysis Statistical tests appropriate
possible biases and limitations taken Multiple comparisons taken into consid-
into consideration eration
Funding or sponsor- Type and sources of support for study Modeling and multivariate techniques
ship appropriate
Power calculation provided
From Agency for Healthcare Research and Quality. Systems to rate Assessment of confounding
the strength of scientific evidence. AHRQ Publication No. 02-E016, Dose-response assessment, if appropriate
April 2002. Rockville, MD: Agency for Healthcare Research and Qual- Results Measure of effect for outcomes and
ity. Available at: http://www.ahrq.gov/clinic/strevinv.htm. Accessed appropriate measure of precision
May 2003, with permission.
Adequacy of follow-up for each study
group
Discussion Conclusions supported by results with
relevance were considered in establishing sample size and in biases and limitations taken into con-
drawing conclusions. sideration
Funding or sponsor- Type and sources of support for study
ship
Observational Studies
From Agency for Healthcare Research and Quality. Systems to rate
In a study of disease etiology, a randomized experiment is sel- the strength of scientific evidence. AHRQ Publication No. 02-E016,
dom possible. When a randomized trial is not feasible, the April 2002. Rockville, MD: Agency for Healthcare Research and Qual-
ity. Available at: http://www.ahrq.gov/clinic/strevinv.htm. Accessed
reader should determine whether the investigator has used the May 2003, with permission.
strongest alternative observational design possible. Important
domains and elements to assess for the quality of observational
360 III. Research Methods

studies are outlined in Table 17.4. The reader should look for randomly assign patients to groups with and without a prog-
additional evidence of a causal relationship as well, such as the nostic factor. Therefore, it is essential that potential confound-
consistency of findings across studies, confirmation that the ers be controlled for in the analysis.
cause preceded the effect temporally, and the presence of a
dose-response effect. As with an experimental study, measure-
Studies Evaluating Diagnostic Tests
ments of cause and outcome should be objective and unbiased,
and both clinical and statistical relevance should be considered In reviewing a study evaluating a diagnostic test, the
in the analysis. Finally, because available designs do not achieve reader’s chief concerns are subject selection and measure-
balance between study groups with respect to confounders, it ment (Table 17.5). The study sample should be representa-
is important that the investigators identify and control for tive of the population of interest in terms of spectrum of
potential confounders in the analysis. disease. Regarding measurement, it is essential that the can-
In evaluating an observational study of the prognosis of dis-
ease, the reader should focus on subject selection, measure-
ment, and analysis. The primary purpose of this type of study TABLE 17.6. IMPORTANT DOMAINS AND ELEMENTS
is to describe the clinical course of patients with a particular FOR ASSESSING STRENGTH OF EVIDENCE IN
disease. As in studies of etiology, the investigator might also SYSTEMATIC REVIEWS
seek to identify factors influencing outcome. It is essential that Domain Element
those studied be representative of such patients, including
Study questions Question clearly specified and appropri-
both those who will eventually recover and those who will die. ate
The reader’s first task, therefore, is to seek evidence that the Search strategy Sufficiently comprehensive and rigorous
study sample was selected early in their disease. The next task is with attention to possible publication
to determine the adequacy of subject follow-up. As the num- biases
Search restrictions justified (e.g., lan-
ber of subjects lost to follow-up increases, the representative-
guage or county of origin)
ness of the sample becomes suspect. Assuming a representative Documentation of search terms and data-
sample and adequate follow-up, the reader should then deter- bases used
mine whether patient outcomes were measured reliably in Sufficiently detailed to reproduce study
ways that can be related to the reader’s own practice. Finally, Inclusion and exclu- Selection methods specified and appro-
sion criteria priate, with a priori criteria specified if
the reader should determine whether the investigator con-
possible
trolled for potential confounders in assessing the role of prog- Interventions Intervention(s) clearly detailed for all
nostic factors. As in studies of etiology, it is impossible to study groups
Outcomes All potentially important harms and ben-
efits considered
Data extraction Rigor and consistency of process
TABLE 17.5. IMPORTANT DOMAINS AND ELEMENTS Number and types of reviewers
FOR ASSESSING STRENGTH OF EVIDENCE IN Blinding of reviewers
DIAGNOSTIC STUDIES Measure of agreement of reproducibility
Domain Element Extraction of clearly defined interven-
tions/exposures and outcomes for all
Study population Subjects similar to populations in relevant subjects and subgroups
which the test would be used and Study quality and Assessment method specified and appro-
with a similar spectrum of disease validity priate
Adequate description of Details of test and its administration Method of incorporation specified and
test sufficient to allow for replication appropriate
of study Data synthesis and Appropriate use of qualitative and/or
Appropriate reference Appropriate reference standard analysis quantitative synthesis, with consider-
standard (“gold standard”) used for com- ation of the robustness of results and
parison heterogeneity issues
Reference standard reproducible Results Narrative summary and/or quantitative
Blinded comparison of Evaluation of test without knowl- summary statistic and measure of preci-
test and reference edge of disease status, if possible sion, as appropriate
Independent, blind interpretation of Discussion Conclusion supported by results with pos-
test and reference sible biases and limitations taken into
Avoidance of verification Decision to perform reference stan- consideration
bias dard not dependent on results of Funding or sponsor- Types and sources of support for study
test under study ship

From Agency for Healthcare Research and Quality. Systems to rate From Agency for Healthcare Research and Quality. Systems to rate
the strength of scientific evidence. AHRQ Publication No. 02-E016, the strength of scientific evidence. AHRQ Publication No. 02-E016,
April 2002. Rockville, MD: Agency for Healthcare Research and Qual- April 2002. Rockville, MD: Agency for Healthcare Research and Qual-
ity. Available at: http://www.ahrq.gov/clinic/strevinv.htm. Accessed ity. Available at: http://www.ahrq.gov/clinic/strevinv.htm. Accessed
May 2003, with permission. May 2003, with permission.
 17. Clinical Investigation 361

didate test be compared with a “gold standard,” that the Meinert CL. Clinical trials: design, conduct and analysis. New
comparison is unbiased, and that the reliability of the test is York: Oxford University Press, 1986.
established. From a practical standpoint, it is important Schiesselman JJ. Case-control studies: design, conduct, analysis. New
that the diagnostic test procedures, as evaluated in the York: Oxford University Press, 1982.
research, can be used in clinical practice.
General Statistic References
Systematic Reviews Dawson-Saunders B, Trapp RG. Basic and clinical biostatistics, 3rd
As opposed to a traditional invited review, a systematic review ed. New York: Lange Medical Books–McGraw-Hill, 2001.
is an organized method of locating, assembling, and evaluating Fleiss JL. Statistical methods for rates and proportions. New York:
a body of literature on a particular topic using a set of specific John Wiley & Sons, 1981.
Glaser AN. High yield biostatistics. Pennsylvania: Williams &
predefined criteria. If the systematic review includes a quanti-
Wilkins, 2001.
tative pooling of data, then it is referred to as a metaanalysis. Hollander M, Wolfe DA. Nonparametric statistical methods, 2nd
As systematic reviews have been more frequently used in recent ed. New York: John Wiley & Sons, 1999.
years as part of the development of practice guidelines, criteria Kleinbaum DG, Kupper LL. Applied regression analysis and other mul-
for evaluating the quality of systematic reviews have also been tivariate methods, 3rd ed. Pacific Grove: Duxbury Press, 1998.
developed (Table 17.6). An example of a systematic review in Tabachnick BG, Fidell LS. Using multivariate statistics. 4th ed.
pediatric nephrology is the evidence-based assessment of treat- Boston: Allyn and Bacon, 2001.
ment options for children with IgA nephropathy by Hogg and Zar JH. Biostatistical analysis, 4th ed. Upper Saddle River, NJ:
Wyatt (37). Prentice-Hall, 1999.
In summary, the busy clinician can afford to be selective in
reviewing the literature. In rating the strength of scientific evi-
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 18

GENETICS
LISA M. GUAY-WOODFORD

The principles of classic genetics were established by Gre- PATTERNS OF INHERITANCE

gor Mendel in 1865. Based on controlled breeding experi-
ments with the garden pea, Mendel determined that Pedigree Analysis
physical characteristics (phenotypes) were transmitted by
The family history is a critical tool for assessing inherited
“hereditary units” from one generation to the next in a pre-
disorders and determining inheritance patterns. The key
dictable fashion. These hereditary units (genes) exist in
issues to investigate are (a) whether anyone in the family has
alternate forms (alleles) that determine the expression of a
had a condition similar to the patient and, if so, whether
particular phenotype. Mendel’s first law of heredity
this condition “runs in the family”; and (b) whether the
describes the segregation of alleles: Alleles have no perma-
family has suffered fetal losses late in gestation or whether
nent effect on one another when present in the same organism,
there have been unexplained infant deaths. In the case of
but they segregate unchanged by passing into different gametes.
rare disorders, the possibility of consanguinity should be
An organism is homozygous if both alleles of a given gene
explored by inquiring whether the parents are related,
are identical, and it is heterozygous if the alleles are differ-
whether their grandparents or great-grandparents may have
ent. Alleles typically have dominance properties. For exam-
intermarried, or whether the parental families originate
ple, one dominant allele determines the phenotype
from the same village or small community.
regardless of the dominance characteristics of the other
With the details obtained from a careful family history,
allele. The recessive phenotype only appears when the
the disease distribution within the family or kindred can be
organism is homozygous for the recessive allele. Mendel
evaluated. The pattern of inheritance is documented by
also determined that alleles of one gene and alleles of
constructing a pedigree (Fig. 18.1) in which the first identi-
another gene are transmitted independently to gametes;
fied patient is designated as the proband or index case. Ped-
thus he formulated his second law of heredity: The assort-
igree analysis indicates whether the disease behaves as a
ment (transmission) of one gene does not influence the assort-
single mendelian trait (i.e., whether it is transmitted
ment of another (1).
according to Mendel’s principles of segregation and inde-
Mendel’s laws were first applied to human disease by
pendent assortment). Alternatively, if the inheritance pat-
Sir Archibald Garrod in 1909. Based on his clinical obser-
tern does not conform to mendelian principles, the genetic
vations, Garrod reasoned that alkaptonuria, albinism,
trait may arise from (a) polygenic inheritance, (b) genomic
cystinuria, and pentosuria were transmitted as mendelian
imprinting, (c) uniparental disomy, or (d) mitochondrial
recessive traits. With alkaptonuria, he postulated that an
inheritance.
essential enzyme was defective, a so-called inborn error of
metabolism (2). The one gene–one enzyme concept later
postulated by George Beadle and Edward Tatum had
Mendelian Inheritance
immediate implications for the inborn errors of metabo-
lism described by Garrod. Indeed, shortly thereafter, the Single-gene disorders that follow a mendelian pattern of
era of biochemical genetics began with the identification inheritance are manifested as (a) autosomal-dominant traits,
of specific enzyme defects as the genetic basis of recessive (b) autosomal-recessive traits, (c) X-linked dominant traits,
methemoglobinemia, von Gierke disease, and phenylketo- or (d) X-linked recessive traits. Genes involved in human
nuria (2). Subsequent studies in the 1940s and early autosomal disorders are carried on any one of the 22 pairs of
1950s determined that the genetic information in each nonsex chromosomes (autosomes). The terms X-linked domi-
cell is encoded in double-stranded DNA molecules. These nant and X-linked recessive refer to trait expression in females.
findings set the stage for the modern era of molecular In comparison, males carry only one X chromosome and,
genetics. therefore, are hemizygous for X-linked traits.
364 III. Research Methods

through successive generations. Each child of an affected

individual has a 50% risk of inheriting the mutant allele.
The sine qua non of autosomal-dominant inheritance is
father-to-son transmission.
Many autosomal-dominant disorders are characterized by
delayed clinical onset as well as variability in the disease phe-
notype between families and even within families. These
issues are important (e.g., when evaluating fetuses and neo-
nates with presumed polycystic kidney disease). In ADPKD,
clinical disease is typically manifested after the third decade
of life. Given this delayed clinical presentation, individuals
who carry a mutant ADPKD allele may be clinically asymp-
FIGURE 18.1. Standard pedigree symbols. tomatic throughout their child-bearing years. However,
ADPKD can be diagnosed in utero; for approximately 30%
of these fetuses, parental sonographs can identify the previ-
Dominant disorders are expressed in heterozygotes, hem-
ously unaware, transmitting parent (4).
izygotes, as well as homozygotes, whereas recessive disorders
Occasionally, the expression of the dominant trait is so
are only expressed in homozygotes and hemizygotes. It is
minimal that clinical disease appears to have “skipped” a
important to note that the terms dominant and recessive refer
generation. Individuals who carry a mutant allele for an
to the inheritance of the disease phenotype rather than to the
autosomal-dominant trait but who have little evidence of
expression of the mutant gene. For example, in the case of
clinical disease are said to demonstrate decreased or incom-
autosomal-dominant polycystic kidney disease (ADPKD),
plete penetrance (Fig. 18.2). In contrast, when an individ-
the disease phenotype is inherited as an autosomal-dominant
ual inherits a mutant allele for an autosomal-dominant trait
trait. However, in cystic epithelia of PKD1 and PKD2
from each parent, the clinical phenotype can be very severe.
patients, there appears to be a “second hit,” which results in
For example, clinical disease in individuals with the domi-
the somatic cell loss of the normal allele. These data therefore
nant disorder familial hypocalciuric hypercalcemia may
suggest that PKD1- and PKD2-associated diseases behave in
proceed undetected. However, the union of two such het-
a recessive fashion at the molecular level (3).
erozygotes can produce a child with two mutant alleles who
then manifests the often fatal disorder neonatal severe
Autosomal-Dominant Traits hyperparathyroidism (5).
Although each child of an individual with a specific
Dominant traits are expressed when only one mutant allele
autosomal-dominant disorder has a 50% risk of inheriting
is inherited. The characteristic pattern of autosomal-domi-
the disease trait, not every affected child necessarily has an
nant inheritance is shown in Figure 18.2. When large num-
affected parent. This is because in every autosomal-domi-
bers of patients are evaluated, an equal number of males
nant disorder, affected individuals can develop the disease
and females are affected. An affected child typically has an
phenotype as the result of a new (spontaneous) mutation
affected parent, and vertical transmission of the trait occurs
rather than through inheritance of a mutant allele. A rea-
sonable estimate of the frequency of mutation is on the
order of 10–6 mutations per allele per generation (1). Many
new mendelian mutations occur in the gametes of fathers
who are of relatively advanced age, the so-called paternal
age effect (2). Most of these mutations either do not impair
the function of the gene product or behave as recessive
alleles. However, others cause a defective gene product that
gives rise to a dominant trait. In these situations, the parent
in whose gametes the mutation arose remains clinically
unaffected. Similarly, the siblings of the affected individual
are also clinically unaffected because spontaneous muta-
tions typically affect only one or a few germ cells.
Before concluding that a dominant trait in a given indi-
vidual is the result of a spontaneous mutation, two other
possibilities must be considered. First, the disease gene may
be carried by one parent in whom the disease phenotype is
FIGURE 18.2. Pedigree illustrating an autosomal-dominant pat-
tern of inheritance. Note the male-to-male transmission (individ- either nonpenetrant or incompletely penetrant. Second, the
ual II:4 to individual III:6) and nonpenetrance (individual IV:8). father may be someone other than the designated man.
 18. Genetics 365

Studies in various cultures around the world have detected

nonpaternity in approximately 3 to 5% of children (2).
A number of different molecular mechanisms may cause
the disease phenotype associated with dominant traits. One
mechanism involves haploinsufficiency, in which 50% of
the normal gene product is insufficient to maintain a
normal phenotype. This mechanism may be operative in
disorders involving cellular receptors (e.g., familial hyper-
cholesterolemia) and key regulatory enzymes (e.g., porphy-
rias). A second mechanism involves a dominant-negative
effect in which the incorporation of an abnormal subunit
disrupts the function of a multimeric protein (e.g., enzyme FIGURE 18.3. Pedigrees illustrating autosomal-recessive patterns
pumps, transport proteins, and complex ion channels). of inheritance. A: A simplex family. B: A consanguineous family.
This mechanism may be operative in autosomal-dominant
forms of nephrogenic diabetes insipidus (6). A third muta-
tional mechanism involves a gain of function in which the recessive alleles generally cause a complete or nearly complete
mutation causes overexpression of the protein product or loss of protein function. For example, a specific monomeric
results in a new, aberrant function of the protein. This enzyme is rendered nonfunctional in the majority of inborn
mechanism may be responsible for one form of autosomal- errors of metabolism. Affected individuals may be homozy-
dominant hypocalcemia, in which a mutation in the gene gous for the same mutant allele, as in the case of parental
encoding the calcium-sensing receptor causes expression of consanguinity, or they may have inherited a different mutant
an overactivated receptor (7). A fourth mechanism for a allele from each parent. In the latter case, the affected indi-
dominant phenotypic effect results when a heterozygous vidual is referred to as a compound heterozygote.
mutation becomes homozygous at the cellular level due to a
second somatic mutation or a “second hit.” This mecha-
X-Linked Inheritance
nism appears to underlie the molecular pathogenesis of
ADPKD (3) and tuberous sclerosis (8). Diseases or traits that result from genes located on the X
chromosome are termed X-linked. Because the female has
two X chromosomes, the terms X-linked dominant and X-
Autosomal-Recessive Disorders
linked recessive refer only to expression of the trait in females.
Autosomal-recessive conditions are clinically apparent only Affected males transmit their X chromosome to all of their
in the homozygous state (i.e., when both alleles of a partic- daughters, rendering them all obligate carriers of an X-linked
ular gene carry mutations). In most autosomal-recessive disease trait. Affected males do not transmit an X chromo-
disorders, the clinical presentation tends to be more uni- some to their sons. Thus, an important feature of X-linked
form than in dominant diseases, and the onset is often early inheritance is the absence of father-to-son transmission.
in life. In recessive pedigrees, the parents are clinically unaf- The female carries two X chromosomes in each cell, but
fected; on average, one-fourth (25%) of the children are only one is expressed per cell. Both X chromosomes are
affected, with an equal gender distribution. Given the small apparently active early in development. However, through
family sizes typical in many societies, pediatricians usually the process of lyonization, one of the X chromosomes is
see isolated or sporadic cases of a recessive disorder. A typi- randomly inactivated in each cell during differentiation (9).
cal simplex pedigree is shown in Figure 18.3A. In these sit- Once inactivated, the cell’s X chromosome is permanently
uations, a high index of suspicion is required to make the nonfunctional such that all of its daughter cells inherit the
correct genetic diagnosis. same inactivated X chromosome. Therefore, depending on
Multiply affected families do occur, particularly in the the proportion of mutant and normal X chromosomes in
context of parental consanguinity (Fig. 18.3B). In general, key tissues, a genetically heterozygous female may be clini-
the less frequent the disease phenotype, the more likely it is cally normal, may have mild disease, or, in rare cases, may
that an affected child resulted from a consanguineous have even severe disease manifestations.
union. On the other hand, certain recessive disorders have a Recognizing new mutations can be more complicated in
relatively high carrier frequency in specific populations X-linked disorders than in autosomal disorders. A new
(e.g., cystic fibrosis in whites and hemoglobin S in blacks). mutation may arise in one of the mother’s two X chromo-
In these populations, recessive diseases usually do not somes, and a single affected male may be born to a woman
involve consanguinity in the parents. who is not a known carrier. Alternatively, the new mutation
New mutations for recessive diseases occur, but because may arise in the maternal grandfather’s X chromosome. In
they generate an asymptomatic heterozygote, these muta- this scenario, a woman without a known family history of
tions are not clinically detectable. In the homozygous state, an X-linked disease may have one or more affected sons.
366 III. Research Methods

sion in autosomal-dominant inheritance and the horizontal

pattern of autosomal-recessive inheritance. Heterozygous
females are usually phenotypically normal. Depending on
the pattern of lyonization in key tissues, these females may
occasionally exhibit mild features of the disease and may
rarely be severely affected.

Nonmendelian Inheritance
Polygenic Inheritance
FIGURE 18.4. Pedigree illustrating an X-linked dominant pat-
tern of inheritance. Note lethality in hemizygous males. In general, most phenotypic traits are determined by the
interaction of several genes rather than by single gene
effects. In terms of genetic disease, polygenic inheritance
X-Linked Dominant Traits provides the basis for understanding many diseases that
have a genetic component but lack a clear-cut pattern of
The X-linked dominant pattern is a rare mode of inheri- mendelian inheritance. These disorders include congenital
tance characterized by a disease frequency in females that is malformations (e.g., most forms of congenital heart dis-
often twice that evident in males. Vitamin D–resistant rick- ease), meningomyelocele and other neural tube defects, and
ets is an example of an X-linked dominant trait (10). Het- vesicoureteral reflux, as well as many familial adult-onset
erozygous females transmit the trait to offspring of both diseases (e.g., essential hypertension, type II diabetes, and
genders, whereas hemizygous males transmit the trait to all coronary artery disease) (2).
of their daughters and none of their sons. The disease In multifactorial genetic diseases, multiple different
expression is more variable and generally less severe in het- genes interact with one another and with certain environ-
erozygous females than in hemizygous males. In other rarer mental factors to cause disease. An individual who inherits
X-linked dominant disorders (e.g., oral-facial-digital syn- a particular combination of interacting genes has a relative
drome type I) (11), the phenotype may be expressed only risk of developing disease. When this genetic component
in the heterozygous female because the condition is lethal interacts with a set of environmental factors, a biologic
in the hemizygous male (Fig. 18.4). threshold is crossed and disease expression occurs (2). For
example, maternal folate deficiency is an important envi-
X-Linked Recessive Traits ronmental contributor to defective neural tube develop-
ment in the fetus. Population studies have demonstrated
In comparison to X-linked dominant inheritance, X-linked that folic acid treatment in women before and during preg-
recessive inheritance is relatively common. These disorders nancy reduces the incidence of neural tube defects by
are fully expressed in the hemizygous affected male. The nearly half. These observations prompted efforts to identify
pedigree pattern tends to be oblique in that affected males variations in genes encoding enzymes of the folic acid met-
have affected uncles and nephews (Fig. 18.5). This oblique abolic pathway. Recent studies have determined that
pattern is readily distinguished from the vertical transmis- women carrying specific variants in several of these genes
have an increased risk of bearing a child with neural tube
defects (12).

Genomic Imprinting
Genomic imprinting refers to the phenomenon in which the
phenotypic abnormalities of a given disorder differ depend-
ing on the sex of the transmitting parent. Genomic
imprinting reflects a functional modification in which there
is a temporary change in the function or expression of a
gene depending on whether it is maternally or paternally
derived (9).
Strong evidence for genomic imprinting in humans has
resulted from molecular studies of two chromosomal dele-
tion syndromes, Prader-Willi syndrome and Angelman syn-
FIGURE 18.5. Pedigree illustrating an X-linked recessive pat- drome. Although clinically quite distinct, both syndromes
tern of inheritance. share similar cytogenetic deletions involving chromosome
 18. Genetics 367

15q11-13. The key factor that determines the specific phe- and wild-type mtDNAs within a given cell and the degree to
notype is not the size of the deletion but the parental origin which that cell depends on oxidative phosphorylation. The
of the deleted chromosome. Prader-Willi syndrome occurs central nervous system is most dependent on mitochondrial
when the deletion is on the paternally derived chromosome energy, followed by the heart, skeletal muscle, liver, and kid-
15, and Angelman syndrome occurs when the deletion is ney. Therefore, defects in the mitochondrial genome and the
on the maternally derived chromosome 15. These parent- associated impairment of oxidative phosphorylation typically
of-origin differences are believed to occur through methyla- manifest as neuromuscular disease but may also cause renal
tion or other modifications of DNA, which differentially tubular dysfunction or glomerular disease (16).
modify the expression of a small number of genes. There-
fore, instead of the typical situation in which both maternal
and paternal alleles of a gene are expressed, genomic
MEDICAL GENETICS
imprinting acts by silencing the expression of one of the
parental alleles. This differential expression of certain genes
Genomic Organization and the
depending on the parent of origin appears to be effaced and
Transmission of Genetic Information
reestablished during gametogenesis at each generation (13).
The genetic complement of each individual is arrayed in 23
pairs of chromosomes (the diploid genome). One-half of
Uniparental Disomy
each individual’s chromosomes, or one haploid genome, is
Uniparental disomy refers to the phenomenon in which maternally derived, and the other half is of paternal origin.
both chromosomes of a given pair are inherited from only Each diploid genome is composed of 22 pairs of auto-
one parent. This phenomenon appears to result from errors somes, numbered in order of descending size, and one pair
in chromosome segregation during gamete formation. Uni- of sex chromosomes (females, XX; males, XY). Chromo-
parental isodisomy occurs when both chromosomes in the somes are stored in the cell nucleus and distributed to
pair are completely identical, and uniparental heterodisomy daughter cells by either mitotic or meiotic cell division. In
results when the two chromosomes come from one parent both processes, the chromosomes are unwound and the two
but are different (14). Uniparental disomy can cause DNA strands are copied. The replicated strands, called
genetic disease either by rendering a mutant allele from the chromatids, remain joined at the centromere. The chroma-
transmitting parent homozygous in the child or by tids, which can be visualized at this stage by light micros-
unmasking an imprinted gene. copy, are composed of two asymmetric arms. The shorter of
the two is termed the p arm, whereas the longer of the two
is called the q arm. When chromosomes are arranged sys-
Mitochondrial Inheritance
tematically in pairs 1 to 23, they comprise the karyotype.
The inheritance of nuclear chromosomes forms the basis During somatic cell division, the process of mitosis dis-
for mendelian inheritance. In contrast, mitochondrial tributes one chromatid of each chromosome pair to the
genes are inherited only from the mother. Mitochondria are daughter cells; thus, the full diploid complement of 23
passed from generation to generation via the cytoplasm of chromosome pairs is transmitted. In contrast, germ cells
the oocyte. Because sperm do not contribute mitochondria undergo a reduction division called meiosis, in which only
to the zygote, mitochondrial genes have a strictly maternal one chromatid is transmitted to each gamete. The diploid
pattern of inheritance (15). In theory, if penetrance were germ cell therefore generates haploid oocytes or sperm that
complete, then all of the sons and daughters of an affected contain 23 individual chromosomes. During fertilization,
female would be affected. In reality, penetrance is rarely the union of the oocyte and sperm constitutes a diploid
complete; thus, confusing inheritance patterns can result. zygote with 23 pairs of chromosomes.
Most cells contain hundreds or thousands of mitochon-
dria, and each mitochondrion contains two to ten mitochon-
Recombination and Genetic Mapping
drial DNA (mtDNA) molecules (15). The mitochondrial
genome is a small, circular, 16.6-kilobase, double-stranded Meiosis is the process by which the chromosome number of
DNA molecule that contains no introns. In the normal situ- diploid germ cells (N = 46) is reduced to the haploid num-
ation, each mtDNA molecule in a given individual is identi- ber (N = 23) in the gametes. The independent assortment of
cal. However, the mutation rate in mtDNA is five to ten chromosomes into gametes during meiosis produces a
times greater than in nuclear DNA (16). Cells that contain a remarkable variety of possible genotypes in the progeny. For
mixture of wild-type and mutant mtDNA are termed hetero- a given set of parents, there are 223 different chromosome
plasmic, whereas cells with a single identical mtDNA are combinations possible in their offspring; thus, the likeli-
termed homoplasmic. Because mitochondria partition to hood that this set of parents will produce two independent
daughter cells in an entirely random fashion, the cellular offspring with an identical complement of chromosomes is
phenotype depends on the relative proportions of mutant 1 in 84 million (1 in 223) (2). The phenomenon of genetic
368 III. Research Methods

recombination during meiosis adds further to the enormous (1). Most of these mutations occur in noncoding sequences
genetic diversity in humans and provides the basis for and typically are detected as variations or polymorphisms
genetic mapping. in the population. In comparison, mutations in coding
During the first meiotic division, chromatids pair with sequences often cause disease. From an evolutionary per-
their homologues (e.g., the maternally derived chromosome spective, these coding sequence mutations appear to play an
1 chromatids pair with the paternally derived chromosome important role in generating genetic diversity and thereby
1 chromatids). This close proximity allows the exchange of permit environmental adaptation of the species through the
segments between maternal and paternal homologues, a process of natural selection.
process termed crossing over. These exchanges, which result Single base changes that occur in coding regions cause
in genetic recombination, occur on average approximately three types of mutations: (a) a silent mutation, in which the
50 times during meiosis, with a slightly higher frequency in base change results in a different three-base code (codon)
female germ cells than male germ cells. With the comple- for the same amino acid; (b) a missense mutation, in which
tion of this first meiotic division, each daughter cell has 46 the base change results in a new codon (i.e., the wild-type
chromosomes but only one copy of each chromatid pair. amino acid is replaced by a new amino acid); and (c) a non-
This separation of maternal and paternal chromosome sense mutation, in which the base substitution changes the
homologues is referred to as a reduction division. The second amino acid codon to a termination (stop) codon. The dele-
meiotic division proceeds without further chromosome rep- tion or insertion of a single base or a few bases within the
lication. The resulting gametes have a haploid chromosome coding region can cause a frame shift mutation in which
complement. the reading frame of the genetic code is altered. Frame shift
Due to genetic recombination, two genes that are mutations alter the amino acid sequence and often lead to
located on opposite ends of a given chromosome typically premature truncation of the peptide by generating a new
behave as if they are on different chromosomes (i.e., they stop codon.
segregate in an entirely random fashion). In comparison, Germ-line mutations can also involve millions of base
recombination events are less likely between genes that are pairs and cause gross chromosomal alterations, including
located close to one another. The distance between two duplications, insertions, deletions, or translocations. Such
such genes can be quantitated either in terms of genetic dis- alterations may disrupt the coding sequence and cause
tance (i.e., the probability of recombination events per gen- either an absence of the protein product, as in juvenile
eration) or physical distance [i.e., millions of base pairs nephronophthisis (NPH1) (17), or the fusion of two
(megabases)]. Genetic distance is measured in centimor- closely related protein products, as in glucocorticoid-reme-
gans (cM). For two genes that are 1 cM apart, there is a 1% diable aldosteronism (18). Finally, sequence alterations in
chance of a recombination event occurring between them the DNA flanking the coding regions may lead to changes
in a given meiotic division. Although on average in the in RNA splicing, transcriptional efficiency, or regulated tis-
human genome, 1 cM corresponds to approximately 1 sue expression.
megabase of DNA, the frequency of recombination may
vary in specific chromosomal regions and between female
and male germ cells (2). STRATEGIES FOR GENE IDENTIFICATION

After the seminal report by Watson and Crick in 1953

Mutations
describing the structure of DNA, identification of disease-
A mutation is a stable alteration of the DNA sequence that causing genes was predicated on first identifying the defec-
can be passed from a parent cell to daughter cells. Alter- tive protein (functional cloning). Perhaps the best known
ations of DNA sequences in cells other than germ cells are successes of the functional cloning approach involve disease
termed somatic mutations. Although these alterations are gene identification in phenylketonuria and sickle cell dis-
not transmitted to the next generation, the mutations are ease. However, for most human diseases, our level of insight
passed on to the progeny of those mutated cells. When a into the fundamental pathogenic mechanisms remains
somatic mutation occurs early in development, the result- rather limited; thus, functional cloning has not been a
ing individual may be a mosaic or a combination of normal broadly applicable approach.
cells and mutant cells. Once development is complete, To a certain extent, the candidate gene approach also
somatic mutations can be silent (i.e., they may not be asso- depends on functional insight into disease pathogenesis. By
ciated with a specific phenotype). Alternatively, somatic itself, this knowledge is not sufficient to precisely pinpoint
mutations (e.g., those involved in ADPKD) play a central the gene; rather, it provides a framework to form a series of
role in disease pathogenesis. educated guesses. For example, clinical studies of Bartter
In comparison, germ-line mutations are transmitted syndrome patients indicated a primary defect in transepi-
from one generation to the next. On average, the rate of thelial sodium chloride transport in the thick ascending
mutation is approximately 10–6 per locus per generation limb of the loop of Henle. With the cloning of genes
 18. Genetics 369

encoding the key transport proteins in the thick ascending for those disease genes that have yet to be cloned; (c) the size
limb [e.g., the bumetanide-sensitive sodium-potassium- and the complexity of the disease genes; and (d) the number
chloride cotransporter (NKCC2), the apical adenosine tri- and distribution of different mutations within the disease
phosphate–regulated potassium channel (ROMK ), and the genes. In addition to the technical challenges of genetic test-
kidney-specific basolateral chloride channel (ClC-Kb)], ing, issues such as phenotype-genotype correlation, sensitiv-
direct molecular studies became possible. Loss-of-function ity and specificity, proper informed consent, and access to
mutations in the genes for each of these transport proteins genetic counseling must be addressed before a test is ready
have been demonstrated in patients with either antenatal for clinical use (21).
Bartter syndrome or the more classic phenotype (19). It is important to stress that genetic tests differ from
Beginning in 1986, positional cloning or map-based other medical tests. They have direct implications for fam-
gene discovery became the leading method for identifying ily members and for reproductive decision making. They
disease susceptibility genes in inherited disorders. With the often raise issues of privacy, independence, insurability, and
availability of the human genome sequence, the positional discrimination, and the test results may have dire emotional
candidate approach has rapidly emerged as the preferred consequences (21). Therefore, genetic counseling should
method for elucidating the molecular basis of genetic dis- precede genetic testing to allow informed decision making.
ease. This strategy combines genetic mapping, usually by
linkage analysis, with information from databases that cata-
Linkage Analysis
logue the human genomic sequence and its transcripts [e.g.,
those maintained by the National Center for Biotechnol- Linkage analysis is an indirect method for diagnostic
ogy Information (http://www.ncbi.nlm.nih.gov)]. Once a genetic testing. It is useful in mendelian disorders in several
candidate disease-susceptibility gene is mapped to a chro- contexts: (a) when the disease gene has not yet been identi-
mosomal subregion, the databases can then be surveyed to fied; (b) when the disease gene is very large or complex; and
identify attractive candidate genes within the interval. (c) or when the mutations are too heterogeneous to be
To date, the successes in gene discovery have primarily readily identified. In linkage-based testing, the disease gene
involved single-gene disorders. Investigators have deter- is not directly analyzed. Rather, its presence is inferred by
mined that the phenotypic expression of these disorders is family studies that track the cosegregation of flanking poly-
due to different mutations within the disease-susceptibility morphic markers and a specific disease phenotype.
gene. In addition, disease expression appears to be influ- The principal advantage of linkage-based testing is that it
enced by the modifying effects of other genes (modifying can be applied to any mapped mendelian trait. The disad-
genes) that are involved in the same molecular pathway(s) vantages of linkage analysis include (a) the absolute depen-
(20). In comparison to single-gene disorders, multifactorial dence of the test on correct phenotypic diagnosis; (b) the
genetic diseases involve genetic susceptibility and environ- requirement for DNA typing of an affected proband as well
mental factors. In these disorders, the expression of the dis- as other affected and unaffected family members; and (c) the
ease phenotype depends on the complement of risk genes limitation of the maximum confidence level to 95 to 99%.
that is inherited and the interaction of these risk genes with Such confidence limits are inherent to linkage analyses
certain environmental factors. The major challenge facing because of the possibility that recombination events could
molecular geneticists is to elucidate and characterize the occur between the marker and the mutant gene, with the
molecular components involved in both single-gene and probability directly related to the genetic distance between
multifactorial disorders and to determine how these molec- them. The risk of possible error can be minimized when
ular factors influence the disease phenotype. intragenic and multiple flanking markers are used for testing.
Although linkage-based testing is a powerful diagnostic tool
in many disorders, diagnoses based on such an indirect
GENETIC TESTING methodology are estimates and should be treated as such.

The immediate clinical impact of gene mapping and identifi-

Direct Mutation Analysis
cation is the potential for gene-based diagnostic testing. DNA-
based diagnosis of single-gene disorders is well established in Once a disease gene is cloned, direct mutation detection
prenatal, pretransplant, and presymptomatic contexts. For strategies can be designed to identify the genetic defect
mendelian traits, such genetic testing can be performed in one that is causally responsible for the disease phenotype.
of two ways: (a) indirect testing, which usually involves linkage Direct mutation analysis can be performed by several
analysis, or (b) direct gene-based testing. methods. The details of these testing modalities are
Before genetic testing can be offered, the molecular “spe- beyond the scope of this chapter and the reader is referred
cifics” for a given disorder must be evaluated. The practicality to several excellent reviews (22–25). In general, the choice
of genetic testing depends on a number of factors, including of method depends on the types and numbers of muta-
(a) the number of genes/loci involved; (b) the genetic map tions that cause the disorder.
370 III. Research Methods

TABLE 18.1. GLOSSARY OF TERMS

Term Definition

Allele An alternative form of a gene. Humans carry two sets of alleles for every gene or genetic marker, one
from each parent.
Alternative splicing A regulatory mechanism by which exons are variably incorporated into the mRNA, leading to the produc-
tion of more than one related protein or isoform.
Autosomes All of the chromosomes except for the sex chromosomes and the mitochondrial chromosome.
cDNA A DNA sequence made from an mRNA molecule. cDNAs contain only exonic sequences.
Cloning The process of generating multiple copies of a particular piece of DNA.
Codon A three-base sequence of DNA or RNA that specifies a single amino acid.
Conservation Sequence similarity for genes that are present in two distinct organisms. Conservation can be detected by
measuring the sequence similarity at the nucleotide (DNA or RNA) or the amino acid level.
Epigenetic A term describing nonmutational phenomena (e.g., methylation and histone modification) that modify
the expression of a gene.
Euchromatin The gene-rich regions of the genome.
Exon A region of a gene that encodes a portion of its protein product.
Genomics The study of the functions and interactions of all the genes in the genome, including their interactions
with environmental factors.
Genotype The set of genes that an individual carries; usually refers to the particular pairs of alleles that the individ-
ual has at a given region of the genome.
Haplotype A group of nearby alleles that are closely linked (i.e., likely to be inherited together).
Heterochromatin Compact gene-poor regions of the genome that are difficult to clone and thus are usually ignored in
genome sequencing.
Intron A region of a gene that does not code for a protein.
mRNA The template for protein synthesis. A precursor RNA is constructed from the gene sequence and then pro-
cessed with removal of the introns by splicing. Spliced RNAs contain only exonic sequences from which
proteins are synthesized.
Microarray High-dimensional tool that allows expression analysis of thousands of genes (or proteins) in a single
hybridization experiment.
Monogenic disease Caused by mutations in a single gene.
Multifactorial disease Caused by the interaction of multiple genetic and environmental factors.
Mutation An alteration in an individual’s genomic sequence compared to a reference sequence. These sequence vari-
ants can have harmful effects (pathogenic mutations) or no harmful effects (silent mutations or poly-
morphisms).
Penetrance The likelihood that a person carrying a particular mutant gene will have an altered phenotype.
Phenotype The observable properties and physical characteristics of an individual.
Polymorphism A region of the genomic sequence that varies between individual members of a population.
Proteomics The study of the functions and interactions of all the proteins encoded in the genome.
SNP A polymorphism caused by the change of a single nucleotide. The human genome is estimated to contain
approximately 10 million SNPs.
Transcription The process of copying a gene into RNA.
Transcriptosome The complete set of RNAs transcribed from the genome.
Translation The process of using an mRNA sequence to build a protein.

cDNA, complimentary DNA; mRNA, messenger DNA; SNP, single-nucleotide polymorphism.

Modified from Venter J, Adams M, Myers E, et al. The sequence of the human genome. Science 2001;291:1304–1351; and Guttmacher A, Collins F.
Genomic medicine—a primer. N Engl J Med 2002;347:1512–1520.

When compared with linkage-based testing, direct newer DNA sequencing technologies as well as the develop-
mutation analysis has several advantages. Most notable ment of microarray-based genomic and proteomic screening.
among these are (a) a previously affected sibling is not
essential, and (b) studies on other family members are not
typically required. The pitfalls can include (a) locus hetero- INSIGHTS FROM THE HUMAN
geneity, in which the same phenotype can be caused by GENOME PROJECT
mutations in more than one gene; (b) no a priori knowl-
edge of the specific mutation segregating in a family or In February 2001, the draft sequence of the human
present in a given individual; and (c) the inability of a given genome was published by two groups, the International
technique to detect all mutations. Human Genome Sequencing Consortium (26) and Celera
With current technologies, detection of mutations can be Genomics (27). The estimated size of the human genome is
time-consuming and expensive. In the postgenomic era, the 3.2 × 109 base pairs or gigabases. The sequence is approxi-
search for clinically useful assays will emphasize the use of mately 90% complete for the 2.95 gigabases of euchro-
 18. Genetics 371

FIGURE 18.6. Schema of a typical eukaryotic gene

and RNA splicing. The nontranslated portion of the
gene contains DNA control regions that specify the
RNA transcription start site and define the specificity of
tissue expression. The sequences that encode the
polypeptide product are organized into discrete units
(exons) and specific sequences within the exons deter-
mine the translation start site (ATG) and stop site
(TGA). The exons are interrupted by noncoding
sequences (introns). The DNA sequence is transcribed in
the nucleus into an RNA product, and the intervening
introns are removed or “spliced out.” By alternative
splicing, a single gene can produce multiple related
proteins. mRNA, messenger RNA. 1–5, exons.

matic or weakly staining gene-rich regions of the human postgenomic era, methods for automated mutation detec-
genome (Table 18.1). Based on initial analyses, investiga- tion, array-based systems for gene and protein expression,
tors predict that the human genome contains 30,000 to and polymorphism profiles of disease susceptibility will all
35,000 protein-encoding genes (Fig. 18.6), which comprise become feasible. These advances will facilitate both the
1.1 to 1.4% of the total sequence (28). The number of cod- diagnosis of mendelian disorders (e.g., autosomal-recessive
ing genes in the human sequence compares with 6000 for polycystic kidney disease) and the evaluation of genetic sus-
the yeast Saccharomyces cerevisiae; 13,000 for Drosophila; ceptibility to more common, multifactorial disorders (e.g.,
18,000 for the roundworm Caenorhabditis elegans; and vesicoureteral reflux).
26,000 for the mustard weed Arabadopsis (29). Therefore, Detailed understanding of the molecular pathophysiol-
our complexity as a species derives from more than just our ogy of human disease will provide new opportunities for
complement of genes. Factors such as variations in gene diagnosis and treatment of both rare and common disor-
regulation, gene expression, the splicing of gene transcripts, ders (Fig. 18.7). Some of these treatments may entail
and modifications of proteins after translation probably all
contribute to our complexity.
The human genome sequence is providing new tools
and new challenges for genetic investigators (29). This
information has already begun to revolutionize biologic
investigation by allowing researchers to examine global
gene expression in different cells, tissues, and disease states
using high dimensional tools, such as microarrays. The
power of these genomic tools will be enhanced as the
genomic sequence is annotated so that the full complement
of genes as well as their regulatory regions is defined. By
extension, the genomic sequence will inform our under-
standing of the full complement of human proteins, the
proteome. Ultimately, this information will permit detailed
characterization of the cellular pathways and networks in
which these proteins interact.
In addition, the human genome sequencing effort has dem-
onstrated that individual humans differ from one another by
approximately one base pair per thousand. By examining the
patterns of these single-nucleotide polymorphisms in patients FIGURE 18.7. Advances in molecular genetics and the clinical
and controls, researchers can evaluate the contribution of indi- implications. In the postgenomic era, positional candidate
approaches can be applied to identify disease-susceptibility
vidual genes to relative susceptibility or resistance to disease. genes for mendelian and, eventually, polygenic disorders. Gene
This genetic variation may also provide insights into individual discovery can be translated into DNA-based diagnostic tests. In a
responses to pharmaceutical treatment (30). growing number of instances, improved diagnostic specificity
can lead to the identification of specific genetic risk factors and
ultimately facilitate the development of preventative medicine
strategies. In addition, gene discovery will open up new treat-
ment options involving pharmaceuticals that target specific
FUTURE CONSIDERATIONS aspects of the molecular pathogenesis. Increasingly, the efficacy
and risk of these new pharmaceuticals for a given individual will
be assayed using single-nucleotide polymorphism (SNP)-based
To date, genetic discoveries have largely focused on identi- profiles. (Modified from Collins F. Positional cloning moves from
fying the genes involved in rare mendelian disorders. In the perditional to traditional. Nat Genet 1995;9:347–350.)
372 III. Research Methods

sophisticated gene therapy techniques, but others will lase/formyltetrahydrofolate synthetase is a maternal genetic
involve the development of targeted pharmaceuticals based risk factor for neural tube defects: report of the Birth
on more precise understanding of molecular physiology. Defects Research Group. Am J Hum Genet 2002;71:1207–
Single-nucleotide polymorphism profiling will afford risk 1215.
assessment for an individual’s genetic susceptibility to cer- 13. Paulsen M, Ferguson-Smith A. DNA methylation in genomic
imprinting, development, and disease. J Pathol 2001;195:97–
tain illnesses (e.g., type II diabetes), and specific preventa-
110.
tive medicine programs will be developed to target this risk. 14. Robinson W. Uniparental disomy and their clinical conse-
In addition, it will become standard practice to use single- quences. Bioessays 2000;22:452–459.
nucleotide polymorphism-based profiles to predict drug 15. Grossman L, Shoubridge E. Mitochondrial genetics and
responses in individual patients (31). Pediatricians and human disease. Bioessays 1996;18:983–991.
pediatric subspecialists must become familiar with the 16. Niaudet P. Mitochondrial disorders and the kidney. Arch Dis
advances in the postgenomic era to incorporate these Child 1998;78:387–390.
changes into their practices. 17. Hildebrandt F, Omram H. New insights: nephronophthisis-
medullary cystic kidney disease. Pediatr Nephrol 2001;16:
168–176.
18. Lifton RP, Dluhy RG, Powers M, et al. A chimaeric 11-B-
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Adv Pediatr 1995;42:91–111. human genome. Science 2001;291:1304–1351.
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 19

TISSUE ENGINEERING
ANTHONY ATALA

Regenerative medicine, a recently defined field, involves the tion of the biomaterial is critical in the development of
diverse areas of tissue engineering, stem cells, and cloning engineered genitourinary tissues. The biomaterial must be
toward the common goal of developing biologic substitutes capable of controlling the structure and function of the
that would restore and maintain normal tissue and organ engineered tissue in a predesigned manner by interacting
function. with transplanted cells and/or the host cells. Generally, the
Tissue engineering follows the principles of cell transplan- ideal biomaterial should be biocompatible, promote cellu-
tation, materials science, and engineering toward the devel- lar interaction and tissue development, and possess proper
opment of biologic substitutes that restore and maintain mechanical and physical properties.
normal function. Tissue engineering may involve matrices In general, three classes of biomaterials have been used
alone, wherein the body’s natural ability to regenerate is for engineering genitourinary tissues: naturally derived
used to orient or direct new tissue growth, or the use of materials (e.g., collagen and alginate), acellular tissue matri-
matrices with cells. ces (e.g., bladder submucosa and small intestinal submu-
When cells are used for tissue engineering, donor tissue cosa), and synthetic polymers [e.g., polyglycolic acid
is dissociated into individual cells that are either implanted (PGA), polylactic acid, and poly(lactic-co-glycolic acid)].
directly into the host or expanded in culture, attached to a These classes of biomaterials have been tested in respect to
support matrix, and reimplanted after expansion. The their biocompatibility with primary human urothelial and
implanted tissue can be heterologous, allogeneic, or autolo- bladder muscle cells (7,8). Naturally derived materials and
gous. Ideally, this approach might allow lost tissue function acellular tissue matrices have the potential advantage of
to be restored or replaced in toto and with limited compli- biologic recognition. Synthetic polymers can be produced
cations (2). The use of autologous cells avoids rejection, reproducibly on a large scale with controlled properties of
wherein a biopsy of tissue is obtained from the host, the their strength, degradation rate, and microstructure.
cells are dissociated and expanded in vitro, reattached to a
matrix, and implanted into the same host.
One of the initial limitations of applying cell-based
TISSUE ENGINEERING OF
tissue-engineering techniques to urologic organs had been
GENITOURINARY STRUCTURES
the previously encountered inherent difficulty of growing
genitourinary-associated cells in large quantities. In the
Urethra
past, it was believed that urothelial cells had a natural senes-
cence that was hard to overcome. Normal urothelial cells Various strategies have been proposed over the years for the
could be grown in the laboratory setting but with limited regeneration of urethral tissue. Woven meshes of PGA have
expansion. Several protocols were developed over the last been used to reconstruct urethras in animals (9,10). PGA
two decades that improved urothelial growth and expan- has been also used as a cell transplantation vehicle to engi-
sion (3–6). Using these methods of cell culture, it is possi- neer tubular urothelium in vivo (11). When using cells for
ble to expand a urothelial strain from a single specimen that transplantation, it has been shown that cells from an abnor-
initially covers a surface area of 1 cm2 to one covering a sur- mal environment, if genetically stable, are able to be engi-
face area of 4202 m2 (the equivalent area of one football neered into normal tissues (12). A homologous free graft of
field) within 8 weeks (3). acellular urethral matrix was used in a rabbit model (13).
Biomaterials in genitourinary tissue engineering may All tissue components were seen in the grafted matrix after
function as an artificial extracellular matrix and elicit bio- 3 months, with further improvement over time; however,
logic and mechanical functions of native extracellular the smooth muscle in the matrix was less than in normal
matrix found in tissues in the body. The design and selec- rabbit urethra and was not well oriented.
374 III. Research Methods

(17). Total urethral replacement is possible with the use of

tissue-engineered constructs composed of urothelial and
muscle cell seeded matrices.

Bladder
Currently, gastrointestinal segments are commonly used as
tissues for bladder replacement or repair. However, gas-
trointestinal tissues are designed to absorb specific solutes,
whereas bladder tissue is designed for the excretion of sol-
utes. When gastrointestinal tissue is in contact with the uri-
nary tract, multiple complications may ensue, such as
infection, metabolic disturbances, urolithiasis, perforation,
increased mucous production, and malignancy (18).
Owing to the problems encountered with the use of gas-
trointestinal segments, numerous investigators have
attempted alternative methods, materials, and tissues for
bladder replacement or repair.
Seromuscular grafts and deepithelialized bowel seg-
ments, either alone or over a native urothelium, have been
attempted (19–26). The concept of demucosalizing organs
is not new to urologists. More than four decades ago, in
1961, Blandy proposed the removal of submucosa from
intestinal segments used for augmentation cystoplasty to
FIGURE 19.1. Urethrogram 6 months postoperatively of a
insure that mucosal regrowth would not occur (19). Hypo-
patient who had a portion of his urethra replaced using tissue- thetically, this would avoid the complications associated
engineering techniques. with using bowel in continuity with the urinary tract
(20,21). Since Blandy’s initial report, 25 years transpired
before there was a renewed interest in demucosalizing intes-
Acellular collagen matrices obtained from donor bladder tinal segments for urinary reconstruction (22). Since 1988,
submucosa have proven to be suitable grafts for repairing several other investigators have pursued this line of research
urethral defects both experimentally and clinically at our (23–26). These investigative efforts have emphasized the
institution. Rabbit neourethras reconstructed with acellular complexity of both the anatomic and cellular interactions
matrices demonstrated a normal urothelial luminal lining present when combining tissues with different functional
and organized muscle bundles without any signs of strictures parameters. The complexity of these interactions is empha-
or complications (14). These results were confirmed clini- sized by the observation that the use of demucosalized
cally in a series of patients with a history of failed hypospa- intestinal segments for augmentation cystoplasty is limited
dias reconstruction, wherein the urethral defects were by either mucosal regrowth or contraction of the intestinal
repaired with human bladder acellular collagen matrices in patch (23,24). It has been noted that removal of only the
an onlay fashion, with the size of the created neourethras mucosa may lead to mucosal regrowth, whereas removal of
ranging from 5 to 15 cm (Fig. 19.1) (15). The same tech- the mucosa and submucosa may lead to retraction of the
nique was used to repair urethral strictures on more than 40 intestinal patch (27,28).
adult patients (16). One of the advantages over nongenital Some researchers have combined the techniques of
tissue grafts used for urethroplasty is that the collagen-based autoaugmentation with enterocystoplasty. An autoaug-
acellular material is “off the shelf.” This eliminates the neces- mentation is performed, and the diverticulum is covered
sity of additional surgical procedures for graft harvesting, with a demucosalized gastric or intestinal segment. In a
which may decrease operative time as well as the potential series of autoaugmentation (25) enterocystoplasty, patients
morbidity due to the harvest procedure. with a neurogenic bladder had either incorporation of
It has also been noted that although acellular collagen- stomach or colon. In both groups of patients, the mucosa
based grafts may be suitable for partial onlay urethral of the enteric segment was dissected away from the
replacement, they are not effective for the replacement of underlying muscle, and the resulting mucosa-free graft
tubularized segments, as this results in the collapse of the was used to cover a newly created bladder diverticulum.
grafts, with subsequent stricture formation (17). Tubular- A satisfactory increase in bladder capacity and compli-
ized urethral repairs require the application of collagen- ance was achieved in most patients. In another series of
based grafts seeded with both urothelial and muscle cells patients who underwent seromuscular colocystoplasty,
 19. Tissue Engineering 375

mL. Histologically, the muscle layer was not fully devel-

oped. A large amount of collagen was interspersed
between a smaller number of muscle bundles. A comput-
erized assisted image analysis demonstrated a decreased
muscle-to-collagen ratio with a loss of the normal archi-
tecture in the small-intestinal submucosa–regenerated
bladders. In vitro contractility studies performed on the
small-intestinal submucosa–regenerated dog bladders
showed a decrease in maximal contractile response by
50% from those of normal bladder tissues. Cholinergic
and purinergic innervation was present (34).
In multiple studies using different materials as an acellu-
lar graft for cystoplasty, the urothelial layer was able to
regenerate normally, but the muscle layer, although present,
was not fully developed (30–33). Engineering tissue using
selective cell transplantation may provide a means to create
functional new bladder segments (11). The success of using
cell transplantation strategies for bladder reconstruction
depends on the ability to use donor tissue efficiently and to
FIGURE 19.2. Bladders augmented with a collagen matrix
provide the right conditions for long-term survival, differ-
derived from bladder submucosa seeded with urothelial and entiation, and growth.
smooth muscle cells [allogenic bladder submucosa (ABS) + cells] Urothelial and muscle cells can be expanded in vitro,
showed a 100% increase in capacity compared to bladders aug- seeded onto the polymer scaffold, and allowed to attach
mented with the cell-free ABS, which showed only a 30%
increase in capacity within 3 months after implantation. (See and form sheets of cells. The cell-polymer scaffold can then
Color Plate 19.2.) be implanted in vivo. A series of in vivo urologic-associated
cell-polymer experiments were performed in mice, rabbits,
and dogs (3,11,31,35).
the bladder capacity increased an average of 2.4-fold in To better address the functional parameters of tissue-
14 patients (26). Ten patients had a postoperative blad- engineered bladders, an animal model was designed that
der biopsy. Seven patients demonstrated urothelium cov- required a subtotal cystectomy with subsequent replace-
ering the augmented portion of the bladder, two had ment with a tissue-engineered organ (36). Dogs under-
regrowth of colonic mucosa, and one showed a mixture went a trigone-sparing cystectomy. The animals were
of colonic mucosa and urothelium. Although colonic randomly assigned to one of three groups. Animals under-
mucosal regrowth is seen, there is a subset of patients went closure of the trigone without a reconstructive
who may benefit from these procedures, wherein mucous procedure, underwent reconstruction with a cell-free blad-
secretion may be reduced or eliminated (26). der-shaped biodegradable matrix, or underwent recon-
Bladder grafts, initially used experimentally in 1961, struction using a bladder-shaped biodegradable matrix
have been used recently by various investigators (29–32). that delivered autologous urothelial and smooth muscle
The allogenic acellular bladder matrix has served as a scaf- cells. The cell populations had been separately expanded
fold for the ingrowth of host bladder wall components. The from a previously harvested autologous bladder biopsy.
matrix is prepared by mechanically and/or chemically Preoperative and postoperative urodynamic, radiographic,
removing the cellular components. gross, histologic, and immunocytochemical analyses
Allogenic bladder submucosa was used as a biomaterial were performed serially, at 1, 2, 3, 4, 6, and 11 months
for bladder augmentation in dogs (31). Biomaterials pre- postoperatively (36).
loaded with cells before their implantation showed better The cystectomy only controls and polymer-only grafts
tissue regeneration compared to biomaterials implanted maintained average capacities of 22% and 46% of preoper-
with no cells in which the tissue regeneration depended on ative values, respectively. An average bladder capacity of
the ingrowth of the surrounding tissue. The bladders 95% of the original precystectomy volume was achieved in
showed a significant increase in capacity of 100% when the tissue-engineered bladder replacements. These findings
augmented with scaffolds seeded with cells, compared to a were confirmed radiographically. The subtotal cystectomy
capacity of 30% for scaffolds without cells (Fig. 19.2). reservoirs, which were not reconstructed, and polymer-only
Small-intestinal submucosa derived from pig small reconstructed bladders showed a marked decrease in blad-
intestine has been used for augmentation cystoplasty in der compliance (10% and 42%). The compliance of the
dogs (33). Preoperative mean bladder capacity was 51 tissue-engineered bladders showed almost no difference from
mL compared to a postoperative mean capacity of 55 preoperative values that were measured when the native
376 III. Research Methods

FIGURE 19.3. Hematoxylin and eosin histologic results 6 months after surgery (original magnifi-
cation: ×250). A: Normal canine bladder. B: The bladder dome of the cell-free polymer recon-
structed bladder consists of normal urothelium over a thickened layer of collagen and fibrotic
tissue. Only scarce muscle fibers are apparent. C: The tissue-engineered neo-organ shows a histo-
morphologically normal appearance. A tri-layered architecture consisting of urothelium, submu-
cosa, and smooth muscle is evident.

bladder was present (106%). Histologically, the polymer- neurogenic, exstrophic). The results of this study were
only bladders presented a pattern of normal urothelial cells also consistent with prior findings that, in diseased blad-
with a thickened fibrotic submucosa and a thin layer of ders, a large portion of the pathologic effects seen are
muscle fibers. The retrieved tissue-engineered bladders due to increased fibrosis, whereas the cells retain their
showed a normal cellular organization, consisting of a genetic stability.
trilayer of urothelium, submucosa, and muscle (Fig. 19.3).
Immunocytochemical analyses for desmin, α-actin, cyto-
Genital Tissues
keratin 7, pancytokeratins AE1/AE3, and uroplakin III
confirmed the muscle and urothelial phenotype. S-100 Reconstructive surgery is required for a wide variety of
staining indicated the presence of neural structures. The pathologic penile conditions, such as penile carcinoma,
results from this study showed that it is possible to tissue trauma, severe erectile dysfunction, and congenital condi-
engineer bladders that are anatomically and functionally tions like ambiguous genitalia, hypospadias, and epispa-
normal (36). Human bladders have been created using the dias. The creation of autologous functional and structural
same techniques. corporal tissue de novo would be beneficial clinically.
Clinically, cells used for tissue engineering may be Studies conducted in our laboratory showed that cultured
harvested from abnormal bladders. We investigated the human corporal smooth muscle cells may be used in con-
contractility of tissue-engineered bladder smooth muscle junction with biodegradable polymers to create corpus
derived from patients with functionally normal bladders cavernosum tissue de novo. The formation of normal cor-
and functionally abnormal neuropathic and exstrophic poral cavernosa tissue is enhanced with the addition of
bladders (12). The tissue-engineered cells showed similar corporal endothelial cells (37,38).
expression of smooth muscle marker proteins (α-actin Further studies show that it is possible to replace
and myosin) regardless of their origin. All scaffolds entire cross-sectional segments of both corporal bodies in
showed similar muscle formation and were α-actin posi- the protruding penis in vivo by interposing autologous
tive. At retrieval, the muscle cell–seeded scaffolds exhib- engineered tissue (39). Rabbits had a small corporal
ited contractile activity to electrical field stimulation and biopsy. Cavernosal smooth muscle and endothelial cells
carbachol. There was no statistical difference between were harvested and expanded. Matrices were obtained
the three different types of muscle cells seeded (normal, using cell lysis techniques from donor penile tissues. Cav-
 19. Tissue Engineering 377

ernosal smooth muscle and endothelial cells were seeded rods were implanted into the corporal spaces of rabbits. All
on the matrices. The entire cross section of the protrud- animals tolerated the implants for the duration of the study
ing rabbit phallus was excised, leaving the urethra intact. (6 months) without any complications. Gross examination
The experimental corporal bodies demonstrated intact at retrieval showed the presence of well-formed milky-white
structural integrity by cavernosography and only a slight cartilage structures within the corpora at 1 month. All
decrease in pressure by cavernosometry when compared polymers were fully degraded by 2 months. There was no
to the controls. Mating activity in the animals with the evidence of erosion or infection in any of the implant sites.
engineered corpora normalized by 3 months. Mating The animals could copulate and impregnate their female
activity in the animals with the matrix alone was low partners without problems.
throughout the study. The presence of sperm was con- We further investigated the feasibility of engineering
firmed during mating, and was present in all the rabbits biomechanically adequate large autologous human carti-
with the engineered corpora but in only two with the lage rods that could be used clinically as penile prosthe-
matrix alone. Gross examination of the corporal implants ses (43). Chondrocytes were isolated from human
with cells showed continuous integration of the graft into auricular cartilage and expanded in vitro. The chondro-
native tissue. The acellular matrices demonstrated the cytes were seeded onto preformed rod-shaped biode-
presence of fibrosis. Histologically, sinusoidal spaces and gradable polymer scaffolds (1.2 cm in diameter, 6.0 cm
walls lined with endothelium and smooth muscle were in length) and maintained in a cell culture bioreactor for
observed in the engineered grafts. However, grafts with- 1 month. Subsequently, the cell-seeded scaffolds were
out cells contained fibrotic tissue and calcifications with implanted into the subcutaneous space of athymic rats.
sparse corporal elements. Each cell type was identified The cell-seeded scaffolds formed milky-white cartilagi-
immunocytochemically using antibodies to smooth muscle nous rods that maintained the same size and shape as the
and endothelium. These studies demonstrate the possibil- initial implants. Histologic analyses using hematoxylin
ity of engineering autologous penile corpora cavernosa and eosin, toluidine blue, and Alcian blue demonstrated
tissue. The engineered tissue is able to achieve adequate mature and well-formed chondrocytes. Tensile tests showed
structural and functional parameters. This technology that the engineered cartilaginous rods were readily elas-
may be potentially applicable to patients who require tic and could withstand high degrees of tensile force
additional tissues for phallic reconstruction; however, (1.78 ± 0.29 kg), which was not significantly different
additional studies involving larger phallic structures need from the Jonas (2.64 ± 0.24 kg) or AMS 700 inflatable
to be completed. These studies are currently being per- prostheses (2.35 ± 0.21 kg). Dynamic bending tests
formed in our laboratory. demonstrated that the cartilaginous rods were flexible
Other alternative tissues are also being studied as possi- and durable. None of the cartilage rods ruptured during
ble replacements for silicone penile prostheses. Although the continuous strenuous mechanical tests. The results
silicone is an accepted biomaterial for penile prostheses, from the above studies demonstrate that autologous
biocompatibility is a concern (40). The use of a natural human auricular cartilage tissue can serve as a cell source
prosthesis composed of autologous cells may be advanta- for engineering cartilaginous penile prostheses. Clini-
geous. A feasibility study for creating natural penile pros- cally, a cartilage biopsy would be obtained from the
theses made of cartilage was performed (41). Cartilage, patient’s ear, and the cells would be expanded and
harvested from the articular surface of calf shoulders, was seeded onto biodegradable rod-shaped scaffolds sized for
isolated, grown, and expanded in culture. The cells were the patient. One month after being in a bioreactor, the
seeded onto preformed cylindrical PGA polymer rods. engineered cartilage rods could be implanted surgically
The cell-polymer scaffolds were implanted in the subcuta- into the patient. Further long-term functional studies
neous space of athymic mice and retrieved up to 6 months are currently being performed to apply this technology
postimplantation. At retrieval, the polymer scaffolds to the clinical setting.
seeded with cells formed milky-white, rod-shaped, solid
cartilaginous structures, maintaining their preimplanta-
Kidney
tion size and shape. Biomechanical analyses showed that
the engineered cartilage rods possessed the mechanical Although the kidney was the first organ to be substituted
properties required to maintain penile rigidity. Histologic by an artificial device and was the first successfully trans-
examination showed the presence of mature and well- planted organ (1), current modalities of treatment are far
formed cartilage. from satisfactory. In addition to the inherent shortage of
In a subsequent study using an autologous system, the transplant organs, complications associated with renal
feasibility of applying the engineered cartilage rods in situ transplantation are yet to be resolved. The kidney is prob-
was investigated (42). Autologous chondrocytes harvested ably the most challenging organ to reconstruct using tis-
from rabbit ears were grown, expanded in culture, and sue-engineering techniques in the genitourinary system
seeded onto biodegradable polymer rods. The cell-polymer owing to its complex structure and function. Extracorpo-
378 III. Research Methods

real assist devices may be improved by implanting isolated generation of functional nephron units. Renal cells were
renal cell lines on bioartificial hemofilters (44). This may harvested and expanded in culture. The cells were seeded
aid in the replacement of specific aspects of renal function, onto a tubular device constructed from a polycarbonate
such as improved selected metabolic and endocrinologic membrane, connected at one end with a silastic catheter,
parameters. Eventually, reconstruction of a tissue-engineered which terminated into a reservoir. The device was implanted
kidney by renal cell expansion in vitro and subsequent in the subcutaneous space of athymic mice (47). Histo-
transplantation in vivo may be possible. To achieve this logic examination of the implanted device revealed exten-
prospect, there are still many technical challenges to be sive vascularization, formation of glomeruli, and highly
solved. organized tubule-like structures. Immunocytochemical
The feasibility of achieving renal cell growth, expan- staining with anti-osteopontin antibody, which is secreted
sion, and in vivo reconstitution using tissue-engineering by proximal and distal tubular cells and the cells of the
techniques was explored in our laboratory. In one set of thin ascending loop of Henle, stained the tubular sections.
experiments, distal tubules, glomeruli, and proximal Immunohistochemical staining for alkaline phosphatase
tubules from donor rabbit kidneys were plated separately stained proximal tubule-like structures. Uniform staining
in vitro, seeded onto biodegradable PGA polymer scaf- for fibronectin in the extracellular matrix of newly formed
folds, and implanted subcutaneously into host athymic tubes was observed. Yellow fluid was collected from inside
mice. At retrieval, the scaffolds histologically showed the implant, and uric acid and creatinine levels were deter-
progressive formation and organization of the nephron mined. The fluid collected from the reservoir contained 66
segments within the polymer fibers over time (45). To mg/dL uric acid (as compared to 2 mg/dL in plasma). The
examine the tubular reconstitution process, nephron ele- creatinine assay performed on the collected fluid showed
ments from animal kidneys were harvested and the cells an 8.2-fold increase in concentration (27.91 ± 7.56 mg/
were expanded in vitro, seeded on biodegradable poly- dL), as compared to serum (4.49 ± 0.08 mg/dL). These
mers with a single-cell suspension, and reimplanted into results suggested that the reconstituted tubules are capable
syngeneic hosts (46). Sequential retrieval of the seeded of unidirectional secretion and concentration of solutes.
polymers over time revealed that the renal epithelial cells The fluid retrieved was consistent with the make-up of
first organized into a cord-like structure with a solid cen- dilute urine, as evidenced by its creatinine and uric acid
ter. Subsequent canalization into a hollow tube could be concentrations. Similar studies were performed with renal
seen by 2 weeks. Histologic examination with nephron cells obtained through nuclear transfer (48). The renal
segment–specific lectins showed successful reconstitu- cells were expanded, seeded onto polycarbonate membrane
tion of proximal tubules, distal tubules, loops of Henle, devices, and implanted into steers. The seeded constructs
collecting tubules, and collecting ducts. were able to form well-defined renal structures that
In a third set of experiments, an attempt was made to secreted dilute urine. The retrieved structures demon-
harness the reconstitution of renal epithelial cells for the strated the presence of nephron units (Fig. 19.4).

FIGURE 19.4. Tissue-engineered renal units. A: Retrieved tissue-engineered renal unit shows the
excretion of yellow fluid in the reservoir. B: The engineered tissue showed a clear unidirectional
continuity between the mature glomeruli, their tubules, and the reservoir. (See Color Plate 19.4.)
 19. Tissue Engineering 379

The results of these studies demonstrate that renal cells can use (50). Long-term studies were conducted to determine
be successfully harvested, expanded in culture, and implanted the effect of injectable chondrocytes in vivo (51). It was ini-
in vivo. The single cells form multicellular structures and tially determined that alginate, a liquid solution of gluronic
become organized into functional renal units that are able to and mannuronic acid, embedded with chondrocytes, could
excrete high levels of solutes through a urine-like fluid. Further serve as a synthetic substrate for the injectable delivery and
challenges await this technology, including the expansion of maintenance of cartilage architecture in vivo. A biopsy of
this system to larger three-dimensional structures. the ear could be easily and quickly performed, followed by
chondrocyte processing and endoscopic injection of the
autologous chondrocyte suspension for therapy.
Fetal Tissue Engineering
Chondrocytes can be readily grown and expanded in cul-
There are several strategies that may be pursued using ture. Neocartilage formation can be achieved in vitro and in
today’s technologic and scientific advances that may facili- vivo using chondrocytes cultured on synthetic biodegradable
tate the future prenatal management of patients with uro- polymers (51). This system was adapted for the treatment of
logic disease. Having a ready supply of urologic-associated vesicoureteral reflux in a porcine model (52). Chondrocytes
tissue for surgical reconstruction at birth may be advanta- were harvested from the left auricular surface of surgically
geous. Theoretically, once the diagnosis of the pathologic created refluxing mini-swine and expanded. The animals
condition is confirmed prenatally, a small tissue biopsy underwent endoscopic repair of reflux with the injectable
could be obtained via ultrasound guidance. These biopsy autologous chondrocyte solution on the right side only.
materials could then be processed and the different cell Serial cystograms showed no evidence of reflux on the treated
types expanded in vitro. Using tissue-engineering tech- side and persistent reflux in the uncorrected control ureter in
niques, reconstituted structures in vitro could then be all animals. The harvested ears had evidence of cartilage
readily available at the time of birth for reconstruction. regrowth within 1 month of chondrocyte retrieval.
Toward this end, a series of experiments were conducted At the time of sacrifice, gross examination of the bladder
using fetal lambs. Bladder exstrophy was created surgically injection site showed a well-defined rubbery to hard carti-
in 90- to 95-day gestation fetal lambs. A small fetal bladder lage structure in the subureteral region. Histologic exami-
specimen was harvested via fetoscopy. The bladder speci- nation of these specimens showed evidence of normal
men was separated, and muscle and urothelial cells were cartilage formation. The polymer gels were progressively
harvested and expanded separately. Seven to 10 days before replaced by cartilage with increasing time. Aldehyde fuch-
delivery, the expanded bladder muscle cells were seeded on sin–Alcian blue staining suggested the presence of chon-
one side and the urothelial cells on the opposite side of a droitin sulfate.
biodegradable polymer scaffold. After delivery, one-half of Using the same line of reasoning as with the chondro-
the lambs had surgical closure of their bladder using the cyte technology, the possibility of using autologous muscle
tissue-engineered bladder tissue. No fetal bladder harvest cells was also investigated (53). In vivo experiments were
was performed in the other lambs, and bladder exstrophy conducted in mini-pigs, and reflux was successfully cor-
closure was performed using only the native bladder. Cysto- rected. In addition to its use for the endoscopic treatment
grams were performed 3 and 8 weeks after surgery. The of reflux and urinary incontinence, the system of injectable
engineered bladders were more compliant (p = .01) and had autologous cells may also be applicable for the treatment of
a higher capacity (p = .02) than the native bladder closure other medical conditions, such as rectal incontinence, dys-
group (49). Similar prenatal studies were performed in phonia, plastic reconstruction, and wherever an injectable
lambs, engineering skin for reconstruction at birth. In addi- permanent biocompatible material is needed.
tion to being able to manage the bladder exstrophy com- Recently, the first human application of cell-based
plex in utero with tissue-engineering techniques, one could tissue-engineering technology for urologic applications has
also manage patients after birth in a similar manner when- occurred with the injection of chondrocytes for the correc-
ever a prenatal diagnosis is not assured. In these instances, tion of vesicoureteral reflux in children (Fig. 19.5) and for
bladder tissue biopsies could be obtained at the time of the urinary incontinence in adults. The clinical trials are cur-
initial surgery. Different tissues could be harvested and rently ongoing (50,54–56).
stored for future reconstruction, if necessary. The potential use of injectable, cultured myoblasts for
the treatment of stress urinary incontinence has recently
been investigated in preliminary experiments (57). Primary
Injectable Therapies
myoblasts obtained from mouse skeletal muscle were trans-
Both urinary incontinence and vesicoureteral reflux are duced in vitro to carry the β-galactosidase reporter gene
common conditions affecting the genitourinary system, and were then incubated with fluorescent microspheres that
wherein injectable bulking agents can be used for treat- serve as markers for the original cell population. Cells were
ment. The goal of several investigators has been to find then directly injected into the proximal urethra and lateral
alternate implant materials that would be safe for human bladder walls of nude mice with a microsyringe in an open
380 III. Research Methods

FIGURE 19.5. A: Preoperative voiding cystourethrogram of a patient showing bilateral reflux.

B: Postoperative radionuclide cystogram of the same patient 6 months after the injection of
autologous chondrocytes.

surgical procedure. Tissue was harvested up to 35 days well as allowing for the long-term physiologic release of
postinjection, analyzed histologically, and assayed for β- testosterone.
galactosidase expression. Myoblasts expressing β-galactosi- Purified Leydig cells were isolated, characterized, sus-
dase and containing fluorescent microspheres were found at pended in an alginate solution, and extruded through an air
each of the retrieved time points. In addition, regenerative jet nozzle into a 1.5% CaCl2 solution in which they gelled
myofibers expressing β-galactosidase were identified within and were further coated with 0.1% poly-L-lysine. The
the bladder wall. By 35 days postinjection, some of the encapsulated cells were pulsed with human chorionic
injected cells expressed the contractile filament a–smooth gonadotropin every 24 hours. The medium was sampled at
muscle actin, suggesting the possibility of myoblastic differ- different time points after human chorionic gonadotropin
entiation into smooth muscle. The authors reported that a stimulation and analyzed for testosterone production. Cell
significant portion of the injected myoblast population per- viability was confirmed daily. The encapsulated Leydig cells
sisted in vivo. The fact that myoblasts can be transfected, were injected into castrated animals, and serum testosterone
survive after injection, and begin the process of myogenic was measured serially. The castrated animals receiving the
differentiation further supports the feasibility of using cul- microencapsulated cells were able to maintain testosterone
tured cells of muscular origin as an injectable bioimplant. levels long term (58). These studies suggest that microen-
capsulated Leydig cells may be able to replace or supple-
ment testosterone into situations in which anorchia or
Testicular Hormonal Replacement
testicular failure is present. A similar system is currently
Leydig cells are the major source of testosterone production being applied for estrogen.
in males. Patients with testicular dysfunction require
androgen replacement for somatic development. Conven-
tional treatment for testicular dysfunction consists of peri- Gene Therapy and Tissue Engineering
odic intramuscular injections of chemically modified
Genetically Engineered Cells
testosterone or, more recently, of skin patch applications.
However, long-term nonpulsatile testosterone therapy is Cells can be engineered to secrete growth factors for various
not optimal and can cause multiple problems, including applications, such as for promoting angiogenesis for tissue
erythropoiesis and bone density changes. regeneration. Angiogenesis, the process of new blood vessel
A system was designed wherein Leydig cells were formation, is regulated by different growth factors. These
microencapsulated for controlled testosterone replace- growth factors stimulate endothelial cells that are already
ment. Microencapsulated Leydig cells offer several advan- present in the patient’s body to migrate to the implanted
tages, such as serving as a semipermeable barrier between area of need, where they proliferate and differentiate into
the transplanted cells and the host’s immune system, as blood vessels (59). One of the major molecules that pro-
 19. Tissue Engineering 381

mote and regulate angiogenesis is vascular endothelial vitro genetic modification of corporal smooth muscle cells
growth factor (VEGF) (60). Several methods have been harvested from an impotent patient, resulting in either a
used experimentally to deliver VEGF in vivo. The growth reduction in the expression of the transforming growth fac-
factor protein can be directly injected into tissues (61); tor-1 gene or the overexpression of genes responsible for
however, the rapid clearance of VEGF proteins from the prostaglandin E1 production, could lead to the resumption
vascular system limits its effect to only minutes. The VEGF of erectile functionality once these cells were used to repop-
gene could be delivered to tissues using various techniques; ulate the diseased corporal bodies.
however, the transfection efficiency is low, the onset of
action is delayed for up to 48 to 72 hours after the VEGF
Stem Cells for Tissue Engineering
cDNA is incorporated, and the effect is transient, lasting
only several days (61,62). Most current strategies for engineering urologic tissues
An approach that has been pursued in our laboratory to involve harvesting of autologous cells from the host diseased
increase and stimulate rapid vascularization in vivo was to organ. However, in situations in which extensive end-stage
engineer a cell line to secrete high levels of VEGF proteins organ failure is present, a tissue biopsy may not yield enough
by gene transfecting the cells with the VEGF cDNA. The normal cells for expansion. Under these circumstances, the
VEGF-secreting cells were encapsulated in polymeric availability of pluripotent stem cells may be beneficial. Pluri-
microspheres. The microspheres would allow nutrients to potent embryonic stem cells are known to form teratomas in
reach the cells, while the VEGF proteins secreted from the vivo, which are composed of a variety of differentiated cells.
cells diffused into the surrounding tissues. The micro- However, these cells may be immunocompetent and may
spheres protect the coated cells from the host immune envi- require immunosuppression if used clinically.
ronment. This novel system of neovascularization was The possibility of deriving pluripotent cells from post-
tested in vitro and in vivo in an animal model. The degree natal mesenchymal tissue from the same host and inducing
of VEGF secretion and the period of delivery can be regu- their differentiation in vitro and in vivo was investigated.
lated by modulating the number of engineered cells that are Pluripotent cells were isolated from human foreskin-
encapsulated per microsphere, as well as the number of derived fibroblasts. Adipogenic, myogenic, and osteoblastic
microspheres injected. A similar strategy has also been pur- lineages were obtained from these progenitor cells. The cells
sued for the genetic engineering of antiangiogenic factor– were grown, expanded, seeded onto biodegradable scaf-
secreting cells (63). These strategies could be useful for folds, and implanted in vivo, where they formed mature tis-
antitumor therapy in urology. sue structures. This was the first demonstration that stem
cells can be derived from postnatal connective tissue and
can be used for engineering tissues in vivo ex situ (65).
Gene Therapy for Tissue-Engineered Constructs
Based on the feasibility of tissue-engineering techniques in
Therapeutic Cloning for Tissue Engineering
which cells seeded on biodegradable polymer scaffolds form
tissue when implanted in vivo, the possibility was explored Recent advances with the cloning of embryos and newborn
of developing a neo-organ system for in vivo gene therapy animals have expanded the possibilities of this technology
(64). In a series of studies conducted in our laboratory, for tissue engineering and organ transplantation. There are
human urothelial cells were harvested, expanded in vitro, many ethical concerns with cloning in terms of creating
and seeded on biodegradable polymer scaffolds. The cell- humans for the sole purpose of obtaining organs. However,
polymer complex was then transfected with PGL3-luc, the potential for retrieving cells from early-stage cloned
pCMV-luc, and pCMVβ-gal promoter-reporter gene con- embryos for subsequent regeneration is being proposed as
structs. The transfected cell-polymer scaffolds were then an ethically viable benefit of therapeutic cloning (Fig.
implanted in vivo, and the engineered tissues were retrieved 19.6). The feasibility of engineering syngeneic tissues in
at different time points after implantation. Results indicate vivo using cloned cells was investigated.
that successful gene transfer may be achieved using biode- Unfertilized donor bovine eggs were retrieved, and the
gradable polymer scaffolds as a urothelial cell delivery vehi- nuclear material was removed. Bovine fibroblasts from
cle. The transfected cell/polymer scaffold formed organ-like the skin of a steer were obtained. The nuclear material
structures with functional expression of the transfected was removed from the fibroblast and microinjected into
genes (64). This technology is applicable throughout the the donor eggshell (nuclear transfer). A short burst of
spectrum of diseases, which may be manageable with tissue energy was delivered, initiating neo-embryogenesis.
engineering. For example, one can envision the use of These techniques replicate what was performed to clone
effecting in vivo gene delivery through the ex vivo transfec- the first mammal—Dolly the sheep. However, instead of
tion of tissue-engineered cell/polymer scaffolds for the implanting the embryo into a uterus, the goal would be
genetic modification of diseased corporal smooth muscle to harvest stem cells from the embryo, which was created
cells harvested from impotent patients. Theoretically, the in not from the union of a sperm and an egg but rather
382 III. Research Methods

techniques of cell harvest, culture, and expansion. Polymer

scaffold design and manufacturing resources are essential
for the successful application of this technology.
The first human application of cell-based tissue engi-
neering for urologic applications occurred at our institution
with the injection of autologous cells for the correction of
vesicoureteral reflux in children. The same technology has
been recently expanded to treat adult patients with urinary
incontinence. Trials involving urethral tissue replacement
using processed collagen matrices are in progress at our cen-
ter for both hypospadias and stricture repair. Bladder
replacement using tissue-engineering techniques is being
explored. Recent progress suggests that engineered urologic
FIGURE 19.6. Schematic diagram of therapeutic cloning
approach for the isolation of genetically matched tissues. (See tissues may have a wider clinical applicability in regenera-
Color Plate 19.6.) tive medicine.

from a skin cell and an eggshell, devoid of any genetic REFERENCES

material. To achieve a proof of principle, bypassing the
in vitro differentiation, the embryos were placed in the 1. Murray JE, Merrill JP, Harrison JH. Renal homotransplan-
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which the cells were cloned. The implants were retrieved lia expanded in vitro. J Urol 1994;152:655.
4. Scriven SD, Booth C, Thomas DF, et al. Reconstitution of
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human urothelium from monolayer cultures. J Urol 1997;158
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These studies demonstrated that cells obtained through urothelial cells produce cytokines and express an activated cell
nuclear transfer can be successfully harvested, expanded surface antigenic phenotype. Semin Urol 1991;9(2):124–130.
in culture, and transplanted in vivo with biodegradable 6. Puthenveettil JA, Burger MS, Reznikoff CA. Replicative
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55:33–39.
skin cell from a patient and having the ability to gener-
8. Pariente JL, Kim BS, Atala A. In vitro biocompatibility eval-
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10. Olsen L, Bowald S, Busch C, et al. Urethral reconstruction
with a new synthetic absorbable device. Scand J Urol Neph-
CONCLUSION rol 1992;26:323–326.
11. Atala A, Vacanti JP, Peters CA, et al. Formation of urothelial
Tissue-engineering efforts are currently being undertaken structures in vivo from dissociated cells attached to biode-
gradable polymer scaffolds in vitro. J Urol 1992;148:658.
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12. Lai JY, Yoon CY, Yoo JJ, et al. Phenotypic and functional
tem. Primary autologous cells, stem cells, and therapeutic characterization of in vivo tissue engineered smooth muscle
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growth factor biology, a cell culture facility designed for 13. Sievert KD, Bakircioglu ME, Nunes L, et al. Homologous
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Urol 1996;155:374–378. cytes: preliminary results. J Urol 1999;162:1185.
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165. 62. Takeshita S, Tsurumi Y, Couffinahl T, et al. Gene transfer of
57. Yokoyama T, Chancellor MB, Watanabe T, et al. Primary naked DNA encoding for three isoforms of vascular endo-
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detrusor contractility; long term survival without significant 63. Joki T, Machluf M, Atala A, et al. Continuous release of
cytotoxicity. J Urol 1999;161:307. endostatin from microencapsulated engineered cells for
58. Machlouf M, Orsola A, Atala A. Controlled release of thera- tumor therapy. Nat Biotechnol 2001;19(1):35–39.
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59. Polverini PJ. The pathophysiology of angiogenesis. Crit Rev 65. Bartsch GC, Yoo JJ, De Coppi P, et al. Dermal stem cells for
Oral Biol Med 1996;6:230. pelvic and bladder reconstruction. J Urol 2002;167:59a.
 S E C T I O N

IV

CLINICAL METHODS
 20

CLINICAL EVALUATION
T. MARTIN BARRATT
PATRICK NIAUDET

Section IV is concerned with methods for evaluating chil- ultrasound scan that is part of routine obstetric care and is
dren with kidney disease. This chapter addresses their pre- now the most common route for infants to reach pediatric
sentation, the relevant features in their clinical history, and urologic services (see Chapter 4). Some abnormalities such
the findings to be sought on physical examination. This as unilateral hydronephrosis are relatively minor; others like
wide canvas is illustrated by didactic algorithms and fre- renal agenesis or severe bladder outflow obstruction are
quent reference to other chapters in the book rather than accompanied by oligohydramnios, a consequence of intra-
by conventional citations of the medical literature, and we uterine renal failure, and pulmonary hypoplasia (the Potter
have drawn on our personal experience in the selection of sequence) (see Chapter 5).
material presented.
The clinical evaluation should follow the classic path of
Neonates
history, physical examination, and urinalysis, leading to a
problem list with differential diagnoses, plan of investigation, Congenital renal abnormalities and inherited disorders are
and proposals for immediate management. It must, however, present at birth but may not be immediately apparent, in part
be modified to take into account the age of the child, the because the placenta is an efficient excretory organ masking
socioeconomic circumstances and medical resources avail- renal failure and in part because the low glomerular filtration
able, the local epidemiology of renal disease, and the mode of rate of the neonatal kidney may conceal genetically deter-
presentation of the problem. mined disorders of renal tubular function. The congenital
The proper recording of the clinical findings in the medi- nephrotic syndrome is present in utero: Affected infants are
cal notes is essential for the management of the case, audit, edematous at birth and have a high placenta/body weight ratio
retrospective review, and medicolegal purposes. It is the duty (see Chapter 25). Polycystic kidneys, particularly the autoso-
of the medical staff to maintain accurate records and of the mal recessive variety, are easily palpable (see Chapter 36).
clinical director of the service to ensure that this is done. Congenital abnormalities such a posterior urethral valve
may be missed in a midtrimester scan and present in the
neonatal period with urinary retention and overflow. Uro-
AGE AND RENAL DISEASE logic malformations may be associated with absent abdom-
inal wall muscles and undescended testicles. Congenital
The span of pediatric nephrology runs from conception to abnormalities in other systems may suggest a syndrome
adolescence. At one end there is an overlap of responsibility with renal involvement (see Chapter 6).
with the geneticist and obstetrician, at the other with the The immaturity of glomerular and tubular function in
“adult” nephrologist, and throughout with the pediatric the neonate is exaggerated in the premature infant, predis-
urologist. posing them to acidosis and hyponatremia. Perinatal crises
may be associated with renal venous thrombosis, character-
ized by hematuria, firm enlarged kidneys, and renal failure
Fetus (see Chapter 64). A urinary tract infection (UTI) may be a
The risk of inherited renal disease in the child may be evi- pointer to an underlying urologic abnormality.
dent from the family history, and several techniques are
available for confirming the diagnosis in early pregnancy Children
(see Chapter 21).
Structural malformations of the fetal kidneys or urinary The clinical evaluation of young children requires special
tract are frequently revealed by the midtrimester maternal pediatric skills. They cannot articulate their symptoms: A
388 IV. Clinical Methods

UTI may present as fever or vomiting (see Chapter 53) and PRESENTATION
chronic renal failure (CRF) as failure to thrive. The presen-
tation of renal disease in older children is similar to adults, A renal or urologic disorder may present with symptoms
but it must be remembered that growth failure may be a such as hematuria or dysuria, obviously pointing to the uri-
pointer to occult renal disease (see Chapter 22). The psy- nary tract, or the kidneys may be involved as part of a more
chosocial problems of chronic renal disease may be particu- widespread syndrome (e.g., systemic lupus erythematosus).
larly troublesome in adolescents, and the possibility of Similarly, a disordered pattern of micturition or change in
noncompliance with therapy must be borne in mind. urine volume or composition may indicate renal pathology.
However, serious renal disease may be present without any
symptoms at all or be associated with symptoms and signs
SOCIAL AND ECONOMIC CIRCUMSTANCES that do not point directly to the urinary tract.

The clinical practice of pediatric nephrology described in

this book is largely derived from and in reality only applica- Abnormalities of Urine Appearance
ble to the 10% of the World’s children who inhabit the
Hematuria
industrialized countries of what might be called (without
entering the political debate) the Developed World (see The addition of even small amounts of blood to urine
Chapter 75A). Three patterns can be discerned on the basis results in obvious macroscopic hematuria (Fig. 20.1). How-
of the development of the specialty itself and the availabil- ever, apparently clear urine may contain an abnormal num-
ity of renal replacement therapy for children. In the Devel- ber of red blood cells (RBCs), referred to as microscopic
oped World there are approximately one to two pediatric hematuria. If the bleeding is brisk, the urine will be red and
nephrologists per million total population, and renal trans- may even contain clots, but usually the pigment changes to
plantation is available for all children for whom it is clini- the more brown color of acid hematin. The presence of
cally appropriate; in the Developing World, such services are blood in the urine should be confirmed by dipstick testing
patchy, and in the Underdeveloped World they are inappro- with Hemastix (Bayer Diagnostics, Pittsburgh) (see Hema-
priate in relation to the other medical needs of children. stix). Microscopy will distinguish hematuria from hemo-
The sick in poor countries are doubly disadvantaged: The globinuria and myoglobinuria, which are also characterized
gross national product is low, and a smaller proportion of it is by dark brown Hemastix-positive urine but without RBCs.
made available for health care. Medical equipment and drugs Bleeding may originate from the glomeruli or from the
are expensive, access to medical centers for children in rural urinary tract: A distinction may be made by careful
communities is difficult, and civil war may disrupt services. microscopy of the urine, with “glomerular hematuria”
Children fare badly in the competition with adults for being characterized by deformed, misshapen RBCs. Red
limited resources. Those with renal disease may be lost cell casts and proteinuria also point to a glomerular ori-
among the many who die from malnutrition and diarrhea: gin, but a urologic disorder, particularly tumor, hydrone-
They tolerate salt and water depletion poorly. Protein-calorie phrosis, or stone, must always be ruled out by careful
malnutrition predisposes to postinfectious glomerulone- ultrasound examination, even if there is obvious evidence
phritis, aggravates the edema of the nephrotic syndrome, of glomerular disease (see Chapters 57 and 58). Hema-
and is a major factor in the growth failure of CRF (see turia may be associated with hypercalciuria even without
Chapter 68). evident calculi. Blood in the initial part of the urinary
stream suggests a urethral origin of the bleeding; terminal
hematuria, particularly if associated with suprapubic pain
EPIDEMIOLOGY or a disturbance of micturition, points to a bladder cause
requiring investigation by cystourethroscopy. However, in
There are considerable differences in the pattern of renal most affected children, hematuria has its origin in the
disease in children around the world. A major distinction upper urinary tract, and endoscopy is not necessary as a
exists between tropical and temperate regions, arising from routine investigation unless other features suggestive of
racial variations in the susceptibility to renal disease, com- lower tract pathology accompany the bleeding.
pounded by the socioeconomic factors described earlier. If the hematuria is persistent and accompanied by heavy
Thus, sickle cell nephropathy is restricted to Afro-Carib- proteinuria or reduced renal function, it is usually neces-
bean children, whereas vesicoureteric reflux (VUR) and sary to proceed to renal biopsy to determine the glomer-
associated UTI are more common in white children. The ular lesion. Recurrent episodes of isolated macroscopic
high incidence of glomerulonephritis in children in tropical hematuria with normal urine in between are usually
countries is related to frequent bacterial and viral infec- benign and suggestive of immunoglobulin A nephropa-
tions, and human immunodeficiency virus nephropathy is thy (see Chapter 31). Persistent microscopic hematuria,
emerging as a major problem (see Chapter 51). particularly with a suggestive family history, should prompt
 20. Clinical Evaluation 389

FIGURE 20.2. Urinary tract infection (UTI). (Hemastix + ve.)

DMSA, dimercaptosuccinic acid.

cessed urine sample are essential in making the diagnosis

of UTI, and screening tests are unreliable. Children with
a proven UTI should have a careful abdominal ultra-
sound examination to exclude a urologic abnormality;
boys and younger girls or those with recurrent UTI may
require a cystogram to exclude VUR or other lower uri-
nary tract abnormality. If VUR is present, a technetium-
99m dimercaptosuccinic acid is the most sensitive
method for determining the extent of associated renal
scarring, but there is currently debate about protocols
for the investigation of children with UTI (see Chapters
FIGURE 20.1. Hematuria. (Hemastix + ve.) RBC, red blood cell.
23, 53, and 54).

Stone
urine screening of all first-degree relatives and is best evalu-
ated by renal biopsy, provided full facilities for its evalua- The passage of a stone is an unusual event in childhood
tion (including electron microscopy) are available (see (see Chapter 57) (Fig. 20.3). Renal calculi may cause
Chapters 24 and 26). hematuria or UTI and, if obstructive, renal colic. They
may consist of calcium salts (phosphates or oxalates),
struvite (the triple salt of magnesium, ammonium, and
Cloudy, Offensive Urine
phosphate with organic matrix), cystine, uric acid, or
Cloudy, offensive urine suggests a UTI, particularly if rarely one of the other purines (xanthine or dihydroxy-
associated with local symptoms of dysuria and frequency adenine). In the United Kingdom, the most common
or systemic symptoms of fever and loin pain, although variety is struvite, typically found in the upper urinary
significant bacteriuria may be asymptomatic (see Chap- tract of young boys with a UTI caused by Proteus. In the
ter 53) (Fig. 20.2). However, normal concentrated urine, United States, the most common variety is calcium-
when cooled, will form a precipitate of calcium phos- containing stones in older children living in the “stone
phate; conversely, significant bacteriuria may be present belt.” Children with renal calculi, especially if associated
without obvious macroscopic abnormality of the urine. with nephrocalcinosis, require full investigation to rule
Microscopy and culture of a properly collected and pro- out an underlying metabolic cause, particularly distal
390 IV. Clinical Methods

FIGURE 20.3. Calculi. UTI, urinary tract infection.

renal tubular acidosis (see Chapter 39) and primary

hyperoxaluria (see Chapter 42). By comparison with
adults, primary hyperparathyroidism is rare.
FIGURE 20.4. Acute renal failure. Oliguria <500 mL/1.73 m 2 SA/
Abnormalities of Urine Volume 24 hr.

Oligoanuria
Anuria is the complete suppression of urine; oliguria is primary polydipsia and primary polyuria may be inferred
the passage of an insufficient volume to maintain from the relative osmolality of plasma and urine and the
homeostasis, usually taken to be less than 500 mL/24 response to controlled water deprivation. The distinc-
hr/1.73 m2 surface area, which in infants approximates tion between pituitary and renal causes of polyuria may
1.0 mL/hr/kg body weight. Urine volume in breast-fed, be made by the plasma arginine–vasopressin concentra-
full-term babies averages 20 mL/24 hr in the first 2 days tion and the urinary response to des-D-amino-arginine
of life, rising to 200 mL by the tenth day. In newborn vasopressin.
infants, 92% pass urine in the first 24 hours, and 98% A defect of urine concentration should be suspected
do so within the first 48 hours of birth (see Chapter 56). in any infant with unexplained irritability, failure to
Oliguria with acute renal failure may represent the phys- thrive, fever, dehydration, or hypernatremia. Older chil-
iologic response of a healthy kidney to inadequate perfu- dren are more likely to have nocturnal enuresis accom-
sion (“prerenal”), parenchymal renal disease (“renal”), or panied by a clear history of polyuria and polydipsia.
obstruction of the urinary tract (“postrenal”) (Fig. 20.4) However, it is difficult to define the threshold of poly-
(see Chapter 64). uria with accuracy, but it is usually not apparent as an
isolated clinical symptom unless the urine is persistently
hypotonic to plasma. Under these circumstances there is
Polyuria
increased thirst, and infants in particular are at risk of
Polyuria results from an excessive intake of fluid, a fail- dehydration. The principal renal diseases that result in
ure of release of antidiuretic hormone, osmotic diuresis, antidiuretic hormone–resistant polyuria of this severity
or renal resistance to the action of antidiuretic hormone are nephrogenic diabetes insipidus (see Chapter 40), cys-
(see Chapter 40) (Fig. 20.5). The distinction between tinosis (see Chapter 41), Bartter’s syndrome (see Chap-
 20. Clinical Evaluation 391

Poor Urinary Stream

Infrequent or difficult micturition may indicate inadequate
bladder contraction or a functional failure of coordination
between detrusor contraction and sphincter relaxation, sug-
gesting a neuropathic bladder. A poor urinary stream in a
male infant, particularly if associated with a full bladder,
strongly suggests an obstructive pathology such as a poste-
rior urethral valve. Observation of the urinary stream is an
essential part of the physical examination of a child sus-
pected of having a disorder of the urinary tract. Urinary
retention is an uncommon problem in childhood and
demands urgent and expert urologic attention to exclude a
congenital anomaly, neuropathic bladder, tumor, or stone.

Frequency and Dysuria

Children of both genders between 5 and 14 years of age
pass urine approximately four to eight times per day.
Increased urinary frequency is seldom a primary complaint
and is usually associated with other symptoms, such as
urgency. An increased frequency of micturition may be due
to polyuria, a reduced bladder capacity, or, most com-
monly, bladder irritability as with a UTI. Painful passage of
urine (dysuria) usually indicates urethritis or cystitis secon-
FIGURE 20.5. Polyuria (>200 mL/1.73 m2 SA/24 hrs). dDAVP, des-
D-amino-arginine vasopressin. dary to UTI, but dysuria may also be a consequence of vul-
vovaginitis in girls and balanitis in boys.

ter 38), obstructive nephropathy (see Chapter 55), and

Abnormalities of Hydration
juvenile nephronophthisis (see Chapter 35).
Dehydration
Abnormalities of Micturition Pure water depletion, resulting, for example, from defects in
urine concentration, is associated, particularly in infants,
Incontinence and Enuresis
with feeding difficulties and fever. If there is no concomitant
Daytime urinary continence usually is achieved by 3 years salt depletion, there will be hypernatremia. The extracellular
of age (see Chapter 56). At the age of 5 years, approxi- fluid (ECF) volume may be relatively well maintained, with
mately 15% of children wet the bed at night, falling to the brunt of the fluid depletion being borne by the intracell-
approximately 7% at 10 years. Nocturnal enuresis on its ular fluid (ICF) compartment, in which case the principal
own is usually a benign self-limiting condition, whereas symptom is thirst. With salt depletion as well, ECF volume
incontinence by day is commonly a behavioral problem. is depleted, resulting in hypotension and sensations of faint-
The development of incontinence in a child who has pre- ness and fatigue. Chronic salt-wasting is often associated
viously been dry raises the possibility of either organic dis- with an increased salt appetite and a preference for savory
ease or a psychological disturbance. Diurnal incontinence rather than sweet foods (see Chapter 7).
requires careful evaluation for the possibility of a neuro-
pathic bladder, especially if associated with chronic con-
Edema
stipation. Persistent dribbling incontinence associated
with otherwise apparently normal micturition suggests an ECF expansion results in edema, often first evident as swell-
ectopic ureter opening below the bladder neck. ing of the eyelids and sometimes mistaken for an allergic
Giggle incontinence is usually a complaint of girls and phenomenon; more generalized edema is characterized by
younger women and is often associated with urgency. The pitting of the legs and abdominal swelling caused by ascites
definition is implicit in the name and is often a pointer to (Fig. 20.6). There are two distinct renal mechanisms of
an unstable bladder with inappropriate detrusor contrac- edema formation. In the first, characteristic of the acute
tions during filling. Younger girls with this complaint may nephritic syndrome and renal failure, a primary failure to
squat on their heels to prevent voiding, a trick known as excrete salt and water results in edema accompanied by
Saint Vincent’s curtsy. expansion of the intravascular volume, hypertension, and
392 IV. Clinical Methods

mia, chronic salt and water depletion, and uremia per se, as
well as (in some cases) a disordered pattern of growth hor-
mone release (see Chapters 67 and 68). CRF may be over-
looked as a cause of growth failure in young children
because the plasma creatinine concentration may not be
significantly elevated if muscle bulk is poor.

Gastrointestinal Problems
Feeding difficulties, vomiting, and anorexia dominate the
clinical picture of CRF in young children. Again, the situa-
tion is complex: There is a disturbance of foregut motor
function, often with gastroesophageal reflux; taste sensation
is disturbed, leading to anorexia because of chronic salt and
water depletion; and there may well have been a seriously
disturbed weaning period. These factors combine to pro-
duce a situation in which inadequate caloric intake
becomes a major factor in the failure to grow.
FIGURE 20.6. Edema. CVP, central venous pressure.

Asymptomatic Presentation
Renal disease is often asymptomatic, only coming to light
pulmonary congestion (see Chapter 30). In the second, as in as a consequence of routine examination or screening pro-
the nephrotic syndrome, edema results from hypoproteine- grams. This has led to various proposals for population
mia, the diminished colloid osmotic pressure of plasma lead- screening. However, the issue is complicated because it can
ing to seepage of fluid from the intravascular into the be assumed neither that the natural history of a disorder
interstitial compartment, with contraction of the plasma vol- detected in this way is necessarily the same as one that pre-
ume secondarily causing salt and water retention. In the sented symptomatically nor that it requires the same proto-
nephrotic syndrome, there is also evidence for a primary col of investigation and treatment.
increase in distal tubular sodium reabsorption. Hypopro-
teinemic edema is more gravity dependent than the edema of
renal failure and may be accompanied by the symptoms of Antenatal Ultrasound
faintness and abdominal pain attributable to hypovolemia The routine use of diagnostic ultrasound in pregnancy
(see Chapter 27). The distinction between the edema of the has led to the detection of urologic abnormality in many
acute nephritic syndrome, with its accompanying hematuria fetuses (see Chapter 4). The most common abnormality
and hypertension, and the nephrotic syndrome is usually detected is simple hydronephrosis caused by presumed
straightforward, but intermediate cases occur. pelviureteric junction obstruction, followed by multicys-
tic dysplastic kidney. Bilateral renal dysplasia or agenesis
General Symptoms associated with oligohydramnios may be detected, and
severe infravesical obstruction has led to attempts of
Malaise uncertain benefit to drain the urinary tract in utero.
The symptoms associated with uremia are vague ill health, However, the antenatal diagnosis of posterior urethral
lassitude, anorexia, and vomiting. It is now rare for a child valves may be missed unless a scan is undertaken late in
to reach such a condition without having come to medical pregnancy. There is still much to be learned about the
attention, but an occasional case presenting as acute renal natural history and implications for postnatal investiga-
failure will in fact be found to have CRF. Anemia may con- tion and management of urologic disorders diagnosed
tribute to the symptomatology, as attested by the improve- antenatally.
ment in general well-being with erythropoietin therapy.
Routine Neonatal Examination
Growth Failure The routine examination of the neonate should include
Children with CRF and some renal tubular disorders grow abdominal palpation, which may reveal evidence of renal
poorly and may indeed present with this problem. The disease (e.g., a palpable bladder or renal masses caused by
causes are numerous and their interaction complex: poor hydronephrosis, cystic disease, venous thrombosis, or
calorie intake, acidosis, uncontrolled osteodystrophy, ane- tumor). Renal agenesis or severe dysplasia should be sus-
 20. Clinical Evaluation 393

pected if there is a history of oligohydramnios associated nosis of autosomal dominant polycystic kidney disease may
with compression deformities [e.g., crumpled ears, Potter’s only become clear when the parents have had an ultra-
facies, talipes, dislocated hips, or pulmonary hypoplasia sound, and a family history of diabetes in a child with
(sometimes with pneumothorax)] (see Chapter 5). An cystic kidneys may be a pointer to a hepatocyte nuclear fac-
absence of abdominal wall muscles (prune belly syndrome) tor-1β mutation (see Chapter 36).
is associated with bilateral cryptorchidism and a wide range Disorders transmitted as autosomal recessive traits are
of urologic abnormalities. There is an increased incidence usually readily apparent and should be considered if there is
of renal anomalies in association with other congenital parental consanguinity. They may have escaped notice if
defects, particularly those of the cardiovascular system, gas- the expression of the disease is mild, as in some cases of
trointestinal tract (especially imperforate anus), and genita- recessive polycystic kidney disease, or in the early stages
lia (see Chapter 6). (e.g., in younger siblings of patients with juvenile nephro-
nophthisis or familial idiopathic nephrotic syndrome).
Clues to X-linked recessive disorders may sometimes
Urine Screening Programs
only be revealed on inquiry into the history of previous
Because of the association between UTI, VUR, and renal generations, such as the loss of the male infant relative of a
scarring, a large number of studies have been directed at the mother of a child with nephrogenic diabetes insipidus or a
role of screening for bacteriuria to prevent renal damage. male relative with deafness and renal failure in an Alport
Because some 5% of schoolgirls will at some stage develop kindred (see Chapter 26).
bacteriuria, the yield of such surveys is large. However, it is Polygenic inheritance, genetic heterogeneity, or incom-
by no means established that the widespread application of plete penetrance of a dominant gene may be operative in
screening will reduce the incidence of end-stage renal fail- the etiology of VUR, with at least 10% of first-degree rela-
ure. Regular urinalysis of school children is practiced in tives being affected (see Chapter 54).
Japan (see Chapter 75C).
Obstetric and Neonatal History
Family Studies
The role of antenatal ultrasonography in the detection of
The diagnosis of an inherited disorder in a child, particu- urologic abnormalities and oligohydramnios is described
larly if dominant or X-linked, should lead to a review of the earlier. An edematous or hypertrophied placenta, more
extended family, which may identify several individuals than 25% of the child’s birth weight, is a feature of the con-
previously unaware of having a renal abnormality. This genital nephrotic syndrome (see Chapter 25). Perinatal
raises ethical problems (see Chapter 18). asphyxia, particularly if associated with macroscopic hema-
turia, may have been responsible for renal venous thrombo-
sis and should be considered in the differential diagnosis of
Routine Biochemical Evaluation
an older child with renal scarring, particularly if there is no
The kidney is involved in a wide variety of multisystem dis- suggestion of VUR.
orders, and, with the modern generation of multichannel
biochemical analyzers, the serendipitous discovery of renal
General Medical History
disease in a sick child is now not an uncommon occurrence.
Because the kidney or urinary tract is commonly involved in
syndromes of congenital malformation or multisystem disor-
ders, attention must be paid to all aspects of the child’s
HISTORY
medical history. Consultation with the department of oto-
laryngology for a deaf child may point to Alport’s or the
Family History
branchiootorenal syndromes; at the ophthalmology depart-
A detailed family history is an essential part of the clinical ment, a diagnosis of retinitis pigmentosa may suggest Lau-
evaluation because it may well provide an important clue to rence-Moon-Biedl syndrome or juvenile nephronophthisis.
the diagnosis. Genetic factors are relevant in a wide variety The orthopedic department may be involved because of
of renal diseases such as inherited disorders of renal tubular bony deformity as a result of osteodystrophy; slipped femoral
function, familial glomerulonephritis, and cystic disease of capital epiphysis; or Perthes’ disease, which is more common
the kidneys. The relevance may not be immediately obvi- with renal abnormalities even without renal failure. Renal
ous. Knowledge of the genetic abnormality carries implica- involvement may be part of a multisystem disorder, such as
tions for counseling the family. It is good practice to obtain systemic lupus erythematosus, juvenile rheumatoid arthritis,
the family tree for the case records. or other vasculitic disorders (see Chapter 46). Drug nephro-
A clue to dominantly inherited conditions may some- toxicity may arise in many different groups of patients, par-
times come from minor abnormalities in a parent: the diag- ticularly from antimicrobials, diuretics, and anticancer drugs
394 IV. Clinical Methods

(see Chapter 52). In a major children’s hospital, the nephrol- to the child’s size than chronologic age. Later, toward
ogist may be as much involved with internal as with external puberty, there will always be concern as to whether or when
referrals and must be familiar with the renal complications of dialysis and transplantation will be necessary. A detailed
systemic disorders and iatrogenic problems. educational history should be obtained and communica-
tion with the school authorities established. A children’s
renal unit should have a dedicated teacher whose opinion
Urine and Micturition
should be sought in individual cases, and an appropriate
A micturition history with specific detail about urinary educational program should be worked out for each child.
stream is essential. The ages at which daytime and noctur- Expertise in this area, as much as experience in organic
nal urinary continence were achieved should be docu- renal disease in childhood, distinguishes skilled pediatric
mented. A history of hematuria or features suggesting UTI nephrologists from their colleagues in internal medicine.
may be relevant to the current renal problem.

Dietary History PHYSICAL EXAMINATION

A review of nutrition and feeding problems, beginning with
General Assessment
the neonatal period, should be undertaken: whether the child
was breast- or bottle-fed, episodes of vomiting or anorexia, A preliminary rapid assessment should be made of how sick
thirst, or dietary preferences. Chronic salt-wasting, a feature of the child is, whether there is circulatory failure or shock, if
CRF, is commonly associated with an increased salt appetite ventilation is adequate, what the level of consciousness is,
and a preference for savory rather than sweet foods. For chil- and whether the child is in pain. The child’s mood and
dren with growth retardation or renal failure, a formal assess- demeanor should be noted (e.g., whether happy or frac-
ment of protein, energy, and other nutrient intake should be tious, alert and lucid, or somnolent and obtunded). The
undertaken by a dietitian skilled in the management of prob- child’s state of care should be assessed (i.e., does the child
lems of renal disease in childhood (see Chapter 67). appear to be kempt or disheveled?). Inspection of the
mucous membranes, conjunctivae, and nailbeds may provide
evidence of anemia. Other features suggesting long-stand-
Gastrointestinal Symptoms
ing CRF are growth retardation and skeletal deformity
A prodrome of diarrhea strongly suggests infection with a caused by uremic osteodystrophy.
verotoxin-producing Escherichia coli as the cause of
hemolytic uremic syndrome (see Chapter 47).
Growth and Nutrition
Proper assessment of the child’s growth necessitates accu-
Psychosocial Review
rate measurement of height with a stadiometer and weight
Many children with renal disease come from families with with scales, both accurate to 0.1%, together with charts of
severe social problems. Some disorders (e.g., nephritis sec- height and weight percentiles appropriate for the child’s
ondary to skin sepsis) are more common in deprived social age, race, and gender (see Chapter 22). Pubertal develop-
classes. However, more important is the effect that social ment must be formally assessed. An impression should be
circumstances have on the ability of the family to cope with formed of muscle bulk and body fat, but nutritional status
such minor symptoms as enuresis or to participate in the is difficult to assess. The relationship between weight and
major upheaval of a renal replacement program. The height may underestimate the degree of loss of muscle tis-
involvement of a skilled social worker is essential in all sue because there is often subclinical expansion of ECF.
cases. The commitment of the family to the child must be
assessed. If the disease is congenital, particularly if pro-
State of Hydration
longed hospitalization has been required or an inappropri-
ately pessimistic prognosis has been given, problems may Assessment of the state of hydration is a skilled matter. Con-
arise from inadequate mother-child bonding, and the opin- sideration should first be given to total body water and its
ion of a family-oriented child psychiatrist is often helpful. partition between ECF and ICF, then to ECF and its parti-
The impact of the disease on the child’s intellectual, tion between intravascular and interstitial compartments,
emotional, and social progress must be assessed. In general, and finally to intravascular volume and circulatory status (see
renal disease does not retard cognitive development, Chapters 7 and 13). There is, unfortunately, no simple clini-
although there may be some delay if renal failure is severe in cal way to estimate total body water, although some clues
early life. A more common feature is emotional immaturity may be obtained from a review of recent changes in body
amounting to infantilism as a consequence of growth retar- weight, which largely reflect changes in total body water. In
dation, eliciting a response from society more appropriate isotonic states (i.e., with normal plasma sodium concentra-
 20. Clinical Evaluation 395

tion), dehydration of moderate severity implies a deficit of

total body water of some 5% of body weight and severe
dehydration, a deficit of 10%. The partition of fluid between
ICF and ECF is determined by osmotic forces, in effect by
sodium; changes in body water alone without changes in
body sodium principally affect ICF volume and are difficult
to detect clinically. Both ICF contraction and expansion
(water intoxication) principally affect the brain because of
the volume constraints imposed by the skull.
Changes in ECF volume are easier to detect clinically than
changes in ICF volume, although edema is not usually evident
until the ECF has been expanded by some 10%. The partition
of fluid between the intravascular and interstitial compartments
is determined by colloid osmotic forces, in effect the plasma
albumin concentration; and the integrity of the capillary wall.
Expansion of the interstitial compartment results in peripheral
edema, most easily detected by pretibial pitting; expansion of
the intravascular compartment results in hypertension, raised
central venous pressure (CVP), and pulmonary edema. Con-
versely, contraction of the interstitial compartment causes
diminished skin turgor, and a reduction of the intravascular
compartment (hypovolemia) results in hypotension and circu-
latory failure with a low CVP. Occult hypovolemia may be
FIGURE 20.7. Hypertension.
revealed by an exaggerated fall in blood pressure on standing.

should not be delegated to the most junior doctor or nurse.

System Review Technical aspects of measuring the blood pressure are impor-
tant, along with standards of blood pressure for age and
Circulatory Status
height (see Chapter 61). Twenty-four-hour monitoring gives
Hypotension and poor peripheral perfusion, characterized a more accurate assessment of the situation. Sustained hyper-
by cold extremities and a peripheral-to-central temperature tension in children requires full investigation (Fig. 20.7).
gap greater than 2°C indicate poor cardiac output. If the
CVP is low, hypovolemia is the cause; if the CVP is raised, Precordium
the problem is cardiac. Assessment of the CVP is an impor- Cardiomegaly may reflect volume overload or long-stand-
tant observation. In the older child, inspection of the jugu- ing hypertension. Murmurs may indicate a congenital car-
lar venous pressure in the semirecumbent position may be diac anomaly that might be the seat of subacute bacterial
helpful, but it is often impossible to be confident of the sit- endocarditis or may simply reflect a high-output state aris-
uation in a younger child. Cardiac size as shown on chest ing from anemia. Pericarditis with rub is a feature of a
radiograph may be helpful, but the sick child under inten- severe uremic state.
sive care usually requires formal CVP monitoring.
Lungs
Adequacy of ventilation should be assessed clinically by
Cardiorespiratory System
cyanosis, but the anemia that accompanies uremia may
Pulse mask desaturation. If there is any doubt, particularly with
The pulses should be examined and the rate, rhythm, and incipient or actual pulmonary edema, pulse oximetry or
volume recorded. The femoral pulses should always be pal- formal blood gas analysis is essential. Hyperventilation sug-
pated; coarctation of the aorta may present with renal fail- gests metabolic acidosis (Kussmaul respiration). The lungs
ure and/or hypertension. Auscultation for bruits over major should be examined in the conventional manner, the most
arteries (e.g., carotid and renal) may indicate extensive vas- important physical sign being the diffuse fine crepitations
cular disease or renal artery stenosis. at both lung bases, which indicate pulmonary edema.

Blood Pressure
Skin
Frequent observation of the blood pressure is an integral part
of the management of the child with renal disease. It is one Examination of the skin may reveal the maculopapular rash
of the most difficult aspects of the physical examination and of Henoch-Schönlein syndrome typically most severe on
396 IV. Clinical Methods

the buttocks, the painful purple lesions involving the fin- as systemic lupus erythematosus or juvenile rheumatoid
gers or toes of a vasculitis, or the butterfly rash across the arthritis; an infection, such as bacterial endocarditis or shunt
nose and cheeks characteristic of systemic lupus erythema- nephritis; a neoplastic process, perhaps complicated by uric
tosus (see Chapters 45 and 46). Multiple café-au-lait spots acid nephropathy; or hypersplenism as a consequence of the
point to neurofibromatosis, which may be associated with hepatic fibrosis associated with the autosomal recessive form
renal artery stenosis and hypertension; adenoma sebaceum of polycystic kidney disease.
and shagreen patches point to tuberous sclerosis; and
angiokeratoma to Fabry’s disease. Dystrophic nails suggest Bladder
the possibility of the nail-patella syndrome. Ichthyosis is Enlargement of the bladder should be sought by palpation and
associated with a lethal variant of Fanconi syndrome. percussion. If detected, the child should be asked to pass urine
Inspection of the mouth is an important part of the and then be reexamined. It is sometimes difficult to detect
physical examination. Poor or mixed primary and secon- bladder enlargement in the neonate when the umbilical cord is
dary dentition is a feature of CRF; gingival hypertrophy still in situ; an ultrasound scan easily resolves the issue.
may be a consequence of cyclosporin toxicity, a tongue too
large for the mouth may point to Beckwith-Wiedemann Genitalia
syndrome (associated with renal medullary dysplasia), and There is a natural but inexcusable tendency to omit exami-
lumps on the tongue suggest the orofaciodigital syndrome nation of the genitalia. The presence of the foreskin in a
(associated with polycystic kidneys). male and the position of the urethral meatus must be ascer-
tained in both genders because anomalies are common.
However, hypospadias is only rarely associated with upper
Abdomen
tract abnormalities. Bilateral cryptorchidism is a feature of
Abdominal Distention prune belly syndrome. Male pseudohermaphroditism with
Abdominal distention in relation to renal disease may be a cryptorchidism is associated with nephroblastoma and glo-
consequence of ascites, renal enlargement because of hydro- merular disease in the Denys-Drash syndrome. Female
nephrosis or tumor, or retention of urine in the bladder. pseudohermaphroditism suggests the possibility of adrenal
Absent abdominal wall musculature, characteristic of the hyperplasia with salt-wasting.
prune belly syndrome and a consequence of intrauterine
abdominal distension, is obvious in infancy but is some- Anus
times less evident in older children or in cases of partial In the neonate, an imperforate or abnormally positioned
absence, and it may be revealed only by asking the recum- anal opening may represent only one feature of complex
bent child to put his arms across the chest and raise his congenital abnormalities, including those affecting the
shoulders off the bed. In affected cases, upward movement renal tract.
of the umbilicus will be observed.
Nervous System
Renal Enlargement
Detection of renal enlargement by palpation is a skilled Neurologic Examination
physical maneuver. The kidneys can often be palpated in a A careful examination of the central nervous system is always
healthy neonate but not in an older child. Palpable enlarge- appropriate, particularly in children who have severe hyper-
ment of the kidneys has the following characteristics: the tension, multisystem disorders, or are in intensive care. Con-
mass can be lifted from the loin, and in contrast to the vulsions may be due to uremia, hypertension, hyponatremia
spleen, it is possible to get above it; there is not much or hypernatremia, hypocalcemia, hypomagnesemia, or disor-
movement of the mass with respiration; and it is resonant ders such as hemolytic uremic syndrome or a systemic vascu-
to percussion anteriorly because the bowel intervenes. litis, which affects both the kidney and the brain. Tetany may
Some inferences can be drawn from the nature of the mass, be due to hypocalcemia or alkalosis.
although it is difficult to distinguish clinically between the Severe hypertension may present with a facial palsy, may
firm enlargement of polycystic disease and tumor or, in an cause a cerebral hemorrhage, and, if the blood pressure is
infant, between the soft enlargement of hydronephrosis and controlled too vigorously, may be associated with anterior
a multicystic kidney. visual pathway infarction, blindness, and abnormal pupil-
lary responses.
Hepatosplenomegaly
The causes of hepatic enlargement are legion, among which Eyes
glycogen storage disease should be noted for its association The eyes offer many clues in the evaluation of renal dis-
with glomerulosclerosis (see Chapters 50). Hepatospleno- ease, and the assistance of an experienced pediatric oph-
megaly in association with renal disease has a more restricted thalmologist is helpful. Scleral calcification may be a
differential diagnosis, suggesting a multisystem disorder, such consequence of hypercalcemia or uncontrolled hyper-
 20. Clinical Evaluation 397

phosphatemia. The crystal deposits in the cornea charac- dislocation of the hips is common in infants with renal
teristic of cystinosis are best detected by slit lamp disease and should be sought with great care. Slipped fem-
examination, but the earliest manifestation in this disor- oral capital epiphysis is a feature of uremic osteodystrophy,
der is a peripheral retinal pigmentary disorder. Alport’s and avascular necrosis of the head of the femur may com-
syndrome may be associated with keratoconus or, more plicate corticosteroid therapy, particularly in transplanted
commonly, with a macular abnormality. Buphthalmos is a patients (see Chapter 69). Polydactyly is found in many
feature of Lowe’s syndrome. Aniridia is accompanied by syndromes with a renal component (see Chapter 6) and
an increased incidence of nephroblastoma. Iridocyclitis may be missed because the accessory digit was removed in
may complicate juvenile rheumatoid arthritis and associ- infancy.
ated amyloid nephropathy or may be found in association Deformities of the spine, such as meningomyelocele, are
with idiopathic interstitial nephritis (see Chapter 43). obvious, but subtle signs, such as a patch of pigmentation or
Cataracts occur in many disorders, among which galac- a tuft of hair, may point to an underlying spinal dysraphism
tosemia may be noted for its association with renal tubu- responsible for a neuropathic bladder. Sacral agenesis, in par-
lar dysfunction. Posterior lenticular opacities may be a ticular, is easily overlooked. Unequal leg growth may be a
consequence of long-term corticosteroid therapy and with consequence of spinal abnormalities. Hemihypertrophy is
careful examination are commonly found, but they rarely occasionally associated with nephroblastoma, with or with-
give rise to visual handicap. out Beckwith-Wiedemann syndrome, or with medullary
The examination of the fundus may reveal the ravages of sponge kidney.
hypertension with arterial narrowing, hemorrhages, exu-
dates, and papilledema. Papilledema is also the major sign of Joints
raised intracranial pressure, as, for example, with cerebral Arthropathy is a classic feature of systemic lupus erythemato-
edema in acutely ill children or after corticosteroid with- sus and the Henoch-Schönlein syndrome. It is occasionally
drawal. Diabetic retinopathy tends to parallel nephropathy observed in children with idiopathic membranous nephropa-
and is rarely observed in childhood. Tapetoretinal degenera- thy. Juvenile rheumatoid arthritis may be complicated by
tion is found in some cases of juvenile nephronophthisis (see amyloid or interstitial nephritis or associated with drug
Chapter 35), and retinitis pigmentosa is characteristic of nephrotoxicity. The Lesch-Nyhan syndrome may present as a
Laurence-Moon-Biedl syndrome in which renal failure renal disorder, either as calculi or renal failure in infancy, and
caused by medullary dysplasia is the principal cause of early may be associated with a gouty arthropathy affecting the
death. The retinal changes in infantile oxalosis are striking interphalangeal joints.
(see Chapter 42).

Ears ROUTINE URINALYSIS

Deformities of the external ear may result from oligohy-
dramnios and even in its absence may be associated with Examination of the urine is an integral part of the clini-
renal anomalies. Preauricular tags and branchial fistulae cal evaluation and is the responsibility of the clinician.
are features of the branchiootorenal syndrome. High- In today’s increasingly busy clinical practice, dipstick
tone sensorineural deafness is characteristic of Alport’s testing of the urine is frequently delegated to the clinic
syndrome and may be insidious in its onset, detectable at nurse and microscopy to the microbiology laboratory.
first only by audiography. Nerve deafness is also associ- Nonetheless, the clinician must be conversant with the
ated with some cases of distal renal tubular acidosis (see methods of urinalysis, their interpretation, and their
Chapter 39). Deafness may be a consequence of the pre- sources of error and ensure that the correct procedures
vious administration of aminoglycoside antimicrobials, are followed.
particularly in the neonatal period, or of high dosages of
furosemide.
Dipstick Tests
Urine samples should be inspected and routinely tested for
Skeletal System
blood, protein, and glucose, using commercially available
Bones dipsticks.
The classic features of rickets may be a consequence of the
early osteomalacic phase of renal osteodystrophy or of
Hemastix
renal tubular disease. Hypophosphatemic vitamin D–resis-
tant rickets is a feature of several genetically determined Hemastix rely on the peroxidase-like activity of hemoglobin
renal tubular disorders (see Chapter 11). Genu valgum is to catalyze the reaction of a hydroperoxide with tetramethyl-
common with renal osteodystrophy and should be quanti- benzidine to give a green-blue color; myoglobin also gives a
fied by measuring the intermalleolar distance. Congenital positive reaction. The test is capable of detecting as little as
398 IV. Clinical Methods

150 μg/L of free hemoglobin and is so sensitive that a nega-

tive test excludes significant hematuria.

Albustix
Protein in urine is most conveniently detected by the so-
called protein error of indicators principle with Albustix
(Ames Company). These reagent strips are impregnated with
tetrabromphenol blue buffered with citrate. Protein binds
with the dye and causes a color change from yellow to green
by displacement of the transformation range of the indicator.
Free light chains (Bence-Jones protein) and other low-molec-
ular-weight “tubular proteins” are not readily detected by
Albustix. A dilute urine specimen may give a false-negative
result. False-positive results occur with very alkaline urine,
with concentrated samples, and with those contaminated
with quaternary ammonium salts such as chlorhexidine. If
the stick is left to soak in the urine or if there is a delay in
reading the strip, false-positive readings can also occur.
Occasionally, protein is excreted in the urine in the
upright posture but not when recumbent. Such postural
proteinuria does not indicate renal disease and should be
ascertained by examination of an overnight specimen of
urine. Sustained proteinuria requires full investigation (Fig.
20.8) (see Chapter 21).

Clinistix
Clinistix (Ames Company) detect glucose in urine by a glu-
cose oxidase-peroxidase linked reaction. The system is sen-
sitive to a glucose concentration of approximately 0.5
mmol/L, but it is inhibited to some degree by glutathione FIGURE 20.8. Proteinuria.
and other substances in the urine, so the trace amounts of
glucose in normal urine are below the threshold of the test.
Microscopy and Culture
Urine microscopy in wards or clinics used to be part of the
Multistix
repertoire of every clinical nephrologist. Now, the availabil-
Multistix (Ames Company) combine the above with tests ity of dipsticks for routine urinalysis and the sophistication
for pH (based on a double-indicator principle), osmolality of equipment have tended to transfer urine microscopy to
(based on the change of pKa of certain polyelectrolytes in the laboratory. There are some disadvantages in this: The
relation to ionic concentration), ketones, bilirubin, urobi- immediate relevance of the findings to the clinical situation
linogen, and nitrite. The pH and osmolality are not suffi- is lost, and it is difficult to ensure that the sample is fresh.
ciently accurate for diagnostic purposes; pH should be Midstream urine specimens should be collected and dis-
measured in the laboratory by a pH meter and osmolality patched to the laboratory for microscopy and culture with-
with an osmometer (see Chapter 21). out delay (see Chapter 53).
 21

LABORATORY INVESTIGATIONS
JEAN-PIERRE GUIGNARD
FERNANDO SANTOS

The validity of the recommendation by the American Acad- as insulin and parathormone. Smaller peptides, such as angio-
emy of Pediatrics that all asymptomatic children have screen- tensin and vasopressin, are directly catabolized by peptidases
ing urinalyses at four intervals from infancy through located in the proximal luminal plasma membranes.
adolescence has been questioned recently (1). The usefulness
of routine multidipstick has, on the contrary, been empha-
sized by experienced pediatric nephrologists (2). Routine Types of Proteinuria
dipstick urinalysis is discussed in Chapter 20. The child with
In normal individuals, the major constituent of nonplasma
positive dipstick screening should be investigated to exclude
proteins is the Tamm-Horsfall protein (Uromodulin) (5). It
or confirm the presence of renal disease.
is a glycoprotein (MW, 95 kDa) actively secreted by the
tubular cells in the ascending thick limb. The Tamm-Hors-
fall protein has no clinical significance. Significant pro-
PROTEINURIA
teinuria can be classified into (a) glomerular proteinuria,
(b) tubular proteinuria, and (c) overflow proteinuria.
Pathophysiology of Proteinuria
The normal glomerulus restricts the filtration of proteins on
Glomerular Proteinuria
both size and electrical charge. The size selectivity of the glo-
merular capillary is largely determined by the cellular layers Altered permselectivity of the glomerular barrier results in
rather than by the glomerular basement membrane (GBM) (3). the abnormal passage of an increased fraction of the glo-
With a molecular weight (MW) of 69 kDa, albumin passes the merular ultrafiltrate through the large nonselective pores
filter in small quantities, so that the ultrafiltration in Bauman’s (3). The altered permselectivity may be the consequence of
capule contains 10 mg/L or less of albumin. The permeability physical damage to the filtering membranes or may be tran-
of the glomerular capillary to large protiens also depends on the sient and hemodynamically or hormonally mediated (6).
electrical change. The glomerular capillary wall is indeed Glomerular proteinuria may be selective or nonselective.
charged negatively due to its rich heparan sulfate content (4). Selective proteinuria is characterized by the predominance
Because of the electrostatic interaction with the filtration bar- of albumin as compared to other proteins of higher MW.
rier, negatively charged macromolecules are more restricted Albumin, immunoglobulin G (IgG), and transferrin
than are neutral or positively charged molecules of similar MW. are proteins used to characterize the selectivity of glo-
The electrical charge does not influence the filtration of small merular proteinuria. A permeability index based on the
molecules of ions. The combined impairment of both bar- IgG/albumin or the IgG/transferrin clearance ratios can
rier charge selectivity and size selectivity is required to be used. An index below 0.1 is indicative of highly selec-
account for the massive proteinuria observed in nephrotic tive proteinuria (7). The clinical usefulness of this index
patients. is questionable. A high selectivity index is usually found
Almost all of the filtered proteins are reabsorbed in the prox- in the steroid-sensitive minimal changes nephrotic syn-
imal tubule by endocytosis at the luminal membrane. Signifi- drome as well as in the Finnish-type congenital neph-
cant proteinuria occurs when this energy-requiring mechanism rotic syndrome. The selectivity is low in orthostatic
is saturated. In the tubular cells, the proteins are degraded by proteinuria (8). The excretion rate of urinary proteins
lysosomal enzymes to low-molecular-weight (LMW) fragments and/or albumin is probably the best independent pre-
and amino acids. By this mechanism, the kidney plays a major dictor of end-stage renal disease in nondiabetic pro-
role in the catabolism of many polypeptide hormones such teinuric chronic nephropathies (9).
400 IV. Clinical Methods

TABLE 21.1. MARKERS USED FOR THE CLASSIFICATION OF PROTEINURIA

Excretion/creatinine

Diagnosis Marker MW (d) g/mol creatinine mg/mol creatinine

Glomerular proteinuria Proteins — <20 <180

Glomerular selectivity Albumin 67'000 <1.17 <0.21
Transferrin 80'000 — —
Immunoglobulin G 150'000 <5.8 <50
Tubular proteinuria β2-microglobulin 11'800 <0.040 <0.35
α1-microglobulin 30'000 <2.2 <19
Retinol-binding protein 21'400 <0.024 <0.21
Tubular injury Lysozyme 14'400 <0.065 <0.6

The loss of charge selectivity of the GBM can be assessed by two enzymes is increased in various conditions associated
the clearance of two proteins of similar MW but with different with acute tubular necrosis. N-acetyl-glucosaminidase is sta-
electric charges. Two pairs of molecules can be used: (a) the bler than alanine-aminopeptidase. Other enzymes may
nonglycated and the negatively glycated albumin and (b) the detect an injury of the proximal tubular cells (α-glutathione
neutral IgG and the negatively charged IgG4 subclass. The transferase) or the distal tubular cells (π-glutathione trans-
ratio of their clearances is modified when the GBM has lost its ferase). Lysozyme (MW, 14.4 kDa) is a marker of tubular
charge selectivity (3). Loss of glomerular anionic charges may proteinuria and of acute tubular necrosis (11) but is less sen-
be the first step in the development of some nephropathies, as, sitive than other markers.
for instance, type 1 diabetic nephropathy.

Quantification of Urinary Proteins

Tubular Proteinuria
Reagent Test Strips
Most LMW proteins (MW below 40 kDa) are filtered and
reabsorbed in the proximal tubule. Impaired proximal Positive dipstick screening for proteinuria warrants quanti-
tubular reabsorption of LMW proteins results in tubular fication of the proteinuria.
proteinuria. This is observed in tubulopathies such as the
Fanconi syndrome. Drug (aminoglycosides) or heavy-metal Protein/Creatinine Ratio
intoxication may induce tubular proteinuria.
The exclusive presence of LMW proteins in the urine The protein/creatinine (prot/creat) ratio is measured in a
characterizes the tubular proteinuria. Three markers are random urine sample. The second morning urine specimen
commonly used (Table 21.1) (10). is most suitable. The urine prot/creat ratio correlates well
with the 24-hour urine protein excretion (13,14). Compar-
1. β2-microglobulin, which is not bound to proteins, is ison of the ratio measured in the first morning urine speci-
easy to measure, but is unstable in acid urine. men with that of urine collected during the day is used for
2. α1-microglobulin is stabler than β2-microglobulin but the diagnosis of orthostatic proteinuria. Normal values of
has a higher MW and thus a more variable filterability. the urine prot/creat ratio observed at different ages are
3. Retinol-binding protein is stable at low urine pH, but given in Table 21.2. By definition, nephrotic range pro-
it strongly binds to plasma proteins. teinuria is superior to 400 g/mol creatinine. The albumin-
uria can also be expressed as an albumin/creatinine ratio.
The upper-normal value is 1.17 g/mol creatinine.
Overflow Proteinuria
An unusual increase in the protein filtered load results in
Quantitative Proteinuria
significant proteinuria. Overflow proteinuria may be seen
in multiple myeloma (Bence-Jones proteins), hemoglobin- Quantitative urine protein excretion can be made using a
uria, and myoglobinuria. 24-hour urine collection, an overnight collection, or a
shorter urine collection over a few hours. The 24-hour
collection in children is cumbersome for the patient and
Markers of Acute Tubular Necrosis
subject to errors. Normal values are defined as less than 4
Two enzymes synthesized in the proximal tubular cells, the mg/m2/hr or 100 mg/m2/day. Values above 40 mg/m2/hr
lysosomal enzyme N-acetyl-glucosaminidase and alanine- define the nephrotic range proteinuria. Microalbuminuria
aminopeptidase, present in the brush border are urinary is defined as an albumin excretion between 0.7 and 7.0
markers of tubular necrosis. The urinary excretion of these mg/m2/hr or 170 mg/m2/day.
 21. Laboratory Investigations 401

TABLE 21.2. NORMAL VALUES OF PROTEIN nal absorption, extrarenal losses, and internal distribution of
EXCRETION AS A FUNCTION OF AGE the solute in the body. Solute excretion varies with age.
Proteinuria Urine solute excretion can be expressed in mg or mmol/
day/kg or per m2, by the solute/creatinine concentration ratio,
Age (yr) g/mol creatinine g/g creatinine
and/or by calculating the solute fractional excretion (FE). Sol-
0.1–0.5 80 0.70 ute/creatinine concentration ratios and FE can be calculated in
0.5–1.0 60 0.55 random urine samples without the need for timed urine col-
1–2 45 0.40 lection. Calculation of FE requires drawing a blood sample at
2–3 30 0.30
3–5 20 0.20
the time of urine collection. The FE is calculated as follows:
5–7 19 0.15 FES = (US/PS) × (PCr/UCr) × 100, where US and PS represent
7–17 18 0.15 the urine and plasma concentrations of a solute (S) and PCr
and UCr represent the plasma and urinary concentrations of
creatinine, respectively. FES expresses the percent of filtered
solute that is excreted in the urine. This concept is equivalent
Analytic Procedures
to the fractional clearance of S (FCS), which is calculated with
The urinary proteins are usually measured by an autoana- the same formula but expressed as mL/dL of glomerular fil-
lyzer-adapted turbidimetry micromethod that uses benze- trate. Calculation of glomerular filtration rate (GFR) by means
thonium chloride to precipitate the urinary proteins at an of the creatinine clearance is GFR = (UCr / PCr) × V, where V
alkaline pH. The quantification of albuminuria is achieved represents the mL of urine eliminated by minute and GFR is
by enzyme-linked immunoabsorbent assays, immunoturbi- expressed in mL/min. For a GFR = 100 mL/min, V = 100 ×
dimetry, or laser nephelometry (12). (PCr / UCr) and represents the mL of urine eliminated by 100
Electrophoretic separation of urinary proteins by means mL of GF. Thus, FCS may be calculated by the formula FCS =
of polyacrylamide or agarose gel electrophoresis is used to US / PS × (PCr / UCr) × 100, which is identical to that used to
distinguish glomerular, tubular, and mixed proteinuria. calculate FES. FCS represents the mL of each 100 mL of GF
Different markers are used to differentiate glomerular from that have been cleared of S and is expressed in mL per 100 mL
tubular proteinuria (Table 21.1). GFR. As noted in the above formula, its calculation is inde-
pendent of the volume of urine and, therefore, does not
require timed urine collection.
Etiology of Proteinuria For those solutes not undergoing tubular secretion, the dif-
ference 100 – FES represents the percentage of solute that is
Orthostatic Proteinuria
reabsorbed. Thus, the tubular reabsorption of phosphate
Orthostatic proteinuria is only present when the patient is (TRP) equals 100 – FEP and represents the percentage of fil-
upright. The proteinuria is usually mild and not selective tered phosphate that is reabsorbed in the convoluted proximal
(8). Orthostatic proteinuria is often seen in slender adoles- tubule. The TRP is frequently expressed as TmP/GFR. TmP,
cent patients. The prognosis of orthostatic proteinuria is representing the maximum tubular phosphate reabsorption,
excellent. No treatment is needed. may be calculated by using phosphate infusions (see Glucose
Titration Test and Fig. 21.1). However, TmP can also be
deduced using a more practical approach. TRP is the differ-
Transient Proteinuria ence between filtered phosphate and excreted phosphate. Indi-
Transient proteinuria is usually associated with exercise, viduals on a normal Western diet ingest a large amount of
stress, fever, and dehydration. It does not reflect renal disease. phosphate. Therefore, in the presence of normal GFR, the rate
of phosphate reabsorption approaches the maximal capacity of
proximal tubules to reabsorb phosphate. Thus, TmP = GFR ×
Persistent Proteinuria SP – UP × V, where SP and UP are the phosphate concentra-
tions in serum and urine. In this equation, SP represents the
Persistent proteinuria indicates renal disease. Proteinuria
renal threshold for phosphate (as explained in the section Glu-
may per se also lead to renal injury (15) and should be thor-
cose Titration Test) when UP × V is equal to 0. Therefore,
oughly investigated (16).
TmP/GFR value equals the threshold concentration for serum
phosphate and is considered as the best indicator of the renal
tubular handling of phosphate (17).
URINE SOLUTE EXCRETION
According to Bijvoet et al., TmP/GFR can be calculated as
follows: TmP/GFR = TRP × SP, or TmP/GFR = [1 – (UP × SCr
Quantification
/ SP × UCr) ] SP, or TmP/GFR = SP – (UP × SCr / UCr) (18).
The renal elimination of a given solute is highly dependent on A nomogram for the determination of TmP/GFR based
a number of factors such as diet content, water intake, intesti- on the plasma phosphate concentration and TRP has been
402 IV. Clinical Methods

the extracellular fluid volume is contracted (i.e., dehydration,

nephrotic syndrome), sodium can be virtually undetectable
in the urine. Although there is no predictable relationship
between the plasma sodium concentration and urinary
sodium excretion, mild hypernatremia has recently been
shown to induce natriuresis in humans (24). Normal urinary
sodium/creatinine ratios in children are shown in Table 21.3.
Urinary chloride excretion is closely associated with that
of sodium. Chloride excretion is dependent on the salt con-
tent of diet, and it is also retained during volume depletion.
The measurement of urinary chloride concentration may be
particularly useful in the diagnostic approach to metabolic
alkalosis, because urine sodium and chloride concentrations
may be dissociated. A urinary chloride concentration above
10 mmol/L is indicative of metabolic alkalosis of renal ori-
FIGURE 21.1. Representative renal titration curve for a sub- gin, whereas lower concentrations of urinary chloride or a
stance (X) with an active and saturable mechanism of proximal reduced chloride/creatinine concentration ratio (25) suggests
tubular reabsorption, like that of glucose or phosphate.
volume contraction as the cause of the metabolic alkalosis.
However, even in the presence of volume contraction and
developed by Walton and Bijvoet (19). The usefulness of active renal reabsorption, urinary sodium may be high
this nomogram in children has been questioned, the use of because it is eliminated in the urine with the bicarbonate
TmP/GFR or the simple use of TP/GFR in well-nourished anion to keep electroneutrality. Normal urinary chloride/cre-
children appearing more relevant (20–23). atinine values are given in Table 21.3.
Urinary potassium excretion ranges from 1 to 2 mmol/
kg/day in a child on a normal diet. The kidney regulates
Sodium, Chloride, and Potassium
potassium balance by changing the secretion rate of potas-
The daily urinary excretion of sodium varies widely in sium in the distal nephron. This regulation is mostly
response to change in dietary salt intakes. On a normal West- dependent on aldosterone and the amount of sodium and
ern diet, children excrete approximately 3 to 4 mmol/kg/day water delivered to the distal nephron. The renal ability to
of sodium. Body fluid composition requires independent retain potassium is not as efficient as that of sodium, and,
regulation of sodium and water balances as intake of water in the face of potassium depletion, urinary potassium does
and sodium may vary separately. Thus, concentration of not decrease to less than 15 mmol/L. Normal urinary
sodium in the final urine is highly related to the degree of potassium/creatinine ratios are shown in Table 21.3.
urinary concentration or dilution. The extracellular fluid vol- The excretion of sodium, chloride, and potassium is fre-
ume is regulated by changes in sodium excretion and when quently expressed as FE rates. In European children older

TABLE 21.3. URINARY SOLUTE/CREATININE (CREAT) RATIOS (FIFTH AND NINETY-FIFTH PERCENTILES) AS A
FUNCTION OF AGE
Age (yr)

Solute/creat 1/ –1 1–2 2–3 3–5 5–7 7–10 10–14 14–17

12

mol/mol P5 P95 P5 P95 P5 P95 P5 P95 P5 P95 P5 P95 P5 P95 P5 P95

Sodium/creat 2.5 54 4.8 58 5.9 56 6.6 57 7.5 51 7.5 42 6 34 — 28

Potassium/creat 11 74 9 68 8 63 6.8 48 5.4 33 4.5 22 3.4 15 — 13
Calcium/creat 0.09 2.2 0.07 1.5 0.06 1.4 0.05 1.1 0.04 0.8 0.04 0.7 0.04 0.7 0.04 0.7
Magnesium/ 0.4 2.2 0.4 1.7 0.3 1.6 0.3 1.3 0.3 1 0.3 0.9 0.2 0.7 0.2 0.6
creat
Phosphate/ 1.2 19 1.2 14 1.2 12 1.2 18 1.2 5 1.2 3.6 0.8 3.2 0.8 2.7
creat
Oxalate/creat 0.06 0.17 0.05 0.13 0.04 0.1 0.03 0.08 0.03 0.07 0.02 0.06 0.02 0.06 0.02 0.06
Urate/creat 0.7 1.5 0.5 1.4 0.47 1.3 0.4 1.1 0.3 0.8 0.26 0.56 0.2 0.44 0.2 0.4

Adapted from Matos V, Melle G van, Boulat O, et al. Urinary phosphate/creatinine, calcium/creatinine, and magnesium/creatinine ratios in a
healthy pediatric population. J Pediatr 1997;131:252–257; and Matos V, Melle G van, Werner D, et al. Urinary oxalate and urate to creatinine
ratios in a healthy pediatric population. Am J Kidney Dis 1999;34:1–6.
 21. Laboratory Investigations 403

than 1 year of age, not fasted, and on a normal diet, FENa has 21.3 (31). Calculation of FEMg allows an assessment of mag-
been shown to range from 0.3 to 1.6%, this variation being nesuria in relation to the level of plasma magnesium. Under
the result of the different dietary salt intakes (26). Calcula- normal conditions, approximately 5% of filtered magnesium is
tion of FENa is widely used to explore whether the kidney excreted in urine, but in the face of magnesium deprivation,
responds adequately to extracellular volume depletion. In FEMg quickly falls below 1% (38). Thus, an FEMg equal to or
this situation, FENa is expected to fall as a result of compensa- greater than 5% with simultaneous hypomagnesemia indicates
tory avid sodium reabsorption. Thus, in patients with prere- inappropriate loss of magnesium in the urine.
nal acute renal failure, FENa is expected to fall below 1%,
whereas it is generally above 3% in acute tubular necrosis. In
Uric Acid and Oxalate
normal conditions, FEK approximates 10 to 15%. Values of
FEK above 100% can be found in renal failure patients as a Elevated urinary concentrations of uric acid and oxalate and
compensatory mechanism to maintain potassium balance. decreased levels of urinary citrate are significant risk factors
for the development of urolithiasis and/or nephrocalcinosis.
It has been shown that the urinary excretion of uric acid cor-
Calcium
rected to 1.73 m2 is independent of age in children aged 2 to
Urinary elimination of calcium is highly dependent not 15 years, mean values being 520 ± 147 (SD) mg/1.73 m2/
only on the diet but also on the child’s age. Although the day (3093 ± 874 μmol/1.73 m2/day) (39). Calculation of
range of normal urinary calcium elimination may vary in urine uric acid concentration corrected for creatinine clear-
different geographical regions, hypercalciuria is usually ance in single fasting morning urine and blood specimens
defined as urinary calcium excretion rates above 4 to 5 mg/ (urine uric acid × plasma creatinine/urine creatinine) has
kg/day (0.100 to 0.125 mmol/kg/day) and is suspected been used as a diagnostic criterion of hyperuricosuria,
when the calcium/creatinine ratio is above 0.20 mg/mg defined by a value above 0.53 mg/dL of GFR (32 μmol/L of
(0.60 mol/mol) (27–29). The upper-normal limit of the GFR) in children between 2 and 12 years of age (40,41).
urinary calcium/creatinine ratio is much higher in infants Mean values of urinary oxalate have been reported to be 36.9
(30). Recent reference values for urinary calcium to creati- ± 13.7 (SD) mg/1.73 m2/day (0.42 ± 0.16 mmol/1.73 m2/
nine concentration ratios in spot urines of children of dif- day) and 1.0 ± 0.6 (SD) mg/kg/day (0.01 ± 0.06 mmol/kg/
ferent ages have been provided by Matos et al. and are day) in Spanish and Italian children older than 3 years,
shown in Table 21.3 (31). Under identical dietary condi- respectively (33,42). Reference values for urinary oxalate and
tions, urine calcium has been shown to be lower in black urate to creatinine ratios in infants, children, and adolescents
than in white children (32). are shown in Table 21.3 (43). There is a high positive correla-
tion (R = 0.75) between oxalate excretion measured in 24-
hour urine and the oxalate/creatinine ratio measured in the
Phosphate
first morning urine sample (44).
Similarly to calcium, phosphaturia expressed per unit of
weight, as well as phosphatemia, markedly decreases with
Citrate
age. In adults, normal urinary phosphate excretion equals
10 to 15 mg/kg/day (0.3 to 0.5 mmol/kg/day), whereas val- Values of citrate excretion in healthy children aged 3 to 14
ues up to 20 to 25 mg/kg/day (0.6 to 0.8 mmol/kg/day) are years followed a normal distribution and were equal to 9.62
found in young children. Recent reference values for uri- ± 4.05 (SD) mg/kg/day (45.78 ± 19.28 μmol/kg/day) (45).
nary phosphate to creatinine concentration ratios in chil- A moderate correlation (R = 0.49) was found between
dren are shown in Table 21.3 (31). Values of TP/GFR vary citruria determined in 24-hour urine specimens and cit-
from 6.9 in neonates to 4.4 at 5 years of age and 3.2 mg/dL rate/creatinine concentration ratios in the second morning
GFR at 6 years of age (21). urine samples. A citrate to creatinine concentration ratio
measured in 24-hour urine below 300 mg/g (0.176 mol/
mol) in girls and 125 mg/g (0.074 mol/mol) in boys has
Magnesium
been proposed as indicative of hypocitruria (41,46). Blau et
Daily elimination of urinary magnesium has been reported to al. have recently reported the simultaneous determination of
decrease from a mean value of 2.44 ± 0.69 (SD) mg/kg/day oxalate, glycolate, citrate, and sulfate in spot urine samples
(0.10 ± 0.03 mmol/kg/day) in Italian children 3 to 5 years of dried on filter papers (47). This procedure may facilitate the
age to 1.77 ± 0.65 (SD) mg/kg/day (0.07 ± 0.03 mmol/kg/day) study of these anions in large series of children.
in children 15 to 16 years old (33). These values are quite simi-
lar to those obtained in other European studies and markedly
Anion Gap
higher than those found in children from Oriental origin (34–
37). Reference values for urinary magnesium to creatinine con- Metabolic acidosis may be classified according to whether it
centration ratios in a pediatric population are shown in Table is associated with elevated or normal serum anion gap
404 IV. Clinical Methods

(AG). To keep the electroneutrality of the extracellular In adults, a TTKG value greater than 5 indicates that
fluid, the sum of the cation concentrations must equal that aldosterone is acting, whereas a value at or below 3 indi-
of the anions. This can be expressed by the equation: Na+ + cates lack of mineralocorticoid activity (52). In the face of
UC = Cl– + HCO3– + UA, where UC and UA represent hyperkalemia, a TTKG below 4.1 in children or below 4.9
unmeasured cations and unmeasured anions, respectively. in infants should be taken as indicative of a state of relative
UC includes K+, Ca2+, and Mg2+, whereas PO43–, SO42–, hypo- or pseudohypoaldosteronism. A TKKG higher than
proteins, and organic anions form the UA. The serum AG 2 in a hypokalemic child should be taken as indicative of
is defined by the formula AG = Na+ – (HCO3– + Cl–) = UA lack of aldosterone suppression (53).
– UC. The normal values range from 8 to 16 mmol/L.
A related concept is that of the urine AG, which can be
calculated by the formula: Urine AG = (Na+ + K+) – Cl–, GLOMERULAR FILTRATION RATE
where Na+, K+, and Cl– represent the urinary concentrations
of these solutes in mmol/L (48–50). During metabolic aci- GFR is the best estimate of the functional renal mass. It is
dosis, urine AG is a rough and inverse estimate of the ammo- the most widely used indicator of kidney function in
nium (NH4+) eliminated in the urine. If large amounts of patients with renal disease. The severity and the prognosis
NH4+ are present in urine, the urinary chloride concentra- of the disease are predicted on this parameter. The assess-
tion is greater than the sum of sodium and potassium con- ment of GFR is thus invaluable in the follow-up of patients
centrations, and urinary AG becomes negative. Thus, in with progressive renal failure. It is also of great value in a
subjects with type II proximal renal tubular acidosis (RTA) variety of clinical conditions, as an estimate of GFR may be
or in patients with metabolic acidosis associated with diar- required to rationally prescribe fluids, electrolytes, or drugs
rhea, urine AG is negative. In acidotic patients with impaired excreted by the kidney.
NH4+ excretion, such as those with type I RTA, type IV
RTA, or chronic renal failure, urine AG remains positive.
Physiology of Glomerular Filtration
Ultrafiltration occurs through the permselective capillary wall.
Transtubular Potassium
Ultrafiltration is driven by Starling forces across the glomerular
Concentration Gradient
capillaries. Changes in renal perfusion and in the Starling
The transtubular potassium concentration gradient (TTKG) forces alter GFR. Both renal blood flow (RBF) and GFR are
provides an estimation of the regulatory mechanisms of dis- held within narrow limits by the phenomenon of autoregula-
tal potassium secretion, thus facilitating the clinical approach tion. Autoregulation is an intrarenal mechanism with two
to disorders of potassium excretion. The measurement of components: (a) the “myogenic mechanism” and (b) the “tub-
the final concentration of potassium in urine gives limited uloglomerular feedback mechanism.” Both mechanisms repre-
information because this concentration depends not only sent an effective means for uncoupling renal perfusion and
on the distal secretion of potassium induced by aldosterone GFR from abrupt changes in blood pressure.
but also on the degree of water reabsorption along the med- The glomerular barrier filters molecules on the basis of
ullary collecting duct. size and electric charges. It behaves as if it were a filtering
The TTKG index attempts to evaluate the gradient membrane containing aqueous pores with a diameter of 7.5
between luminal and peritubular potassium concentrations to 10.0 nm. Molecules with a diameter smaller than 3.6 nm
in the late distal tubule and the cortical collecting duct as a are filtered freely. Molecules with a diameter larger than 7.2
reflection of aldosterone bioactivity. The test assumes that nm are not filtered. Albumin, with an effective diameter of
the systemic venous plasma potassium equals the peritubu- 7.1 nm, is filtered in minute amounts. The electrical
lar potassium concentration in the renal cortex and that the charges of the molecules play an important role in their fil-
luminal potassium concentration in the distal tubule and terability. Filtration of large anionic molecules is restricted
the cortical collecting duct can be deduced by dividing the by the presence of negatively charged glycoproteins on the
urine potassium concentration by the urine to plasma surface of all components of the glomerular membrane bar-
osmolality ratio—that is, adjusting the urinary potassium rier. In the glomerulus, the MW cut-off for the filter is
concentration for medullary water reabsorption. These approximately 70 kDa. With an MW of 69 kDa, albumin
assumptions require that sufficient sodium be delivered to passes through the filter in minute quantities. The glomer-
the distal nephron for exchange with potassium and that ular filter is freely permeable to those molecules with an
urine be hypertonic (51,52). MW less than 7 kDa. Filtration of proteins, with conse-
Based on the preceding rationale, TTKG can be calcu- quent proteinuria, is increased in a number of glomerular
lated as follows: diseases associated with the loss of negative charges on the
TTKG = UK/(UOSM/POSM)/PK , or glomerular filtration barrier.
The GFR is proportional to the sum of the Starling
TTKG = (UK × POSM)/(PK × UOSM) forces across the glomerular capillaries (ΔP – Δπ) times the
 21. Laboratory Investigations 405

ultrafiltration coefficient (Kf): GFR = Kf × (ΔP – Δπ), Standard Urinary Clearance

where ΔP and Δπ represent the capillary transglomerular
Endogenous Markers
hydrostatic and oncotic pressure difference, respectively.
The urinary excretion of the endogenous marker is mea-
Kf is the product of the intrinsic permeability of the glo-
sured over a few hours, and its excretion rate is divided by
merular capillary and the glomerular surface area available
the plasma concentration present during the urine collec-
for filtration. The permeability of the glomerular capillaries
tion period. When urine flow rate is expressed in mL/min/
is 100 times higher than that of the capillaries elsewhere in
1.73 m2, the standard urinary clearance (UV/P) formula
the body.
also expresses GFR in mL/min/1.73 m2. Duration of the
collection may last from 3 to 24 hours. Short periods are
Concept of Clearance best in children to avoid inaccuracies in urine collection.
Creatinine is the endogenous marker that is used for assess-
The most common measurement of GFR is based on the ing GFR.
concept of clearance, which relates to the volume of plasma
that in a given unit of time would be completely “cleared” Exogenous Markers
of a substance. The clearance of substance (x) is expressed The exogenous marker is infused at a constant rate over 3 to
by the formula Cx = Ux × V/Px, where V represents the 4 hours to achieve constant plasma concentrations. To
urine flow rate and Ux and Px the urine and plasma concen- shorten the time of equilibration, a priming dose of the
tration of substance x, respectively. marker is administered at the onset of the clearance study.
For its plasma clearance to be equal to the rate of glomeru- When stable plasma concentrations are attained, urine and
lar filtration, a marker must have the following properties: blood samples are collected at regular intervals, and the UV:P
(a) it must be freely filterable through the glomerular capillary ratio is calculated. The ideal exogenous marker is inulin.
membranes—that is, not bound to the plasma proteins or Other markers include iohexol and unlabeled iothalamate.
sieved in the process of ultrafiltration; (b) it must be biologi-
cally inert and neither reabsorbed nor secreted by the renal
tubules; (c) it must not be toxic; and (d) it must not alter Constant Infusion Technique
renal function when infused in quantities that permit ade- without Urine Collection
quate quantification in plasma and urine. Several substances, The constant infusion technique assumes that the rate of
endogenous or exogenous, have been claimed to have the infusion (IR) of a marker (x) needed to maintain constant
preceding properties: creatinine, inulin, iohexol, and three its plasma concentration is equal to the rate of its excretion
compounds labeled with radioisotopes: diethylenetriamine- (54,55). After equilibration of the marker in its distribution
penta-acetic acid (DTPA), ethylenediaminetetra-acetic acid space, the excretion rate must thus be equal to the IR,
(EDTA), and sodium iothalamate. The experimental evi- hence the formula:
dence that this is true has only been produced for inulin.
Although inulin is the most accurate marker, creatinine is the Cx = Ux × V/Px = IRx/Px
most commonly used in children. The flow rate of the test solution containing the marker is
expressed in mL/min/1.73 m2 and so is the clearance Cx.
To accelerate the achievement of a steady concentration
Methods Available for Assessing
of the marker, a loading dose precedes the constant intrave-
Glomerular Filtration Rate
nous (i.v.) infusion. The constant infusion method may
A variety of markers (Table 21.4) can be used to assess GFR overestimate GFR if steady concentration of the marker is
using different methods, the principles of which are not achieved when the plasma concentration is measured.
described in the following sections. Depending on the conditions, the equilibration of the

TABLE 21.4. CHARACTERISTICS OF THE GLOMERULAR MARKERS

Inulin Creatinine Iohexol DTPA EDTA Iothalamate

Molecular weight 5200 113 811 393 292 637

Elimination half-life 70 200 90 110 120 120
(min)
Plasma protein binding 0 0 <2 5 0 <5
(%)
Space of distribution EC TBW EC EC EC EC

DTPA, diethylenetriaminepenta-acetic acid; EC, extracellular space; EDTA, ethylenediaminetetra-acetic

acid; TBW, total body water.
406 IV. Clinical Methods

marker within its distribution space may only be achieved an estimate of GFR derived from the child’s creatinine
after several hours of constant infusion. In newborn plasma concentration and height.
infants, the proportionally greater extracellular space and
GFR = k × Height/Pcreat
the low GFR require a much longer period for steady
plasma concentrations to be attained. Inulin is the marker where k is a constant, Height represents the body height,
of choice for this technique, which is used when urine col- and Pcreat the plasma creatinine concentration. This formula
lection is not possible. is based on the assumption that creatinine excretion is pro-
portional to body height and inversely proportional to
plasma creatinine (57). The value of factor k can be
Plasma Disappearance Curve (Single
obtained from the formula k = GFR × Height/Pcreat. Under
Injection Technique)
steady state conditions, k should be directly proportional to
The mathematical model for the plasma disappearance curve the muscle component of body weight, which corresponds
(single injection technique) is an open, two-compartment reasonably well to the daily urinary creatinine excretion
system (56). The glomerular marker is injected in the rate.
first compartment, equilibrates with the second com- The serum concentration of cystatin C has been pro-
partment, and is excreted from the first compartment by posed to assess GFR. Although the serum concentration of
glomerular filtration. The plasma disappearance curve of cystatin C may be superior to Pcreat in distinguishing nor-
the marker follows two consecutive patterns. In the first, mal from abnormal GFR, a rational numerical estimate of
the plasma concentration falls rapidly but at a progres- GFR cannot be derived from its plasma concentration (58).
sively diminishing rate. This reflects the diffusion of the
marker in its distribution volume as well as its renal
excretion. In the second, the slope of the decline of the Estimation of Glomerular Filtration Rate in
plasma concentration reflects its renal excretion rate Clinical Practice with Different Markers
only. During this phase, the marker concentration decreases
Inulin
at the same exponential rate in all the compartments
where it is distributed. The Marker
To obtain a well-defined plasma disappearance curve, Inulin, a fructose polysaccharide derived from dahlia roots
and therefore an accurate calculation of the plasma clear- and Jerusalem artichokes, has an Einstein-Stokes radius of
ance, numerous blood samples are required. Extension of 1.5 nm and an MW of approximately 5.2 kDa. It diffuses
the sampling period to 4 to 5 hours improves the precision as would a spherical body of such radius. Inulin is inert, not
of the results. Simplified techniques have been proposed metabolized, and can be recovered quantitatively in the
that are based on a single-compartment model. They obvi- urine after parenteral administration. Evidence that inulin
ate the need for frequent blood sampling but result in in neither reabsorbed nor secreted by the renal tubules has
greater accuracy. been well demonstrated in experimental micropuncture
The plasma disappearance curve is most often used studies (59,60).
when assessing GFR with radionuclides. Infusion of radio- The rate of excretion of inulin is directly proportional to
nuclides for several hours is indeed undesirable, and they the plasma concentration of inulin. Its clearance (UV/P) is
are best reserved for single injection studies in which the consequently independent of its plasma concentration.
radioisotope is more rapidly eliminated from the kidney. Because the renal excretion of inulin occurs exclusively by
Commonly used markers include technetium 99m (99mTc)- glomerular filtration, its clearance is the most accurate index
DTPA, chromium 51 (51Cr)-EDTA, and iodine 125 (125I)- of GFR. Estimates of inulin clearance provide the basis for a
iothalamate, and, more recently, cold iohexol. Clearance standard reference against which the route or mechanism of
values derived from the plasma disappearance curve should excretion of other substances can be ascertained.
be analyzed critically, particularly in patients with reduced Sinistrin is a readily soluble preparation of polyfructose
GFR and those with edema and circulatory disturbances. with side branching (extracted from the bulb of Urginea mar-
itima), which is more suitable for clinical use and readily
available in Europe. This compound is identical to inulin.
Plasma Concentration
The concentration of endogenous markers, such as creati- Measurement of Inulin
nine, increases when GFR decreases. The increase in In the classical anthrone method, inulin is hydrolyzed to
plasma creatinine is not linear, however. Several attempts fructose by sulfuric acid, and the fructose is then measured
have thus been made to develop reliable methods that will by colorimetry (61). Carbohydrates present in the plasma
allow a correct estimate of creatinine clearance from its and urine also react in the assay (62). The method has a vari-
plasma concentration (Pcreat) alone, without urine collec- ation coefficient close to 6%. A specific, highly sensitive,
tion. The following formula has been developed that allows enzymatic method has been described by Kuehnle et al. (63).
 21. Laboratory Investigations 407

The use of sinistrin has been associated with anaphylac-

tic reaction (65) in a 45-year-old white patient, whereas
anaphylaxis from inulin in vegetables has been reported in a
39-year-old man (66).
The rise in GFR from birth to adulthood is illustrated in
Figure 21.2 and given in Table 21.5. Mature values of GFR
range from 100 to 120 mL/min/1.73 m2. In a study by Koop-
man et al. in adult volunteers, GFR, as assessed by inulin clear-
ance, had a circadian rhythm (67). It started to decrease during
late afternoon or evening and rose again at the end of the
night. The mean amplitude of the rhythm was 36 mL/min.

FIGURE 21.2. Inulin clearance as a function of age X ± SD. Constant Infusion Technique of Inulin
without Urine Collection
The constant infusion technique of inulin without urine col-
In this method, inulin is hydrolyzed by insulinase, with lection yields reproducible results, as long as sufficient time is
simultaneous oxidation of the native glucose using glucose given for steady concentrations to be reached (54,55,68).
oxidase and H2O2. The fructose generated by inulinase is This may take as long as 12 hours in adults and even longer
then converted to glucose-6-phosphate. This method has a in infants (68,69). During the equilibration period, inulin is
variation coefficient below 5% in serum and urine samples. excreted by filtration, while at the same time diffusing into
A fully automated enzymatic inulin assay, with minimal sam- the extracellular space. Calculations of the infusion (IR/P)
ple prehandling and capable of complete sinistrin hydrolysis, clearance before equilibration overestimate clearance values
has been described (64). The coefficient of variation was up to 30% above the urinary clearance. The shorter the time
reported at 4.4%. of infusion, the larger is the overestimation. The constant
infusion method is useful when timed urine collections are
Inulin Urinary Clearance difficult or unreliable, such as in small babies, children with
Inulin or sinistrin urinary clearance (UV/P) is the gold stan- hydronephrosis, or those with neurogenic bladder.
dard for the assessment of GFR. In the classical method, inulin The result of the study comparing the “3-hour constant
is administered as a priming dose to achieve plasma concentra- infusion” with the urinary clearance of inulin in 60 children
tions close to 200 to 400 mg/L. It is then constantly infused to older than 1 year showed a significant correlation, but the
maintain constant levels over the 3-hour clearance study. After calculated regression line differed significantly from the line
an equilibration period of 90 minutes, urine and blood sam- of identity, and the scatter of points was considerable (70).
ples are collected at intervals of 30 minutes. The mean value of These results did not confirm an earlier report by Cole et al.
three to four clearance periods is considered as representing the demonstrating virtual superposition of inulin clearance val-
child’s GFR. In small children, the urine samples are collected ues generated with and without urine collection (55). The
through an indwelling bladder catheter. In preschool and constant infusion technique should be reserved for studies
school children, urine is collected by spontaneous voiding. when the precise collection of urine cannot be performed
Generous oral water loading (~300 mL/m2/hr) is given to and its result interpreted with caution.
maintain high urine flow rates. Sugar (e.g., glucose, fructose,
and saccharose)-containing drinks must be avoided, as these Plasma Disappearance Curve of Inulin
glucosides interfere with the measurement of inulin in both The total body clearance of inulin can be estimated from
the plasma and urine. the plasma disappearance curve after a bolus i.v. short infu-

TABLE 21.5. NORMAL VALUES OF THE GLOMERULAR FILTRATION RATE (GFR) AS MEASURED BY CREATININE
CLEARANCE AND RENAL PLASMA FLOW (RPF) AS MEASURED BY P-AMINOHIPPURIC ACID
AND REFERENCE VALUES OF MAXIMAL URINE OSMOLALITY
Neonate 1–2 wk 6–12 mo 1–3 yr Adult

GFRa mL/min × 1.73 m2 26 ± 2 54 ± 8 77 ± 14 96 ± 22 118 ± 18

RBFa mL/min × 1.73 m2 88 ± 4 154 ± 34 352 ± 73 537 ± 122 612 ± 92
Maximal urine osmolalitya mOsm/kg H2O 543 ± 50 619 ± 81 864 ± 148 750 ± 1330 825 ± 1285

aMean values ± SEM.

Adapted from García-Nieto V, Santos F. Pruebas funcionales renales. In: Garcia-Nieto V, Santos F, eds. Nefrología pediátrica. Madrid: Aula Médica,
2000; and García-Nieto V, Santos F, eds. Grupo aula medica. Madrid: Aula Médica, 2000;15–26.
408 IV. Clinical Methods

sion (<5 minutes) of inulin at a dose of 100 mg/kg. The

amount of inulin injected is such as to acutely increase the
plasma concentration to 0.5 to 1.0 g/L (71). The concen-
tration of inulin is ideally followed for at least 4 hours after
the injection. Studies comparing the single injection with
the urinary clearance of inulin have reported significant
correlations between the two methods (72,73), but the sin-
gle injection significantly overestimated the urinary clear-
ance in a study by Florijn et al. (72). Pharmacokinetic
adaptation of the standard technique can probably reduce
the overestimation, but the procedure implies the collection
of a blood sample for as long as 10 hours after the bolus
injection in patients with impaired renal function (74).

Inulin as a Marker of Glomerular FIGURE 21.3. Postnatal increase in glomerular filtration rate in
Filtration Rate in the Neonate term and preterm infants. Open part in columns represents
mature levels of glomerular filtration rates. (Adapted from
Studies comparing the clearance of inulin with that of other Guignard JP, Torrado A, Da Cunha O, et al. Glomerular filtration
glomerular markers have led to the hypothesis that glomer- rate in the first three weeks of life. J Pediatr 1975;87:268–272.)
ular pore size could be related to body size and that inulin
may not be freely filtered by the immature glomerulus (75).
This hypothesis has not been confirmed by studies of inulin duration, as well as careful supervision of the test. Using the
handling in rats or fetal lambs, both failing to demonstrate 24-hour constant infusion method in preterm infants, Van
any restriction to the filtration of inulin (76,77). The same der Heijden et al. confirmed that GFR increases rapidly
conclusion was reached from clinical studies in preterm after birth (83).
infants showing that high-molecular-weight inulin or Results comparing data obtained by the single injection
polysaccharides did not accumulate in the plasma of very technique with those obtained with the urinary clearance of
immature babies infused with these glomerular markers inulin are conflicting. Early optimistic results have not been
for several days, thus excluding any retention of the larger confirmed (84). A 30% overestimation of the true GFR
molecules (78,79). was described by Fawer et al. in neonates 1 to 3 days old
Studies on standard urinary inulin clearance in neonates (85). These results were confirmed by Coulthard in pre-
are scarce. Those performed during the first 2 days of life of term babies, who also described a large coefficient of varia-
preterm and term neonates have shown that GFR at birth tion for the single injection technique (69).
approximates 20 mL/min/1.73 m2 in term neonates and 12
to 13 mL/min/1.73 m2 in preemies of 28 to 30 weeks of
Creatinine
gestation (80). The GFR matures rapidly in the early post-
natal period, doubling during the first 2 weeks of life (80) The Marker
(Fig. 21.3). The velocity of the maturation is somewhat Creatinine is the anhydrase of creatine, a compound
slower in the most premature infants. present in skeletal muscle as creatine phosphate. It has an
Conflicting results have been observed in neonates stud- MW of 113 d. The serum creatinine levels reflect total
ied by the inulin constant infusion technique over a few body supplies of creatinine and correlate with muscle mass.
hours. Although Cole et al. and Leake et al. found an excel- After initial decrease during the first month of life (Table
lent correlation between the constant infusion clearance 21.6), it increases steadily with age (Table 21.7). The excre-
and the urinary clearance of inulin (R = 0.999), Alinei and tion rate varies according to body weight and age, both
Guignard found the constant infusion technique to greatly reflecting muscle mass. The renal excretion pattern of endo-
overestimate (~30%) the urinary clearance of inulin in genous creatinine is similar to that of inulin in humans and
infants (55,81,82). The overestimation declined with time several animal species. However, in addition to being fil-
but remained substantial after 3 hours of infusion. The tered through the glomerulus, creatinine is secreted in part
same conclusion was reached by Coulthard, who also by the renal tubular cells.
observed an overestimation of GFR by the constant infu- The validity of creatinine as a marker of GFR has been
sion technique in spite of the fact that the plasma inulin questioned because creatinine is not only secreted by the renal
concentration was apparently stable (69). Reliable estimates tubular cells, but could also be reabsorbed under certain condi-
of GFR could, however, be obtained when inulin was con- tions. Such reabsorption has been shown to occur in rats and
stantly infused for 24 hours, with or without a bolus injec- dogs at low urine flow rates (86,87). Substantial tubular secre-
tion at the start of the test. The main disadvantage of the tion and reabsorption of creatinine has been suggested in
method is that of requiring a constant infusion of long humans in relation to the degree of hydration (88,89).
 21. Laboratory Investigations 409

TABLE 21.6. MEAN VALUES OF PLASMA CREATININE cotrimoxazole (91). The noncreatinine chromogens do not
DURING THE FIRST WEEKS OF LIFE OF TERM AND interfere significantly in the urine.
VERY LOW BIRTH WEIGHT NEONATES
Modified kinetic Jaffe techniques have improved the
Plasma creatinine (μmol/L)a specificity of creatinine measurement. Enzymatic methods
have subsequently been developed that are more specific
Postnatal period (d)
than the Jaffe reaction. Their cost and questionable preci-
Birth weight (g) 1–2 8–9 15–16 22–23 sion have limited their widespread use.
1001–1500 95 ± 5 64 ± 5 49 ± 4 35 ± 3 Determination of serum creatinine by isotope dilution
1501–2000 90 ± 5 58 ± 7 50 ± 8 30 ± 2 mass spectrometry has been described as the potential
2001–2500 83 ± 5 47 ± 8 38 ± 8 30 ± 10 “definitive” method (92). High-performance liquid chroma-
Term 66 ± 3 40 ± 4 30 ± 8 27 ± 7 tography (HPLC) will undoubtedly become the reference
aMean
method for creatinine determination (93,94), even if its
values ± SEM.
routine use is still not possible in most clinical laboratories.

Creatinine Urinary Clearance

Overestimation of GFR by creatinine clearance is more
The urinary clearance of creatinine (UV/P) is the most com-
pronounced at low GFR. Indeed, as GFR falls progressively
monly used method for assessing GFR in children. The urine
during the course of renal diseases, the renal tubular secre-
is collected over 4 to 24 hours, with the plasma sample col-
tion of creatinine contributes to an increasing fraction of
lected at the middle of the urine collection period. The
urinary excretion, so that creatinine clearance may substan-
plasma creatinine concentration can be significantly increased
tially exceed the actual GFR. Diffusion of creatinine into
by cooked-meat feeding (95). Drugs, such as trimethoprim
the gut may also interfere with the accuracy of its clearance
and cimetidine, increase the plasma creatinine by interfering
in uremic patients. At a normal plasma concentration, the
with the renal tubular secretion, presumably by competition
amount of creatinine entering the gut is negligible. It may
for the organic cation secretory passageway (96,97).
become significant during renal failure when the plasma
Estimation of GFR by measuring the urinary clearance
creatinine concentration increases (90). This phenomenon
of creatinine yields values that have been shown to correlate
explains in part why creatinine clearance overestimates true
variably with inulin clearance. The best correlation is seen
GFR in patients with renal failure.
when GFR is normal. This results from the balance
between two artifacts: (a) the excretion rate of creatinine is
Measurement of Creatinine
higher than the filtered rate because of the tubular secretion
Creatinine concentration in plasma and urine is usually
of creatinine and (b) the measured plasma creatinine is
based on the Jaffe reaction, characterized by the production
higher than the true creatinine because of the presence of
of an orange-red color when creatinine reacts with alkaline
noncreatinine chromogens that interfere with a standard
sodium picrate. The method is not very specific, noncreati-
colorimetric analysis of creatinine in the Jaffe reaction.
nine chromogens generating sufficient color to account for
Overestimation of GFR by the urinary creatinine clear-
0.2 to 0.3 mg/dL (~30 μmol/L) of “false creatinine.” The
ance is usually maximal at low levels of GFR (98). The ratio
interference of noncreatinine chromogen obviously is highest
of the urinary creatinine clearance to urinary inulin clear-
at the lowest values of creatinine, as present in newborn
ance has been shown to vary between 1.14 and 2.27 in adult
infants (91). Negative interference by conjugated and uncon-
subjects. Dodge et al. and Kim et al. have long ago sug-
jugated bilirubin makes the use of the Jaffe reaction ques-
gested that creatinine clearance should not be used for the
tionable in neonates. Other interference substances include
estimation of GFR, as this clearance varies with tubular
acetoacetate, pyruvate, uric acid, the cephalosporins, and
secretion of creatinine, which follows no apparent pattern
(99,100). In the latter study, 42% of patients with decreased
TABLE 21.7. PLASMA CREATININE IN CHILDREN renal function would have been diagnosed as normal using
urinary creatinine clearance. Like those of Rosenbaum, data
Plasma creatinine Creatininuria
obtained by Guignard et al. in 72 children older than 1 year
Age showed a substantial overestimation of GFR by creatinine
(yr) μmol/L mg/dL μmol/kg/d mg/kg/d clearance at all levels of GF (70,101).
<2 35–40 0.4–0.5 62–88 7.1–9.9
2–8 40–60 0.5–0.7 108–188 12.2–21.2 Assessment of Glomerular Filtration Rate
9–18 50–80 0.6–0.9 132–212 14.9–23.9 by the Formula (2 Ccreat + Curea)/3
In children undergoing simultaneous inulin and creatinine
Adapted from García-Nieto V, Santos F. Pruebas funcionales renales. urinary clearance studies, the overestimation of GFR by
In: Garcia-Nieto V, Santos F, eds. Nefrología pediátrica. Madrid: Aula
Médica, 2000; and García-Nieto V, Santos F, eds. Grupo aula medica. creatinine clearance was “corrected” when the clearance of
Madrid: Aula Médica, 2000;15–26. urea was also taken into account (70). When using the for-
410 IV. Clinical Methods

TABLE 21.8. VALUES OF K FOR VARIOUS AGE GROUPS

k values when Pcreat expressed in:

μmol/L mg/dL

Low-birth-weight infants 29 0.33

<2.5 kg
Normal infants 0–18 mo 40 0.45
Girls 2–16 yr 49 0.55
Boys 2–13 yr 49 0.55
Boys 13–16 yr 62 0.70

Note: Values of k for estimating creatinine clearance by the k × height/

Pcreat formula.
Adapted from Schwartz GJ, Brion LP, Spitzer A. The use of plasma
creatinine concentration for estimating glomerular filtration rate in
infants, children, and adolescents. Pediatr Clin North Am 1987;34:
571–590.

FIGURE 21.4. Relationship between standard urinary clearance

of inulin and the (2 Ccreat + Curea) / 3 formula. (Adapted from Guig- ity, 407 ± 61 mOsm/kg H2O) or undergoing water diuresis
nard JP, Torrado A, Feldmann H, et al. Assessment of glomerular (urine flow rate, 8.5 mL/min/1.73 m2; mean urine osmolal-
filtration rate in children. Helv Pediatr Acta 1980;35:437–447.) ity, 163g ± 20 mOsm/kg H2O). In hydropenic children, the
values of k were identical when derived from Cinulin or Ccreat
(49 ± 2 and 50 ± 3, respectively). They differed significantly
mula (2 Ccreat + Curea)/3, the regression line correlating this when k was derived from urinary creatinine clearance in
formula to inulin clearance was indistinguishable from the children undergoing water diuresis (50 ± 3 vs. 64 ± 4; p
line of identity (Fig. 21.4). The scatter of points around the <.001). Increased secretory rates of creatinine at high urine
regression line was, however, not negligible. flow rates probably account for the elevated value of k calcu-
Clearly, creatinine clearance alone is not a good alterna- lated from Ccreat in well-hydrated children. Extensive tubu-
tive to inulin clearance when a precise measurement of lar secretion and reabsorption of creatinine in relation to the
GFR is needed. The use of the formula (2 Ccreat + Curea)/3, degree of hydration has been well described in humans (86).
calculated over 3 to 4 hours in well-hydrated children, is In spite of its relative inaccuracy, the k × height/Pcreat for-
recommended when the clinical situation does not warrant mula has proved valuable as a rapid rough estimate of GFR
the cumbersome measurement of inulin clearance or when in clinical practice.
the technique is not available.
Creatinine as a Marker of Glomerular
The GFR = k × Height/Pcreat Formula Filtration Rate in the Neonate
Numerous studies have used the GFR = k × Height/Pcreat In tiny premature neonates, the clearance of creatinine
formula, first described in 1976 by Counahan et al. and underestimates inulin clearance (105,106). Studies in piglets
Schwarz et al. (102,103). A critical review of the use of this and newborn rabbits suggest that the filtered creatinine is sig-
formula has been published by Haycock (104). The formula nificantly reabsorbed by the immature tubule (107,108).
provides useful data when used cautiously. It cannot be used Creatinine reabsorption by the immature kidney probably
in obese or malnourished children in whom body height occurs by passive back-diffusion of filtered creatinine across
does not accurately reflect muscle mass. Ideally, the exact leaky tubules. Significant reabsorption of filtered creatinine
value of k should be derived from the laboratory where the supposedly accounts for the transient increase in plasma crea-
plasma creatinine is measured and from inulin clearance as tinine in the first 3 days of life of very low birth weight
the reference method for estimating GFR. The values of k, infants (Fig. 21.5) (109–111). After the neonatal period,
as derived from creatinine clearance in different age groups Pcreat rises steadily throughout infancy and childhood toward
by Schwarz et al., are given in Table 21.8 (57). In a study adult levels (Table 21.7).
involving 200 patients aged 1 month to 23 years, Haenggi In spite of these drawbacks in the assessment of GFR by
et al. compared the values of k derived from the urinary inu- creatinine clearance in newborn infants, the latter method
lin clearance to that derived from simultaneous urinary crea- has been used commonly in this age group. Studying very
tinine clearance (89). The value of k derived from creatinine low birth weight infants, Stonestreet et al. have reported a
or inulin clearance differed significantly, being lower when correlation coefficient of 0.78 when values of creatinine
calculated from Cinulin. The effect of the state of hydration clearance were compared to those of inulin clearance (106).
was assessed in 18 children in a “hydropenic state” (urine In recent studies in premature and term neonates, creati-
flow rate, 3.5 ± 0.6 mL/min/1.73 m2; mean urine osmolal- nine clearance has been shown to be low at birth and to rise
 21. Laboratory Investigations 411

gested that the diffusion of iohexol in its distribution vol-

ume is slow, and that it may take more than 4 to 6 hours to
reach equilibrium after an i.v. injection of the agent (117).
This may limit the usefulness of iohexol as a marker of
GFR, even if significant correlations have been demon-
strated between standard inulin clearance and values gener-
ated by the plasma disappearance curve of iohexol (74).

Cystatin C
Cystatin C, a nonglycosated, 13-d basic protein, is a pro-
teinase inhibitor involved in the intracellular catabolism of
proteins (118). It is produced by all nucleated cells at a con-
stant rate apparently independent from inflammatory con-
ditions, muscle mass, and gender (119). It is freely filtered
across the glomerular capillaries, almost completely reab-
sorbed, and catabolized in the proximal tubular cells (120).
Being reabsorbed, cystatin is not a classical glomerular
marker as strictly defined (121). Fully automated assays
using particle-enhanced turbidimetry or particle-enhanced
FIGURE 21.5. Changes in plasma creatinine (A) and urea (B) con- nephelometry are available for the measurement of cystatin
centrations during the first 100 hours of life of premature neonates in plasma and serum (122,123). The assays are precise,
of variable gestational age. The shaded area represents 95% Cis for
the mean plasma creatinine or urea of all infants. (Adapted from rapid, and usable in clinical routine practice.
Miall LS, Henderson MJ, Turner AJ, et al. Plasma creatinine rises dra- Cystatin C does not cross the placenta, and there is no cor-
matically in the first 48 hours of life in preterm infants. Pediatrics relation between maternal and neonatal serum cystatin C
1999;104:e76.)
levels (124). Cystatin C concentrations are high at birth and
then decrease to stabilize after 12 months of age (119) (Fig.
rapidly after birth (111,112). The slope of maturation was 21.6). Whether cystatin C is significantly higher in premature
steeper in the most mature infants (112). The increase in infants as compared to term infants is not yet clear (119,125).
creatinine clearance correlated with the postnatal increase Bökenkamp et al. found serum cystatin levels between 0.70
in systemic blood pressure as well as with gestational and and 1.38 mg/L in children older than 1 year (126).
postnatal age (111–113). Creatinine clearance close to 42 Serum cystatin C concentrations are closely related to
mL/min/1.73 m2 in term neonates and 27 mL/min/1.73 GFR as are serum creatinine levels. Serum cystatin C
m2 in premies younger than 27 weeks of gestation were increases when GFR decreases. The reciprocal values of cys-
recorded on the fifty-second day of life (111). Mature levels tatin C correlate linearly with GFR, and cystatin C has
close to 100 mL/min/1.73 m2 are reached at the end of the been claimed to be at least as good as serum creatinine for
first year of life. the evaluation of GFR in adults (127). In children aged 1.8
to 18.8 years with various levels of GFR, serum cystatin C
has been found to be broadly equivalent or even superior to
Iohexol
serum creatinine as an estimate of GFR (58,126). The fact
Iohexol is a nonionic contrast agent with an MW of 821 d. that cystatin C is independent of age, gender, height, and
It does (almost) not bind to plasma proteins, diffuses into body composition has been considered an advantage (126).
the extracellular space (114), and is eliminated exclusively The major drawbacks in using cystatin C are that it is not
via the kidneys (115). Iohexol has an elimination half-life a true glomerular marker and its clearance cannot be calcu-
of approximately 90 minutes. Total urine recovery of lated. A recent study by Martini et al. compared the reliabil-
iohexol after i.v. injection occurs within 12 hours (114). ity of different estimates of GFR to distinguish impaired
Iohexol can be measured by x-ray fluorescence and HPLC from normal GFR, with a cut-off at 100 mL/min/1.73 m2
methods (74). More recently, iohexol has been successfully (128). Although plasma cystatin was slightly superior to
measured by the capillary electrophoresis technique, with plasma creatinine in diagnosing renal insufficiency, it was sig-
results similar to those obtained by the HPLC method nificantly less sensitive than both urinary creatinine clearance
(74). The capillary electrophoresis technique is less expen- and the estimated k × Height / Pcreat. The authors concluded
sive, more rapid, and more sensitive than both the HPLC that simply measuring the child’s height in addition to the
and x-ray fluorescence methods. Good correlations have plasma creatinine was a simpler, cheaper, and better means of
been reported between plasma iohexol clearance and that of rapidly assessing GFR in children than measuring the plasma
51Cr-EDTA (116). Clinical studies have, however, sug- cystatin C. The recent observation that the urine cystatin C/
412 IV. Clinical Methods

minimal. Its clearance approximates that of inulin (132). The

occurrence of extrarenal elimination of DTPA is, however,
suggested by the observation that the plasma clearance of
99mTc-DTPA exceeds the urinary clearance of the compound

by a variable but significant amount (130,132). A significant

drawback in using 99mTc-DTPA as a glomerular marker is
the fact that the 99mTc-DTPA can significantly dissociate
from DTPA during a clearance study (133). In the neonate,
99mTc-DTPA scans are characterized by poor visualization of

the kidneys and a relatively flat renal curve. Better-quality

dynamic isotope scans are generally only achieved at the end
of the neonatal period, when GFR has reached higher values.
51Cr-EDTA

EDTA is a glomerular marker similar to DTPA. EDTA has

an MW of 292 d and is used as a chelate of 51Cr. Its plasma
disappearance curve measured after a bolus i.v. injection
exceeds its urinary clearance by an average of 6 mL/min, thus
suggesting an extrarenal route of elimination (134). This
phenomenon is particularly evident at low filtration rates.

Iothalamate
Iothalamate sodium has an MW of 637 d. Iothalamate has
been used as 125I-radiolabeled or without radioactive label,
its plasma concentration being then assessed by x-ray fluo-
FIGURE 21.6. Box plot distributions showing (A) cystatin C and rescence or by HPLC, or, more recently, by capillary elec-
(B) creatinine values (tenth, twenty-fifth, fiftieth, and ninetieth trophoresis (135). A recent study showed an excellent
percentiles) across the age groups. The categories of 24 to 36
and 29 to 36 weeks refer to gestational ages of preterm babies. correlation between the clearance of 125I-iothalamate and
Dotted lines indicate 95% confidence interval of adult range. cold iothalamate, as assessed by capillary electrophoresis
Preterm babies born between 24 to 36 weeks’ gestation were 1 (136). Critical studies have, however, unequivocally dem-
day old. (Adapted from Finney H, Newman DJ, Thakkar H, et al.
Reference ranges for plasma cystatin C and creatinine measure- onstrated that iothalamate is actively secreted by renal
ments in premature infants, neonates and older children. Arch tubular cells, and perhaps also undergoes tubular reabsorp-
Dis Child 2000;82:71–75.) tion in human and animal species (137). The urinary clear-
ance of iothalamate significantly exceeds that of inulin in
creatinine ratio could provide information on both glomeru- patients with normal renal function (138). When present,
lar function and tubular injury casts doubts on the real value the agreement of iothalamate clearance with inulin clear-
of cystatin C as the best estimate of GFR (129). ance appears to be a fortuitous cancellation of errors
between tubular excretion and protein binding (139). The
use of iothalamate as a glomerular marker is presently not
Radionuclides
recommended.
Radionuclides, such as 51Cr-EDTA, 99mTc-DTPA, and 125I-
iothalamate, have been used as alternative filtration markers
Renal Functional Reserve
(Table 21.4). They permit accurate detection of minute con-
centrations of the markers in plasma and urine samples. It has been considered for a long time that GFR in healthy
Their urinary clearance, as calculated after a constant infu- individuals is remarkably stable from day to day over a
sion of the marker, was initially reported as fairly similar to period of years (121). This concept has been challenged by
that of inulin (130). Prolonged radioactive exposure during the observation that GFR in normal subjects could vary
the infusion of radiolabeled markers has limited the use of with their diet protein content (140). Alterations in protein
these compounds to single injection studies only. Even in intake thus result in variations in GFR that are indepen-
these conditions, radioactive markers are best avoided in dent of changes in renal mass. Patients with renal disease on
pregnant women and small children. a low-protein diet may thus have a reduction of GFR unre-
lated to the progression of renal disease (140). Variations in
99mTc-DTPA GFR also occur during the circadian rhythm, and upsurges
DTPA has an MW of 393 d. It appears to be excreted mainly in GFR are observed in patients with burns, women during
by glomerular filtration (131). Protein binding of DTPA is pregnancy, and patients infused with amino acids (67).
 21. Laboratory Investigations 413

The baseline or “resting” GFR theoretically depends on reabsorption capacity is required for protein-stimulated
the working level of the intact nephrons and is the conse- renal vasodilatation and hyperfiltration (145).
quence, in part, of the amount of proteins ingested per day.
The renal functional reserve represents the potential of the
Protocol for Measuring the Functional
kidney to increase GFR from a baseline or resting value to a
Reserve Capacity
maximal one (the so-called maximal filtration capacity) in
response to increased physiologic demands. Depending on The functional reserve is assessed during a standard clear-
the daily protein intake, GFR may vary from 30 to 143 ance test, using inulin and p-aminohippuric acid (PAH) as
mL/min/1.73 m2 in normal individuals (140). markers of GFR and effective renal plasma flow (ERPF),
The maximal filtration capacity can be measured in respectively. After a priming dose of inulin 25% [64 mg/kg
patients undergoing inulin clearance studies before and body weight (BW)] and PAH 20% (9 mg/kg BW), a con-
after an oral load of proteins, during the infusion of amino tinuous infusion of 1 to 2 mg/kg/min inulin and 0.15 to
acids, or during the combined administration of amino 0.30 mg/kg/min PAH is given intravenously. Water diuresis
acids and dopamine (see section Protocol for Measuring the is induced by an oral ingestion of 20 mL/kg BW (maxi-
Functional Reserve Capacity). The maximal filtration mum, 1200 mL) during the first hour, and then 5 mL/kg
capacity in young adults has been shown to reach values BW (maximum, 300 ml) every 30 minutes. Urine samples
close to 160 mL/min/min (140,141). are collected by spontaneous micturition at 30-minute
The renal functional reserve represents the difference intervals and blood samples drawn midway through each
between the resting GFR and the maximal GFR. The mag- urine collection period. After an equilibration period of 60
nitude of the renal functional reserve obviously depends on minutes and 3-minute baseline periods, an oral protein
the resting GFR (142). Normal individuals with the lowest load of 1.5 mg/kg BW (mixed meal containing meat with
protein intake have the highest renal reserve. In patients milk protein as a supplement) is given within 30 minutes.
with renal disease, the maximal filtration capacity decreases After the meal, the creatinine test is continued for an addi-
as the disease progresses. The resting GFR is insensitive as tional 3-hour period (6 periods of 30 minutes each).
an indicator of renal damage. On the contrary, the filtration The baseline GFR and ERPF corresponds to the mean
capacity is a good indicator of renal damage as it decreases values of the three periods before the protein-rich meal.
proportionally to the extent of injury to the kidney. In The maximal filtration capacity is defined as the peak values
patients with GFR below 40 mL/min/1.73 m2, the renal observed after the protein-rich meal. The functional reserve
functional reserve is usually negligible or absent, indicating is calculated by subtracting the baseline GFR and ERPF
that at this level of function, all nephrons are working max- from their maximal values observed after the protein-rich
imally at all times. In most conditions, the increase in GFR meal. The renal function reserve is also expressed as frac-
observed after protein loading or i.v. amino acid infusion is tional increase (%) from the baseline values. Another
associated with a parallel increase in RBF. The increase in method for assessing the functional reserve consists of an
RBF is probably the consequence of decreased intrarenal i.v. infusion of a 7.5% solution of amino acids at a rate of
vascular resistance, mainly at the level of the afferent and 0.08 mL/min/kg. Dopamine (2 μg/kg BW/min) can be
efferent arterioles. infused concomitantly to the infusion of amino acids.
Several mechanisms may play a role in mediating the
renal response to protein loading or amino acid infusion,
including a number of circulating or locally acting hor-
RENAL BLOOD FLOW
monal factors and intrinsic intrarenal mechanisms (143).
Experimental data suggest that growth hormone and
Physiology
atrial natriuretic peptide are not involved in the response to
protein or amino acid administration, but that prostaglan- RBF approximates 20% of resting cardiac output—that is,
dins, nitric oxide, and the kallikrein-kinin system could 1200 mL/min/1.73 m2. Most of this flow supplies the met-
play key roles (143). Intrinsic renal mechanisms, namely, abolic active renal cortex, whereas 10% of RBF is directed
tubular transport and tubuloglomerular feedback mecha- toward the outer medulla and only 1 to 2% into the capil-
nisms, could also play key roles in mediating the amino lary tissue. A small part goes to the capsule and perirenal
acid or the protein-stimulated renal vasodilatation and fat. Several factors, such as a high-protein diet, can increase
hyperfiltration. Amino acids could cause a primary stimula- RBF both acutely and chronically. RBF also increases dur-
tion of proximal tubular sodium reabsorption and in turn ing pregnancy under the influence of gestational hormones.
cause renal vasodilatation and hyperfiltration (144). The RBF is proportional to the pressure gradient across the
finding that the response to a meat meal is inhibited by acet- renal vasculature (renal artery pressure minus renal vein pres-
azolamide, an inhibitor of sodium transport in the proxi- sure) and inversely proportional to the total renal vascular
mal tubule, but not by amiloride provided strong support resistance. Most of the resistance resides in three major seg-
for the hypothesis that normal proximal tubular sodium ments: (a) the intralobular arteries, (b) the afferent arterioles,
414 IV. Clinical Methods

and (c) the efferent arterioles. The renal vascular tone is way. The extraction efficiency of PAH may also be depressed
affected by neural impulses and by circulating intrarenal by drugs commonly used in renal patients (i.e., angiotensin-
vasoactive compounds such as angiotensin II, prostaglandins, converting enzyme inhibitors and cyclosporine) (147,148).
nitric oxide, endothelin, bradykinin, and adenosine.
RBF is autoregulated by an intrinsic myogenic mecha-
Measurement of p-Aminohippuric
nism. The vessels have the capacity to constrict when blood
Acid Clearance
pressure rises and to vasodilate when it decreases. RBF is
thus independent of the perfusion pressure over the range The clearance of PAH is usually measured concomitantly
of autoregulation (~80 to 180 mm Hg). with that of inulin. A priming dose of PAH is given at a
The major role of blood supply to the kidney is to pro- dose of 9 mg/kg BW, followed by a continuous infusion of
vide blood for filtration. The fraction of plasma that is fil- 0.15 to 0.30 mg/kg/min to maintain plasma concentration
tered through the glomeruli, the so-called filtration between 10 and 20 mg/L. Water intake is administered as
fraction, approximates 20% in normal conditions. When for the inulin clearance test (20 mL/kg BW during the first
RBF is reduced, GFR is usually maintained by angiotensin hour, and then 5 mL/kg BW every 30 minutes). Urine and
II–induced efferent arteriolar vasoconstriction. This is evi- blood samples are collected as described for the inulin
denced by an increase in the filtration fraction. clearance test.

Measurement of Renal Blood Flow Determination of p-Aminohippuric Acid

Measurement of RBF is based on the Fick principle. PAH in plasma and urine can be measured by the classical
According to this principle, any substance completely colorimetric reaction of Bratton and Marshall (149). Sev-
extracted from the blood during a single pass through the eral compounds interfere with the reaction, in particular
kidney would allow the measurement of true RBF. In addi- those with a para-amino ring, including acebutolol, sotalol,
tion to being completely extracted from the blood, the sub- procaine, cotrimoxazole, and furosemide (62). PAH can be
stance should not be metabolized by the kidney, not bound measured more accurately by HPLC (150).
to proteins, and accurately measurable in blood and urine.
PAH is the only known compound that meets the required
Adverse Effects of p-Aminohippuric Acid
properties. It has an MW of 194 d, is filtered through the
glomerular capillaries, and is secreted by the proximal tubu- The administration of PAH is usually well tolerated but
lar cells (146). It is almost completely removed from the may be associated with nausea, vomiting, vasomotor distur-
plasma by a single pass. At low arterial plasma concentra- bances, flushing, tingling, cramps, and a feeling of warmth,
tions (~20 mg/L) the concentration in the renal vein is neg- and sometimes the urge to urinate or defecate.
ligible. The plasma perfusing the kidney being completely
cleared of PAH and the extracted PAH quantitatively
Drawbacks in the Interpretation of CPAH
excreted in the urine, the clearance of PAH can be used to
estimate renal plasma flow (RPF). By measuring the hemat- The main drawbacks in the use of PAH clearance to assess
ocrit, RBF can be derived from RPF by the formula RBF = RPF resides in the fact that the extraction of PAH is not
RPF/1-hematocrit. complete and that it can vary under the influence of disease
The clearance of PAH underestimates slightly the true states, drug administration, and age. Newborn infants have
RPF, as PAH extraction is only approximately 90% com- low extraction ratios that limit the usefulness of PAH in the
plete because part of the blood delivered to the kidney goes first month of life (151).
to sites not available for secretion in the proximal tubule
(medulla, capsule, perinatal fat). The PAH clearance thus
Other Cold Markers of Renal Plasma Flow
assesses cortical plasma flow more than true total RPF. The
term effective renal plasma flow is often used to described 5-Hydroxyindoleacetic acid (5-HIAA) is the major metabo-
the value derived from PAH clearances. The extraction lite of serotonin. Its renal elimination, analogous to that of
ratio can vary in disease states or under the influence of cer- PAH and other weak organic acids, is achieved by glomeru-
tain drugs, thus making the measurement of renal vein con- lar filtration and by probenecid-sensitive active secretion in
centration necessary for accurate determination of RPF. the proximal tubule (152). A renal extraction ratio of 78%
Because PAH undergoes acetylation by the liver, its clear- has been measured in hypertensive adult patients with nor-
ance estimated by the constant infusion technique without mal renal function (153). This ratio is lower than the PAH
urine collection or by the plasma disappearance curve signifi- extraction ratio measured in human subjects at plasma
cantly overestimates the urinary clearance. The clearance of PAH concentrations below the maximum transport. The
PAH can be affected by drugs, such as penicillin, salicylates, or efficient renal tubular secretion of 5-HIAA is further illus-
sulfinpyrazone, that compete for the same secretory passage- trated by the 5-HIAA to inulin ratio as high as 6.3 ± 0.66
 21. Laboratory Investigations 415

(SEM) in healthy subjects and in patients with reduced has been found in dogs when blood flow calculated from
GFR (153). Plasma levels of 5-HIAA have been shown to contrast ultrasonography was compared with direct measure-
correlate inversely (R = 0.85) with the PAH clearance. ment obtained with an electromagnetic flow probe (161).
The extraction of 5-HIAA in newborn animals, like that of
PAH, is well below its level in mature subjects. The low extrac-
Quantification with Color Flow and Pulsed
tion of PAH in neonates has been ascribed to the shunting of
Doppler Ultrasound
blood to the nonextracting renal tissues, to the relative decrease
in tubular mass, and to the immaturity of the renal tubular Estimates of total RBF have been produced in neonates by
secretory passageways (154). This endogenous marker, easily estimating the vessel diameter from color flow diameter
assayed in plasma by using liquid chromatography and electro- and flow velocity from pulse Doppler ultrasound measure-
chemical detection (155), may prove useful for a quick orien- ments (162).
tation on RPF in clinical practice (153).
Electron-Beam Computed Tomography
Radionuclides as Markers of From studies in anesthetized dogs, comparing data from
Renal Plasma Flow electron-beam computed tomography with those of elec-
Orthoiodohippurate (Hippuran) tromagnetic flow probe, Lerman et al. (163) concluded that
electron-beam computed tomography could provide credi-
Hippuran is eliminated by the kidney in a way similar to ble quantification of RBF.
PAH. Its extraction ratio is, however, significantly lower
than that of PAH (70 to 85% as compared to 90%) (156).
Protein binding of Hippuran is not negligible. The urinary Dynamic Magnetic Resonance Imaging and
clearance of Hippuran underestimates RPF more than does Gadolinium-DTPA
that of PAH (157). Hippuran can easily be labeled by a By the use of a T1-weighted fast gradient sequence after an
simple iodine exchange reaction; most often it is labeled i.v. bolus injection of gadolinium-DTPA, dynamic images of
with either 131I or 123I. 123I-Hippuran can be used in chil- the kidney have been obtained in patients presenting with
dren in larger doses than 131I-Hippuran, while still impart- well-functioning native or transplanted kidneys, patients
ing a lower radiation burden to the patient. Its short half- with significant artery stenosis, or patients with renal failure
life and greater cost limit its widespread use. (164). The authors concluded that realistic quantitative data
on renal perfusion could be obtained by standard magnetic
99mTc-Mercaptoacetyltriglycerine resonance imaging sequence after injection of a contrast
medium. This method awaits confirmation.
In spite of early claims, mercaptoacetyltriglycerine (MAG-
3) is not a marker of RPF. In normal volunteers, 99mTc-
MAG-3 and 123I-Hippuran showed comparable image TESTS TO ASSESS TUBULAR FUNCTION
quality, renal extraction, blood clearance, and time to peak
height of the renogram curve. Overall, the clearance of Functional tests explore specific aspects of renal function
99mTc-MAG-3 is, however, approximately only 80% of the
under conditions in which the tubule must develop an
simultaneous clearance of 131I-Hippuran (158). adaptive response aimed at preserving body homeostasis.
The use of functional tests often allows nephron disorders
not apparent or hardly recognizable under basal conditions
Newer Imaging Techniques for the
to be disclosed. Some tests are cumbersome, thus limiting
Assessment of Renal Perfusion
their use in the clinical setting.
Newer promising imaging techniques have been developed Description of the tests includes (165) (a) the function
in the last years, both in animals and humans (159). None that is tested, (b) the rationale of the test, (c) the method
can be a substitute to the clearance of PAH to precisely and normal results, and (d) the indications for and limita-
assess RPF. These techniques may, however, provide reliable tions of the test.
estimates of total renal or regional renal blood perfusion.
Glucose Titration Test
Contrast-Enhanced Ultrasound
Function tested: Characteristics of glucose tubular
An excellent correlation has been found in dogs between cor- reabsorption.
tical nutrient blood flow using microbubbles and ultrasonic Rationale: The amount of glucose filtered per minute is a
flow probe–derived RBF values (R = 0.82; p <.001) over a linear function of its plasma concentration provided that
wide range of flows (160). A similar correlation (R = 0.84) GFR remains steady (Fig. 21.1). Below a threshold level, all
416 IV. Clinical Methods

filtered glucose is reabsorbed, and no glucose is detected in with spontaneous or induced metabolic acidosis. For diag-
the urine. When the load of filtered glucose saturates the nostic purposes, the calculation of the plasma threshold for
mechanism of transport in all nephrons, the maximum HCO3– is seldom required. It is usually sufficient to calcu-
tubular reabsorption is achieved. From then on, the urinary late FEHCO3– in the presence of normal bicarbonatemia—
elimination of glucose runs parallel to the filtered glucose. that is, 20 to 22 mmol/L in infants and 22 to 24 mmol/L
The same rationale may be applied to other substances with in older children—and to measure the PCO2 in alkaline
an active and saturable mechanism of proximal tubular urines (pH above 7.45 to 7.60 and bicarbonaturia higher
reabsorption (Fig. 21.1). than 80 mmol/L) induced by a single oral dose of 3 to 4
Method and normal results: Starting from a plasma con- mmol/kg of NaHCO3– (1 g = 12 mmol). Normal values are
centration below the threshold—that is, when glucosuria is FEHCO3– less than 5% and urinary PCO2 above 70 mm Hg,
negative—3 mL/min/1.73 m2 of i.v. 5% dextrose is infused. which is equivalent to a urine minus blood PCO2 difference
Dextrose solution concentration is increased by 5% each 30 (Δ PCO2) greater than 20 to 30 mm Hg. Urine HCO3– can
minutes until glycemia reaches 350 to 400 mg/dL (19 to 22 be calculated by the Henderson-Hasselbalch equation:
mmol/L). The maximal concentration of dextrose that can
pH = pK + log (HCO3–/H2CO3)
be infused in a peripheral vein is 35%. Urine samples col-
lected during the last 15 minutes of each 30-minute period Knowing that
and plasma samples drawn in the midpoint of each urine
1. H2CO3 = CO2 + H2O = 0.03 PCO2, where PCO2 is
collection period are used for calculation. Normal plasma
the partial pressure of CO2 measured in mm Hg, as
threshold is approximately 180 mg/dL (9.9 mmol/L). Mean
determined by a pH/blood gas analyzer in urine col-
reference values of the maximum tubular reabsorption are
lected and processed in anaerobic conditions, and
375 and 303 mg/min/1.73 m2 (2.1 and 1.7 mmol/min/
0.03 is the solubility constant of CO2 in aqueous
1.73 m2) for males and females, respectively.
solutions.
Indications and limitations: The test may be used for
2. The pK of the NaHCO3– buffer system in urine is
diagnostic classification of renal glucosurias. In some cases,
6.33 – 0.5 multiplied by the square root of the sum of
the renal threshold for glucose is so low that it cannot be
the urine sodium and potassium concentrations
achieved without risk to the patient. Accurate collection of
expressed in mol/L.
frequent and timed urine samples may require bladder
catheterization in young children. Indications and limitations: The test may be used in the
differential diagnosis of the types of RTA. FEHCO3– is below
5% in type I distal RTA, above 15% in type II proximal
Bicarbonate Loading Test
RTA, and between 5% and 15% in type IV RTA. ΔPCO2 is
Function tested: Characteristics of tubular reabsorption of well below 20 mm Hg in children with primary forms of
bicarbonate (HCO3–) and hydrogen ion (H+) distal secretory type I RTA. Calculation of FEHCO3– in the face of normal
capacity in the face of a favorable chemical gradient. bicarbonatemia may give a clue to the dose of alkali to be
Rationale: The test assesses the tubular reabsorption of recommended in recently diagnosed RTA children. The
HCO3– on the basis of a theoretical background similar to measurement of PCO2 in alkaline urine has been proposed
that described above for glucose. The difference between as a sensitive method to detect subtle defects of distal acidi-
filtered and excreted HCO3– accounts for the HCO3– reab- fication (167,168). An accurate assessment of FEHCO3–
sorbed along the renal tubule, mostly in the proximal requires a nonexpanded extracellular fluid volume. Other-
tubule. In addition, HCO3– loading will give rise to alka- wise, proximal tubular reabsorption is inhibited, giving rise
line urine with pH higher than that of blood. This stimu- to high values of NaHCO3– excretion. Because of the mas-
lates H+ secretion by alpha-intercalated cells of the sive bicarbonaturia, normal bicarbonatemia is hard to
collecting duct. Hydrogen ions so secreted combine with achieve and maintain in children with proximal RTA.
HCO3– anions to form carbonic acid. As at this level of the
nephron, there is no carbonic anhydrase in the luminal side
Phosphate Loading Test
of the tubule; carbonic acid dehydrates very slowly into car-
bon dioxide (CO2) and water. The medullary trapping of Function tested: Secretory capacity of H+ by the distal
CO2 and the unfavorable surface-to-volume relationship in nephron in the face of large availability of intraluminal
the lower urinary tract limit the diffusion of CO2 out of the buffer. The use of phosphate infusions to assess phosphate
tubular lumen. As a result, the measurement of the partial tubular reabsorption has largely been replaced by the indi-
pressure of CO2 (PCO2) in alkaline urine can be used as a ces described above.
reliable index of distal H+ secretion (166). Rationale: A high luminal concentration of phosphate
Method and normal results: An i.v. solution of 0.3 to 0.5 stimulates H+ secretion. When urine pH is close to the uri-
mol/L of NaHCO3– is infused at a rate calculated to nary pK of the phosphate buffer system, disodium phos-
increase bicarbonatemia by 2 to 3 mmol/L/hr in a subject phate combines with the secreted H+ to form acid
 21. Laboratory Investigations 417

phosphate that, in turn, reacts with NaHCO3– to form threshold. Subjects with type IV RTA show normal ability to
H2CO3 and to increase PCO2 in the final urine. Measure- decrease urinary pH and defective excretion of ammonium.
ment of urinary PCO2 reflects distal H+ secretion. A urine pH equal to or below 6.0 in the second morning
Method and normal results: Concentrations of urinary urine in fasting conditions has been proposed as a reliable
phosphate greater than 20 mmol/L can usually be induced screening tool to exclude incomplete or minor forms of distal
by oral administration of 50 mg/kg/day of elemental phos- RTA. However, the range of urine pH determined in the
phorus given as a mixture of monosodium and disodium early morning urine in normal children ranges from 5.16 to
phosphate salts over a 3-day period. Alternatively, 0.6 7.07 (170). As pH is a measurement of free H+ concentra-
mmol/kg of neutral sodium phosphate diluted in 180 mL tion, patients unable to properly concentrate urine may in
of normal saline may be infused at 1 mL/min for 3 hours. turn be unable to decrease urine pH to normal minimal val-
Urinary PCO2 measured in urine samples with pH of ues. Similarly, in subjects with contracted extracellular vol-
approximately 6.80 should normally exceed blood PCO2 by ume, the urinary acidification defect may be a result of the
20 to 25 mm Hg (169). lack of sodium at the lumen of the distal nephron. Accord-
Indications and limitations: The test may be used to ingly, a proper evaluation of urinary acidification requires the
explore the mechanism of defective acidification in patients measurement of urinary sodium and urine osmolality as well
with distal type I RTA. A normal Δ PCO2 in individuals as the simultaneous determination of urine pH and NH4+
with distal RTA suggests that the acidification defect is due because a low pH associated with reduced ammoniuria does
to back-leak of secreted acid. not exclude a defective distal acidification, and, on the con-
trary, a high ammoniuria may be responsible for the fact that
pH does not decrease below 5.5.
Acidification Test
Minimal Urinary pH and Net Acid Excretion Furosemide Test
Function tested: Secretory capacity of H+ by the distal Function tested: Secretory capacity of H+ by the distal
nephron in the face of metabolic acidosis. nephron in the presence of large amounts of sodium deliv-
Rationale: Metabolic acidosis stimulates distal secretion of ered to the distal nephron and a favorable electronegative
H+. The secreted H+ will be found in the urine in free form, lumen potential.
estimated by the urinary pH, and will be bound to the two Rationale: Furosemide inhibits reabsorption of sodium
major urinary buffers, phosphate, and, mainly, ammonia chloride in the thick ascending limb of the loop of Henle.
generated by the proximal tubule. Net acid excretion is equal Early distal reabsorption of sodium cation leaves behind the
to the sum of titratable acid plus NH4+ minus bicarbonate. chloride anion, enhancing the electronegativity of tubular
Titratable acid refers to the amount of alkali needed to bring lumen, which stimulates H+ secretion.
the pH of an acid urine back to a value of 7.4. Method and normal results: Furosemide is given at a dose
Method and normal results: The measurements are done of 1 mg/kg, orally or intravenously. In normal subjects, a
in situations of spontaneous or exogenously induced meta- minimal urine pH below 5.5, associated with a kaliuretic
bolic acidosis. Ammonium chloride is the agent of choice response, is usually found within 3 to 4 hours after furo-
for inducing metabolic acidosis. It is given at a dose of 75 to semide administration. Mean (SD) values of minimal uri-
100 mmol/m2 (1 g = 18.7 mmol) either intravenously over nary pH and maximal FEK reported in 20 normal children,
4 to 6 hours in a 0.9% solution, or by oral route in enteric aged 2 to 14 years, given 1 mg/kg of i.v. furosemide are
coated capsules given over 1 hour. Ammonium chloride is 4.96 (0.32) and 34.4% (8.9%), respectively (171). The H+
an irritating agent and may produce vomiting. To avoid this secretion stimulated by furosemide also increases ammoni-
effect, 1.9 mmol/kg/day of ammonium chloride may be uria above 30 μmol/min/1.73 m2 (172).
given in a 3- to 5-day period, which, in addition, represents Indications and limitations: The test is used in the differen-
a much more potent stimulus of ammonia production than tial diagnosis of RTA. Type 1 distal RTA patients with an
the short test. A state of moderate metabolic acidosis (blood intrinsic defect in the proton pump cannot maximally decrease
pH less than 7.33 and bicarbonatemia below 16 to 18 urine pH in response to furosemide. Subjects with type IV
mmol/L) is usually sufficient to assess urine acidification. RTA and those with subnormal distal H+ secretion secondary
Normal individuals lower urine pH below 5.5 and increase to low distal delivery of sodium (i.e., nephrotic syndrome) or
net acid excretion above 70 μmol/min/1.73 m2. reversible impairment of sodium distal reabsorption (i.e., sickle
Indications and limitations: The test is useful in the differ- cell anemia or lithium administration) respond normally.
ential diagnosis of RTA. Patients with type I distal RTA are
unable to both maximally decrease urinary pH and increase
Acetazolamide Test
net acid excretion up to normal values. In type II proximal
RTA, minimal urinary pH and normal net acid excretion are Function tested: Ability of distal nephron to secrete H+ in
achieved once plasma bicarbonate falls below the renal the face of a favorable chemical gradient.
418 IV. Clinical Methods

Rationale: By inhibiting carbonic anhydrase, acetazola- Rationale: As urinary osmolality is strongly influenced
mide reduces proximal tubular reabsorption of bicarbonate by plasma concentrations of vasopressin, administration of
and causes bicarbonaturia and alkaline urine. Measurement pharmacologic amounts of a vasopressin analogue results in
of PCO2 in urine samples with pH above that of blood maximally concentrated urine even in the absence of fluid
reflects the ability of the distal nephron to secrete H+. restriction.
Method and normal results: Oral acetazolamide is given at Method and normal results: The compound most often
a dose of 0.5 to 1.0 g/1.73 m2. A normal response is charac- used is the 1-desamino-8-D-arginine vasopressin (DDAVP).
terized by urinary PCO2 above 70 mm Hg or Δ PCO2 above It can be given intranasally at doses of 10 μg in infants and
20 to 30 mm Hg. 20 μg in children (175). In infants, usual fluid intake must
Indications and limitations: The test is used to explore be restricted to 50% in the 12 hours after DDAVP adminis-
the mechanism of defective acidification in distal type I tration because of the risk of overhydration and hyponatre-
RTA. Children with primary defects of the distal proton mia. Osmolality is measured in individual urine samples
pump do not increase urine PCO2. Alon et al. found similar collected over a 6- to 8-hour period after DDAVP adminis-
response to oral bicarbonate (2.5 mmol/kg) or acetazola- tration. Normal values of maximal urine osmolality are
mide (17 ± 2 mg/kg) in terms of urine bicarbonate concen- shown in Table 21.5 (172,176). Recently, it has been pro-
tration and PCO2 (173). posed that assessment of renal concentrating capacity by
administration of DDAVP at bedtime and measurement of
osmolality in one urine sample taken the following morning
Concentrating Ability may be reliable and easy to perform in children (177).
Indications and limitations: The DDAVP test represents a
Urinary Concentration during Hydropenia
good alternative to thirst stimulus in infants and polyuric
Function tested: Ability to concentrate urine in response subjects in whom, as stated above, fluid suppression is hardly
to fluid suppression. feasible. The test may also be used in moderately polyuric
Rationale: Fluid restriction increases blood tonicity, thus subjects sequentially after a period of fluid restriction when
stimulating vasopressin secretion. As a result, free water is maximal urine osmolality obtained by hydropenia has not
reabsorbed, and the urine becomes concentrated. achieved normal values. In these cases, a DDAVP-induced
Method and normal results: Urinary osmolality is mea- increment of urine osmolality below 10% suggests tubular
sured at the end of a 12- to 16-hour period of fluid depriva- resistance to antidiuretic hormone, whereas a 40 to 60%
tion. During this period, children are allowed to eat solid increment in urinary osmolality indicates defective vaso-
foods except those having a high content in salt and water. pressin release or primary polydipsia. In these sequential
In healthy children older than 2 years of age, mean (SD) tests, a correct interpretation of the results requires that after
values of urinary osmolality in the second and third urine DDAVP administration the child be allowed to drink an
voided in the morning during fluid suppression since noon amount of fluid similar to that of urine voided. Thus, the
have been reported to be equal to 1035 and 1127 (128) degree of plasma hypertonicity induced by hydropenia
mOsm/kg H2O (174,175). When fluid intake is suppressed remains unchanged.
since 5 or 6 p.m., the urinary osmolality measured in the
second urine of the following morning is normally higher
than 725 mOsm/kg H2O. If the determination of osmolal- Dilution Ability (Free Water Clearance)
ity is not available, urinary density values above 1025 are
Hypotonic Saline Loading
usually indicative of normal renal concentration capacity.
Indications and limitations: The test is used to investigate Function tested: Chloride, sodium, and potassium han-
the origin of polyuric states as well as to disclose subclinical dling along the nephron under conditions of maximal uri-
defects of urine concentrating ability. Prolonged fluid nary dilution and in the absence of hormonal influences.
restriction may be harmful in infants and poorly tolerated Rationale: Maximal urine dilution and suppression of
in subjects with chronic primary polydipsia. In addition, aldosterone and antidiuretic hormone are induced by
these patients have a hypotonic medullary interstitium and extracellular volume expansion with hypo-osmolar fluid
may require much longer periods of hydropenia to maxi- loading. Under these conditions, maximal excretion of
mally concentrate the urine. In these cases, a progressive electrolyte-free water is generated in the diluting seg-
reduction in fluid intake over days or weeks is necessary ments of the tubule. An indirect estimate of solute reab-
before exposing the child to a thirst test. sorption in the different segments of the nephron may
be obtained by calculation of several urinary indices, as
shown in Table 21.9.
Vasopressin Administration
Method and normal results: After oral administration of
Function tested: Ability to concentrate urine in response 20 mL/kg of water, a constant i.v. infusion of 2000 mL/
to vasopressin administration. 1.73 m2 of 0.45% saline is administered over 2 hours. Cal-
 21. Laboratory Investigations 419

TABLE 21.9. NORMAL VALUES OF MAXIMAL FREE WATER CLEARANCE

INDICES DURING HYPOTONIC SALINE DIURESIS
Infants Children
(n = 22) (n = 17)

Urine osmolality (mOsm/kg) 51.8 ± 12.8 54.1 ± 13.3

Urine volume (mL/dL GF) 22.8 ± 3.6 17.2 ± 2.7 a
Osmolar clearance (Cosm) (mL/dL GF) 4.3 ± 1.3 3.2 ± 0.7a
Free water clearance (CH O) (mL/dL GF) 18.5 ± 2.9 14.0 ± 2.6a
2
Sodium clearance (CNa) (mL/dL GF) 1.9 ± 0.8 1.4 ± 0.4a
Potassium clearance (CK) (mL/dL GF) 19.9 ± 12.0 12.9 ± 5.2a
Chloride clearance (CCl) (mL/dL GF) 2.7 ± 1.1 2.1 ± 0.7a
Percentage of distal sodium reabsorption: 100 × CH O / 90.8 ± 4.5 90.9 ± 3.3
2
(CH O + CNa) (%)
2
Percentage of distal chloride reabsorption: 100 × CH O / 87.2 ± 5.3 86.7 ± 4.1
2
(C H2O + C Cl) (%)
Creatinine clearance (mL/min/1.73 m )2 88.5 ± 27.1 124.8 ± 25.2a

GF, glomerular filtration (values are × ± SD).

aSignificantly different from infants.

CH2O + CNa: Sodium delivery to the distal nephron. CH2O + CCl: Chloride delivery to the distal nephron. CH2O:
Reabsorption of sodium chloride in the distal nephron. CH2O / (CH2O + CNa): Fraction of sodium reabsorbed in
the distal nephron. CH2O / (CH2O + CCl): Fraction of chloride reabsorbed in the distal nephron.
Adapted from Rodríguez-Soriano J, Vallo A, Castillo G, Oliveros R. Renal handling of water and sodium
in infancy and childhood: a study using clearance methods during hypotonic saline diuresis. Kidney Int
1981;20:700–704.

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Renal Disease Study. Am J Kidney Dis 1990;16:224–235. mented conscious dog. Am J Physiol 1996;271:F269–F274.
139. Russell CD. Radiopharmaceuticals used to assess kidney 158. Durand E, Prigent A. The basics of renal imaging and func-
function and structure. In: Tauxe WN, Dubowski EV, eds. tion studies. Q J Nucl Med 2002;46:249–267.
Nuclear medicine in clinical urology and nephrology. Nor- 159. Young LS, Regan MC, Barry MK, et al. Methods of renal
walk, CT: Appleton-Century-Crofts 1985;5–31. blood flow measurement. Urol Res 1996;24:149–160.
140. Bosch JP. Renal reserve: a functional view of glomerular fil- 160. Wei K, Le E, Bin JP, et al. Quantification of renal blood
tration rate. Semin Nephrol 1995;15:381–385. flow with contrast-enhanced ultrasound. J Am Coll Cardiol
141. Regazzoni B, Genton N, Pelet J, et al. Long-term follow-up 2001;37:1135–1140.
of renal functional reserve capacity after unilateral nephrec- 161. Aronson S, Wiencek JG, Feinstein SB, et al. Assessment of
tomy in childhood. J Urol 1998;160:844–848. renal blood flow with contrast ultrasonography. Anesth
142. Molina E, Herrera J, Rodriguez-Iturbe B. The renal func- Analg 1993;76:964–970.
tional reserve in health and renal disease in school age chil- 162. Visser MO, Leighton JO, Van de Bohr, et al. Renal blood
dren. Kidney Int 1988;34:809–816. flow in neonates: quantification with color flow and pulsed
143. Woods LL. Intrarenal mechanisms of renal reserve. Semin Doppler US. Radiology 1992;183:441–444.
Nephrol 1995;15:386–395. 163. Lerman LO, Bell MR, Lahera V, et al. Quantification of
144. Claris-Appiani A, Assael BM, Tirelli AS, et al. Proximal global and regional renal blood flow with electron beam
tubular function and hyperfiltration during amino acid computed tomography. Am J Hypertens 1994;7:829–837.
infusion in man. Am J Nephrol 1988;8:96–101. 164. Vallee JP, Lazeyras F, Khan HG, et al. Absolute renal blood
145. Woods LL, Smith BE, De Young DR. Regulation of renal flow quantification by dynamic MRI and Gd-DTPA. Eur
hemodynamics after protein feeding: effects of proximal and Radiol 2000;10:1245–1252.
distal diuretics. Am J Physiol 1993;264:R337–R344. 165. Santos F, Orejas G, Foreman JW, Chan JCM. Diagnostic
146. Elbourne I, Lumbers ER, Hill KJ, et al. The secretion of workup of renal disorders. Curr Probl Pediatr 1991;21:48–74.
organic acids and bases by the ovin fetal kidney. Exp Physiol 166. DuBose TD Jr. Hydrogen ion secretion by the collecting
1990;75:211–221. duct as a determinant of the urine to blood PCO2 gradient
147. Wenling GJ, Derkx FH, Tan-Tjiong LH, et al. Risks of in alkaline urine. J Clin Invest 1982;69:145–156.
angiotensin converting enzyme inhibitors in renal artery ste- 167. Batlle DC, Grupp M, Gaviria M, Kurtzman NA. Distal
nosis. Kidney Int 1987;31:S180–S183. renal tubular acidosis with intact capacity to lower urinary
148. Myers BD, Ross J, Newton L, et al. Cyclosporine-associated pH. Am J Med 1982;72:751–758.
chronic nephropathy. N Engl J Med 1984;311:699–705. 168. Strife CF, Clardy CW, Varade WS, et al. Urine-to-blood car-
149. Bratton AC, Marshall EK Jr. A new coupling component for bon dioxide tension gradient and maximal depression of uri-
sulfanilamide determination. J Biol Chem 1939;128:537– nary pH to distinguish rate-dependent from classic distal
550. renal tubular acidosis in children. J Pediatr 1993;122:60–65.
150. Prueksaritanont T, Chen ML, Chiou WL. Simple and micro 169. Batlle DC, Sehy JT, Roseman MK, et al. Clinical and
high-performance liquid chromatographic method for the pathophysiologic spectrum of acquired distal renal tubular
simultaneous determination of p-aminohippuric acid and acidosis. Kidney Int 1981;20:389–396.
iothalamate in biological fluids. J Chromatogr 1984; 306:89– 170. Skinner R, Cole M, Pearson ADJ, et al. Specificity of pH and
97. osmolality of early morning urine sample in assessing distal
151. Calgagno PL, Rubin MI. Renal extraction of para-amino- renal tubular function in children. BMJ 1996;312:1337–338.
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1632–1639. in childhood. Pediatr Nephrol 1988;2:498–509.
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172. García-Nieto V, Santos F. Pruebas funcionales renales. In: 175. Aronson AS, Svenningsen NW. DDAVP tests for estimation
García-Nieto V, Santos F, eds. Nefrología pediátrica. Madrid: of renal concentrating capacity in infants and children. Arch
Grupo Aula Médica, 2000;15–26. Dis Child 1974;49:654–659.
173. Alon U, Hellerstein S, Warady BA. Oral acetazolamide in the 176. Marild S, Jodal U, Jonasson G, et al. Reference values for
assessment of (urine-blood) PCO2. Pediatr Nephrol 1991;5: renal concentrating capacity in children by the desmo-
307–311. pressin test. Pediatr Nephrol 1992;6:254–257.
174. Edelmann CM Jr, Barnett HL, Stark H, et al. A standard- 177. Marild S, Rembratt A, Jodal U, Norgaard JP. Renal concen-
ized test of renal concentrating capacity in children. Am J trating capacity test using desmopressin at bedtime. Pediatr
Dis Child 1967;114:639–644. Nephrol 2001;16:439–442.
 22

EVALUATION OF GROWTH
AND DEVELOPMENT
SANDRA L. WATKINS
GAIL E. RICHARDS

NORMAL GROWTH meatus describing a line that is perpendicular to the back

board of the stadiometer of the infant-measuring board.
Growth is such a valuable biologic indicator of health For standing measurements, the neck should be gently
during childhood that careful assessment of growth stretched while being sure that feet remain flat on the
should be a routine part of initial and ongoing care of ground. Socks and shoes should be removed so that any
children with documented or suspected renal disease. To lifting of the feet can be seen and corrected. Balance beam
appreciate the implications of growth perturbation caused scales should not be used for the assessment of height, as
by renal disease, it is necessary to understand normal the measurements are not accurate owing to the flexibility
growth and the uses and limitations of methods of growth of the measurement arm.
evaluation. Inaccurate plotting of measurements can lead to signifi-
cant misinterpretations of growth data. It is crucial to plot
the height as close to the fractional age as possible rather
MEASUREMENT OF GROWTH
than plotting on the nearest year or half-year. Use of a
growth chart appropriate to the measurement technique is
Linear Growth
also important. Standard charts for infants that often
The growth charts that are commonly used in clinical set- extend to 36 months are meant to be used with supine
tings are derived from cross-sectional data and are useful measurements taken on an infant-measuring board. Use of
to determine how the growth of an individual compares standing heights on these charts can lead to an erroneous
to age- and gender-matched peers (Fig. 22.1). To see the conclusion that growth has slowed, because standing
impact over time of both normal physiology and superim- heights are shorter than recumbent lengths. Similarly,
posed growth abnormalities, it is crucial to have accurate growth charts that include 2 years to 18 years or more are
longitudinal data. The importance of accurate measure- meant to be used for standing heights only and should not
ments cannot be overemphasized. Accurate measuring be used to plot recumbent lengths.
equipment and accurate measuring technique are crucial. Most growth charts are expressed in terms of percentiles for
Inaccurate positioning of a child on an infant-measuring a reference population. This is useful if a child’s measurement
board or stadiometer can lead to significant misinterpreta- lies within the percentiles. If, however, height is below the per-
tion of growth trends. Figure 22.2 demonstrates proper centiles, an alternative method of height quantitation is
positioning of an infant (Fig. 22.2A) and an older child needed. The SD score (SDS or Z score) is the number of SDs
(Fig. 22.2B). Special attention should be paid to position- below or above the age- and gender-appropriate mean for a
ing of the feet and head. Feet must be flat and positioned given height measurement. The concept of SDS is useful for
against the same board as the head. Usually an assistant most biologic variables and can be used for weight and body
should hold the feet in position to assure that the patient mass index (BMI) as well as for height and height velocity to
does not rise as the measurement is being taken. Buttocks compare individuals to the reference population and to follow
and shoulder blades should be touching the back board. change accurately. To use SDS accurately, a computer program
For infants, it is usually necessary for an assistant to hold is required that will convert height, weight, birth date, and
the knees down and hold the feet flat against the end measurement date to SDS scores. These programs are readily
board. The head should be in the Frankfurt plane, with available and should be part of the standard armamentarium
the outer canthus of the eye and the external auditory of pediatric nephrologists.
426 IV. Clinical Methods

FIGURE 22.1. A: Height percentiles in boys 2 to 20 years of age. (continued)

 22. Evaluation of Growth and Development 427

FIGURE 22.1. (continued) B: Height percentiles in girls 2 to 20 years of age.

428 IV. Clinical Methods

FIGURE 22.1. (continued) C: Height percentiles in girls from birth to 36 months.

 22. Evaluation of Growth and Development 429

FIGURE 22.1. (continued) D: Height percentiles in boys from birth to 36 months. BMI, body
mass index. (From Centers for Disease Control and Prevention Web site. Available at: http://
www.cdc.gov. Accessed April 2003, with permission.)
430 IV. Clinical Methods

tion should be used in interpretation of growth rates

calculated over less than a 6-month period. It is crucial
when plotting growth velocities on charts to draw bars
around the midpoint of the growth velocity as plotted to
indicate the time over which the velocity was calculated. It
is also important, especially when assessing therapies, to
choose time intervals for growth velocity calculation that
represent significant events. For example, the expectation
and interpretation for a growth velocity in the first year of
growth hormone (GH) treatment is different from the
expectation for the second year of treatment and so forth.
Intervals of treatment should not be mingled with intervals
without treatment in calculation of growth velocities.
Growth velocity data that correspond to the height stan-
dards shown in Figure 22.1 are not available. Thus, stan-
dards for growth velocity that were derived from
longitudinal data should be used (Fig. 22.3).
Interpretation of growth velocities differs from interpre-
tation of absolute height. Because a growth velocity of
around the fiftieth percentile is required to maintain an
absolute height around the fiftieth percentile, a growth
velocity that is consistently at the tenth percentile over
years cannot be considered normal. That differs signifi-
cantly from an absolute height that could be considered
quite normal if it remained at the tenth percentile over
years. A child who has a growth velocity at the tenth (or
even twenty-fifth percentile) over many years will gradually
fall lower on the percentiles for absolute height.

Sitting Height
Measurement of sitting height can be helpful when there is
reason to suspect that the patient has a condition or disease
that differentially affects either the spine or the long bones.
Examples might be hypophosphatemic rickets or a patient
FIGURE 22.2. Proper positioning of an infant (A) and child (B)
for accurate measurement. with scoliosis or those who have had irradiation of the spine
or spina bifida. Sitting height is best measured with a device
specifically constructed for this purpose consisting of a box
of appropriate width for comfortable sitting of a precise
Growth Velocity
height. The patient can then sit on the box and position the
Growth velocity is an extremely useful tool for assessing upper body and head as for a standing measurement. The
health and for determining the effectiveness of various ther- height of the box is subtracted from the stadiometer mea-
apies. The accuracy and interpretation of growth velocity surement. Standards for sitting height are provided in Fig-
calculations depend on the accuracy of the underlying mea- ure 22.4. In circumstances in which it is informative to
surement and on the choice of an appropriate interval for a separately assess growth of the spine and long bones, height
velocity calculation. Growth velocity varies throughout the and sitting height can be measured. The difference between
year in healthy children; thus, the most accurate calculation the two can be used to assess growth of long bones in con-
of growth rate is that done with measurements that are junction with measurement of arm span.
exactly a year apart. Sometimes practical considerations,
such as availability of measurements and the need to make
Other Measurements of Linear Growth
therapeutic decisions in a timely manner, preclude using
measurements over a complete year. Because multiplying To assess very small changes in growth over short periods of
the time over which two measurements were taken by the time, very precise methods have been devised for measure-
appropriate factor to calculate an annual growth rate neces- ment of the lower leg. This technique is referred to as kne-
sarily multiplies any measurement inaccuracy, extreme cau- mometry. Although the precision of the technique is very
 22. Evaluation of Growth and Development 431

FIGURE 22.3. A: Height velocity in boys 2 to 19 years of age. (continued)

432 IV. Clinical Methods

FIGURE 22.3. (continued) B: Height velocity in girls 2 to 19 years of age. [From Tanner JM,
Davies PS. Clinical longitudinal standards for height and height velocity for North American chil-
dren. J Pediatric 1985;107(3):317–329, with permission.]
 22. Evaluation of Growth and Development 433

FIGURE 22.4. A: Standards for sitting height for boys. (continued)

434 IV. Clinical Methods

FIGURE 22.4. (continued) B: Standards for sitting height for girls. [From Prader A, Largo RH,
Molinari L, et al. Physical growth of Swiss children from birth to 30 years of age. Helv Pediatr Acta
1988;43(Suppl 52):3–125, with permission.]
 22. Evaluation of Growth and Development 435

high, and significant advance in growth can be detected ion in specific bones. In clinical practice, the atlas by Greulich
over time periods as brief as a single day, in practice, this and Pyle is almost universally used (7). This method displays a
technique is not widely applied in clinical settings and is series of standards for boys and girls at various ages. An accom-
mainly reserved for research use (1–3). panying text describes the changes in specific bones because the
last standard is provided. The bone age is reported as the stan-
dard that looks most like the radiograph of the patient. To use
Head Circumference
this information appropriately, it is important to recognize that
Occipitofrontal circumference is an important measurement the standards were derived approximately 50 years ago and
in young children up to about 3 years while brain size is may not entirely reflect current secular trends. There are often
increasing. Occipitofrontal circumference is affected by nutri- gaps of an entire year between standards, making it difficult to
tional conditions and chronic illness only in extreme situations assess less dramatic changes in bone maturation. Finally, this
and generally does not give much insight into either the pres- system does not take into account that not every bone in every
ence or progression of renal disease. As occipitofrontal circum- patient matures at the same pace as the radiographic standards.
ference reflects brain growth, which is generally independent It is quite common to find a significant discrepancy among the
of somatic growth, it is an independent marker of whether an maturity of the bones of the hand and wrist.
insult or multisystem disease that may have caused renal dis- A more precise and useful method for bone age assessment
ease has also affected central nervous system development. requires 20 bones to be scored into one of (usually) eight cate-
gories of maturity and a score given for each of the categories
(8). The numerical sum of the scores for each of the 20 bones
Body Mass Index
is translated to a bone age. This method has the first advantage
Weight is commonly measured, and the measurement is of a validated weighting system to account for the fact that
usually quite precise if care is taken to weigh the patient in some bones are more reliable predictors of maturity than oth-
a standard and minimum amount of clothing, such as a ers. A second advantage is the ability to assess the bone age as
light hospital gown (or diaper for a young infant). Weight an almost continuous variable of 0.1-year increments. Using
can be plotted for age and compared to height. Generally, it this system, it is possible to detect more subtle changes in mat-
is thought to be optimum if the weight and height percen- uration. When applied by very experienced interpreters, these
tiles are the same. This is not the case for most patients, so two methods give quite comparable results. In situations of
an alternative measure of the weight to height relationship discordant clinical and radiologic findings, it is sometimes
is needed to determine whether a patient is at risk from advantageous to use both methods. The 20-bone method can
either too much or too little body mass (weight). BMI is be much more time consuming, however, and is unlikely to be
calculated as weight in kg/height in m2. Children who have commonly used by radiologists.
a BMI higher than the seventy-fifth percentile or lower The issue of what a bone age means is both intuitively
than the fifth percentile have increased health risks, which obvious, yet potentially open to misinterpretation. On the
will be additive to any underlying renal disease (4–6). one hand, a delay in bone age can imply that the individual
Thus, calculating BMI and either plotting or determining in question has more potential for growth than would be
BMI SDS is another valuable health indicator. Standards assumed by plotting a height measurement alone. On the
for BMI percentiles are provided in Figure 22.5. other hand, the tendency of films to be interpreted as either
BMI is generally perceived to be a proxy for body com- normal or abnormal based on whether they fall within two
position, but there is significant variation in the weight of SDs of a mean reading can cause unwarranted consterna-
bone and muscle among individuals, making the correla- tion. It is perhaps more helpful to think of a bone age as a
tion between BMI and other determinations such as fat descriptor of maturation that cannot be abnormal in and of
mass are not precise. If techniques such as isotope dilution itself. A bone age is only one clue contributing to a decision
or quantitative dual-energy x-ray absorptiometry are not about whether a growth pattern is indicative or a conse-
practical, a good approximation of fat mass can be deter- quence of an underlying abnormality or treatment.
mined by bioelectrical impedance. If it is important to doc-
ument whether changes in weight are due to fat or fat-free
Pubertal Stages
mass, bioelectrical impedance can be quite helpful. It is
important to use regression equations derived specifically Just as accurate assessment of height and growth velocity
for children, however, rather than relying on the immediate informs assessment of health, accurate staging of the pubertal
output of commercial machinery. process and monitoring of the progression of pubertal devel-
opment can be a good indicator of general health and ade-
quacy of treatment for underlying renal disease. Figure 22.6
Bone Age
describes the pubertal staging method of Tanner that is the
Physiologic maturity of the skeleton can be estimated by assess- accepted method to quantify pubertal development. It should
ing specific radiologic findings that progress in an orderly fash- be noted that pubertal stage is not described in one number,
436 IV. Clinical Methods

FIGURE 22.5. A: Standards for body mass index (BMI) percentiles for boys. (continued)
 22. Evaluation of Growth and Development 437

FIGURE 22.5. (continued) B: Standards for body mass index (BMI) percentiles for girls. (From
Centers for Disease Control and Prevention Web site. Available at: http://www.cdc.gov. Accessed
April 2003, with permission.)
438 IV. Clinical Methods

FIGURE 22.6. Stages of pubertal development for boys and

girls (A–C). Stages of genital development for boys (A): (1) Pre-
adolescent—testes, scrotum, and penis are of about the same
size and proportions as in early childhood. (2) Enlargement of
scrotum and of testes—the skin of the scrotum reddens and
changes in texture; little or no enlargement of penis at this
stage. (3) Enlargement of penis, which occurs at first mainly in
length—further growth of testes and scrotum. Increased size
of penis with growth in breadth and development of glans;
further enlargement of testes and scrotum; increasing darken-
ing of scrotal skin. Stages of breast development for girls
(B): (1) Preadolescent—elevation of the papilla only. (2) Breast
bud stage—elevation of papilla as a small mount; enlargement
of areolar diameter. (3) Further enlargement and elevation of
breast and areola, with no separation of their contours. (4) Pro-
jection of areola and papilla to form a secondary mound; the
development of the areolar mound does not occur in all girls;
in one-fourth it is absent and in a further one-fourth relatively
slight. (5) Mature stage—projection of papilla only, caused by
recession of the areola to the general contour of the breast.
Stages of pubic hair development for boys and girls (A,C): (1) Pre-
adolescent—the vellus over the pubes is not developed further
than over the abdominal wall. (2) Sparse growth of long, slightly
pigmented downy hair, or only slightly curled, appearing
chiefly at the penis or along the labia. (3) Considerably darker,
coarser, and more curled; the hair spreads sparsely over the
junction of the pubes. (4) Hair now resembles adult in type but
the area covered is still considerably smaller than in the adult;
no spread to the medial surface of the thighs. (5) Adult in
quantity and type with distribution of the horizontal (classi-
cally feminine) pattern; spread to the medial surface of thighs
but not up linea alba or elsewhere above the base of the
inverse triangle. (From Tanner J. Growth at adolescence.
Oxford: Blackwell, 1962, with permission.)
 22. Evaluation of Growth and Development 439

but, rather, a separate stage should be given for pubic hair of the child’s own normal endocrine system. The growth
development and breast or genital development. In some situ- velocity curves demonstrate well that growth velocity is con-
ations, these numbers are discordant. It is particularly impor- stantly declining during this phase. Growth velocity for any
tant to accurately assess testicular size by comparison to a given child is not always in a consistent percentile. In fact, a
standard orchidometer with a gradation of sizes from 1 to 25 healthy child with a growth velocity with a negative SDS
mL. This volume should be recorded in addition to the pubic (i.e., below the mean) in 1 year is statistically most likely to
hair stage and the genital stage. A description of pubertal stage have a growth velocity with a positive SDS (i.e., above the
should be part of every physical examination. mean) in a subsequent year. Thus, in addition to the useful-
ness of calculating growth velocities over a full year, addi-
tional conclusions about the normality or abnormality of a
NORMAL GROWTH—PHASES AND DRIVERS growth pattern can require reliable data over several years.
OF GROWTH The fourth phase of growth is the pubertal growth spurt.
The time of onset of the pubertal growth spurt varies
The first phase of growth is intrauterine growth. Although pre- between genders but also among individuals of the same
natal growth can be influenced by factors such as chromo- gender. Growth velocity increases dramatically and more in
somal constitution and fetal hormone production, the single girls than in boys before declining as pubertal development
most significant contributor to adequate fetal growth seems to is complete. Because the timing of the onset of puberty is
be adequate nutrition. Adequate nutrition, in turn, depends variable, it is appropriate to take pubertal stage into consid-
on adequate maternal nutrition and adequate delivery of nutri- eration when assessing growth rate. If puberty has not
ents through a competent placenta. If either of these aspects of begun, growth velocity will continue to decline until
nutrition fails, intrauterine growth retardation or restriction pubertal onset. Thus, growth velocity can be quite low in a
(IUGR) results. Approximately 85% of children with intra- child who has delayed onset of puberty at an age when
uterine growth retardation or restriction exhibit catchup most of his or her peers are having a pubertal growth spurt.
growth in the preschool years and ultimately achieve normal This “dip before the spurt” phenomenon can be differenti-
adult stature. The observation that approximately 15% of ated from a significant abnormality of growth by assessing
intrauterine growth retardation or restriction babies do not pubertal stage and possibly bone age (see later).
catch up has lead to the hypothesis of intrauterine growth pro- After several years of exposure to sex steroids, the epi-
gramming. This hypothesis and the mechanisms that might be physes of the long bones fuse, and linear growth is no
responsible for it are under active investigation (9). longer possible. This effect on bones is believed to be medi-
The second phase of growth is the rapid growth in both ated by estrogen in both boys and girls (13). It is this matu-
length and weight that occurs during the first 2 years or so rational effect that is responsible for the decline in growth
after birth. Through mechanisms that are poorly understood velocity after the peak of the pubertal growth spurt and for
and may be related to nutritional programming of growth, this the ultimate cessation in growth. Thus, individuals who
infantile growth also seems to be driven to a significant extent have significant delay of their bone maturity (bone age) also
by intrauterine nutrition. During this phase of growth, a have residual growth potential. If the delay in bone matu-
healthy baby would be expected to grow approximately 25 rity is due to either the effect of serious illness or due to
cm/year. During the later part of this growth phase, most com- treatment with glucocorticoids or other medication, the
monly between approximately 9 and 24 months, many chil- growth potential inherent in a delayed bone age is not
dren who were well nourished in utero but whose genetics always completely achieved (14).
suggest they are likely to be shorter children cross percentiles in
a downward fashion to bring their length more in line with
genetic expectations. This is a normal growth adjustment in DETERMINATION OF GENETIC TARGET
this phase and does not necessarily indicate an abnormality of RANGE AND CORRECTION OF GROWTH
growth. EXPECTATIONS FOR PARENTAL HEIGHTS
There have been a number of studies associating growth
during this phase with plasma growth factors, such as insulin- In the third or childhood phase of growth, height is signifi-
like growth factor (IGF)-1, IGF-2, IGF-binding protein cantly impacted by genetics. Any assessment of absolute
(IGFBP) 3, and IGFBP1 (10–12). It is not clear, however, that height must take into account the target height range,
these associations reflect a causative influence of the growth which is best estimated statistically by parental heights. To
factors or a common driver of both growth and the growth plot a genetic target range, the height of the same sex par-
factors, such as nutrition or programming of the growth fac- ent should be plotted on the right edge of the growth chart.
tors by either nutrition or other unknown factors. The height of the opposite sex parent should be plotted
The third phase of growth lasts from approximately 2 either by transferring the percentile on the appropriate sex
years until the time of the pubertal growth spurt. During this chart to the child’s chart (i.e., plotting a twenty-fifth per-
phase, growth is driven by genetics with the permissive input centile mother at the twenty-fifth percentile on a boy’s
440 IV. Clinical Methods

A diagram of the functioning of the somatotropic axis is

shown in Figure 22.7 (15). Multiple inputs from a variety of
neurotransmitters and neuropeptides influence the hypotha-
lamic output of GH releasing hormone, which stimulates,
and somatostatin, which inhibits GH release from the pitu-
itary. A newly described hormone, ghrelin, appears to be an
additional stimulant to pituitary release of GH, but its role in
physiologic regulation of growth is not yet clear (16).
GH combines with a specific GH receptor to initiate a sig-
nal transduction cascade in multiple tissues that leads to synthe-
sis of IGF-1. IGF-1 in turn interacts with multiple IGFBPs and
the type 1 IGF receptor to affect multiple other signal transduc-
tion cascades that ultimately lead to tissue growth and other
metabolic effects of IGF-1. Thus, it is possible for perturbations
in this axis at any point to cause an abnormality of growth.
Increasing understanding of the complex interrelationship of
nutrition and the somatotropic axis makes a clear distinction
between primary nutritional abnormalities and primary abnor-
malities of the somatotropic axis very difficult. A recent review
of the regulation and abnormalities of the somatotropic axis
outlines the many subtleties of the interpretation of hormone
levels in the context of nutritional perturbation (17).

GROWTH IN CHRONIC RENAL INSUFFICIENCY

Growth failure has long been recognized in children with

FIGURE 22.7. Normal physiology of the somatotropic axis.
chronic renal failure (18). Recent advances in dialysis and
GHRH, growth hormone-releasing hormone; IGFBP, insulin-like transplantation offer the potential for the child with end-
growth factor-binding protein; SMS, somatostatin. (From Rosen- stage renal disease to live well into adulthood. This magni-
feld R, Rosembloom A, Guevara-Aguirre J. Endocrine Reviews fies the problem of growth failure, as they are unlikely to
1995;15:369–390, with permission.)
reach normal adult stature, leading to the need for investi-
gation of the etiology of growth delay and development of
chart) or by adding 13 cm to the mother’s height and plot- strategies to overcome short stature in children with
ting it on a boy’s chart or subtracting 13 cm from a father’s decreased renal function. The need for adequate, sustained
height and plotting it on a girl’s chart. Next, the midpoint growth is the single most important element that sets chil-
of these two parental heights should be calculated and plot- dren apart from adults with chronic renal failure.
ted. A range of 10 cm above and below this midpoint will Most children with renal insufficiency exhibit profound
include the fifth to ninety-fifth percentiles of the target growth retardation. The classic depiction of a typical growth
range for that set of parents. pattern in a child with renal failure is shown in Figure 22.8
Further use can be made of these data to calculate a Z (19). Infants demonstrate severe statural height failure,
score adjusted for parental height. For example, if a child whereas older children parallel the growth curve of the nor-
has a height Z score of –2 and the midpoint of the genetic mal child but remain below it, and the adolescent again falls
target range has a Z score of –1.5, the child has a Z score of away from the curves demonstrating an attenuated pubertal
–0.5 when adjusted for genetic target range. growth spurt (19,20). The Growth Failure in Children with
Renal Diseases Study looked at children with renal failure
from 23 centers, grouped by degree of insufficiency and age
PHYSIOLOGY OF GROWTH (Table 22.1). Children with more severe renal failure and
HORMONE SECRETION those with congenital disease and early onset of renal failure
tended to have more growth delay (21).
Although a comprehensive discussion of the physiology and In a study of children requiring dialysis (22), Fine found
pathophysiology of GH secretion is beyond the scope of this severe height delay with 70% of children below the age of 5
text, it is useful to bear in mind current understanding of the years more than 2 SD below the mean for age (Fig. 22.9).
regulatory mechanisms of the somatotropic axis when assess- The effect of long-term dialysis on the growth of children
ing the growth of children with renal dysfunction. with chronic renal failure was studied by Kleinknecht et al. in
 22. Evaluation of Growth and Development 441

TABLE 22.1. GROWTH FAILURE IN CHILDREN WITH

CHRONIC RENAL FAILURE
Category No. of patients Height SD score

Age (yr)
1–3 31 –1.4
4–6 23 –1.0
7–10 28 –0.7
Disease
Acquired 23 –0.9
Congenital 59 –1.1
Renal failurea
Mild 34 –0.9
Moderate 29 –1.2
Severe 12 –1.1

aMild, creatinine clearance 50–75 mL/min/1.73 m2; moderate, creati-

nine clearance 30–49 mL/min/1.73 m2; severe, creatinine clearance

<30 mL/min/1.73 m2.
Adapted from Abitbol C, Warady B, Massie M, et al. Linear growth
and anthropometric and nutritional measurements in children with
mild to moderate renal insufficiency: a report of the growth failure
in children with renal diseases study. J Pediatr 1990;116:S46–S62.

Several studies indicate that children on chronic ambula-

tory peritoneal dialysis grow better than those receiving
chronic hemodialysis or transplantation (28–34). However,
these studies show little, if any, statistical significance of the
data and are not controlled for age, underlying disease, nutri-
tional intake, and other factors affecting health and growth.
Often, the hemodialysis data are derived from historical con-
FIGURE 22.8. Schematic representation of growth in children trols. This can be misleading, as studies have shown general
with renal insufficiency. A represents the first 2 years of life. B improvement in SD scores on hemodialysis when comparing
represents 2 years of age until onset of puberty. C represents the successive time periods (35). Some of the children had expe-
pubertal period. (From Betts P, Magrath G. Growth pattern and
dietary intake of children with chronic renal insufficiency. BMJ rienced failure of peritoneal dialysis before the institution of
1974;2:189–193, with permission.) hemodialysis, further confounding the analyses. Even under
optimal conditions, children on peritoneal dialysis still do
not exhibit catchup growth, with several studies showing
the 1970s (23). Seventy-six children were followed for 12 to more than one-half of the children continue to lose growth
111 months (mean 30 months) on their individualized dialy- percentiles while on dialysis (30,36–38).
sis regimens (23). In the 50 prepubertal children there was a Transplantation remains the ultimate goal for the pedi-
mean height loss of 0.38 SD/year. The mean height gain atric end-stage renal disease patient. Recent data from the
during puberty in 11 pubertal children was only 2 cm/year North American Pediatric Renal Transplant Cooperative
in girls and 2.7 cm/year in boys, although some subjects Study show a mean standardized height of –2.16 SDS in
exhibited a prolonged growth period (up to age 21 years in nearly 400 transplant recipients (26). Younger children
one child). A more recent study by van Diemen-Steenvoorde tend to be more severely growth retarded, reflecting the
and Donckerwolcke of 35 prepubertal children receiving impact of chronic renal failure during the early growth
hemodialysis for more than 1 year (mean, 2.5 years) revealed spurt. After transplantation, children younger than 5 years
a loss of 0.31 SD/year (24). The pubertal growth spurt was demonstrate some catchup growth, but children older than
3.2 cm/year for boys and 2.1 cm/year for girls at this center. 6 years have no acceleration of growth rate and may even
The multicenter report from the European Dialysis and continue to lose ground, becoming more growth retarded
Transplant Association in 1978 of prepubertal children fol- (26,34,39). Thus, it is clear that growth failure cannot be
lowed for more than 4 years on hemodialysis cited a loss of corrected by transplantation and must be addressed earlier
0.43 SD/year in height (25). More recent data from the in the course of renal failure.
North American Pediatric Renal Transplant Cooperative Final adult height is greatly affected by the growth fail-
Study (26), a registry of children with renal failure in North ure seen with chronic renal failure in children. The Euro-
America, continues to demonstrate a loss of 0.32 height SDS pean Dialysis and Transplant Association found in 1981
over a 2-year period in children receiving dialysis (27). (40) that the mean height of adults who had suffered from
442 IV. Clinical Methods

TABLE 22.2. FACTORS CONTRIBUTING TO GROWTH

FAILURE IN CHILDREN WITH RENAL INSUFFICIENCY
Age at onset of primary renal disease
Etiology of primary renal disease
Degree of renal impairment
Inadequate caloric and protein intake
Renal osteodystrophy
Metabolic acidosis
Tubular defects
Polyuria
Salt wasting
Potassium depletion
Other losses (calcium, phosphorous, etc.)
Corticosteroid administration
Anemia
Infection
Psychosocial factors
Uremic inhibitors of growth hormones and related proteins

Adapted from Stewart C, Fine R. Growth in children with renal insuf-

ficiency. Semin Dial 1993;6:37–45; Rigden S, Rees L, Chantler C.
Growth and endocrine function in children with chronic renal fail-
ure. Acta Paediatr Scand 1990;370:S20–S27; and French C, Genel M.
Pathophysiology of growth failure in chronic renal insufficiency. Kid-
ney Int 1984;30:S59–S64.

poorly understood (41–43). As noted in Figure 22.9, chil-

dren with onset of renal failure earlier in life have more severe
growth impairment than those diagnosed later. Figure 22.10
depicts the severe growth failure of a group of children with
obstructive uropathy, which is usually congenital, thus exert-
ing an influence on growth from birth (22).
A correlation between degree of renal failure and growth
dysfunction has been reported, although this correlation
has not been demonstrated in all studies (21,35,42,44).
FIGURE 22.9. Relationship between degree of growth retarda- Poor nutritional intake can certainly influence growth in
tion and age at onset of renal insufficiency. (From Fine R.
Growth in children with renal insufficiency. In: Nissenson AR,
any setting, and chronic renal failure is no exception (45).
Gentile DE, eds. Clinical dialysis, 2nd ed. Norwalk, CN: Appleton Some authors report improvement in height SD scores with
& Lange, 1990:667–675, with permission.) nutritional intervention (42,46–48).
Renal osteodystrophy, intercurrent infections, metabolic
acidosis, and tubular defects have been correlated to poor
chronic renal failure during childhood was 3 SD below the growth, and specific therapy for these defects can be growth
mean (–3.0 SDS). In 1985, the European Dialysis and enhancing (21,44,45,49). Corticosteroids are well known
Transplant Association (35) further reported 62% of men to inhibit statural growth, but are not usually required ther-
and 41% of women who had renal replacement therapy apeutically once dialysis is initiated (50). Although chronic
before the age of 15 had final adult heights below the nor- anemia was long thought to be a contributing factor to
mal range. This confirms the expectation that children growth failure, early reports of correction of anemia with
with renal failure at an early age never demonstrate recombinant erythropoietin have been disappointing
catchup growth and thus reach adulthood significantly regarding improvement in nutrition or growth (51–54).
height compromised. Psychosocial factors have also been shown to affect the
growth of children with impaired renal function (35).

ETIOLOGY OF GROWTH DELAY IN CHRONIC

RENAL FAILURE EFFECTS OF UREMIA ON GROWTH HORMONE/
INSULIN-LIKE GROWTH FACTOR AXIS
Many factors, listed in Table 22.2, are thought to contribute
to the growth failure associated with chronic renal insuffi- It was thought that abnormalities of GH and IGF-1 played
ciency, but the mechanisms responsible for their effects are no role in the pathogenesis of growth failure in renal insuf-
 22. Evaluation of Growth and Development 443

uremic inhibitors (66–68). More recent reports, using

improved extraction techniques, indicate that IGF-1 activ-
ity is near normal in uremia (64,69–71).
Current research is focusing on the physiology and
pathophysiology of GH-binding proteins and IGFBPs in
normal and abnormal growth (71–77). Uremic children
have been shown to have a low serum level of GH-binding
protein (75). This may play some role in end-organ effect.
At least six IGFBPs have been identified and characterized
(55,78). IGFBP1, IGFBP3, and IGFBP4 have been shown
to be elevated in uremic children and the excess binding
protein, especially IGFBP3, acts as an IGF-1 inhibitor
(55,72,73,76,79).

TREATMENT OF THE GROWTH FAILURE OF

CHRONIC RENAL INSUFFICIENCY

The alterations in GH-binding proteins and IGFBPs in

chronic renal failure provide a rationale for the use of
recombinant human growth hormone (rhGH) to treat the
growth failure in prepubertal uremic children. Theoreti-
cally, excess ligand might be expected to overcome any
inhibitory effect of increased binding. Landmark studies by
Mehls and colleagues demonstrated a stimulatory effect of
growth hormone on chondrocytes and longitudinal growth
in uremic rats (80–82). Several other investigators con-
firmed the growth-enhancing effects of GH in this animal
model (83–86). These studies also provided reassurance
regarding stability of renal function and serum lipids
(83,84,86), although concern has been raised regarding a
trend toward reduced survival and glomerular hypertrophy
with glomerulosclerosis in treated animals (83).
The favorable results of the use of rhGH in the rat
model led to subsequent trials in uremic children. In 1988,
Lippe et al. reported a twofold increase in height velocity in
five growth-retarded, uremic children given rhGH (87).
FIGURE 22.10. Relationship between degree of growth retar- This center has since reported growth data from nine
dation and primary renal disease. (From Fine R. Growth in chil-
dren with renal insufficiency. In: Nissenson AR, Gentile DE, eds. patients followed for up to 36 months on GH therapy (88–
Clinical dialysis, 2nd ed. Norwalk, CN: Appleton & Lange, 90). SD scores in these nine patients improved from mean
1990:667–675, with permission.) –3.18 to –2.50 at 12 months, whereas the four subjects
treated for 36 months improved from –2.78 to –1.29. The
individual growth chart of a typical subject is shown in Fig-
ficiency. However, recent studies have begun to elucidate ure 22.11. Bone age did not advance disproportionately to
perturbations of the GH axis in chronic renal failure, which chronologic age in these patients. There were no significant
brings this aspect of growth failure into focus and lays the changes in serum chemistries, calculated creatinine clear-
groundwork for therapeutic interventions (55–64). ances, thyroid function tests, or glucose tolerance. IGF-1
GH functions by stimulating production and release of levels increased in all patients (90).
IGF-1 as well as by more direct effects on target tissues These encouraging preliminary results have been sup-
(64,65). Early reports indicated reduced (66) or high (62) ported by Tönshoff and the German Study Group who
IGF-1 levels in uremia when measured by radioimmunoas- have now studied 61 uremic children (20 predialysis, 24
say. Bioassays have also found reduced activity of IGF in dialysis, 17 transplanted) receiving daily subcutaneous
uremia, with some investigators finding a slight rise in rhGH for 1 to 2 years (91,92). The 17 prepubertal, predial-
activity after a dialytic session and others demonstrating ysis subjects improved from a pretreatment median SDS of
higher levels with transplantation, raising the possibility of –3.0 to –2.0 after 1 year of therapy. This study reports a
444 IV. Clinical Methods

Concerns regarding potential long-term side effects have

not been borne out by experimental data. Fine and col-
league’s randomized, placebo-controlled study should pro-
vide excellent data on both long-term effectiveness and
adverse effects (99). Specific concerns raised regarding avas-
cular necrosis of the femoral head, progression of renal fail-
ure, and lipid and insulin abnormalities have not been
borne out in subsequent investigations (104–109).
Reports of the use of rhGH in growth-retarded children
with renal allografts are encouraging with some improvement
in height SDS despite the concomitant use of corticosteroids
to suppress allograft rejection (92–95,110–114). However,
concerns about a deleterious effect on the allograft demand
caution in this patient population (115,116). Further random-
ized, controlled studies are necessary to determine the safety of
treatment in this setting. Until then, the best approach appears
to be normalization of height using growth hormone before
transplantation, as final adult height is best predicted by height
achieved before transplantation (39,117).
Many of the above studies included patients receiving
dialysis. Fine et al. reported five children undergoing
chronic cycling peritoneal dialysis who received rhGH
(118). Only three showed improvement in height SDS, but
length of treatment was less than 1 year in the other two.
Tönshoff et al. found improvement in the height velocity
FIGURE 22.11. Growth of individual patient before and during
treatment with recombinant human growth hormone (rhGH). ↓, SDS in eight children undergoing dialysis treated with
rhGH thrice weekly; ↑, rhGH daily; D, dialysis initiated; D/C, rhGH rhGH (1 hemodialysis, 7 peritoneal dialysis), but the
discontinued. [From Fine R, Pyke-Grimm K, Nelson P, et al. change was not as marked as in a single predialysis patient
Recombinant human growth hormone treatment of children
with chronic renal failure: long-term (1- to 3-year) outcome. (91). This trend continued in the larger German study,
Pediatr Nephrol 1991;5:477–481, with permission.] with improvement in SDS from –3.2 to –2.7 in ten dialysis
patients treated for 1 year (96). However, in Hokken-Koel-
ega and colleagues’ placebo-controlled, double-blind, cross-
significant improvement in height in all patient groups over trial there was no difference found in the response of
with no untoward effects, except an elevation in insulin dialysis patients compared to conservatively managed pre-
levels. This has been found to be a transient abnormality in dialysis patients, with mean increases in height velocity of
the longer-term study (90). Mehls et al. also found an 2.9 cm/6 months versus 3.0, respectively (96). An encour-
increase in IGF-1 after treatment, with no further rise in aging report by Tom et al. demonstrates some catchup
IGFBP3 concentration (55). Johansson et al. and Van Es growth in children on maintenance hemodialysis with
and the European Study Group have followed 94 prepuber- increased intensity of hemodialysis and closely monitored
tal children (41 predialysis and 53 transplanted) for up to 2 nutritional intake, raising the possibility of overcoming the
years of rhGH therapy (93,94). In children treated for 2 poor growth response in the dialysis population (119).
years, height SDS was improved from –2.8 to –1.8 in pre- Significant controversy exists regarding the safety and
dialysis patients. No significant side effects were reported. efficacy of rhGH after transplantation. Thus, it remains to
Multiple other reports have confirmed the effect of be seen whether any height deficit remaining after success-
rhGH in overcoming the short stature associated with ure- ful transplantation will be amenable to rhGH therapy.
mia in the pediatric population (95–101), including two Children treated with GH may have their growth limited
reports of improved height in children with nephropathic by fusion of the epiphyses as a result of normal pubertal devel-
cystinosis (100,101). Encouraging data exist about opment. Because of some evidence that delaying puberty in
improvement in height SDS and head circumference in children with GH deficiency can increase height prognosis
infants treated with GH. The role of the GH/IGF-1 axis in (17), such therapy might be considered for children under-
this age group still requires more clarification, especially going GH treatment for chronic renal insufficiency. The bene-
whether treatment could relate to improved neurodevelop- fit to be derived from such treatment in patients with chronic
ment (98,99). Although more difficult to assess, pubertal renal insufficiency has yet to be documented.
uremic children are also believed to respond favorably to There are early indications that therapy with GH can have a
GH treatment (92–95,97,102,103). positive effect on ultimate adult height. Recently, the German
 22. Evaluation of Growth and Development 445

Study Group for Growth Hormone Treatment in Chronic 10. Fall C, Pandit A, Law, et al. Size at birth and plasma insulin
Renal Failure demonstrated that mean final adult height for line growth factor-1 concentrations. Arch Dis Child 1995;
children with chronic renal failure treated with growth hor- 73:287–293.
mone was 1.6 SD below normal, a marked improvement over 11. Ong K, Kratsch J, Kiess W, et al. Size at birth and cord
historical controls who were untreated (120). blood levels of insulin, insulin-like growth factor I (IGF-I),
IGF-II, IGF-Binding Protein-1 (IGFBP-1), IGFBP-3 and
the soluble IGF-II/mannose-6-phosphate receptor in term
human infants. J Clin Endocrinol Metab 2000;85:4266–
CONCLUSION 4269.
12. Ong K, Kratzsch J, Kiess W, et al. Circulating IGF-I levels
The growth retardation of children with chronic renal fail- in childhood are related to both current body composition
ure is a major problem for the pediatric nephrologist. With and early postnatal growth rate. J Clin Endocrinol Metab
the current success of dialysis and transplantation in mini- 2002;87:1041–1044.
mizing the mortality of early-stage renal disease, these chil- 13. Smith E, Boyd J, Frank G, et al. Estrogen resistance caused
dren should live well into adulthood. Allowing them to by a mutation in the estrogen receptor gene in a man. N
appreciate their full adult height potential is a realistic goal. Engl J Med 1994;331:1056–1061.
The use of rhGH to enhance growth during the early years 14. Ackland F, Preece M. Catch-up growth. Pediatrician
1987;14:226–233.
of renal insufficiency and during dialysis has been shown to
15. Rosenfeld R, Rosembloom A, Guevara-Aguirre J. Growth
be effective. Further long-term studies are needed to deter- hormone (GH) insensitivity due to primary GH receptor
mine whether growth will continue and genetic potential deficiency. Endocrine Reviews 1995;15:369–390.
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term side effects also need to be continually monitored in a secretion during fasting in man. Eur J Endocrinol 2002;
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function. Metabolism 1981;30:1091–1095. 86. Santos F, Chan J, Hanna J, et al. The effect of growth hor-
68. Kapila P, Jones J, Rees L. Effect of chronic renal failure and mone on the growth failure of chronic renal failure. Pediatr
prednisolone on the growth hormone-insulin-like growth Nephrol 1992;6:262–266.
factor axis. Pediatr Nephrol 2001;16:1099–1104. 87. Lippe B, Fine R, Koch V, et al. Accelerated growth follow-
69. Powell D, Rosenfeld R, Sperry J, et al. Serum concentra- ing treatment of children with chronic renal failure with
tions of insulin-like growth factor (IGF-I), IGF-II and recombinant human growth hormone (Somatrem): a pre-
unsaturated somatomedin carrier proteins in children with liminary report. Acta Paediatr Scand 1988;343:S127–S131.
chronic renal failure. Am J Kidney Dis 1987;10:287–292. 88. Koch V, Lippe B, Nelson P, et al. Accelerated growth after
70. Phillips L, Fusco A, Unterman T, et al. Somatomedin inhib- recombinant human growth hormone treatment of children
itor in uremia. J Clin Endocrinol Metab 1984;59:764–772. with chronic renal failure. J Pediatr 1989;115:365–371.
71. Hodson E, Brown A, Roy L, et al. Insulin-like growth fac- 89. Fine R. Recombinant human growth hormone treatment of
tor-1, growth hormone-dependent insulin-like growth fac- children with chronic renal failure: update 1990. Acta Pae-
tor-binding protein and growth in children with chronic diatr Scand 1990;370:S44–S51.
renal failure. Pediatr Nephrol 1992;6:433–438. 90. Fine R, Pyke-Grimm K, Nelson P, et al. Recombinant
72. Blum W, Ranke M, Kietzmann K, et al. Growth hormone human growth hormone treatment of children with chronic
resistance and inhibition of somatomedin activity by excess renal failure: long-term (1- to 3-year) outcome. Pediatr
of insulin-like growth factor binding protein in uraemia. Nephrol 1991;5:477–481.
Pediatr Nephrol 1991;5:539–544. 91. Tönshoff B, Mehls O, Heinrich U, et al. Growth-stimulat-
73. Lee P, Hintz R, Sperry J, et al. IGF binding proteins in ing effects of recombinant human growth hormone in chil-
growth-retarded children with chronic renal failure. Pediat- dren with end-stage renal disease. J Pediatr 1990;116:561–
ric Res 1989;26:308–315. 566.
74. Postel-Vinay M, Tar A, Crosnier H, et al. Plasma growth 92. Tönshoff B, Dietz M, Haffner D, et al. Effects of two years
hormone-binding activity is low in uraemic children. Pedi- of growth hormone treatment in short children with renal
atr Nephrol 1991;5:545–547. disease. Acta Paediatr Scand 1991;379:S33–S34.
75. Maheshwari H, Rifkin I, Butler J, et al. Growth hormone 93. Johansson G, Sietnieks A, Janssens F, et al. Recombinant
binding protein in patients with renal failure. Acta Endo- human growth hormone treatment in short children with
crinol 1992;127:485–488. chronic renal disease, before transplantation or with func-
76. Powell D, Liu F, Baker B, et al. Characterization of insulin- tioning renal transplants: An interim report on five Euro-
like growth factor binding protein-3 in chronic renal failure pean studies. Acta Paediatr Scand 1990;370:S36–S43.
serum. Pediatric Res 1993;33:136–143. 94. Van Es A. Growth hormone treatment in short children
77. Tönshoff B, Schaefer F, Mehls O. Disturbance of growth with chronic renal failure and after renal transplantation:
hormone-insulin-like growth factor axis in uraemia. Pediatr combined data from European clinical trials. Acta Paediatr
Nephrol 1990;4:654–662. Scand 1991;379:S42–S49.
78. Powell D. Effects of renal failure on the growth hormone- 95. Rees L, Rigden S, Ward G, et al. Treatment of short stature
insulin-like growth factor axis. J Pediatr 1997;131:S13–S16. in renal disease with recombinant human growth hormone.
79. Ulinski T, Mohan S, Kiepe D, et al. Serum insulin-like growth Arch Dis Child 1990;65:856–880.
factor binding protein (IGFBP)-4 and IGFBP-5 in children 96. Hokken-Koelega A, Stijnen T, de Muinck Keizer-Schrama
with chronic renal failure: relationship to growth and glomeru- S, et al. Placebo-controlled, double-blind, cross-over trial of
lar filtration rate. The European Study Group for Nutritional growth hormone treatment in prepubertal children with
Treatment of Chronic Renal Failure in Childhood. German chronic renal failure. Lancet 1991;338:585–590.
Study Group for Growth Hormone Treatment in Chronic 97. Ortiz A, Placida Garron M, Rovira A, et al. Effect of recombi-
Renal Failure. Pediatr Nephrol 2000;14:589–597. nant human growth hormone in a postpediatric hemodialysis
80. Kreusser W, Weinkauf R, Mehls O, et al. Effect of parathy- patient with delayed growth. Am J Nephrol 1992;12:471–473.
roid hormone, calcitonin and growth hormone on cAMP 98. van Renen M, Hogg R, Sweeney A, et al. Accelerated
content of growth cartilage in experimental uremia. Eur J growth in short children with chronic renal failure with
Clin Invest 1982;12:337–343. both strict dietary therapy and recombinant growth hor-
81. Mehls O, Ritz E. Skeletal growth in experimental uremia. mone. Pediatr Nephrol 1992;6:451–458.
Kidney Int 1983;24:S53–S62. 99. Fine R, Kohaut E, Frane J, et al. Multicenter randomized
82. Mehls O, Ritz E, Hunziker E, et al. Improvement of growth double blind placebo—controlled study of recombinant
and food utilization by human recombinant growth hor- human growth hormone (rhGH) in children with chronic
mone in uremia. Kidney Int 1988;33:45–52. renal failure (CRF) (abstract). Pediatr Res 1993;33:355A.
448 IV. Clinical Methods

100. Wilson D, Jelley D, Stratton R, et al. Nephropathic cystino- with recombinant human growth hormone. Kidney Int
sis: Improved linear growth after treatment with recombinant 1996;49:781–785.
human growth hormone. J Pediatr 1989;115:758–761. 110. Bartosh S, Kaiser B, Rezvani I, et al. Effects of growth hor-
101. Andersson H, Markello T, Schneider J, et al. Effect of mone administration in pediatric renal allograft recipients.
growth hormone treatment on serum creatinine concentra- Pediatr Nephrol 1992;6:68–73.
tion in patients with cystinosis and chronic renal disease. J 111. Fine R, Yadin O, Nelson P, et al. Recombinant human
Pediatr 1992;120:716–720. growth hormone treatment of children following renal
102. Yadin O, Kamil E, Pyke-Grimm K, et al. Recombinant transplantation. Pediatr Nephrol 1991;5:147–151.
human growth hormone in pubertal patients with chronic 112. Fine R, Yadin O, Moulten L, et al. Extended recombinant
renal disease. Contrib Nephrol 1992;100:139–154. human growth hormone treatment after renal transplanta-
103. Hokken-Koelega A, de Jong R, Donckerwolcke R, et al. Use tion in children. J Am Soc Nephrol 1992;2:S274–S283.
of recombinant human growth hormone (rhGH) in puber- 113. Van Dop C, Donohoue P, Bock G, et al. Enhanced growth
tal patients with CRI/dialysis/post-transplant: Dutch data. with growth hormone therapy after renal transplantation.
Dutch Study Group on Growth in Children with Chronic Pediatr Nephrol 1989;3:468–469.
Renal Disease. Br J Clin Prac 1996;85:S5–S6. 114. Van Dop C, Jabs K, Donohoue P, et al. Accelerated growth
104. Watkins S, Johanson A, Winters W, et al. Legg-Perthes in rates in children treated with growth hormone after renal
children with chronic renal failure during growth (abstract). transplantation. J Pediatr 1992;120:244–250.
J Am Soc Nephrol 1990;1:344. 115. Benfield M, Aail A, Waldo F, et al. The effect of rhGH in
105. Catterall A, Roberts G. Association of Perthes’ Disease with vitro on donor-specific hyporesponsiveness in pediatric
congenital anomalies of genitourinary tract and inguinal transplantation. Pediatr Transplant 1997;1:90–97.
region. Lancet 1971;1:996–997. 116. Benfield M, Kohaut E. Growth hormone is safe in children
106. Mehls O, Ritz E, Oppermann H. Femoral head necrosis in after renal transplantation. J Pediatr 1997;131:S28–S31.
uremic children without steroid treatment or transplanta- 117. Rodiguez-Soriano J, Vallo A, Quintela M, et al C. Predictors
tion. J Pediatr 1981;99:926–929. of final adult height after renal transplantation during child-
107. Tönshoff B, Tönshoff C, Mehls O, et al. Growth hormone hood: a single-center study. Nephron 2000;86:266–273.
treatment in children with preterminal chronic renal failure: 118. Fine R, Koch V, Boechat M, et al. Recombinant human
no adverse effect on glomerular filtration rate. Eur J Pediatr growth hormone (rhGH) treatment of children undergoing
1992;151:601–607. peritoneal dialysis. Perit Dial Int 1990;10:209–214.
108. Jedrzejowshi A, Panczyk-Tomaszewska M, Roszkowska- 119. Tom A, McCauley L, Bell L, et al. Growth during mainte-
Blaim M, et al. Growth hormone therapy and lipid profile nance hemodialysis: Impact of enhanced nutrition and
in children on chronic peritoneal dialysis. Pediatr Nephrol clearance. J Pediatr 1999;134:464–471.
2002;17:830–836. 120. Haffner D, Schaefer F, Nissel R. Effect of growth hormone
109. Fine R, Kohaut E, Brown D, et al. Long-term treatment of treatment on the adult height of children with chronic renal
growth retarded children with chronic renal insufficiency, failure. N Engl J Med 2000;343:923–930.
 23

DIAGNOSTIC IMAGING
FRED E. AVNI
MICHELLE HALL

Imaging the urinary tract (UT) plays a significant role in the 0.6 × bladder height × width × transverse diameter
work-up of many nephrouropathies (1,2). The numbers of
US can be used as an alternative method to detect vesi-
techniques that are available to image the UT are numerous;
coureteric reflux (VUR) with sonicated contrast media (Fig.
each has its advantages and drawbacks. The work-up must be
23.4). There is a good correlation with classic voiding cys-
tailored to every patient taking into account the type of dis-
togram for demonstrating reflux (8).
ease, clinical data, and questions to be answered. Hazards
related to the use of a specific technique, such as radiation
hazards, allergy, or postprocedural infection, should also be Voiding Cystourethrogram
taken into account. Whatever technique is chosen, a decision
Voiding cystourethrogram (VCUG) remains the best imag-
tree must be applied in accordance with the ALARA principle
ing technique to detect VUR (Fig. 23.5). It also allows an
(as low as reasonably achievable). This concept implies that
evaluation of the urethra. It can be performed by catheteriz-
imaging techniques have to be chosen to provide the most
ing the bladder or through suprapubic puncture. The proce-
accurate diagnosis using the most adequate and safest tech-
dure should be explained in detail to the parents and to the
nique in a specific indication. Whenever possible, a radiating
patient. It should be performed in a strictly sterile manner.
technique should be replaced by a nonradiating one (3).
To minimize radiation, pulsed fluoroscopy may be used.
Repeated filling of the bladder (cyclic VCUG) increases the
rate of detection of VUR, especially in infants. VUR is
DIAGNOSTIC PROCEDURES
graded according to the international reflux study group
(grades I to V) (Fig. 23.5). Prophylactic antibiotherapy given
Ultrasound
2 days before and 2 days after the examination is controver-
Ultrasound (US) is the central imaging modality for assessing sial, but the examination should be performed only when the
the UT in children. It is easy to perform. The examination is urine has been tested sterile. Postprocedural infection occurs
very accurate in assessing the presence of a kidney and the in 1 to 3% of patients, and hematuria may develop (9).
degree of dilatation. It should include evaluation of the blad-
der and ureters (not visible under normal conditions) (4,5).
Intravenous Pyelography
Kidney size should be compared to normograms; established
according to age, weight, or height; and expressed in standard The use of intravenous pyelography (IVP) has markedly
deviation (6). On US, the kidneys appear with a corticomed- decreased in recent years (Fig. 23.6A). The technique is
ullary differentiation (CMD); the cortex is relatively echo- progressively replaced by US, nuclear medicine, magnetic
genic and the medulla appears hypoechoic (Fig. 23.1). In the resonance (MR) imaging (Fig. 23.6B), and even computed
newborn, the cortex is hyperechoic compared to the liver; this tomography (CT). Known allergic disease and renal failure
is more pronounced in the premature newborn. are the main contraindications (10).
Pulsed Doppler analysis provides information about the
venous and arterial vascularization of the kidneys. Spectral
Computed Tomography
analysis demonstrates the degree of renal vascular imped-
ance by means of the resistive index (RI) and blood velocity CT has gained popularity since the development of helical
(Fig. 23.2). Color Doppler and Doppler energy allow the CT that allows very rapid assessment of the UT. It provides
mapping of renal vascularization (5) (Fig. 23.3). exquisite cross-sectional anatomic and functional details.
The bladder shape, wall, and content are evaluated as Two- and three-dimensional reconstruction helps to better
well. The wall thickness should be inferior to 3 mm. The visualize various anomalies. Yet, it is a radiating technique
volume can be evaluated using the following formula (7): and it necessitates contrast injection. Still, the method is
450 IV. Clinical Methods

FIGURE 23.3. Power Doppler ultrasound of a normal kidney

vascularization. Lack of vascularization of the upper pole is
FIGURE 23.1. Normal ultrasound of the kidney in a child. Sagit- related to a rib artefact (asterisk).
tal scan of the right kidney; the pyramids (arrows) are hypo-
echoic compared to the cortex. Cortical echogenicity is similar to
that of the liver (L).
information on the renal parenchyma, especially after a gado-
linium injection. The measurement of parenchymal
helpful in several conditions (tumors, urolithiasis, or enhancement provides information about the glomerular fil-
abscess) (see below). Known allergic disease and renal fail- tration (Fig. 23.6B).
ure are contraindications for contrast injection (11,12). MR imaging is progressively replacing IVP for the
assessment of dilated UT, particularly for the evaluation of
abnormal duplex systems or suspicion of ectopic extravesi-
Magnetic Resonance Imaging
cal ureteral insertion. Evaluation of tumoral extension or
Thanks to the development of faster sequences, the use of reflux nephropathy (RN) is another good indication.
MR imaging for the evaluation of the UT in children has Another promising field is vascular investigation through
rapidly extended. Structures are defined as appearing hyper- MR angiography of the renal arteries, particularly in
(white) or hyposignal (black). T2-weighted sequences (the patients with hypertension (Fig. 23.7). The drawbacks of
so-called uro-MR sequences), allow the visualization of the the method are its poor accessibility and the need for seda-
collecting system. T1-weighted sequences provide more tion in infants (13,14).

Nuclear Medicine
Recent progress in nuclear medicine techniques includes pro-
duction of new tracers, new applications of known tracers,
development of various algorithms to determine renal func-
tion, and increased quality of instrumentation (15–18).

FIGURE 23.2. Duplex Doppler evaluation of normal renal ves- FIGURE 23.4. Sonographic voiding cystourethrogram. Sagittal
sels. Measurement of the resistive index (IR). The sample is mea- scan of the right kidney before (left) and after (right) instillation
sured at the level of the renal hilum. IR equals 0.68 (normal), of sonicated albumin. The contrast distends the renal pelvis
maximum systolic velocity is 0.25 m/sec, and minimum diastolic (arrows) indicating vesicoureteric reflux. (Courtesy of K. Darge,
velocity is 0.08 m/sec. CAL, calibration; S/D, systolic/diastolic. M.D., Heidelberg, Germany.)
 23. Diagnostic Imaging 451

99mTc–dimercaptosuccinic acid (DMSA) is taken up by

the proximal tubular cells and is only slightly excreted in
the urine. DMSA provides interesting morphologic infor-
mation in diseases affecting the renal cortex (Fig. 23.8).
By means of bladder instillation, 99mTc–pertechnetate
anion can be used for direct cystography and detection of
VUR (18).

Indications
Common indications are unilateral or bilateral hydronephrosis,
UT infections with or without associated vesicoureteral reflux,
small kidney, single kidney, urethral valves, pre- and postopera-
tive follow-up, and follow-up of known VUR (15–21).

Study of Urinary Tract Obstruction

(Standard and Diuretic Renography)
Two aspects of renal function are assessed: renal clearance
and excretion of the tracer. Estimation of relative clearance
(differential renal function) requires the measurement of
GFR by injection of chromium isotope–ethylenediamine-
tetraacetic acid using a simple plasma sample technique.
The most accurate method for evaluation of GFR is based
on the plasma disappearance curve after a single bolus
FIGURE 23.5. Radiologic voiding cystourethrogram. Bilateral injection of a glomerular tracer.
vesicoureteric reflux [grade II (right) and grade III (left)] with The second parameter that may be obtained from a
intrarenal reflux enhancing the right upper and left upper renographic curve is the evaluation of tracer molecule
parenchyma (arrows).
transport through the entire nephron, known as the transit
time. A normal transit time (approximately 3 minutes)
excludes renal obstruction. Abnormal transit time indicates
Renal Tracers
urine stasis. It is not possible on the basis of a prolonged
Chromium isotope–ethylenediaminetetraacetic acid, which transit time to differentiate obstruction from simple dilata-
is eliminated exclusively through glomerular filtration with- tion of the collecting system.
out tubular secretion or reabsorption, is a good tracer for The response to intravenous injection of furosemide pro-
the determination of glomerular filtration rate (GFR) using vides additional information in cases of renal stasis. In cases
blood sampling. of nonobstructive dilatation, the retained radioactivity in the
Technetium 99m (99mTc)–diethylenetriamine pentaacetic UT is washed out rapidly by increased urine flow, whereas
acid is exclusively eliminated by glomerular filtration without renal emptying is slow or nonexistent in the case of obstruc-
tubular secretion or reabsorption. The energy of 99mTc–dieth- tion. However, the degree of washout depends on the degree
ylenetriamine pentaacetic acid is adequate for gamma camera of uptake of the tracer by the kidney. Poor response to furo-
studies and allows studies of individual kidney renographic semide may be observed in kidneys with impaired renal
curves, relative and absolute GFR, determination of renal tran- function in the absence of any obstruction (17).
sit times, and furosemide-augmented tests. Tubular tracers with a high extraction (e.g., iodine 123–
Iodine 131– or iodine 123–hippurate is secreted mainly by hippurate, 99mTc–MAG-3, or 99mTc–ethylene dicysteine)
the tubular cells and by the glomerular filtration (5 to 10%). are recommended. The response depends on the rate of
Hippurate clearance is considered to reflect the renal plasma tracer extraction. Postmicturition images should be acquired.
flow and is therefore called the effective renal plasma flow. During the first months of life, the response to furosemide
99mTc-mercaptotriglycylglycine (MAG-3) is eliminated is often equivocal despite the absence of obstruction because
almost completely by tubular secretion. The advantages of of the low extraction (low clearance values). In the presence
MAG-3 compared to 99mTc–diethylenetriamine pentaace- of a full bladder, drainage from the kidney may be delayed
tic acid are its higher extraction rate, higher signal to noise even in the normal individual.
ratio, and better reproducibility for separate renal clearance The interpretation of drainage in the presence of marked
measurements. 99mTc–MAG-3 offers the advantage of hydronephrosis is more difficult. Only good drainage is eas-
combining image with information about renal excretion ily defined. The definitions of obstruction and risk factors
(Fig. 23.7). of renal deterioration are still controversial.
452 IV. Clinical Methods

FIGURE 23.6. Bilateral urinary tract dilatation. A: Intravenous pyelography showing poor visual-
ization of the right urinary tract. B: Magnetic resonance imaging showing uro–magnetic reso-
nance sequence. There is good visualization of both ureters.

FIGURE 23.7. Mercaptotriglycine renogram in a 1-month-old boy with antenatal diagnosis of

right hydronephrosis and left small cystic kidney. The renogram confirms a nonfunctioning left
kidney and a ureterohydronephrosis with response to furosemide (megaureter on magnetic reso-
nance). A: Curves corresponding to the function of the right kidney. B: Morphologic appearance
of the dilated right urinary tract. (See Color Plate 23.7.)
 23. Diagnostic Imaging 453

FIGURE 23.8. Technetium 99m–DMSA scan in a girl with acute

pyelonephritis. There are defects in the upper pole of the right
kidney (arrow).

Renal Morphology
99mTc-DMSA is the best agent for the demonstration of focal

renal abnormalities because the tracer is retained mainly in the

tubular cells. DMSA scintigraphy is the technique of choice
for the detection of acute pyelonephritis (APN) and renal
sequelae (Fig. 23.8). The DMSA technique is also indicated
for the evaluation of small, ectopic, dysplastic, or infarct kid-
neys and for the confirmation of nonfunctional multicystic FIGURE 23.10. Neonatal work-up of in utero dilatation. +, nor-
kidneys (19,21). Indirect cystogram is highly valuable for the mal US; –, abnormal US; AB, antibistherapy; IVP, intravenous
follow-up of known VUR or in screening siblings (18). pyelography; MR, magnetic resonance; US, ultrasound; VCUG,
voiding cystourethrogram.

IMAGING CONGENITAL URONEPHROPATHIES

diagnosed in utero and evaluated at birth in asymptom-
Antenatal diagnosis has markedly modified the time of atic patients. Obstetric US is able to diagnose accurately
discovery of nephrouropathies. More and more cases are many uronephropathies. In some specific indications
(maternal obesity, oligohydramnios, complex malforma-
tions), fetal MR imaging provides supplementary infor-
mation (22,23).

Neonatal Period
Because of the wider use of obstetric US, the evaluation of
UT dilatation discovered in utero has been standardized
(Figs. 23.9 and 23.10).
US is performed first—very rapidly if there is evidence
of bilateral obstructive uropathy (i.e., pneumothorax, oli-
goanuria) or at the end of the first week in all other cases.
If US examination is abnormal (Fig. 23.11 and Table
23.1), a VCUG is performed. If US is normal, a control
examination is performed at 6 weeks. In the absence of
VUR but with persisting dilatation, the degree of
obstruction is evaluated with isotopes. Whenever surgery
is elected, IVP or, rather, MR imaging should be per-
formed to assess the morphology of the UT. When a con-
FIGURE 23.9. Ureteropyelic junction obstruction in utero servative, nonsurgical approach is preferred, US allows
(ultrasound). Transverse scan of the fetal abdomen at 34 weeks’ the follow-up of the dilatation, which spontaneously
gestation. Marked dilatation of the left renal pelvis and calyces
(4 cm between crosses). Sp, fetal spine. resolves in many cases (22,24–26).
454 IV. Clinical Methods

FIGURE 23.12. Dilated ureter (u) at ultrasound. Sagittal scans

FIGURE 23.11. Ureteropyelic junction obstruction in a newborn of the bladder (B). A dilated ureter is visible behind the bladder.
(ultrasound). Marked dilatation—transverse scan 30 mm Its caliber varies a few seconds apart, due to peristalsis.
between crosses. The calyces (asterisks) are also dilated.

onstrated on the micturition phase of a VCUG (Fig. 23.14).

Urinary Tract Dilatation Isotopes evaluate the degree of renal functional impairment.
Other causes of bladder outlet obstruction include megaloure-
The role of imaging is to determine the level and degree of
thra, anterior valves, and urethral polyps, which are evaluated
obstruction. Ureteropelvic junction (UPJ) obstruction is the
by a VCUG during the micturition phase (27,28).
main cause of UT dilatation in utero (Fig. 23.12) and at birth
Duplex kidneys are commonly found in newborns. On
(Fig. 23.13). US shows a dilated renal pelvis above 7 mm (on
US, a band of parenchyma separates the two pelvic com-
a transverse scan of the kidney) (Figs. 23.12 and 23.13).
plexes. A nondilated duplex kidney should be considered as a
Dilatations can be classified as mild (7 to 10 mm), moderate
normal variant. Complications may occur at the upper or
(10 to 15 mm), and marked (greater than 15 mm). Thinned,
lower poles and consist of obstruction or VUR. An extravesi-
echogenic or cystic cortex suggests associated dysplasia. The
cal ectopic insertion or ureterocele is classically found in rela-
degree of functional impairment is evaluated by isotopic
studies. IVP or better MR imaging helps to demonstrate the
degree of dilatation and the status of the remaining paren-
chyma (Fig. 23.14). On US, UPJ obstruction should be dif-
ferentiated from multicystic dysplastic kidney (see later),
ureterovesical junction obstruction, or dilating VUR.
A ureterovesical junction obstruction is characterized by
the presence of a visible ureter (above 3 mm diameter).
This dilatation is best evaluated behind a filled bladder
(Fig. 23.12). The anomaly may be confirmed using MR
imaging (Fig. 23.13).
The presence on US of a large bladder (above 6 cm
height) suggests a bladder outlet obstruction. The main cause
in boys is posterior urethral valves. The bladder is enlarged, its
wall is thickened, and bilateral ureterohydronephrosis may be
present already in utero. After birth, the anomaly is best dem-

TABLE 23.1. ULTRASOUND CRITERIA OF ABNORMAL

URINARY TRACT POTENTIALLY ASSOCIATED WITH
VESICOURETERIC REFLUX

Renal pelvis dilatation >7 mm

Ureteral dilatation >3 mm
Calyceal dilatation >2 mm
Pelvic wall thickening >1 mm
Dysplasia (small kidney, echogenic irregular cortex, cortical
cysts)
Enlarged bladder >6 cm FIGURE 23.13. Magnetic resonance imaging of a left mega-
ureter. Sagittal view. Uro–magnetic resonance sequence.
 23. Diagnostic Imaging 455

Cystic Renal Diseases

Cystic renal diseases discovered in utero or in the neonatal
period encompass a large panel of diseases and US patterns
(see the section Imaging of Renal Failure and Renal Graft).
Unilateral solitary cortical cyst is an uncommon finding.
US follow-up is usually sufficient to exclude a cystic renal
tumor.
In the case of unilateral multiple cysts, a multicystic dys-
plastic kidney is the most frequent finding (Fig. 23.17). In
most cases, the US diagnosis is straightforward: a mass con-
taining multiple cysts of varying size, without communica-
tion between them and without normal renal parenchyma.
Contralateral anomalies are frequent, especially VUR. A
99mTc–MAG-3 diuretic renogram may assess the nonfunc-

tioning multicystic dysplastic kidney and exclude an anom-

aly in the remaining functioning kidney. Involution may
occur in utero or after birth (Fig. 23.17).
Bilateral cysts have to be separated between cases with
medullary cysts and those with cortical cysts. In the case of
medullary cysts, autosomal-recessive polycystic kidney dis-
ease is the first diagnosis to suspect. Affected kidneys are
usually markedly enlarged (+2 to +4 standard deviation).
The US pattern may be much more heterogeneous (Fig.
23.18A). Liver involvement is difficult to demonstrate in
FIGURE. 23.14. Posterior urethral valves in a neonate—voiding
cystourethrogram. Distended posterior urethra (asterisk) and nar- the neonatal period (33). Other causes of medullary cysts
row anterior urethra (arrow). Megabladder with diverticular wall. include many syndromes (e.g., Bardet Biedl syndrome).
The finding of bilateral cortical cysts is in favor of auto-
somal-dominant polycystic kidney disease (34). Yet, it is
tion to a dilated upper pole. The VCUG is performed after unusual to find significant cysts in the neonatal period (see
birth to visualize the ureterocele, which may prolapse within below). Other causes of (large) neonatal bilateral renal cysts
the urethra and induce bladder outlet obstruction. MR are tuberous sclerosis and von Hippel–Lindau disease.
imaging has proved useful in the morphologic evaluation of Renal cystic disease is often detected in utero in fetuses
complicated duplex systems (29,30) (Fig. 23.15). with bilateral hyperechoic kidneys. Large (+4 standard
In cases of nonsurgical management of urogenital urop- deviation), poorly differentiated kidneys may correspond
athies, imaging allows a close follow-up. US confirms reso- to autosomal recessive polycystic kidney disease (Fig.
lution of the dilatation and sufficient renal growth. Isotope 23.18B) or autosomal dominant polycystic kidney dis-
techniques assess renal function. ease. Hyperechoic kidneys with persisting CMD suggest
autosomal dominant polycystic kidney disease and con-
genital nephrotic syndrome (35,36). US examination of
Abnormal Kidney Number or Location
the parents’ and grandparents’ kidneys may help with the
The role of imaging is either to confirm renal agenesis or to diagnosis (37).
demonstrate an ectopic location. 99mTc-DMSA scanning is
indicated when only one kidney has been identified. US
Congenital Nephrouropathies
can demonstrate pelvic ectopia. In the case of horseshoe
in Older Children
kidney, a DMSA scan might better define the isthmus. In
the case of cross ectopia and fusion, IVP or, rather, MR Some nephrouropathies are discovered later in childhood
imaging is helpful to assess the exact anatomy and the due to complications (infections, lithiasis, trauma, increas-
potential complications (Fig. 23.16). US can find associ- ing obstruction) (see below). They are usually detected on
ated genital malformations in girls (31,32). US. CT, MR imaging, or VCUG is performed according to
US findings and the complication. For instance, UPJ
obstruction may be discovered after a trauma or lithiasis
Kidney Dysplasia and Hypoplasia (Hypodysplasia)
may develop in a previously unrecognized primary mega-
US signs of hypodysplasia include small size, hyperechoge- ureter (38) (Fig. 23.19).
nicity of the cortex, cysts within the parenchyma, and A dysplastic upper pole of a duplex system with an ectopic
thinned parenchyma. extravesical insertion may be discovered because of urinary
456 IV. Clinical Methods

FIGURE 23.15. Complicated duplex kidney. A: Ultrasound of the left kidney. Sagittal scan showing
significant dilatation of the upper pole (arrow). The lower pole (LP) is not dilated. B: Ultrasound of
the bladder (B). A lobulated ureterocele (Uc) is visible within the bladder. C: Voiding cystourethro-
gram, showing intravesical Uc and vesicoureteric reflux into the left lower pole. Right grade II vesi-
coureteric reflux. D: Magnetic resonance imaging. Magnetic resonance urography sequence (coronal
view). Dilated upper pole (asterisk) in relation to an intravesical ureterocele (arrows).

dribbling in a toilet-trained girl. The dysplastic upper pole tion, whether the kidney is affected, and whether there is
may escape the US examination. An MR imaging examina- a VUR (Table 23.2). The US allows the detection of a
tion (or a CT when MR imaging is not available), presently congenital anomaly or a lithiasis. DMSA scintigraphy
the most accurate technique for this indication (39) (Fig. evaluates kidney involvement, and VCUG detects a VUR
23.20), confirms the clinical suspicion. (40–42).

Acute Pyelonephritis
INFECTIONS OF THE URINARY TRACT
The main role of US is to detect a congenital anomaly. In
The role of imaging in the case of UT infection is to addition, several features indicate acute renal infection: The
determine whether there is an underlying favoring condi- main sign is an increased renal length or, more accurately, an
 23. Diagnostic Imaging 457

areas of increased or decreased echogenicity corresponding to

an inflammatory edema and (hemorrhagic) necrosis. Such
areas are typically undervascularized. This can be shown
using color or power Doppler (Fig. 23.22). Echogenic urine
with echogenic fluid level may correspond to pyonephrosis.
It should be stressed that a normal US examination does not
exclude APN (42,45–47).
Renal cortical lesions are detected by 99mTc-DMSA scin-
tigraphy (Fig. 23.8). Decreased uptake is seen in areas of
APN (47). The presence of a renal abnormality during the
acute phase of infection seems to be the best predictor of
renal sequelae. Conversely, the risk of developing scars is
low when early scintigraphy is normal.
CT demonstrates very accurately renal involvement in cases
of APN. It shows an increased renal volume and, after contrast
enhancement, affected parenchymal areas. Yet, it is an irradiat-
ing technique, and it should not be used routinely (48) (see
below). MR imaging has similar diagnostic performances to
CT or DMSA scanning (Fig. 23.23).
A VCUG is performed to detect a VUR. Indirect cystogra-
phy may be obtained by injecting contrast intravenously. It is
also a valuable technique for detecting VUR under physiologic
conditions without using a bladder catheterization. Children
who are not toilet trained (younger than 3 years of age) may
FIGURE 23.16. Intravenous pyelography of a right crossed not be able to undergo this examination. The main indica-
fused ectopia of the left kidney. tion is the follow-up of patients with known VUR.

Evolution of Acute Pyelonephritis

increased renal volume (Fig. 23.21). Another sign is a thick-
ening (above 1 mm) of the renal pelvis wall with a hypere- When treatment is adequate, the inflammatory infiltrates
chogenicity of the perihilar renal fat. These signs suggest resolve progressively without sequelae; in such cases, the renal
pyelitis and ureteritis. The renal parenchyma may also show volume as measured on US returns to normal within 4 to 6

FIGURE 23.17. Involution of a multicystic dysplastic kidney (K). A: At birth; sagittal scan of the
right kidney. Four cysts are visible (C). B: At 1 year of age, only one cyst has remained (arrow).
458 IV. Clinical Methods

FIGURE 23.18. Neonatal autosomal-recessive polycystic kidney

disease. Two different patterns at ultrasound. A: Sagittal scans of
the left kidney (K) showing multiple medullary cysts. B: Left kidney
in another patient. The pyramids appear hyperechoic (arrows).

weeks. This delay should be taken into account for planning

follow-up examinations intended to assess renal growth (49).
In cases of late or inadequate treatment or resistance to anti-
biotherapy, the renal lesions may coalesce and form an abscess.
US demonstrates the abscess as an inhomogeneous mass
within the parenchyma; CT or MR imaging seems to be a bet-
ter technique to demonstrate abscess formation, especially for
bilateral lesions. These techniques better evaluate the extrare-
nal extension of the abscess than does US (46) (Fig. 23.21).
The rationale for early work-up and treatment of APN
is to prevent recurrence and permanent lesions. Scintigra-
phy is performed at least 6 months to 1 year after the
acute episode to detect renal scarring. Six months is the
minimal time before one can consider a lesion as perma-
nent (49,50) (Fig. 23.24A). US and IVP underestimate
the number of scars. MR imaging is a promising tech-
nique for the evaluation of permanent renal damage and
chronic renal scars (RN or chronic pyelonephritis) (Fig.
23.24B) (13,14).

Cystitis and Urethritis

The bladder is more commonly affected by infection than
the kidneys; findings on US are not specific. The bladder
wall may be thickened (above 3 mm), and urine may
appear with echogenic debris. Of note, these findings may
be present without infection (43,44). Urethritis is uncom-
FIGURE 23.19. Horseshoe kidney with left ureteropyelic junc-
mon in children. On VCUG, during the micturition phase, tion obstruction in a 9-year-old boy with acute left flank pain.
the urethral lumen appears irregular. Typical findings on an intravenous pyelography.
 23. Diagnostic Imaging 459

FIGURE 23.21. Acute right pyelonephritis. Sagittal scan of the

right kidney that appears swollen, with patchy hyperechoic
areas corresponding to inflammatory changes (asterisks).

Tuberculosis
The most typical features have been described on IVP:
“drooping Lily” appearance of the pyelocalyceal system. In
more advanced cases, the kidney is completely destroyed,
FIGURE 23.20. Four-year-old girl with urinary dribbling. Mag- small, and calcified (52).
netic resonance imaging. Frontal view showing a right duplex
kidney with dilated upper pole (arrow) associated with a dilated
ectopic ureter (U).
VESICOURETERIC REFLUX

Specific Organism Infections Primary VUR is a frequent cause of in utero dilatation of the
UT (19). The most severe cases are detected in males. VUR
Candidiasis
may be associated with various congenital uropathies. Voiding
Candidiasis develops in immunocompromised patients, dysfunction is another circumstance in which VUR is
premature infants, and patients under chronic antibiother- detected; in such cases, VUR is secondary to the bladder dys-
apy. Candidiasis infection should be suspected on US case function. Although the relationship between UT infection
of: global renal hyperechogenicity, sludge distending the and VUR is still debated, VUR is found in approximately
renal cavities, or the presence of fungus balls (echogenic 45% of cases with UT infection (53,54). Radiologic VCUG
masses partially filling the renal cavities) (51). is the best method for the first assessment. VCUG confirms

TABLE 23.2 IMAGING IN URINARY TRACT INFECTION: RELATIVE CONTRIBUTION OF

IMAGING EXAMINATIONS
Magnetic
Computed resonance Intravenous Voiding Dimercaptosuccinic
Question Ultrasound tomography imaging pyelography cystourethrogram acid

Underlying ++ ++ ++ + 0 0
favoring con-
dition?
Kidney affected? + +++ +++ + 0 +++
Vesicoureteric 0 but + with soni- 0 0 0 ++ 0
reflux? cated contrast
Scars + ++ +++ + 0 +++

0, low; +, little; ++, moderate; +++, very contributive.

460 IV. Clinical Methods

in 20 to 30% of the cases (associated VUR and hypodyspla-

sia), and acquired renal damage seems less frequent (60–62).

VOIDING DISORDERS

Voiding disorders are associated with a wide spectrum of

anomalies. The role of imaging is to differentiate between
organic and functional diseases.
Plain film of the abdomen evaluates constipation or
associated vertebral malformations. US can exclude UT
malformations and measure the bladder wall thickness and
FIGURE 23.22. Acute pyelonephritis. Power Doppler ultra- the vesical content pre- and postvoiding. VCUG is per-
sound showing striking area of absent vascularization due to
inflammatory involvement (arrows). (Courtesy of Dr. Dufour.) formed in the case of persisting symptoms. It detects a
(See Color Plate 23.22.) VUR and shows the bladder volume at maximum filling,
the bladder contours, the type of micturition, and the
appearance of the urethra. It may show a cause of obstruc-
the VUR (including intrarenal VUR) and shows the mor- tion in boys.
phology of the lower UT (including the urethra) (Fig. 23.5). On VCUG, a transient opening of the bladder neck and
It may also show bladder diverticula, reflux into ectopic ure- opacification of the urethra up to the external sphincter,
ters, or ureteroceles. Cyclic filling of the bladder has been along with cessation of contrast material dripping, suggest
shown to improve the rate of detection of VUR by 10 to an uninhibited detrusor contraction. A spinning-top ure-
15%, especially in newborns. Tailored pulsed fluoroscopy and thra or a wide bladder neck anomaly suggests a bladder
adapted filtration material (55) reduce radiation hazards. instability (63,64) (Fig. 23.25).
VUR is graded according to the international grading system. In the case of neurologic symptoms, MR imaging of the
Higher grades of VUR are less likely to resolve spontaneously conus medullaris is helpful for the demonstration of a teth-
and are more often associated with dysplastic kidneys (56). ered, low-positioned cord; MR urography is efficient to
Isotopic direct or indirect VCUG and cystosonography (using demonstrate ectopic ureteral insertion associated with uri-
sonicated contrast media) are better suited for the follow-up nary dribbling (65).
of patients with known VUR or for screening siblings of A particular circumstance of voiding dysfunction is asso-
patients with VUR (18,57,58). US is a poor predictor of a ciated with neurogenic bladder. This anomaly is related, in
VUR. Yet, a completely detailed normal US examination of most cases, to an open spina bifida. Typically, a neurogenic
the UT is rarely associated with high-grade VUR (59). The bladder has a pear-shape appearance, irregular diverticular
parenchymal damage, the so-called RN, is best assessed by margins, and a thickened wall (Fig. 23.26) (66).
isotopes and MR imaging (Fig. 23.24B). It is present at birth

IMAGING OF RENAL FAILURE

AND RENAL GRAFT

In most patients with renal failure, US is important for diag-

nosis and follow-up. The contribution of US varies greatly
from patient to patient. In some cases, US is insufficient and
complementary examinations are necessary (e.g., obstructive
tumoral infiltration of the ureters is evaluated with CT or
MR imaging). All imaging findings must be analyzed accord-
ing to the clinical findings and familial history.

Acute Renal Failure

Imaging is helpful in determining postrenal obstructive
causes of acute renal failure; it is often informative for renal
FIGURE 23.23. Acute pyelonephritis. Demonstration of a peri- causes but seldom contributive for prerenal causes. The
renal abscess on computed tomography (after contrast injec- main US feature is an increased renal echogenicity (67). A
tion). A necrotic abscess has developed in the posterior surface
of the left kidney and has extended into the posterior abdomi- completely normal US examination does not exclude renal
nal wall (arrows). lesions.
 23. Diagnostic Imaging 461

FIGURE 23.24. Pyelonephritic scars. A: Dimercaptosuccinic acid scan showing small, irregular
kidneys. B: Magnetic resonance imaging showing bilateral small, irregular, scarred kidneys.

Hemolytic-Uremic Syndrome 1) (Fig. 23.27) that decreases with recovery. At a later

phase, sequelae may appear as areas of medullar or cortical
During the acute phase, the renal cortex appears hyper- calcifications (related to necrosis), and global renal growth
echoic with increased CMD. During the oligoanuric phase, may be impaired (68). US examination helps to assess
Doppler spectral analysis shows a high RI (approximately extrarenal lesions (digestive wall thickening, hemobilia). In

FIGURE 23.25. Spinning-top urethra on voiding cystourethro- FIGURE 23.26. Neurogenic bladder on voiding cystourethro-
gram, typical for vesicourethral dyssynergia. gram. Filling phase: trabeculated, diverticular bladder.
462 IV. Clinical Methods

FIGURE 23.27. Hemolysis (uremia syndrome), acute phase. A: Ultrasound of the right kidney (k).
Striking hyperechogenicity of the cortex. B: Duplex Doppler ultrasound evaluation of the renal
vessels. Resistive index equals 1; there is no diastolic flow. CAL, calibration; L, liver.

patients with neurologic symptoms, MR imaging of the age. X-rays include (a) anteroposterior view of the hands
brain may be necessary. and chest, including the claviculae, pelvis, long bones; and
(b) dental view. Anomalies suggesting renal osteodystrophy
Medullar or Cortical Necrosis and Shock Kidneys include poor mineralization of the bones, osteolysis at the
clavicular ends, phalangeal resorptions, and long bones
Medullar and cortical necrosis occur mainly in premature
with cortical thinning.
infants and are related to reduced arterial renal perfusion; dur-
ing the acute phase, the renal cortex appears hyperechoic with
persisting CMD, and spectral Doppler analysis demonstrates a Renal Dysplasia and Hypoplasia
high RI. In the case of medullar necrosis, calcifications may Renal dysplasia and hypoplasia may coexist, but dysplasia is
develop within the pyramids. In the case of severe cortical only demonstrated on renal histology. At US, kidneys are
necrosis, the renal parenchyma shrinks, whereas in less severe small usually without CMD. Small cysts may be present
cases, calcifications develop within the parenchyma. These within the parenchyma (71).
conditions must be differentiated from the transient acute
tubular blockade of a dehydrated neonate (Tamm-Horsfall
proteinuria). The latter appears on US with hyperechoic Cystic Renal Diseases
pyramids, resolving within 2 to 3 days (69). Among the diseases without genetic transmission, only
bilateral obstructive uropathies may lead to CRF. In such
Renal Vein Thrombosis conditions, on US, the kidneys are small, with a thin and
Renal vein thrombosis most often occurs in the neonatal hyperechoic cortex without CMD, and the renal cavities
period and may develop in utero. At US, kidneys are are dilated. Several cysts of varying sizes are visualized
enlarged and CMD is absent with hyperechoic streaks. within the parenchyma.
Color Doppler may show the thrombus within the renal Among the hereditary cystic diseases, some display
vein or the inferior vena cava (70). characteristic patterns, and others are difficult to recog-
nize (72). In autosomal recessive polycystic kidney dis-
Obstructive Uropathies ease, kidneys are enlarged, and the cystic lesions are
mainly located in the medulla. The medulla may show
Several forms of obstructive uropathies may be associated macrocysts or may be hyperechoic due to multiple micro-
with acute renal failure: the urethral prolapse of an ectopic cysts. With time, the cysts increase in size; some hyper-
ureterocele; congenital uropathy of a single kidney; and echoic spots may appear and correlate with the
tumoral infiltration of the kidneys, ureters, or bladder neck. development of CRF (Fig. 23.28). The periportal hepatic
Bilateral urolithiases are all circumstances that may lead to fibrosis may progress with periportal hyperechogenicity,
acute renal failure. and inhomogeneous liver echogenicity and signs of portal
hypertension can be observed as well. In the case of auto-
Chronic Renal Failure somal dominant polycystic kidney disease, macrocysts are
most often observed. In nephronophthisis, US changes
Chronic renal failure (CRF) interferes with statural and are observed at approximately 10 to 12 years with the
bone growth. Skeletal x-rays allow the evaluation of bone development of macrocysts (73). Peripheral cortical cysts
 23. Diagnostic Imaging 463

FIGURE 23.28. Autosomal-recessive polycystic kidney disease in FIGURE 23.30. Congenital nephrotic syndrome of the Finnish
a 10-year-old boy. The kidney (K) has a heterogeneous echoge- type in a newborn with proteinuria. Enlarged kidney (6 cm
nicity. Some macrocysts have developed (arrows). between the crosses) with hyperechoic cortex and preserved cor-
ticomedullary differentiation.

can be found in glomerulocystic kidneys (Fig. 23.29).

This entity can be isolated or part of syndromes (72). It
is noteworthy that cysts may appear in patients on Chronic Pyelonephritis or Reflux Nephropathy
chronic dialysis. The relationship between chronic pyelonephritis, RN, and
CRF is controversial, as many parenchymal lesions may
preexist already at birth. Whatever the cause of the lesions,
the role of imaging is the detection of an underlying favor-
ing condition. US may detect UT malformation, but the
technique is less accurate for the demonstration of paren-
chymal lesions, which are best visualized on DMSA scans
or MR imaging (74).

Congenital Nephrotic Syndromes

US shows enlarged kidneys with small hyperechoic pyramids
in newborns with Finnish-type nephrotic syndrome. With
time, the kidney size and echogenicity decrease and the pyra-
mids disappear (Fig. 23.30). In patients with diffuse mesan-
gial sclerosis, the US may show as diffusely heterogeneous
with a patchy appearance. Other congenital nephrotic syn-
dromes do not have typical US appearances (75).

Congenital Tubular Syndromes

FIGURE 23.29. Glomerulocystic kidney. Sagittal scan of the left Congenital tubular syndromes are numerous renal diseases
kidney (K). The parenchyma is hyperechoic without corticomed-
ullary differentiation and with small peripheric cysts (between that include primitive or secondary tubulopathies. Hyper-
crosses). calciuria is often present and is responsible for nephrocalci-
464 IV. Clinical Methods

nosis that is detected by US (see below). US is also helpful

in type I hyperoxaluria (see below) (76).

Renal Transplant
The renal transplant is easily evaluated by US. The kidney is
very close to the abdominal surface; therefore, US is able to
demonstrate its anatomy with good details. A CMD is
always present. Examination includes an evaluation of the
transplant volume (L × l × W × 0.52) and an analysis of the
kidney echogenicity. Spectral analysis of the intrarenal vessels
allows the evaluation of vascularization (77,78).
Complications may occur after surgery, during the first
days, or later during follow-up. In the early posttransplant
period, the role of imaging (mainly US) is to differentiate
between postsurgical complications and early acute rejec-
tion (79).

Postsurgical Complications
US may demonstrate a fluid collection around the kidney.
MR imaging is useful to differentiate a lymphocele, a hema-
toma, or a urinary leak. Doppler (duplex and color) is able to
diagnose renal artery or vein thrombosis and, later, a renal
artery stenosis (Fig. 23.31). Urologic complications may
develop secondary to ureteral stenosis. An opacification may
be necessary to show the exact site of obstruction (79).

Acute Rejection
In the case of acute rejection, the transplant volume is
increased, echogenicity may become heterogeneous, and
FIGURE 23.32. Acute rejection. A: Ultrasound. The renal trans-
plant (RT) is swollen, the collecting system is distended, and
there is perihilar hyperechogenicity. B: Doppler analysis.
Increased vascular resistance (resistive index equals 0.85).

intrarenal RI may increase dramatically. In some cases,

renal hilum becomes markedly hyperechoic, and the pelvic
walls thicken (Fig. 23.32).

Other Complications
Other complications include infection and, rarely, calculi.
Classical US signs usually accompany these complications.
Lymphomas developing in immunocompromised patients
may appear either as localized nodules or as an echogenic
enlarged transplant.

UROLITHIASIS AND NEPHROCALCINOSIS

Urolithiasis
US is effective in detecting lithiasis, even tiny (2 to 3
mm), located in the kidney or the bladder. Urolithiasis
determines, in most cases, a characteristic acoustic shad-
FIGURE 23.31. Aneurysm (arrow) on the vascular anastomotic
owing (Fig. 23.33). The role of imaging is to detect the
site demonstrated by angio–magnetic resonance imaging. lithiasis, to demonstrate a favoring underlying condition
 23. Diagnostic Imaging 465

FIGURE 23.33. Urolithiasis—ultrasound. Sagittal scan of the

left kidney (K). A lithiasis (arrow) is visible within the dilated col-
lecting system.
FIGURE 23.35. Cortical nephrocalcinosis. Highly echogenic kid-
ney (between crosses) in a case of primary hyperoxaluria of type
I. L, liver.
(congenital malformation, nephrocalcinosis), and to fol-
low the lithiasis under treatment. Plain film of the abdo-
men is helpful to demonstrate ureteral calcified stones
that may be difficult to detect on US (80–82). In the case and plain film of the abdomen can be used to follow the
of renal colic, it might be useful to perform unenhanced effects of treatment (83).
CT with two- or three-dimensional reconstruction; this
has been shown to be effective in demonstrating obstruc- Nephrocalcinosis
tive (multiple) ureteral stones (Fig. 23.34). Urethral Ultrasound Patterns
stones may be more difficult to demonstrate; retrograde
urethrography should be used to confirm the urethral Nephrocalcinosis is suspected when hyperechogenic areas
stone. Whenever extracorporeal lithotripsy is applied, US disturb the appearance of the kidney. Most cases of nephro-
calcinosis are bilateral (84).

Cortical Nephrocalcinosis
Cortical nephrocalcinosis is rare in children. It can be
extensive and determine acoustic shadowing behind the
kidney as in neonatal hyperoxaluria of type I, or it can be
more localized within areas of the parenchyma and corre-
spond to sequelae of cortical necrosis or renal vein throm-
bosis (Fig. 23.35). In the case of vascular calcifications, the
nephrocalcinosis can be unilateral.

Medullar Nephrocalcinosis
On US, medullar nephrocalcinosis (MNC) can be graded
into three stages: diffuse and homogeneous, diffuse and
inhomogeneous, or limited to the tip of the pyramids (Fig.
23.36). Grading helps to evaluate the effect of treatment in
specific diseases. US is the best way to detect medullary
nephrocalcinosis. Medullar hyperechogenicity is not always
due to nephrocalcinosis. The differential diagnosis must,
therefore, include the US features as well as clinical, bio-
chemical, and genetic data (85,86).

Imaging Features of Specific Diseases

FIGURE 23.34. Urolithiasis—computed tomography: Reformat-
ted two-dimensional images without contrast demonstrating
MNC is present and may be associated with urolithiasis
multiple lithiasis (arrows) within the kidney and dilated ureter. and medullary cysts in patients with distal tubular acidosis
The inset box indicates the scan plan. (87). The severe neonatal form of primary hyperoxaluria
466 IV. Clinical Methods

FIGURE 23.36. Medullary nephrocalcinosis. Sagittal scan of

the kidney (K). Diffuse medullar, hyperechogenicity (arrows).

type I is associated with a cortical nephrocalcinosis that

induces a markedly increased cortical echogenicity deter-
mining an acoustic shadowing. In later childhood, MNC is
observed. Urolithiasis and renal colic are common compli-
cations demonstrated by US (88). Renal calcifications and
skeletal lesions may be impressive (Fig. 23.37). The rela-
tionship between furosemide and MNC is unclear. The
MNC is inhomogeneous and persists for a long period of
time (89).

TRAUMA TO THE URINARY TRACT

FIGURE 23.37. Primary hyperoxaluria of type I. A: Plain film of
Kidney Trauma the abdomen. Impressive bilateral renal calcifications. B: Pelvic
girdle. Densification and coarse trabeculation of the bones due
Imaging, especially US and CT, has greatly modified the to renal osteodystrophy.
management of abdominal trauma in children. It allows
more conservative, nonsurgical attitudes (90–92). US
shows major renal damage and fluid collections (Fig. persistent bleeding. Angiography must be limited to cases
23.38A). Color and duplex Doppler allows the evaluation for which therapeutic intervention is likely.
of the vessels. Yet, the method has several limitations;
because intestinal air may limit renal visibility, a normal US
Lower Urinary Tract
does not exclude parenchymal damage. Therefore, CT may
be more suited for the first evaluation of children with sus- Bladder laceration is associated with bony pelvis fractures.
pected renal damage (Fig. 23.38B). CT is performed after Such laceration of the bladder should be suspected on US
contrast injection to demonstrate urinary leakage. CT angi- when large amounts of fluid are visualized in the cul-de-
ography two-dimensional reconstruction allows an accurate sac. VCUG can be performed if necessary to visualize the
evaluation of the renal vessels. A control examination may leakage site. The same mechanism explains the lesions to
be useful if clinical evolution is unsatisfactory, suggesting the urethra. Demonstration of the lesion may be difficult,
 23. Diagnostic Imaging 467

FIGURE 23.39. Renal tumors: small right Wilms’ tumor in a case

FIGURE 23.38. Left renal trauma. A: Ultrasound. Sagittal scan of hemihypertrophy. A: Ultrasound of echogenic area (arrow) in
of the kidney (K). A renal fracture is visible (arrows). B: Com- the upper pole of the right kidney (K). B: Computed tomogra-
puted tomography after contrast enhancement. Left perirenal phy after contrast enhancement. The mass is less dense than the
hematic collection. The renal fracture is also visible (arrow). S, rest of the kidney (arrow) L, liver.
spleen.

and a retrograde urethrogram should be performed very peritoneal vessels (Figs. 23.39 and 23.40). CT is performed
cautiously (93). without and with contrast enhancement. CT without contrast
demonstrates the presence of calcifications within the tumor.
Contrast enhancement is aimed to define the limits between
RENAL AND URINARY TRACT TUMORS normal parenchyma and tumor. Urographic-type images can
be obtained at the end of the examination to visualize the ure-
Role of Imaging ters. During the same examination, an evaluation of the lungs
can be obtained to exclude metastasis. A disadvantage of the
Imaging allows the detection and localization of the tumor,
technique is the irradiation hazards that can be associated. MR
the analysis of its content and appearance, and the evalua-
imaging may provide the same information as CT about the
tion of the extent. Evaluation must be performed in con-
tumor without using x-rays (94–96).
junction with clinical and biologic data. It also has to fit
into therapeutic protocols.
US easily localizes small tumors (Fig. 23.39). Large tumors Imaging of Specific Renal Tumors
are more difficult to delineate (Fig. 23.40). The technique eas-
Wilms’ Tumor or Nephroblastoma
ily differentiates cystic from solid-type tumors. CT and MR
imaging are better suited for the evaluation of large tumors. At diagnosis, the tumor is large but well demarcated from
Both are able to assess the tumor’s relationship with the retro- the normal parenchyma. On US, the tumor is echogenic
468 IV. Clinical Methods

FIGURE 23.41. Renal lymphoma. Computed tomography with

contrast. There is a bilateral nodular (n) involvement of both
kidneys. The pancreas (p) was also affected.

whereas neuroblastoma tends to encase them; calcifications

are more frequent in neuroblastoma.

Nephroblastomatosis
There is a loss of CMD on US in the diffuse form. On CT
or MR imaging, the kidneys are large and there is a rim of
thick cortical parenchyma that does not enhance after con-
trast injection. In the focal nodular form, one or more nod-
ules are present; they do not enhance after contrast injection.
They are more homogeneous than Wilms’ tumor (97,98).

Lymphoma
Renal involvement occurs mainly in Burkitt’s lymphoma and
is usually bilateral. Other sites are often affected (e.g., ovaries,
testicles). On US, lymphomatous nodules are hypoechoic
FIGURE 23.40. Renal tumors: large Wilms’ tumor. A: Ultra-
compared to normal parenchyma; they enhance poorly on
sound. The tumor (between crosses) is difficult to delineate. B: CT after contrast injection (99) (Fig. 23.41).
Magnetic resonance imaging transverse scan after gadolin-
ium. The mass (M) is better delineated.
Other Malignant Tumors
Other malignant tumors are rare in children; their diagnosis is
with a solid content (Figs. 23.39 and 23.40). Areas of usually performed at histology rather than on imaging. Their
necrosis or hemorrhage are visible within the mass, espe- rate of metastasis, particularly to the skeleton, is higher (100).
cially after chemotherapy. Adenopathies can be seen next to
the mass; extrarenal metastatic extent can be seen within
Screening Patients at Risk
the vena cava. The contralateral kidney should be checked
for bilateral tumors. On CT or MR imaging, the tumor is As several syndromes (Denys-Drash, Beckwith-Wiede-
more clearly delineated than with US, and bilateral tumors mann, Perlman, aniridia) are associated with a higher risk
are easier to demonstrate (Figs. 23.39 and 23.40). The of Wilms’ tumor, patients affected should be screened regu-
tumor appears heterogeneous due to areas of necrosis, but larly by US (101) (Fig. 23.40).
the solid content is enhanced after contrast injection. The
tumor rarely contains calcifications or fat. It may appear
Benign Renal Tumors
partially or completely cystic.
The main differential diagnosis is neuroblastoma, Mesoblastic nephroma is the most common tumor in the
Wilms’ tumor tends to displace the abdominal vessels, newborn and fetus. On imaging, the mass is well defined
 23. Diagnostic Imaging 469

RENOVASCULAR HYPERTENSION

Imaging may show scars, diffuse nephropathy, or diffuse

sequelae of a vascular disease. Doppler US may show signs of
renal artery stenosis (i.e., increased blood velocity at the renal
artery ostium). A normal US examination does not exclude
an anomaly. MR angiography has great potential for demon-
strating renal vessel anomalies; the method is not yet stan-
dardized, and its accuracy has to be confirmed. 99mTc–
MAG-3 or -DMSA before and after captopril stimulation
can be a sensitive screening method for renovascular disease.
As fibromuscular hyperplasia and osteal stenosis are the
most common causes of hypertension in children, arteriogra-
phy remains the method of choice whenever the renal vascu-
lar origin of hypertension is highly probable. When a stenosis
is present, an angioplasty can be performed immediately
(108–110) (Fig. 23.42).
In a case of pheochromocytoma, metaiodobenzylguani-
dine scan, CT, or MR imaging should be performed to
localize and delineate the mass.

INTERVENTIONAL IMAGING AND

POSTOPERATIVE STUDIES

Interventional Imaging
Nephrostomy (Fig. 23.43) is the main interventional pro-
cedure in children. Pyonephrosis in a dilated collecting sys-
tem is an indication (111).

Specific Postoperative Findings

US is the main imaging technique for the follow-up of sur-
FIGURE 23.42. Hypertension in an 18-month-old boy with
severe left renal artery stenosis. A: Doppler ultrasound. Increased gical procedures (112). Isotopic evaluation allows detection
resistive index and increased velocity at the ostium of the left of progression renal impairment.
renal artery. B: Angiography. Complete occlusion of the left renal
artery (arrow). (Courtesy of F. Brunelle, M.D.)
Ureteropelvic Junction Obstruction
from the normal parenchyma and is generally of solid type Nephrostomy of a very dilated congenital UPJ obstruction
(102). Multilocular cystic nephroma appears as a cystic, allows assessment of kidney function and helps the physi-
septated mass on imaging; it cannot be differentiated from cian decide whether pyeloplasty or nephrectomy should be
cystic nephroblastoma (103). Angiomyolipoma in children performed. Dilatation of the pelvicalyceal system may
is invariably associated with tuberous sclerosis. The lesions increase slightly after surgery due to edema; it decreases
are small and diffuse in early childhood, with a high fatty slowly thereafter within 6 to 8 weeks. Isotopic evaluation 6
content; they are larger and fewer in adolescents and appear months after surgery enables assessment of the evolution of
heterogeneous on US or CT (104). renal function (113,114).

Bladder Tumors Ureterovesical Junction Obstruction

US is the easiest imaging method for the demonstration of Leakage due to necrosis can occur at the site of the vesical
a bladder tumor. Yet the method is unable to differentiate reimplantation, and a hematoma or a urinoma may
malignant from benign or inflammatory pseudotumors develop. The resolution of the ureteral dilatation can be
unless the tumor has extended beyond the bladder wall and monitored on US. At the level of the bladder, a characteris-
metastases are present. CT and MR imaging are performed tic posterior wall thickening can be observed at the level of
to evaluate the spreading of a malignant tumor (105–107). the reimplantation (112,115).
470 IV. Clinical Methods

Duplex Collecting System

Unroofing of ectopic intravesical ureteroceles during endos-
copy has been advocated to relieve obstruction and to allow
functional improvement of the affected upper pole. The
ureterocele collapses and appears as an intravesical pseudo-
mass. The dilatation resolves and can be monitored by US.
Secondary VUR may develop (112,117).

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 24

RENAL PATHOLOGY
AGNES B. FOGO

This chapter reviews the usual circumstances in which Proteinuria

biopsies are obtained, methods of obtaining the biopsy
Isolated proteinuria may be postural or due to tubulointerstitial
material and analyzing the tissue, and the distinct charac-
disease. These possibilities should be evaluated completely over
teristic morphologic findings in various diseases. Finally,
time before renal biopsy is considered. Any glomerular disease
current experimental techniques that may provide impor-
may cause mild to moderate proteinuria as the only manifesta-
tant pathogenic, prognostic, or diagnostic information are
tion, and biopsy may yield the diagnosis even at an early stage.
discussed.

Nephrotic Syndrome
RENAL BIOPSY INDICATIONS Numerous children with nephrotic syndrome (NS) were
studied when renal biopsy first became available. The biop-
The indications for renal biopsy vary according to the eth- sies showed so-called minimal change disease (MCD) in the
nic and age characteristics of the population studied and vast majority of cases. The efficacy of corticosteroids in this
the geographic location because these factors influence the setting has obviated the need for renal biopsies in most of
incidence of various renal diseases. The indications dis- these cases. Therefore, young children with NS typically
cussed below present the most common settings in children undergo a therapeutic trial of corticosteroids without a
for which renal biopsy is undertaken. biopsy. However, in infants with NS, in older children, or in
those with evidence of nephritis (hypertension, hematuria,
low C3, or decreased renal function) or who do not respond
Hematuria
to corticosteroid therapy, renal biopsy is often performed. In
Isolated hematuria (i.e., without proteinuria and with these patients, a disease other than MCD [e.g., focal segmen-
normal function of the kidney) may be due to hypercalci- tal glomerulosclerosis (FSGS), membranoproliferative glo-
uria, familial, or urologic disease (1–3). Once these disor- merulonephritis (MPGN), IgA nephropathy, membranous
ders are ruled out, a glomerular origin of persistent glomerulonephritis, or, more rarely in infants, Finnish-type
isolated hematuria should be considered. When present, NS or diffuse mesangial sclerosis] is often present (4–9).
red blood cell casts or dysmorphic red blood cells indicate
glomerular origin of hematuria. Renal biopsy may define
Acute Nephritis
the underlying abnormality in these patients. The most
common findings are mesangial proliferative disease or The child with acute glomerulonephritis may need a biopsy
immunoglobulin (Ig) A nephropathy (Berger’s disease). when the course is not typical of acute poststreptococcal dis-
Less common disorders include hereditary nephritis ease or if urinary abnormalities persist. Although the primary
(Alport’s syndrome) and thin basement membrane lesion. disease process may be evident in systemic conditions [e.g.,
The latter may be familial (benign familial hematuria) or Henoch-Schönlein purpura or systemic lupus erythematosus
sporadic. One-fourth to nearly one-half of the patients (SLE)], renal biopsy often is indicated to assess severity of
with isolated hematuria have normal biopsies (1–4). injury and to guide therapy and prognosis. Differentiation of
Renal biopsy may, therefore, define the pathology and specific types of proliferative lesions (e.g., MPGN type I and
provide assurance of a benign prognosis in some patients dense deposit disease) is made by renal biopsy. This distinc-
or diagnose a possible hereditary disease, which initiates tion has important implications for eventual treatment
screening of other family members. Finally, the informa- because the morphologic lesions of dense deposit disease
tion obviously is of importance in avoiding further invariably recur in transplants, although the clinical course is
repeated invasive evaluation in the patient. less severe than in the native kidney (10,11).
476 IV. Clinical Methods

Acute Renal Failure tion of specific lupus nephritis World Health Organization
(WHO) class (see below) may be difficult without renal biopsy
The cause of acute renal failure may be clinically obvious, or
(16,17). Overall, evidence indicates that renal biopsy findings
there may be multiple potential culprits. When prerenal and
may be more sensitive than clinical assessment alone in evalu-
obstructive causes are not apparent, renal parenchymal disease
ating the severity of renal involvement in SLE (18,19).
should be considered. When acute renal failure is associated
with nephritis, NS, or evidence of vasculitis or systemic dis-
Follow-Up of Disease
eases, biopsy is usually performed. Other common causes
include acute tubular necrosis or injury, often caused by drug With improved therapeutic modalities available for interven-
or ischemic injury, vascular disease, and interstitial nephritis. tion in chronic progressive renal disease, sequential or fol-
These conditions can often be diagnosed without renal biopsy. low-up biopsies become increasingly necessary to evaluate
However, when the cause remains uncertain after complete therapeutic efficacy. On the other hand, additional cyto-
evaluation, renal biopsy may be necessary for diagnosis (12). toxic therapy with its side effects may be withheld if the
biopsy shows end-stage histology. Intervention with, for exam-
Rapidly Progressive Glomerulonephritis ple, low-protein diets, angiotensin-converting enzyme inhibi-
tors, or angiotensin type 1 receptor blockers has been shown to
Renal biopsy may be considered an urgent procedure in the
alter the course of chronic progressive renal disease (20).
patient with rapidly progressive glomerulonephritis. Various
systemic vasculitides that may be distinguished only by specific
Transplantation
serologic studies (see below) or renal biopsy must be treated
urgently to avoid severe chronic renal damage. Although Renal transplant biopsies are useful in assessing episodes of
anti–glomerular basement membrane (GBM) antibody or clinically suspected rejection, investigating the cause of
antineutrophil cytoplasmic antibody (ANCA) titers may pro- decreased renal function or urine output, and detecting the
vide useful information, the ANCA test in particular is not development of de novo or recurrent disease. Occasionally,
diagnostic of a specific condition; rather, it is a screening test infection may be diagnosed by renal biopsy. Drug toxicity may
for necrotizing vasculitides (13,14). ANCA positivity, whether be diagnosed by morphologic findings. The absence of lesions
in a perinuclear ANCA or cytoplasmic ANCA pattern, was in a patient with a rise in creatinine supports cyclosporine tox-
present in approximately 60% of patients with immune- icity because this drug commonly causes a decline in the glo-
complex glomerulonephritis with crescents in a study of more merular filtration rate by vasoconstriction and not overt
than 200 renal biopsies (15). Furthermore, these specialized structural lesions. The absence of findings of acute rejection in
assays may have a longer turn-around time than the renal renal biopsy or by needle aspiration (see below) can assist in
biopsy, from which preliminary information from immuno- avoiding unnecessary immunosuppressive therapy with its
fluorescence (IF) and light microscopy (LM) studies can be potential for increased morbidity and mortality. Even a diag-
available within hours after biopsy. nosis of chronic rejection, which is not amenable to therapy,
has important therapeutic implications for the patient.
Chronic Renal Insufficiency Diseases that recur in the transplant with high frequency
include IgA nephropathy, MPGN type I, dense deposit dis-
Patients with renal insufficiency of uncertain etiology are
ease (also known as MPGN type II ), FSGS, HUS, and
candidates for renal biopsy. Although renal biopsy of the
membranous glomerulonephritis. The latter two can occur
small, shrunken kidney is riskier because of the greater inci-
de novo in the transplant. Metabolic diseases (e.g., oxalosis
dence of bleeding complications, the diagnosis of primary
and diabetic nephropathy) can also cause recurrent disease
disease can be important. This information allows assessment
in the renal transplant if liver or pancreas transplantation
of existing severity of morphologic lesions, determination of
does not cure the primary abnormality (11). Alport’s syn-
risk of recurrence in eventual renal transplant, and suitability
drome is caused by mutation in one of the type IV collagen
of cadaveric versus living-related donor transplantation. If
genes, resulting in abnormal GBM assembly and structure.
the disease has a familial basis or recurs frequently with resul-
Patients may develop anti-GBM antibody disease in the
tant graft loss, cadaveric transplantation may be preferable to
transplant because of antibodies against its normal type IV
living-related donor transplant (11).
basement membrane collagen (21,22).
Systemic Diseases
OBTAINING TISSUE
The severity of renal involvement in systemic disease [e.g.,
hemolytic-uremic syndrome (HUS)], Henoch-Schönlein pur-
General Considerations
pura, diabetes mellitus, or SLE may not be apparent without
renal biopsy. The trend is now toward early biopsy in patients Percutaneous renal biopsy is the most common method for
with diabetes and renal abnormalities. Severity of lesions and obtaining tissue for the kidney. In large series, major compli-
stage of chronicity and activity impart prognostic information cations are rare. The technique, first done in 1951 by Iverson
and may affect therapeutic decisions (see below). The most and Brun, allows tissue yield in 93 to 95% of biopsies, with
extensively studied disease in this regard is SLE. Differentia- more than 87% of these being adequate (23–25). The biopsy
 24. Renal Pathology 477

TABLE 24.1. DEFINITIONS OF COMMON MORPHOLOGIC TERMS

Term Definition

Light microscopy
Focal Involving some glomeruli
Diffuse Involving all glomeruli
Segmental Involving part of glomerular tuft
Global Involving total glomerular tuft
Lobular Simplified, lobular appearance of capillary loop architecture (membranoproliferative glomerulone-
phritis)
Nodular Acellular areas of mesangial matrix (diabetic nephropathy)
Sclerosis Collapse and scarring of capillary loop
Crescent Proliferation of parietal epithelial cells
Spikes Projections of GBM intervening between subepithelial immune deposits (membranous glomerulo-
nephritis)
Endocapillary proliferation Increase in mesangial or endothelial cells
Hyaline Descriptive of glassy, smooth-appearing material
Hyalinosis Hyaline appearing insudation of plasma proteins (focal segmental glomerulosclerosis)
Mesangium Stalk region of capillary loop with mesangial cells surrounded by matrix
Subepithelial Between visceral epithelial cell and GBM
Subendothelial Between endothelial cell and GBM
Tram-track Double contour of GBM due to deposits or CMIP (see below)
Wire loop Thick, rigid appearance of capillary loop due to subendothelial deposits
Activity Score of possible treatment-sensitive lesions (e.g., based on extent of crescents, cellular infiltrate,
necrosis, proliferation)
Chronicity Score of probable irreversible lesions (e.g., based on extent of tubular atrophy, interstitial fibrosis,
fibrous crescents, sclerosis)
Immunofluorescence
Granular Discontinuous flecks of staining along capillary loop producing granular pattern
Linear Smooth continuous staining along capillary loop
Electron microscopy
Foot process effacement Flattening of foot processes so that they cover the basement membrane
Microvillous transformation Small extensions of visceral epithelial cells with villus-like appearance
CMIP Extension of mesangial cell cytoplasm with interposition between endothelial cell cytoplasm and
basement membrane and underlying new basement membrane formation
Reticular aggregates Organized arrays of membrane particles within endothelial cells
Immunotactoid GP Large, organized microtubular deposits, >30 nm in diameter
Fibrillary GP Fibrils 14–20 nm in diameter without organization

CMIP, circumferential mesangial interposition; GBM, glomerular basement membrane; GP, glomerulopathy.

findings altered diagnoses in half of the cases in one series, kidneys—all are associated with greater risk for complica-
indicating different therapeutic approaches in approximately tions. Open biopsy is preferable if the biopsy information is
one-third of those cases (26). Although some renal diseases crucial in these conditions. Percutaneous biopsy is contrain-
show diagnostic features by LM (Table 24.1), special studies dicated if the kidney has tumors, large cysts, abscesses, or
add to the sensitivity of the study. For the renal biopsy to be pyelonephritis because the needle track may facilitate spread
most useful, it must be evaluated appropriately by an experi- of malignant cells or infection. Open biopsy allows selection
enced renal pathologist. Biopsies must be examined by spe- of specific areas for biopsy in these situations.
cial LM, IF microscopy, and electron microscopy (EM) for
the most accurate diagnosis (27). If the nephrologist’s hospi- Biopsy Technique
tal does not provide these services, arrangements must be
The patient may be admitted on the day of the biopsy.
made to send tissue in appropriate fixatives (see below) to a
Before biopsy is done, ultrasound examination must con-
reference laboratory with these capabilities. If such services
firm that there are two kidneys in normal position. Labora-
cannot be provided, it is doubtful whether the institution
tory evaluation must include a complete blood cell count
should be undertaking renal biopsies.
with normal platelet count, partial thromboplastin and
prothrombin times, fibrinogen level, and bleeding time.
Contraindications
The blood bank should type and hold one unit of blood.
Contraindications to percutaneous biopsy are solitary, The biopsy optimally is timed so that an experienced tech-
ectopic, or horseshoe kidney; bleeding diathesis; abnormal nician or pathologist can attend to ensure prompt process-
renal vascular supply; and uncontrolled hypertension (23– ing of the biopsy tissue.
25,28). Relative contraindications include obesity; uncoop- Food and drink should be withheld for at least 6 hours
erative patients; hydronephrosis; ascites; and small, shrunken before biopsy, and the child should be lightly sedated but
478 IV. Clinical Methods

still able to cooperate during the biopsy. The child lies in be discharged the next day if no complications arise (28).
the prone position with a sandbag or rolled sheet under the An evaluation of the impact of earlier discharge showed
abdomen, and the skin of the flank is prepped and draped that observation for 8 hours or less after renal biopsy
in sterile fashion. Although the left kidney is usually pre- missed more than 20% of complications (32).
ferred, either side can be chosen for biopsy. The lower pole Open biopsy can be performed under local or general
of the kidney is marked on the skin with a pen after local- anesthesia. The kidney can be directly visualized even
ization by any of several imaging techniques (e.g., fluoros- through a small incision. Although a larger sample may be
copy, radionuclide scanning, intravenous urography, obtained with a wedge biopsy, it is preferable to also per-
computed tomography, or ultrasound—the last being the form a needle biopsy to sample deeper cortex and medulla
most commonly used method). Local anesthetic is infil- for assessment of diseases that preferentially involve jux-
trated first in the skin and then in deeper tissues, taking tamedullary glomeruli (see below).
care not to enter the kidney. A small incision is made
through the skin and a spinal needle stylet inserted into the
Aspiration Biopsy
depth of the kidney while the patient holds his or her
breath in midinspiration. When the kidney capsule is Fine-needle aspiration (FNA) biopsy technique is used
punctured, a loss of resistance can be felt. The needle moves most often in the transplant setting for analysis of immu-
with inspiration when the child is asked to breathe, con- nologically activated cells. FNA is especially useful in evalu-
firming that it is within the renal parenchyma. Arterial pul- ating acute rejection (33). FNA can also obtain material for
sations can also be seen when the needle is within the culture. A modified FNA technique has been described for
kidney. The exact depth of the kidney and position of the collection of glomeruli from either native or transplant kid-
needle are noted by ultrasound and marked on the stylet at neys to analyze glomerular lesions. This technique has limi-
the point where these respiratory movements are no longer tations in sample size and is obviously not suitable for study
transmitted to the stylet as it is slowly removed. Conven- of vascular, tubular, or interstitial processes. The less inva-
tional or spring-loaded needles are used for renal biopsies. sive nature of the procedure makes it amenable to serial
Most now use the spring-loaded, so-called biopsy gun. For monitoring of diffuse glomerular lesions in native or trans-
conventional needles, the biopsy needle is inserted to the planted kidneys (34).
desired position as the patient again holds his or her breath,
advancing the cannula over the obturator once the needle is
Complications
in correct position. The entire needle with the core of tissue
is then removed. Important complications occur in 5 to 10% of patients
An automatic spring-loaded biopsy system has been (23,35–43). Major complications, usually bleeding, leading
used widely in the last several years because of the simplic- to nephrectomy occurred in five patients in a review of
ity and ease of the technique (29,30). The kidney is local- 8081 biopsies (41) and one patient in a series of 5120 biop-
ized with ultrasound guidance, and the depth of the sies. In a series totaling 1820 biopsies in children, one
kidney as judged by ultrasound and the spinal needle is nephrectomy resulted (35–40). Transient microscopic
used to place the biopsy needle to reach the kidney cap- hematuria is universal after biopsy, although macroscopic
sule. The patient holds his or her breath for only a few hematuria is seen in only 5.0% and requires transfusion in
seconds while the spring-loaded needle is activated, caus- up to 2.3% of patients. Perirenal hematoma is most often
ing the obturator to automatically advance into the kid- asymptomatic and can be seen by computed tomography in
ney, and the entire needle is then removed with the tissue up to 85% of biopsies. Symptomatic hematoma is rare,
core. It is important to note that the caliber of the needle occurring in less than 2%. Arteriovenous fistulae are symp-
used with any of these techniques directly impacts the tomatic with hematuria, hypertension, or cardiac failure in
adequacy of the specimen (31). When 18-gauge needles only 0.5% of biopsies, although bruits may be detected in
are used with this method, the resulting cores are very as many as 75% of patients. Most fistulae heal within a few
small and there is artifact along the edges. The use of a months. Other complications have been reported, includ-
16-gauge needle, thus, is more likely to provide an ade- ing inadvertent puncture of other viscera or major renal
quate tissue sample without distortion and with fewer vessels, sepsis, renal infection, and seeding of cancer. Death
passes necessary to obtain adequate tissue. is rare, occurring in less than 0.1% in reviews of large series.
Usually, two cores of tissue are necessary for optimum Complication rates appear to be slightly higher in less
evaluation, or three cores if 18-gauge needles are used. If developed countries (35,37). Complications for the spring-
tissue cannot be obtained after several passes, the biopsy loaded needle biopsy system appear to be similar to conven-
should be attempted on another day. After biopsy, a firm tional needles if the same gauge is used (44). Complications
dressing is applied, and the child is kept supine in bed for relate in part to the number of passes made to obtain tissue.
24 hours and monitored with frequent checks of vital signs Therefore, the lower rate of complication with an 18-
and urine for hematuria. The hematocrit should be gauge, spring-loaded needle in some studies is offset largely
rechecked at 4 and 24 hours after the biopsy. The child may by the need for more passes for adequate samples and the
 24. Renal Pathology 479

distortion of tissue and edge artifact with use of an 18- a sponge or gauze pad because this may cause a divot-like
gauge needle (see above). artifact as the unfixed tissue molds to the holes of the
underlying surface. The biopsy specimen is therefore placed
on a clean smooth surface, such as a wax board, for cutting.
ASSESSMENT OF THE RENAL BIOPSY
It may be difficult to identify the cortical end of the
specimen even after inspection with a hand lens or dissect-
Adequacy of Sample
ing microscope when scarring or severe injury is present.
Cores of fat and connective tissue float when placed in saline, Therefore, we recommend cutting two 1-mm pieces with a
but a core of renal parenchyma sinks. The biopsy sample sharp blade from each end of the core for EM studies. The
should also be visually inspected with a dissecting micro- remaining core is then divided into specimens for IF and
scope or hand lens. Glomeruli are visualized as small red dots LM. Of note, use of an 18-gauge needle yields tissue cores
in the biopsy core. Scarred glomeruli may be difficult to that are too thin to divide lengthwise, leading to a greater
identify because they are not perfused. In a diffuse disease, chance of problems in allocation of tissue for each method
such as membranous glomerulonephritis, one glomerulus of study. This tissue should be cut across into two pieces for
may be adequate for diagnosis. However, in other conditions IF and LM. When two cores are obtained, we prefer to
(e.g., crescentic glomerulonephritis, FSGS, or lupus WHO duplicate this process, rather than allocating one complete
class III glomerulonephritis), the lesions may be focal. The core to one study to maximize chances of adequacy of tissue
greater the number of glomeruli sampled, the lower the for each study. When the tissue sample obtained is very
probability of missing a focally distributed lesion (45). If small, the nephrologist and the pathologist should consider
only 10% of glomeruli in the kidney are involved by the the differential diagnosis and allocate tissue accordingly.
focal process, a biopsy sample of only ten glomeruli has a For example, in a case of suspected IgA nephropathy, tissue
35% probability of missing the lesion, decreasing to 12% if for IF is most important. Although EM studies can be done
the biopsy contains 20 glomeruli. When one-fourth of glo- on other portions of tissue (as long as mercury-based fixa-
meruli are involved in the kidney, there is only a 5% chance tives have not been used), IF studies cannot be done reli-
of missing the abnormal glomeruli in a biopsy of ten glomer- ably on fixed tissue. When tissue for EM is inadequate,
uli. A biopsy of 20 to 25 glomeruli is sufficient to distinguish portions of the paraffin-embedded tissue left after LM
between mild disease (less than 20% of glomeruli involved), examination may be cut out from the block and processed
moderate disease (20 to 50% of glomeruli involved), or for EM. Although the quality is not optimal, diagnostic
severe disease (more than 50% of glomeruli involved). How- findings can still be discerned. In special circumstances,
ever, the widespread use of small-gauge, spring-loaded biopsy when no tissue remains in the paraffin block and a focal
needles often results in smaller samples, which make the lesion in one section must be studied, one may attempt to
above assessments of severity and extent of lesions difficult or process the tissue section from a glass slide for EM.
impossible. The sample site must also be considered in evalu-
ating the adequacy of tissue. Even a large biopsy consisting
Light Microscopy
only of superficial glomeruli cannot exclude the presence of
early FSGS in which the initial involvement is in the jux- Numerous fixatives are used for LM examination, and they
tamedullary glomeruli. Likewise, although nephronophthisis vary from institute to institute. Satisfactory results may be
is most often diagnosed clinically, a juxtamedullary biopsy obtained with Zenker’s, Bouin’s, formalin, Carnoy’s, or
would be necessary for morphologic diagnosis. paraformaldehyde. Material for IF studies may be snap fro-
zen immediately at 20°C in solutions of isopentane, dry
ice, acetone, or freon and embedded in Tissue-tech, OCT,
Allotment of Tissue
or other compounds for frozen sections. If tissue cannot
Renal tissue should be studied by LM techniques with spe- immediately be snap frozen, it may be placed in Michel’s
cial stains [hematoxylin and eosin, modified silver stain tissue media, where it may be stored for up to 1 week
(periodic acid and methenamine or Jones’ stain), periodic before freezing. This allows tissue to be sent to reference
acid-Schiff (PAS), IF, and EM] (26,27,46). The tissue is laboratories for appropriate processing. Tissue for EM may
divided so that glomeruli are present in each portion of the be fixed in glutaraldehyde, formaldehyde, or other appro-
sample. Optimally, the pathologist or an experienced histo- priate fixatives. Tissue placed in glutaraldehyde should be
technologist attends the biopsy and inspects and allots tis- promptly processed or, if stored for future possible process-
sue for each study. If this is impossible, tissue may be placed ing, should be transferred to an appropriate buffer solution
in saline and brought directly to the laboratory for prompt within 1 week to avoid artifacts. Tissue for LM is routinely
processing. It is important to handle the tissue gently so processed, embedded in paraffin, and cut into 2- to 3-μ
that artifacts do not occur. The fresh tissue must not be thick sections and stained. Serial sections with multiple lev-
picked up with forceps because this crushes and distorts the els are then prepared for examination.
morphology. The core can be handled carefully with a If water-soluble compounds are expected (e.g., as urate
wooden stick or pipette. The core should not be placed on or uric acid), the tissue should be fixed in ethanol. Lipids
480 IV. Clinical Methods

are best detected in frozen sections because they are exact localization of those deposits (27,46). Lesions of
extracted during xylene processing for paraffin sections. interest should be photographed because fluorescence fades
Hematoxylin and eosin stains are most useful for overall on storage and with light exposure.
assessment of the interstitium, allowing particularly good
visualization of infiltrating cells, especially eosinophils. In
Electron Microscopy
addition, fibrin may be easily visualized by this stain. PAS
stain accentuates basement membranes and matrix material Tissue for EM is processed with postfixation in 1%
and allows definition of the brush border of proximal tubu- osmium tetroxide, which enhances contrast of the tissue,
lar cells. Areas of hyalinosis and protein precipitation, and then dehydrated and embedded. With new, more rap-
including cryoglobulin, are also accentuated with PAS idly polymerizing embedding media (e.g., Spurr), tissue
stain. The basement membrane and areas of deposits are may be ready for examination within 1 day. EM study was
best visualized by silver stain, usually the Jones’ stain. Mas- found to add information in 6 to 11% of renal biopsies in a
son’s trichrome stain may be used to accentuate areas of study from 1983 (24). A more recent study showed that
fibrosis and stains areas of collagen deposition bluish. EM was needed to make a diagnosis in 21% of cases and
Other special stains may be indicated. These include provided important confirmatory data in approximately
Congo red stain for amyloidosis and special stains for bac- 20% of cases (27). EM showed diagnostic pathologic
terial or fungal organisms and acid-fast bacilli. Special tech- abnormalities in 18% of patients with normal LM findings.
niques may also be used on the LM material. These include Larger, so-called thick sections (1 μ) are stained with tolui-
polarization to detect crystals or foreign bodies and mor- dine blue to select smaller areas for thin sectioning for the
phometry to assess glomerular size (see below) and severity electron microscope. Usually, the glomerulus containing
of interstitial fibrosis quantitatively. the most representative lesion is chosen. If there are irregu-
lar or focal lesions, several glomeruli may be sampled. The
60- to 90-Å thin sections are stained with uranyl acetate
Immunofluorescence
and lead citrate before viewing to enhance contrast.
IF studies are most commonly done by direct IF on frozen The characteristic immune deposits are denser than
tissue sections, with application of fluorescein-conjugated basement membrane or matrix materials. In specific dis-
antibodies directed against IgG, IgA, IgM, and comple- eases, such as cryoglobulinemia, amyloid, immunotactoid
ment component C3. Additional antisera may be used as or fibrillary glomerulopathies, or lupus nephritis, specific
clinically indicated. These include antisera to κ or λ light substructure of deposits may be seen. Specific localization
chain, antisera to hepatitis B antigen, thyroglobulin, fibrin- of immune complexes is done by EM examination, indicat-
ogen, C1q, antisera to type IV collagen chains, and C4d. ing whether deposits are subendothelial, subepithelial,
Some of these antigens are recognized by antisera even after mesangial, or in all of the above compartments. In some
fixation and can be detected by immunohistochemical diseases, such as light chain deposition disease or lupus
techniques (e.g., immunoperoxidase) on the formalin-fixed nephritis, deposition may also be seen in vessels and
tissue sections and then studied with a light microscope. tubules. So-called fingerprint deposits, with substructures
This technique requires enzyme pretreatment of tissue, reminiscent of fingerprinting, are present in some cases of
which must be tailored exactly depending on length and lupus nephritis. Reticular aggregates (or so-called tubular
type of fixation, section thickness, and antigen one wishes arrays) of membrane material in endothelial cell cytoplasm
to study. Direct observation of the digestion process, stop- throughout the body are characteristically seen in large
ping when all plasma is removed from capillary loops, has numbers in patients with SLE or human immunodefi-
been used to achieve reliable results (47). These challenges ciency virus (HIV) infection and are believed to reflect a
have prevented widespread use of this technique. response to high levels of interferon (46,48).
Frozen tissue sections stained by the commonly used EM also delineates specific basement membrane abnor-
method of fluorescein-conjugated antibodies are viewed by malities. For instance, small subepithelial deposits without
IF microscopy, evaluating staining in glomeruli, vessels, surrounding new basement membrane material do not
tubules, and interstitium. The pattern of glomerular stain- result in spikes and, therefore, cannot be visualized by
ing is assessed to define granular or linear capillary base- Jones’ stain on LM but can still be detected directly by EM.
ment staining or mesangial deposits. Arteriolar staining, The lamina densa of the GBM is markedly thickened in
especially with C3 in patients with hematuria, may be of diabetic nephropathy. Circumferential mesangial interposi-
diagnostic significance. Tubules may show deposits in lupus tion is defined by extension of monocytes and mesangial
nephritis or light chain deposition disease. Nuclear staining cell cytoplasm into the subendothelial space, with newly
can be seen in lupus or lupus-like diseases as a tissue mani- formed basement membrane interpositioning between the
festation of the patient’s positive antinuclear antibodies. IF advancing mesangial cells and the endothelium, thus pro-
is more sensitive than EM in identifying immune deposits; viding the classic double contours with silver stain seen by
however, EM provides more detailed information on the LM in MPGN. Increased lucent material is present in the
 24. Renal Pathology 481

lamina rara interna in transplant glomerulopathy, HUS, Endothelial cells are opposed to the inner surface of the
toxemia, and other diseases presumed to involve coagulopa- GBM and are fenestrated. At the stalk of the capillary, the
thy. In these conditions, deposition of fibrin, fibrinogen, endothelial cell is separated from the mesangial cells by
and their degradation products may also occur. Fibrin is intervening mesangial matrix. Because the endothelial cell
recognizable by its dense, coarse, sheaf-like structure, with nucleus most often lies in this stalk region, it may be diffi-
periodicity observed in favorable sections. Morphometry of cult to distinguish from the mesangial cell nucleus by LM.
the GBM from EM prints is used to diagnose thin base- The term endocapillary is used to describe proliferation fill-
ment membranes in hereditary nephritides. EM also allows ing up the capillary lumen, contributed to by proliferation
structural assessment of changes of specific cells (see of mesangial, endothelial, and infiltrating inflammatory
below). Diagnostic inclusions are seen in various storage or cells. In contrast, extracapillary proliferation refers to prolif-
metabolic diseases (e.g., Fabry’s disease). eration of the parietal epithelial cells that line Bowman’s
capsule.
The mesangial cell is a contractile cell that also has
BASIC RENAL LESIONS
phagocytic properties. It lies embedded in the mesangial
matrix in the stalk region of the capillary loops, attached to
Normal
anchor sites at the ends of the loop by thin extensions of its
The normal glomerulus consists of a complex branching cytoplasm. Normally, up to three mesangial cell nuclei per
network of capillaries originating at the afferent arteriole lobule are present. The basement membrane consists of
and draining into the efferent arteriole. The glomerulus three layers distinct by EM, the central broadest lamina
contains three resident cell types: mesangial, endothelial, densa, and the less electron-dense zones of lamina rara
and epithelial cells (Fig. 24.1). The epithelial cells cover the externa and interna. Thickening occurs with maturational
urinary surface of the GBM with pseudopod-like exten- growth. Most investigators have found thicker basement
sions called foot processes with intervening filtration slits. membranes in boys, with normal range from 220 to 260

FIGURE 24.1. Schematic illustration of glomerulus with glomerular capillary attached to a

mesangial stalk area. The glomerular endothelium (E) is fenestrated and lines the glomerular
basement membrane (GBM), which covers the mesangium. The outside of the GBM is covered by
the epithelial cell and its foot processes (Ep). The mesangial cell (M) is embedded within the
mesangial matrix (MM), with processes connecting to the GBM. (Courtesy of Professor Wilhelm
Kriz. From Venkatachalam MA, Kriz W. Anatomy of the kidney. In: Heptinstall RH, ed. Pathology
of the kidney, 4th ed. Boston: Little, Brown and Company, 1992:1:35, with permission.)
482 IV. Clinical Methods

and many diseases are characterized by focal lesions. Assess-

ment of severity and patterns of lesions is made, and nor-
mal and affected glomeruli are counted. Lesions are
classified as focal if only some glomeruli are involved, dif-
fuse if all glomeruli are involved, segmental if only portions
of glomeruli are involved, and global if entire glomerular
tufts are involved. Characteristic glomerular disease pat-
terns include lobular proliferation in MPGN, nodular pro-
liferation of mesangial matrix material with paucicellular
areas in characteristic Kimmelstiel-Wilson lesions, focal
and segmental sclerosis, and crescent formation (Table
24.1). The crescent, or proliferation of parietal epithelial
cells, owes its name to its shape in well-established lesions.
FIGURE 24.2. Immature glomerulus with plump, dark epithelial Fibrin and thrombosis are best assessed by Jones’ stain.
cells from biopsy of a baby at 29 weeks’ gestation (periodic acid- Glomeruli are assessed for alterations in size (see below).
Schiff stain, ×670).
It is important to compare with a normal control for a
given age group because glomerular maturational growth is
rapid in children. Glomerular hypertrophy may be an
nm at 1 year of age, 280 to 327 nm at 5 years of age, 329 to
important predictor of increased risk of FSGS in children
370 nm at 10 years of age, and 358 to 399 nm at 15 years
with apparent MCD (see below). Maturational pattern of
of age (49,50). In our laboratory, we found a range of
glomeruli (see above) should be noted.
GBM thickness in children with normal kidneys from
Glomeruli are assessed for glomerulosclerosis (i.e., the
approximately 110 nm at 1 year of age to 222 ± 14 nm at 7
presence of segmental obliteration and scarring of glomerular
years of age.
capillary tufts). Sclerosis may be in a segmental or global pat-
The glomerulus is surrounded by Bowman’s capsule,
tern (Table 24.2). Previous studies suggested that up to 10%
which is lined by parietal epithelial cells. These are con-
of glomeruli are normally globally sclerosed in people
tinuous with the proximal tubule, identifiable by its PAS-
younger than 40 years of age (53). More recent work suggests
positive brush border. The efferent and afferent arterioles
that this number may be even smaller in children, with less
can be distinguished morphologically in favorably ori-
than 1 to 3% global sclerosis expected normally up to age 40
ented sections or by tracing their origins on serial sec-
or 56 years, respectively (54,55). These occasional globally
tions. Segmental, interlobular, and arcuate arteries may
sclerotic glomeruli are believed to represent errors of nephro-
also be present in the renal biopsy specimen. The cortical
genesis. The incidence of global sclerosis increases with
biopsy also allows assessment of the tubulointerstitium.
aging, up to half the patient’s age minus 10 (55). Globally
Proximal tubules are readily identified by their PAS-posi-
sclerosed glomeruli in a greater percentage indicate the possi-
tive brush border lacking in the distal tubules. Collecting
bility of renal disease (focal global sclerosis) (56).
ducts show cuboidal, cobblestone-like epithelium. The
The pattern of tubulointerstitial fibrosis, whether pro-
medulla may also be included in the biopsy.
portional to glomerular sclerosis, whether diffuse or present
During fetal maturation, the glomerular capillary tufts
in a striped pattern after the medullary rays or in broad
are initially covered by large, cuboidal, darkly staining epi-
patchy zones, has diagnostic significance (see below).
thelial cells with only small lumina visible (Fig. 24.2). The
cells lining Bowman’s space undergo a similar change from
initial tall columnar to cuboidal to flattened epithelial cells, Specific Glomerular Cells
except for those located at the opening of the proximal
Podocytes
tubule, where cells remain taller. Immature nephrons may
occasionally be seen in the superficial cortex of children up The podocytes (glomerular visceral epithelial cells) may show
to 1 year of age. Glomerular growth continues until adult- vacuolization in various diseases with severe proteinuria.
hood, with average normal glomerular diameter approxi- Although more extensive vacuolization of podocytes has
mately 95 μ in a group of patients younger than 5 years of been seen in FSGS compared with patients with MCD (57),
age (average age, 2.2 years) and 140 to 160 μ in adulthood these changes are seen only after established sclerotic lesions
(51,52). are identifiable by LM and do not permit distinction of these
two disease processes in the early phase in which segmental
sclerosis may be undetected. Hypertrophy of podocytes is
Overall Pattern
prominent in MCD and FSGS. Effacement of the foot pro-
Assessment of the biopsy specimen must include inspection cesses of the podocytes by EM is common to any disease
of all sections from different levels because additional glo- with marked proteinuria, and the podocyte may also show
meruli may be sampled on deeper cuts of the biopsy core microvillous transformation with long, attenuated pseudo-
 24. Renal Pathology 483

TABLE 24.2. CHARACTERISTIC ABNORMALITIES OF GLOMERULAR DISEASES

Immunofluorescence staining

Disease and typical Light microscopy

clinical presentation pattern Mesangial Subepithelial Subendothelial EM, other findings

Hematuria/nephritis
Alport’s syndrome Early: normal – – – Thin, split GBM
Late: sclerosis
Lupus WHO class II Normal or mesangial + – – Immune deposits by EM, reticu-
(mesangial) proliferation All Igs, C3, lar aggregates in endothelial
C1q cells
Lupus WHO class III Proliferative, <50% of + + (few) + (scattered) Immune deposits by EM, reticu-
(focal prolifera- glomeruli All Igs, C3, C1q lar aggregates in endothelial
tive) cells
Lupus WHO IV (dif- Proliferative, >50% of + + + (wire loop) Immune deposits by EM, reticu-
fuse proliferative) glomeruli All Igs, C3, C1q lar aggregates in endothelial
cells
IgA nephropathy Mesangial proliferation + – – Immune deposits by EM
Predomi-
nantly IgA
Henoch-Schönlein Mesangial prolifera- + +/– +/– Immune deposits by EM
purpura tion, crescents Predominantly IgA
Postinfectious GN Hypercellular, PMNs, +/– + – Irregular, hump-like deposits on
endocapillary prolif- Coarsely top of GBM by EM
eration granular
IgG, C3
Hemolytic-uremic Arteriolar/glomerular – – – Increased lucency and thickness
syndrome thrombosis of lamina rara interna by EM,
swollen endothelial cells, no
deposits by EM
MPGN I Hypercellular, lobular + – + Subendothelial, intramembra-
double contour GBM IgG, C3 nous immune deposits by EM,
circumferential mesangial
interposition
MPGN II (dense Hypercellular, +/– lobu- ± Ribbon-like, discontinuous Intramembranous nonimmune
deposit disease) lar, ribbon-like capil- C3 C3 dense deposits by EM
lary wall
Nephrotic syndrome
Minimal change dis- Normal – – – Effacement of epithelial cell foot
ease processes, no deposits by EM
Focal segmental Segmental glomerulo- +/– – – Effacement of epithelial cell foot
glomerulosclerosis sclerosis, glomerular IgM, C3 processes, no deposits by EM
hypertrophy
Diabetic nephropa- Increased mesangial – – – Thick GBM without deposits
thy matrix, +/– nodular,
thick GBM, hyalin-
ized arterioles
Lupus WHO class V Spikes on Jones’ stain Few + – Immune deposits by EM, reticu-
(membranous) All Igs, C3, C4 lar aggregates in endothelial
cells
Idiopathic membra- Spikes on Jones’ stain – + – Immune deposits by EM
nous GN IgG, C3
Rapidly progressive
glomerulonephritis
Anti-GBM disease Focal segmental necro- – Linear IgG No deposits by EM
sis of
glomeruli, crescents
Wegener’s Focal segmental necro- – – – No deposits by EM
granulomatosis sis of
glomeruli, crescents
Microscopic Focal segmental necro- – – – No deposits by EM
polyangiitis sis of
glomeruli, crescents

+, present; –, absent; +/–, variably present; EM, electron microscopy; Ig, immunoglobulin; GBM, glomerular basement membrane; GN, glomerulo-
nephritis; MPGN, membranoproliferative glomerulonephritis; PMNs, polymorphonuclear neutrophils; WHO, World Health Organization.
484 IV. Clinical Methods

pods. Podocytes are limited in their ability to proliferate. Crescents may occur in a variety of diseases. Diseases with
However, the early sclerotic lesion of FSGS is characterized crescents as a primary manifestation include antibody-medi-
by prominence and apparent increase of the overlying ated injury (anti-GBM antibody disease), immune-complex
podocytes often associated with endocapillary foam cells. diseases (e.g., lupus nephritis), and nonimmune diseases.
The collapsing variant of FSGS or HIV-associated nephropa- The latter are often, but not invariably, associated with posi-
thy both show prominent hyperplasia and protein droplets of tive ANCA tests and may be associated with systemic disease
the podocytes overlying segmental collapse of the glomerular or be renally limited. The perinuclear ANCA pattern is most
capillary tuft. In the situation of recurrent FSGS in the renal often associated with microscopic polyangiitis, whereas the
transplant, NS and foot process effacement may be seen cytoplasmic ANCA pattern is typical in Wegener’s granulo-
within weeks after biopsy, with sclerosis becoming apparent matosis. Of note, positive ANCA tests are not sensitive in
at a later date (58). In Fabry’s disease, there is accumulation distinguishing these categories (13–15). Renal biopsy is,
of glycosphingolipid because of deficiency of α-galactosidase. therefore, critical for accurate diagnosis. Diagnosis and
Podocytes show marked vacuolization by LM with character- appropriate treatment must occur rapidly in this clinical situ-
istic whorled, laminated, electron-dense myelin bodies by ation to optimize chances of recovery of renal function. The
EM. In Fabry’s disease, these inclusions may also be present early lesion of cellular crescents is responsive to cytotoxic
in endothelial cells, tubular epithelial cells, and some intersti- therapy. Biopsy indications of irreversible renal damage
tial cells (24,46). include breaks of Bowman’s capsule and fibrous transforma-
tion of the cellular crescents, periglomerular fibrosis, and
scarred glomeruli and tubulointerstitium.
Mesangial Cells
Hyperplasia of mesangial cells is recognized by LM when
Glomerular Basement Membrane
more than three mesangial cell nuclei are present per
mesangial region. Increased mesangial prominence may be GBM abnormalities are best evaluated by EM. The base-
due to increased cellularity, increased matrix, deposits, or a ment membrane is abnormally thick in diabetic nephropa-
combination. Large mesangial deposits appear on Jones’ thy (46). Diffuse, abnormally thin GBMs, less than 250
stain as pinkish areas surrounded by the light silver-staining nm in adults, are seen in familial hematuria (49–50). In
areas of mesangial matrix. So-called mesangial interposition children, the diagnosis of thin basement membranes is
results when the monocyte or mesangial cell cytoplasm more difficult than in adults because GBM increases in
extends outward between basement membrane and endo- thickness with normal maturation. GBM thickness should
thelial cells and new matrix accumulates between the be compared with normal for age and sex (see above)
mesangial and endothelial cell bodies. (49,59,60). In Alport’s syndrome, the basement membrane
is characterized by irregular thickness, either very thin or
very thick, with splitting and splintering of the basement
Endothelial Cells
membrane (22,61). The GBM in nail-patella syndrome is
Extreme proliferation and swelling of endothelial cells can irregular, thickened, and split, with electron-lucent areas
obliterate capillary lumina in conditions characterized by containing banded collagen type I fibers (46).
abnormalities of coagulation. Endothelial cells usually con- Immune deposits may localize on either side of the
tain characteristic reticular aggregates in lupus nephritis GBM. Subepithelial immune deposits are characteristically
and HIV-associated nephropathy (46,48). Endocapillary seen in membranous glomerulonephritis. Subendothelial
cell proliferation is characteristic of, for example, diffuse immune deposits are seen, for example, in lupus prolifera-
proliferative lupus nephritis and MPGN type I. tive nephritis or MPGN type I.
The basement membrane may appear split by LM in
diseases other than MPGN type I or dense deposit disease.
Crescents
In transplant glomerulopathy, the split appearance results
Crescents consist primarily of proliferating parietal epithe- from varying degrees of mesangial interposition and widen-
lial cells with some infiltrating macrophages and are a man- ing with increased lucent material in the lamina rara
ifestation of severe glomerular injury. The name reflects the interna. This is also a characteristic finding in preeclampsia,
often crescent-shaped sheet of cells filling up part or nearly transplant glomerulopathy, and chronic HUS (46).
all of Bowman’s space. Crescents result from injuries that
break the GBM, leading to exudation of plasma protein
Tubules
and formation of fibrin within Bowman’s space, which then
induces proliferation of the parietal epithelial cells and infil- Morphologically evident tubular necrosis correlates poorly
tration of macrophages. When crescents are a prominent with the clinical extent of acute tubular necrosis. The
histologic feature, the patient most often presents clinically changes vary from nondiagnostic vacuolization to frank
with a rapidly progressive glomerulonephritis. necrosis with sloughing of tubular epithelial cells and flat-
 24. Renal Pathology 485

tened epithelium characteristic of regeneration (24). In cor- cases of idiopathic interstitial nephritis (64). Eosinophils
tical necrosis, zones of cortex, including glomerular can also be part of acute cellular rejection in the transplant.
structures, are necrotic. Tubules are atrophied with dilation Nonnecrotizing granulomas, with or without eosinophils,
and flattened epithelium in chronic renal disease, presum- most often reflect drug-induced hypersensitivity reaction.
ably secondary to lesions affecting the glomerulus, Fibrosis results in increased spacing of tubules because of
although inherent interstitial factors may also be involved the accumulation of PAS-positive collagenous material.
in these changes. Tubular atrophy is also present in primary The connective tissue also stains specifically blue with Mas-
tubulointerstitial diseases. Tubulointerstitial fibrosis is an son’s trichrome stain. Fibrosis in a striped pattern suggests
important manifestation of cyclosporine toxicity. The fibro- chronic cyclosporine toxicity (65,66). Occasionally, the
sis occurs along the medullary rays, resulting in a striped, interstitium is infiltrated by malignancy. Hematopoietic
rather than diffuse, pattern of fibrosis, with intervening neoplasms are especially prone to involve the kidney.
preserved tubules.
Nonspecific casts of Tamm-Horsfall protein are seen in
Vessels
chronic renal disease. Other casts may have a diagnostic
appearance, such as the giant cells surrounding tubular Arterioles and larger segmental and interlobular arteries are
casts in light chain cast nephropathy (so-called myeloma evaluated for changes in the intima and media; the presence
kidney). Casts of myoglobin with characteristic reddish- of deposits, fibrin, hyalin, amyloid, or other material; or the
brown appearance are seen in rhabdomyolysis, often with presence of vasculitis. Larger vessels typically are not sam-
associated acute tubular necrosis. Crystals (e.g., oxalate) pled by a biopsy, and diseases that affect these large vessels
may be identified by examination under polarized light. (e.g., classic polyarteritis nodosa) are, therefore, best evalu-
Tubules contain characteristic inclusions in Fabry’s disease. ated by other methods (e.g., arteriography). Intimal fibrosis
Polymorphonuclear neutrophils within collecting ducts and medial thickening with hyperplasia and hypertrophy of
and proximal tubules are diagnostic of acute pyelonephritis. media are characteristic of hypertensive injury. Specific
In chronic pyelonephritis, there is tubular atrophy and eccentric patterns of medial necrosis with nodular protein
interstitial fibrosis, characteristically in a patchy, regional deposition suggest acute cyclosporine nephrotoxicity (66).
distribution. The combination of segmental glomerular Fibrin thrombi, when present in glomeruli or arterioles, are
sclerosis with ischemic changes of corrugation and thicken- the essential lesions of the thrombotic microangiopathies
ing of the GBM and periglomerular fibrosis and patchy, (e.g., HUS) (46). Fibrin localizes predominantly within
regional interstitial fibrosis and tubular atrophy is charac- glomerular lumina in disseminated intravascular coagula-
teristic of reflux nephropathy (62). tion and hyperacute rejection.
Cysts may be demonstrated by biopsy, although the diag-
nosis of specific cystic diseases is usually made by combina-
tion of clinical and ultrasound findings. The segment of the CLINICAL PATHOLOGIC CORRELATIONS
nephron giving rise to the cysts can be identified by his-
tochemical stains (63). Areas of low cuboidal epithelial-lined After evaluation of the structural changes of the renal
structures surrounded by a cuff of immature mesenchyme biopsy in conjunction with the clinical history, a diagnosis
are present in the dysplastic kidney, often with cartilage, fat, may be obvious. In some cases, the biopsy specimen may
or abnormal blood vessels in the interstitium. The deep show overlap features, or there may be elements that do not
medullary cystic dilation characteristic of nephronophthisis correlate clearly with the clinical setting. Close collabora-
can be identified with a deep biopsy. Dilation of proximal tion by nephrologists and pathologists is essential in arriv-
tubules with microcyst formation in conjunction with scle- ing at the diagnosis. The pathologist must be familiar with
rosis is characteristic of congenital NS of Finnish type. clinical manifestations of renal disease, and the nephrolo-
FSGS in a collapsing pattern with podocyte hyperplasia, gist should be familiar with the terminology used by the
numerous reticular aggregates by EM, and tubular cystic pathologist to describe the biopsy findings (Table 24.2).
dilation and interstitial fibrosis out of proportion to the When lesions are evaluated, the balance of all elements
severity of glomerular lesions is highly suggestive of HIV- must be considered. When a typical disease pattern is not
associated nephropathy (48). present, one must consider whether more than one process
is taking place. For instance, drug-induced interstitial
nephritis may be superimposed on other glomerular dis-
Interstitium
ease. This is especially true in the transplant setting in
Interstitial edema is a nonspecific change present, for exam- which multiple disease processes may occur at one time. In
ple, in early acute transplant rejection, renal vein thrombo- some instances, the biopsy findings do not correlate with
sis, or inflammatory processes. Identification of eosinophils the patient’s renal function. When such apparent discrep-
in an infiltrate is suggestive of drug-induced interstitial ancies are found, one possibility is that the biopsy specimen
nephritis, although eosinophils are also present in some is not representative of all the nephrons of the kidney. The
486 IV. Clinical Methods

number of glomeruli necessary to estimate the severity of

diseases that show focal distribution has been discussed
above. However, the extent of glomerulosclerosis may in
and of itself not correlate with renal function. Tubulointer-
stitial atrophy and fibrosis may be more closely correlated
with extent of renal damage and renal function (67–69).
One must also consider the elements other than structure
that influence the patient’s renal function (i.e., blood pres-
sure, filtration properties of the GBM, and the glomerular
filtering surface area). Patients with enlarged glomeruli,
either caused by compensatory hypertrophy or by a pri-
mary pathologic process, may show less deterioration of
renal function than expected based on the extent of glomer-
ular scarring. Similarly, treatment of the patient with anti-
hypertensive agents that affect glomerular filtration rate
(e.g., angiotensin-converting enzyme inhibitors, which FIGURE 24.3. Minimal change disease. The foot processes are
preferentially dilate efferent arterioles) may actually flattened and appear fused (×11,000).
increase serum creatinine levels in the short term. Compen-
sation by remaining nephrons may mask ongoing severe
disease processes such that creatinine levels may remain sia—shows a rapid progression to end-stage disease (76).
near normal until late in the course of disease when therapy The cellular lesion, with endocapillary proliferation with
is less likely to have an impact on chronic progressive frequent foam cells and often with podocyte hyperplasia,
injury. may represent an early stage of FSGS (77). The tip lesion
(i.e., sclerosis localized to the proximal tubular pole) may
have a better prognosis (78). The perihilar variant, with
Diagnostic Findings in Selected sclerosis and hyalinosis localized to the vascular pole, likely
Renal Diseases more often represents a secondary sclerosing process.
C1q nephropathy is characterized by either no sclerosis
Minimal Change Disease and Focal Segmental
or segmental glomerulosclerosis by LM, with mesangial
Glomerulosclerosis
C1q deposits and lesser Ig components without dominant
MCD is diagnosed only after the exclusion of abnormal or codominant IgA (79). EM shows mesangial and para-
findings at the LM level, with diffuse foot process efface- mesangial dense deposits but a lack of reticular aggregates.
ment as the only abnormality by EM. The disease is charac- Patients typically are adolescents, have steroid-resistant NS,
teristically sensitive to glucocorticoid therapy. However, and do not have clinical evidence of SLE. Although these
repeated renal biopsies in patients with apparent MCD ini-
tially have shown progression to FSGS, which has a high
incidence of progression to end-stage renal disease (70,71). TABLE 24.3. WORKING CLASSIFICATION OF FOCAL
As discussed above, a small sample may not include the seg- SEGMENTAL GLOMERULOSCLEROSIS (FSGS)
mentally sclerotic glomerulus diagnostic of FSGS. In
Key histologic Possible prognostic
FSGS, there is often also hyalinosis, an insudation of Type feature implication
plasma proteins and lipids with a glassy, smooth (hyaline)
appearance on LM. There are no immune deposits, and FSGS, not oth- Segmental sclerosis Typical course
erwise speci-
foot process effacement is present in all glomeruli by EM fied
(Fig. 24.3). Mesangial expansion in the native kidney Collapsing FSGS Collapse of tuft, Poor prognosis
biopsy may be associated with increased risk for recurrence GVEC hyperpla-
in the transplant (72). sia
The presence of IgM in a biopsy that otherwise appears Cellular FSGS Endocapillary pro- ?Early stage lesion
liferation, often
to be MCD (so-called IgM nephropathy) does not have GVEC hyperpla-
prognostic value (73). Some variants of FSGS may have sia
prognostic value (74). A recently proposed working classifi- Tip lesion Sclerosis of tuft at ?Better prognosis
cation of FSGS aims to examine whether morphologic pat- proximal tubule
terns of FSGS have prognostic implications (Table 24.3) pole
Perihilar variant Sclerosis and hyali- ?May reflect a secon-
(75). The usual type is diagnosed when no special features nosis at vascular dary type of FSGS
are present. The collapsing type of FSGS—characterized by pole
collapse of the glomerular tuft, either segmental or global
with associated podocyte hypertrophy and/or hyperpla- GVEC, glomerular visceral epithelial cell.
 24. Renal Pathology 487

findings suggest a distinct clinicopathologic entity, the

prognostic significance of these lesions has not yet been
established. Progression to end-stage renal disease has
occurred in some patients with sclerosis at biopsy, but long-
term outcome of those without sclerosis at presentation has
not yet been established.
Because therapy and prognosis are different for MCD
versus FSGS, early distinction of these two entities is of pri-
mary interest. We studied pediatric patients with steroid-
resistant NS and MCD on renal biopsies and compared
them to patients with apparent MCD on biopsy who sub-
sequently progressed to overt FSGS (51). Morphometric
analysis of initial biopsies showed that glomerular size at
the onset of disease, before sclerosis was apparent, was
remarkably larger in patients who subsequently progressed
to FSGS (Fig. 24.4) (80,81). There was a higher risk for
development of FSGS in patients younger than 5 years of
age with glomerular area greater than 1.5 times that of nor-
mal age-matched controls. On the other hand, glomerular
size equal to or less than normal controls in this group of
patients indicated a good prognosis. Calculated glomerular
diameter for increased risk of FSGS in these patients
younger than 5 years of age was more than 118 μ versus 95
μ glomerular diameter in age-matched controls. Depending
on processing and fixation, these values may vary (our val-
ues are based on paraffin-embedded tissue fixation in for-
malin or Zenker’s fixative). Normal ranges should be
established in each laboratory assessing glomerular size.
From these studies, abnormal glomerular enlargement
suggests a high probability of development of FSGS in FIGURE 24.4. Apparent minimal change disease (MCD) with
pediatric patients with apparent MCD. Causes of abnormal subsequent progression to focal segmental glomerulosclerosis
(FSGS). The first biopsy from this 5-year-old girl (middle) was
glomerular enlargement other than idiopathic FSGS (e.g., indistinguishable from MCD, except for marked glomerular
diabetes mellitus, cyanotic cardiovascular disease, and mas- hypertrophy versus age-matched typical MCD with subsequent
sive obesity) must be excluded before such inferences can benign clinical course (top). The patient’s later biopsy (bottom),
50 months later, showed segmental sclerosis, diagnostic of FSGS
be made. It is interesting to note that the incidence of (Jones’ stain, ×160).
FSGS may be increased in these diseases. The association of
abnormal glomerular growth with development of glomer-
ulosclerosis may reflect a pathogenic linkage in that pro- podocytes and is an integral stomatin protein family mem-
cesses leading to excess matrix and sclerosis may be ber. Its function is not determined. Mutations in NPHS2
manifested as glomerular growth. This view is supported by have been described in sporadic steroid-resistant FSGS. In
the coexistence of these two processes in many other dis- both autosomal dominant and autosomal recessive forms of
eases, including sickle cell diseases, HIV infection, and FSGS, there is genetic heterogeneity. These familial forms
reflux nephropathy (82). do not have specific morphologic features of the segmental
Recently, specific gene mutations of podocyte-specific sclerosis.
genes have been identified in some forms of familial FSGS. In contrast, congenital NS of Finnish type shows mesangial
A gene for autosomal dominant FSGS has now been local- hypercellularity or no glomerular lesion, along with dilated
ized to ACTN-4 at chromosome 19q13. ACTN-4 encodes proximal tubules by LM. The gene, NPHS1, also located in
alpha-actinin-4, and a gain-of-function mutation with pos- the 19q13 region, codes for nephrin and is mutated in this dis-
sible altered actin cytoskeleton interactions has been pro- ease (85). Nephrin localizes to the slit diaphragm of the
posed (83). The prognosis of this form of familial FSGS has podocyte and is tightly associated with CD2-associated pro-
been poor, with progression to renal disease in 50% of tein. Nephrin is believed to function as a zona occludens–type
patients by age 30 years. Recurrence in the transplant has junction protein. CD2-associated protein plays a crucial role
been very rare in autosomal dominant forms of FSGS. in receptor patterning and cytoskeletal polarity, and its absence
Autosomal recessive FSGS with early onset and rapid pro- resulted in sclerosis and foot process effacement in mice, sup-
gression to end stage is caused by mutations in NPHS2, porting a role for CD2-associated protein in the function of
which encodes podocin (84). Podocin is expressed only in the slit diaphragm. The slit diaphragms are crucial for regula-
488 IV. Clinical Methods

FIGURE 24.5. Arteriolar fibrin thrombi with minor areas of FIGURE 24.6. Hemolytic uremic syndrome. The lamina rara
thrombi in capillary loops in hemolytic uremic syndrome (Jones’ interna (endothelial side of the glomerular basement mem-
stain, ×270). brane) is widened with increased lucent material. No immune
deposits are present (×3300).

tion of permselectivity and are decreased in density in pro- ment membrane with entrapped platelets, fibrin, and red cell
teinuric conditions. fragments without immune deposits (Fig. 24.6). De novo
FSGS associated with mitochondrial cytopathy, due to a thrombotic microangiopathy in the renal transplant is indis-
mutation of mitochondrial DNA in tRNAleu(UUR), may tinguishable morphologically from HUS in the native kidney.
show multinucleated podocytes and have abnormal mito- Cyclosporine and FK506 have both been implicated in its
chondria by EM examination. Patients also have unusual pathogenesis (89,90). For further discussion of HUS, see
hyaline lesions in the arterioles. Some patients with FSGS Chapter 47.
without full-blown features of mitochondrial cytopathy
(e.g., myopathy, stroke, encephalopathy, occasionally dia-
Henoch-Schönlein Purpura and Immunoglobulin
betes mellitus, hearing problems, and cardiomyopathy)
A Nephropathy
have also been reported to have this mitochondrial muta-
tion (86). Henoch-Schönlein purpura is often viewed as the systemic
variant of IgA nephropathy (Berger’s disease) (4,91). The
glomerular manifestations are similar, with mild to moder-
Hemolytic-Uremic Syndrome
ate mesangial proliferation with predominance or codomi-
HUS is the most common disease in children that is mani- nance of mesangial IgA by IF studies. IgG, IgM, and C3
fested by injury to the microvasculature. In adults, thrombotic deposits may also be detected. The Ig deposits are present
thrombocytopenic purpura and postpartum renal failure may diffusely, even in glomeruli that appear normal by LM.
produce similar morphologic changes. By LM, thrombi in glo-
meruli and arterioles are present (Fig. 24.5). The renal biopsy
findings, rather than clinical parameters, have recently been
found to best predict long-term prognosis (87). Patients with
cortical necrosis have a particularly ominous prognosis. The
extent of glomerular versus arteriolar involvement is also of
prognostic significance. Generally, both the long-term progno-
sis and the clinical presentation are more severe if larger vessels
are involved. Arterial involvement was not seen in biopsies per-
formed during the first 2 weeks of hospitalization (88). The
glomerular endothelium is markedly swollen, nearly occluding
capillary lumina. Fibrin thrombi are visualized easily. With
chronicity, these areas may progress to segmental collapse with
sclerosis, especially when arterioles are involved. The arterioles
can become completely occluded by thrombi, with necrosis of
vessel walls. IF shows occasional nonspecific entrapment of C3
and IgM in collapsed areas with fibrin and fibrinogen. EM FIGURE 24.7. Mesangial prominence, segmental sclerosis, and
small organizing crescent with adhesion in Henoch-Schönlein
shows extreme swelling of the glomerular endothelium with purpura. Immunofluorescence demonstrated immunoglobulin A
increased lucent material in the lamina rara interna of the base- mesangial deposits (Jones’ stain, ×430).
 24. Renal Pathology 489

FIGURE 24.9. Split basement membrane (tram-tracking) in mem-

branoproliferative glomerulonephritis type I caused by subendo-
thelial/intramembranous deposits and mesangial interposition
(Jones’ stain, ×1125).

States (96,97). This association has not been demonstrated

FIGURE 24.8. Dense deposits surrounding mesangial cell in
Henoch-Schönlein purpura (×3400). in a study of children with apparent idiopathic MPGN (98).
MPGN type I recurs in 20 to 30% of grafts and may lead to
graft loss (11). Secondary MPGN more often demonstrates a
Early lesions show predominantly mesangial hypercellular- focal segmental pattern of proliferation, contrasting the more
ity, with more prominence of matrix and even segmental diffuse involvement seen in idiopathic MPGN.
sclerosis with progressive disease (92) (Fig. 24.7). In severe
cases, there may be necrosis of glomerular tufts with cres- Dense Deposit Disease
cents in Bowman’s space. By EM, electron-dense mesangial
deposits are present, with occasional spillover of deposits to In dense deposit disease (also called type II MPGN), the glo-
subendothelial regions in the regions adjacent to the meruli may appear similar by LM to those of type I MPGN.
mesangium (Fig. 24.8). Deposits are decreased when clini- However, the pathogenesis is entirely different. These patients
cal remission occurs (93). In Henoch-Schönlein purpura, show circulating IgG autoantibodies, also known as C3
deposits are often present in subepithelial areas as well as nephritic factor (10,99,100). The basement membranes are
associated with more severe glomerular lesions, including deeply eosinophilic, often with a ribbon garland or sausage-
crescents, and worse outcome (92). Classification schemas shaped contour. By IF, smooth linear deposits of C3 are found,
analogous to those for lupus nephritis have been proposed typically without Ig staining. The disease is named dense
(94,95). For further discussion of Henoch-Schönlein pur- deposit disease because of the characteristic appearance by EM
pura and IgA nephropathy, see Chapters 31 and 45. with strongly electron-dense deposits underlying the basement
membrane. Studies of the dense deposits indicate that these are
Membranoproliferative Glomerulonephritis Type I
Type I MPGN is characterized by the tram-track appearance
of the GBM on silver stain because of duplication around
intramembranous and subendothelial deposits and interposi-
tion of mesangial cells and macrophages (Fig. 24.9). The glo-
meruli are enlarged and hypercellular with a lobular appearance
by LM (Fig. 24.10). There is marked mesangial hypercellularity
and occasional polymorphonuclear neutrophils, and mononu-
clear cells may be present. By IF, C3 predominates in a coarse
granular pattern along basement membranes with moderate
amounts of IgG and IgM. Subendothelial and occasional
mesangial immune deposits are seen by EM (4,46). MPGN
may be idiopathic or secondary to any of numerous chronic
infections. Hepatitis C positivity, often with associated cryo-
globulins, was present in approximately one-fourth of adult FIGURE 24.10. Lobular appearance of glomeruli in membrano-
cases of MPGN type I in adults in Japan and the United proliferative glomerulonephritis type I (×430).
490 IV. Clinical Methods

FIGURE 24.13. Immunofluorescence of granular capillary and

FIGURE 24.11. Lupus nephritis, diffuse proliferative with mas-
small mesangial immunoglobulin G deposits in diffuse prolifera-
sive dense mesangial and subendothelial deposits and fewer
tive lupus nephritis. The larger segments of capillary loop stain-
deposits in subepithelial areas (×5600).
ing correspond to subendothelial deposits, with a smooth outer
edge where deposits are molded underneath the glomerular
basement membrane (×250).
likely an alteration of basement membrane material and not
deposition of circulating immune complexes. Although less
specific than EM diagnosis, deposits can also be identified by compartments of the glomerulus (i.e., mesangial, subepithelial,
their staining with the fluorescent dye thioflavin T in cases in and subendothelial regions) (17,18) (Figs. 24.11 and 24.12).
which EM examination cannot be performed (100). Renal All Ig classes, C3, and smaller amounts of C4 are usually
survival may be worse than in type I MPGN (median survival, found in lupus nephritis deposits (Fig. 24.13), and dense
8.7 vs. 15.3 years) (101). The distinction between these two immune-complex deposits are seen by EM. Reticular aggre-
diseases is also important because dense deposit disease invari- gates (see above) are typically seen in endothelial cells in any
ably recurs in renal transplantation, although loss of graft is class of lupus nephritis (4,46) (Fig. 24.14).
not always the outcome (10). The WHO classifications, either the original or modified,
are most commonly used (16,17). A recent meeting sponsored
by the International Society of Nephrology and the Renal
Lupus Nephritis
Pathology Society has put forth a revised lupus nephritis classi-
Lupus nephritis is not a single disease but rather a spectrum of fication to clarify some areas of difficulty in the previous ver-
severity of involvement of the kidney by the immune com- sions. In this new classification, class I has minimal mesangial
plexes characteristic of SLE. Most patients with SLE have mor- deposits with normal LM. Class II is characterized by mesangial
phologic manifestation of renal immune deposition. However, expansion and deposits with only scattered peripheral loop
patients who undergo renal biopsy most often have clinical deposits. In class III, focal proliferative lupus nephritis, deposits
renal manifestations and have more pronounced changes. are present in mesangial areas, focal endocapillary proliferation,
Lupus nephritis is characterized by deposits in all anatomic and subendothelial deposits, with or without scattered subepi-

FIGURE 24.12. Membranous glomerulonephritis with subepi-

thelial deposits and intervening lamina densa (seen as spikes by FIGURE 24.14. Endothelial cell containing tubular-shaped retic-
silver stain on light microscopy) (×15,580). ular aggregates in lupus nephritis (×20,000).
 24. Renal Pathology 491

thelial deposits. The process, by definition, involves less than

50% of glomeruli. There is wide heterogeneity in the severity
and activity of lesions, with varying amounts of proliferation.
Foci of necrosis, cellular crescents (indices of activity), and cel-
lular adhesions may be present, which over time may evolve to
sclerosis and fibrous crescents (indicators of chronicity). The
subendothelial deposits result in thick, rigid-appearing capillary
basement membrane by LM, the so-called wire-loop lesions.
When lesions affect more than 50% of glomeruli, lupus
nephritis is characterized as class IV diffuse proliferative glomer-
ulonephritis. Findings are similar to those in class III but with
more endocapillary proliferation and other active lesions.
Hyaline thrombi (aggregates of immune complexes) may fill
capillary lumina. IF demonstrates widespread distribution of
immune complexes. EM confirms the massive and extensive FIGURE 24.15. Antiglomerular basement membrane antibody
immune complex deposition (Fig. 24.11). For classes III and IV, disease with linear staining by immunofluorescence for immu-
the extent of active versus chronic lesions is specified. Evidence noglobulin G. A crescent is present in the glomerulus on the
right (×125).
indicates a particularly poor prognosis of segmental necrotizing
lesions in class IV lupus nephritis; thus, the presence of these
lesions versus global endocapillary proliferation is noted (102). (e.g., microscopic polyangiitis or anti-GBM antibody disease)
Class V membranous lupus nephritis is characterized by (Fig. 24.16). The lesions are focal and segmental. Granulomas
predominance of subepithelial deposits in a pattern similar to are rare in the kidney, and arteritis is rarely found in the small
that of idiopathic membranous glomerulonephritis with sample inherent to the renal needle biopsy. IF studies allow dif-
added mesangial deposits (Fig. 24.12). Subendothelial deposits ferentiation of the lesion from anti-GBM antibody disease. It
are minor components in class V. When there are superim- shows fibrin and fibrinogen in areas of necrosis and nonspe-
posed focal or diffuse proliferative lesions in addition to mem- cific trapping of Ig, especially IgM. By EM, immune deposits
branous changes, both processes are diagnosed (e.g., combined are not identified. Distinction from microscopic polyangiitis
diffuse proliferative and membranous lupus nephritis, WHO cannot usually be made by renal biopsy findings. Clinical
classes IV and V). Widespread chronic sclerosing lesions in a manifestations must be used to distinguish between these two
nonspecific pattern in a case of lupus nephritis are defined as disorders. For further discussion of Wegner’s granulomatosis,
class VI. Tubular basement membrane deposits can occur in see Chapter 34.
any class of lupus nephritis and may account in part for the
tubulointerstitial injury. Vascular lesions include immune
deposits or thrombotic microangiopathy, often related to Postinfectious Glomerulonephritis
antiphospholipid antibodies. For further discussion of lupus Patients with typical poststreptococcal glomerulonephritis do
nephritis, see Chapter 46. not usually undergo renal biopsy. When the diagnosis remains
in question, when abnormalities persist, or when the initial dis-
Anti–Glomerular Basement Membrane
Antibody Disease
LM examination shows crescentic glomerulonephritis with
focal necrotizing lesions. Patients may not always show detect-
able serum levels of anti-GBM antibodies, especially after the
acute phase of illness. Serology is positive in 95% of patients in
the first 6 months after onset. However, in all patients, even
those with negative serology, linear IgG staining by IF of capil-
lary basement membrane is present (Fig. 24.15). EM shows no
immune deposits. Patients with more than 50% crescents have
a worse prognosis (103). For further discussion of anti-GBM
antibody disease, see Chapter 34.

Wegener’s Granulomatosis
FIGURE 24.16. Segmental necrosis and crescent in Wegener’s
By LM, the appearance of Wegener’s granulomatosis is the granulomatosis. Immunofluorescence was negative (Jones’
same as for other crescentic necrotizing glomerulonephritides stain, ×430).
492 IV. Clinical Methods

Diabetic Nephropathy
Diabetic nephropathy affects 30 to 40% of patients with
diabetes mellitus, either type 1 or type 2, with overt clinical
nephropathy manifest 15 to 20 years after onset of diabetes.
Therefore, diabetic nephropathy has been considered a disease
of adults. However, recent studies in adolescents demonstrate
that diabetic lesions may be present even after short duration
of disease (104). In addition, obesity and type 2 diabetes melli-
tus are increasing in children. These structural changes
included GBM thickening and mesangial expansion and were
associated with proteinuria, hypertension, and decline in glo-
merular filtration rate. Overt diabetic nephropathy with nodu-
FIGURE 24.17. Postinfectious glomerulonephritis with endo- lar glomerulosclerosis and afferent and efferent arteriolar
capillary proliferation and polymorphonuclear neutrophil infil- hyalinization was present in several of these patients. For fur-
tration (arrows) (periodic acid-Schiff stain, ×430).
ther discussion of diabetic nephropathy, see Chapter 49.

ease is severe, renal biopsy may be done. Glomeruli are enlarged Alport’s Syndrome and Thin Basement
and hypercellular with prominent endocapillary proliferation Membrane Lesion
and infiltration by neutrophils and mononuclear cells (Fig.
24.17). In severe disease, crescents are present. Occasionally, Early in life, in boys with Alport’s syndrome and in female car-
large subepithelial deposits can be visualized by LM. These dif- riers, the renal biopsy may show no significant LM abnormali-
fer from those typical of membranous glomerulonephritis in ties. At later stages, glomerulosclerosis, interstitial fibrosis, and
being more unevenly distributed along the capillary basement prominent foam cells are typical. These foam cells are not spe-
membrane and larger in size. The deposits lie on top of the cific for this disease and are found in numerous proteinuric
basement membrane, rather than being embedded within it (as states. Glomeruli show varying stages of matrix expansion and
in membranous glomerulonephritis); therefore, spikes are not sclerosis. IF may show nonspecific trapping of IgM. By EM,
usually present. By IF, there are coarsely granular, discontinuous the diagnostic lesion consists of irregular thinned and thickened
areas of IgG and C3 along the capillary basement membrane. areas of the GBMs with splitting and irregular multilaminated
The capillary lumina can be obliterated by infiltrating mono- appearance of the lamina densa, so-called basket weaving. In
nuclear cells, swollen endothelial cells, and polymorphonuclear between these lamina, granular, mottled material is present. At
leukocytes. Electron-dense subepithelial deposits are large, var- early stages of disease (i.e., in children or women), the basement
iegated, dome-shaped or haystack-shaped, and irregularly membrane may show only thinning. Some male patients with
spaced (Fig. 24.18). Occasional mesangial deposits are present classic Alport’s syndrome only have basement membrane thin-
in many biopsies. For further discussion of postinfectious glo- ning even at advanced clinical stages (22,105).
merulonephritis, see Chapter 30. Immunostaining for type IV collagen chains can aid in the
interpretation of thin basement membranes (105). Heterotri-
mers of α3, α4, and α5 type IV collagen are normally present
in the GBM. Mutation of α5 type IV collagen in X-linked
Alport’s syndrome prevents incorporation of the other chains
into the heterotrimer. In kidney biopsies, approximately 70 to
80% of male patients with X-linked Alport’s syndrome lack
staining of GBM; distal tubular basement membrane; and
Bowman’s capsule for α3, α4, and α5 (type IV) chains. In auto-
somal recessive Alport’s syndrome, due to mutations of either
α3 or α4, the GBMs usually show no expression of α3, α4, or
α5 type IV collagen; however, in contrast to X-linked cases,
there is strong expression of α5 and α6 type IV collagen in
Bowman’s capsule, distal tubular basement membrane, and
skin. Female patients who are heterozygous for X-linked
Alport’s syndrome frequently show mosaic staining of GBM
FIGURE 24.18. Electron micrograph of subepithelial large, and distal tubular basement membrane for α3, α4, and α5 type
irregularly spaced, dome-shaped subepithelial deposits in IV collagen chains and skin mosaic staining for α5 type IV col-
postinfectious glomerulonephritis. The deposits are variegated lagen. Patients with autosomal dominant Alport’s syndrome
and lie on top of the glomerular basement membrane (short
arrows). There is endocapillary proliferation with polymorpho- have not been studied immunohistochemically. Of note, occa-
nuclear neutrophil (long arrow) infiltration (×7000). sional cases with Alport’s syndrome clinically and by renal
 24. Renal Pathology 493

biopsy showed apparent normal α5 type IV pattern of skin IF classes (16–18). In lupus nephritis, patients with less severe
staining, and approximately 20% of male X-linked Alport’s proliferative disease, especially segmental necrotizing lesions,
syndrome patients and affected homozygous autosomal reces- appear to have better prognoses.
sive Alport’s syndrome patients show faint or even normal Although the presence of cellular crescents is associated with
staining of the GBM for α3 and α5, likely because the anti- activity of disease clinically, the renal biopsy offers additional
genic site recognized by the antibody has not been altered by prognostic information beyond that gleaned from the clinical
the mutation. presentation (111). Focal and diffuse proliferative lesions
Thinning of the GBM is the characteristic finding in benign (WHO classes III and IV) may present very similarly clinically,
familial hematuria (59,60). The diagnosis of thin basement but only the latter appears to require intense, long-term immu-
membranes is based on morphometric measurements from nosuppression. Lesions of activity in lupus nephritis include
EM prints, revealing marked thinning of the lamina densa of endocapillary proliferation, necrosis, cellular crescents, intersti-
the GBM. LM and standard IF are normal. The GBM thick- tial inflammatory cells, and acute tubular necrosis. Lesions that
ness normally increases with age. Normal thickness in adults in indicate chronicity include tubular atrophy, interstitial fibrosis,
one series was 373 ± 42 nm in men versus 326 ± 45 nm in glomerular sclerosis, and fibrous crescents.
women. GBM thickness less than 250 nm has been used as a Although assessment of activity and chronicity indices is
cutoff in many series (106). In children, the diagnosis of thin useful for population groups, these appear to have less abso-
basement membranes must be made with caution, establishing lute information to guide assessment in individual patients.
normal age-matched controls within each laboratory. In our Nonetheless, in large series, assessment of indices of activity
laboratory, we found a range of GBM thickness in normal chil- and severity in patients with lupus nephritis or other diseases
dren, from approximately 110 nm in 1-year-olds to 222 ± 14 has shown some correlation with prognosis and response to
nm in 7-year-olds. As mentioned above, thin GBM (without therapy. Diffuse proliferative lesions, extensive crescents, seg-
lamellation) may also be an early or only manifestation in some mental necrosis, and tubulointerstitial fibrosis are associated
kindreds with Alport’s syndrome. Thus, the presence of thin with progression to end-stage renal disease (111,112). The
GBM cannot per se be taken to categorically indicate a benign best prognostic indicator in a recent study was the propor-
prognosis. Some patients with benign familial hematuria clini- tion remaining of intact glomeruli (113).
cally have mutations of α4 type IV collagen, suggesting a con- IgA nephropathy was previously believed to have a benign
tinuum of autosomal recessive Alport’s syndrome with benign prognosis. In a large series of adult patients with IgA nephrop-
familial hematuria (107). The immunostaining patterns in such athy, poor prognosis was indicated by segmental glomerulo-
benign familial hematuria patients have not been established. sclerosis, adhesions or crescents, and tubulointerstitial fibrosis
(114). Progression occurs in 11 to 15% of pediatric patients
(4,92,115). Scoring of activity and chronicity of lesions has
PROGNOSTIC IMPLICATIONS OF
been correlated with clinical course. Activity is assessed by
BIOPSY FINDINGS
degrees of crescent formation, mesangial proliferation, and
interstitial infiltrate. Chronicity is scored by degrees of fibrous
When the biopsy sample is adequate, extensive, severe, and
crescents, segmental and global sclerosis, tubular atrophy, and
irreversible lesions signify a dismal prognosis for the patient.
interstitial fibrosis (116). High indices of chronic injury and
Globally sclerotic glomeruli are not amenable to treatment,
focal segmental glomerular changes were associated with a
although evidence from human diabetic nephropathy and ani-
worse prognosis (117,118). Histologic features that predicted
mal studies indicates that the earlier stages of sclerosis may be
progression in a recent multicenter study in children were
affected by some therapeutic interventions and may even be
crescents, tubulointerstitial fibrosis, and glomerulosclerosis in
reversible (108–110). Similarly, active lesions with ongoing
20% or more of glomeruli (115). Predominance of matrix
cellular crescents, necrosis, and inflammatory infiltrate are
expansion appears to be a later stage of injury associated with a
potentially dramatically modulated by therapy, allowing subse-
higher percentage of sclerosis and persistent proteinuria (93).
quent healing. There may be minimal irreversible damage to
Extension of deposits to glomerular basement areas has also
glomerular structures when intervention occurs early.
been reported as a poor prognostic indicator (116).
Although the renal biopsy may yield a diagnosis, there is
Focal glomerulosclerosis superimposed on membranous
less information of prognostic indicators in diseases that have
glomerulonephritis has been associated with more severe
a variable course. Extensive analysis aimed at determining
tubulointerstitial nephritis and a worse outcome. This
histologic features associated with poor prognosis has been
lesion was present in 20% of children with hepatitis B–
done in some diseases discussed below.
associated membranous glomerulonephritis (119).
Classification schemes, especially for lupus nephritis and
membranous glomerulonephritis, imply progression from
Renal Transplant Biopsy
one stage of disease to the next. Although sequential biop-
sies have illustrated progression from focal to diffuse prolif- The primary use of biopsy in the renal transplantation is to
erative glomerulonephritis in lupus nephritis, there is no uncover the reason for altered renal function. Causes of
clear-cut evidence that progression occurs among all WHO renal dysfunction in the transplant can be broadly divided
494 IV. Clinical Methods

Several schemes have been used to diagnose and classify

rejection: the Banff scoring system, based on detailed scoring
of various components of injury; and the Cooperative Clini-
cal Trials in Transplantation criteria (120,121). In both classi-
fication schemes, acute rejection is based on the presence of
tubulitis or endothelialitis (i.e., lymphocytes in the tubule
under the tubular basement membrane or underneath the
endothelium of arteries). Other inflammatory cells (e.g.,
eosinophils, neutrophils, and plasma cells), although much
fewer in number than T lymphocytes, may also contribute to
the infiltrate in acute rejection. Type I rejection in both sche-
mas is diagnosed when interstitial lymphocytic infiltrate and
tubulitis are present (greater than 25% of parenchyma infil-
FIGURE 24.19. Acute rejection, classified as type I by Coopera- trated in Banff; greater than 5% in Cooperative Clinical Tri-
tive Clinical Trials in Transplantation criteria. There is interstitial
lymphocytic infiltrate with tubulitis, activated lymphocytes, als in Transplantation criteria) (Fig. 24.20). Infiltrate and
tubular cell injury, and interstitial edema (Jones’ stain, ×220). tubulitis less than specified for type I is called borderline by
Banff criteria (126). Acute vascular rejection is classified in
the same manner in both schemas. Type II acute vacular
into (a) those related to rejection, drug toxicity, recurrent or rejection is diagnosed when there is mild or moderate endot-
de novo disease; and (b) those related to the procedure itself helialitis (arteritis), and severe acute vascular rejection (type
(e.g., acute tubular necrosis). III) is diagnosed when there is transmural vascular inflamma-
tion or fibrinoid necrosis. These types are differentiated not
Rejection only based on histologic pattern, but also on differences in
Acute rejection is diagnosed by the presence of either intersti- underlying mechanisms and response to therapies; types I
tial inflammation with lymphocytes and plasma cells infiltrat- and II are T-cell dependent processes and are separated based
ing tubules (tubulitis, the hallmark of acute interstitial type on the likely greater severity of any rejection with endotheli-
rejection) (Fig. 24.19) or, when more severe, by extension of alitis, whereas antibody-mediated mechanisms contribute to
this process to vessels, with subendothelial arterial or arteriolar type III changes.
infiltration by lymphocytes (endothelialitis, the hallmark of Identification of acute rejection at earlier stages and, thus,
acute vascular rejection) (Fig. 24.20). The interstitial changes initiation of treatment at milder levels of injury appear to be
of acute rejection are not pathognomonic. In contrast, the clinically important. Thus, mild tubulitis that is borderline
finding of endothelialitis is highly specific for acute vascular by Banff criteria, even in normally functioning grafts, was
rejection. Appropriate stains (e.g., PAS) must be used to allow found to be predictive of higher serum creatinine at follow-
visualization of the tubular basement membrane and identifi- up. In contrast, treatment of such subclinical rejection in the
cation of tubulitis. An adequate specimen for evaluation of early time period after transplantation resulted in better pre-
possible rejection should contain at least two cores with at least served renal function at 24 months (122).
seven glomeruli and two arteries (120). There are no specific IF or EM immune complexes asso-
ciated with acute rejection. The recent surge of exciting
molecular studies indicates the possibility of earlier, more
sensitive, and specific diagnosis of acute rejection using
these techniques (see below) (123). In particular, the pres-
ence in peritubular capillaries of C4d, a complement break-
down product that binds covalently to tissue, is highly
associated with antidonor antibodies (humoral rejection)
(124). Diagnosis of humoral antibody-mediated rejection
has important therapeutic and prognostic implications.
C4d staining can be done on frozen tissue and, most
recently, also on fixed paraffin-processed tissue (125).
The changes of chronic rejection include intimal fibrosis
of arteries, interstitial fibrosis, and transplant glomerulopa-
thy (121,126). A previous or baseline biopsy is necessary to
prove that intimal fibrosis is de novo and potentially repre-
FIGURE 24.20. Acute vascular rejection, classified as type II by sents chronic rejection rather than a preexisting, nonspe-
Cooperative Clinical Trials in Transplantation criteria. There is
subendothelial infiltration by lymphocytes in this artery, so- cific change in the graft. Interstitial fibrosis is also a
called endothelialitis (Jones’ stain, ×220). nonspecific finding and may result from various injuries.
 24. Renal Pathology 495

Transplant glomerulopathy is a more specific lesion indica- indicate an undetermined lesion, EM study should also be
tive of chronic rejection. By LM, the glomeruli show base- performed.
ment membrane splitting, corrugation, and even segmental In children, the most common recurrent diseases
sclerosis with hyalinosis. The latter lesion likely resulted in include, notably, IgA nephropathy, Henoch-Schönlein pur-
erroneous reports of de novo idiopathic FSGS in the trans- pura, MPGN, dense deposit disease, and FSGS (130).
plant. However, in transplant glomerulopathy, there is wid- Although SLE has been reported to recur only rarely, our
ening of the lamina rara interna of the GBM with experience indicates a recurrence rate of approximately
mesangial cell interposition and new basement membrane 30% (131). However, morphologic recurrence of disease
formation by EM. Reduplication of basal lamina of peritu- does not necessarily lead to graft loss (126,130). Dense
bular capillaries is suggested to be more specific of trans- deposit disease recurs morphologically in nearly all patients
plant glomerulopathy but may also occur in some other but with only 10 to 20% resultant graft loss. Although clas-
glomerular diseases and HUS (127). sic diarrhea-associated HUS recurs only very rarely, atypical
HUS (which includes familial forms) has a recurrence rate
of 15 to 25%, with 40 to 50% graft loss. Although IgA
Cyclosporine and FK506 Toxicity
nephropathy recurs in approximately 50% of patients, only
Cyclosporine toxicity may manifest in various ways. Tacroli- 10% of grafts with recurrent disease are lost. MPGN type I
mus (FK506) has much the same spectrum of toxicity as recurs in 20 to 30%, with 10 to 40% graft loss. FSGS
cyclosporine (126,128). The most common morphologic recurs in 20 to 30% of cases, resulting in graft loss in 30 to
lesion in patients with a clinical diagnosis of cyclosporine tox- 50% of these. Of note, in recurrent FSGS, the only mor-
icity, as verified by clinical follow-up, is that of a normal kid- phologic change found in the first weeks after recurrence of
ney biopsy morphologically. In these patients, renal proteinuria is foot process effacement, with early segmental
dysfunction is due to reversible, cyclosporine-induced vaso- sclerosis detectable at 6 to 8 weeks.
constriction and hypofiltration. Morphologic changes of De novo disease may also affect the transplant. Membra-
cyclosporine toxicity include arteriolopathy with injury to the nous glomerulonephritis is the most common de novo glo-
endothelium and vascular smooth muscle cells. In its classic merulonephritis in the transplant. The etiology remains
form, this injury results in nodular IgM IF positivity along the unknown (126). Glomerulonephritis related to infections
apical side of the arteriole, with necrosis and smooth muscle (e.g., hepatitis C–related MPGN) also can occur in the
cell injury demonstrated by EM (126,128). By LM, concen- transplant. Early changes of diabetic nephropathy develop
tric hyalinosis is present, whereas typically eccentric, more seg- much more rapidly in the transplant than in the native kid-
mental hyalinosis is associated with hypertension. Isometric ney and may occur within a few years, whether diabetes
tubular vacuolization in a patchy distribution, although not preexisted or is corticosteroid induced. Thrombotic micro-
specific, is also indicative of cyclosporine toxicity. Chronic angiopathy may be related to drug toxicity (see above) or be
cyclosporine toxicity results in a striped distribution of intersti- idiopathic in the transplant.
tial fibrosis caused by injury along the medullary rays (126). Posttransplant lymphoproliferative disease (PTLD) is due
This pattern often cannot be gleaned by small needle biopsies. to the unrestrained proliferation of B lymphocytes, most
FSGS with ischemic, corrugated GBMs in remaining glomer- often because of transformation by Epstein-Barr virus, and is
uli may also result from cyclosporine toxicity and can be asso- an aggressive process, which if untreated, disseminates and
ciated with significant proteinuria (128). may cause death (132). PTLD may respond to decreased
Cyclosporine has also been associated with thrombotic immunosuppression. An expansile lymphoid infiltrate with
microangiopathy lesions (see above) (126,128). Of note, atypical, transformed lymphocytes and serpiginous necrosis
thrombotic microangiopathy can occur in patients who are are features suggestive of PTLD (132).
recipients of transplants of kidneys or other organs and Immunohistochemical studies can be used to detect
after radiation, with or without cyclosporine treatment. In Epstein-Barr virus to further support this diagnosis. Typing
some patients, collapsing-type glomerulosclerosis may be studies of the lymphocytic infiltrate are not often helpful
associated with cyclosporine toxicity, likely representing a because most PTLD is polytypic, rather than clonal. Of note,
response to severe vascular injury and ischemia (129). acute rejection and PTLD may be present concurrently.
Polyoma (BK) virus nephropathy has increased in the
last years in the transplant and is perhaps related to
Recurrent and De Novo Disease
increased immunosuppression (133). The biopsy shows a
Recurrent and de novo diseases are important causes of pleomorphic infiltrate with lymphocytes, plasma cells, and
renal allograft injury, affecting approximately 10% of polymorphonuclear neutrophils and eosinophils, with
renal allografts (126). Of all graft loss, 2 to 4% is due to enlarged tubular cells with smudgy nuclei. BK infection is
recurrence of disease. IF microscopy should be performed confirmed by immunostaining. The prognosis is poor,
in all transplant biopsies to rule out this possibility. When although there may be some response to decreased immu-
IF or LM findings in conjunction with the clinical setting nosuppression and antiviral therapy.
496 IV. Clinical Methods

NEW METHODS FOR THE FUTURE sary to fully understand pathogenesis. Instead of diagnoses
of morphologic patterns recognized by current techniques
With the recent surge of application of molecular biology in the renal biopsy specimen, molecular techniques may
techniques to the study of renal disease, candidate factors allow more precise diagnosis of the specific diseases and
involved in pathogenesis and progression of disease are being identification of injury mechanisms.
studied in animal models. Studies in human beings have also
commenced. With further development of such studies, we
may identify specific abnormal processes and, thus, target ACKNOWLEDGMENTS
therapy more specifically. Research techniques that have been
advantageously applied to elucidate pathogenesis of disease The author wishes to thank Drs. Tina Kon and Aida Yared,
include immunostaining; identifying specific antigen in for their suggestions, and the late Dr. Alan Glick, for his
deposits of membranous glomerulonephritis in some advice and photographic assistance.
patients (thyroglobulin with Hashimoto’s disease; hepatitis
B, C antigen); identifying light chains or paraproteins in
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 S E C T I O N

GLOMERULAR DISEASE
 25

CONGENITAL NEPHROTIC SYNDROME

CHRISTER HOLMBERG
KARL TRYGGVASON
MARJO K. KESTILÄ
HANNU J. JALANKO

Renal diseases associated with nephrotic syndrome (NS) in clature; both CNF and NPHS1 are used as abbreviations
the first year of life are uncommon and make up a hetero- for the same disorder. In this text, CNF is used to denote
geneous group of disorders with different proposed causes, the clinical entity. The term NPHS1 can be used to refer to
courses, and prognoses. Congenital NS (CNS) is defined as cases known to be caused by NPHS1 mutations.
proteinuria leading to clinical symptoms soon after birth.
An arbitrary age limit of 3 months has been proposed to
Epidemiology
separate CNS from “infantile” NS, which becomes mani-
fest later during the first year of life (1). Although this age CNF is an autosomal recessive disease first described by
limit is useful for statistical purposes, it is not sufficient for Hallman et al. (5). The disease is more frequent in Finland,
the classification of early-onset NS (Fig. 25.1). The most the incidence being 1 in 8200 live births (7). However,
common type of CNS is CNS of the Finnish type (CNF), patients with CNF have been reported all over the world
with a clinical onset before the age of 1 month. The other among various ethnic groups (8–12). A high incidence of
early-onset NSs have a more widespread age of onset, from NPHS1 has been reported among the Old Order Menno-
the first days of life to several months of age. The diagnosis nites in Lancaster County, Pennsylvania (9). In a subgroup
of a patient with early manifestations of NS must be based of “Groffdale Conference“ Mennonites, the incidence is 1
on several criteria, including clinical presentation, family in 500, which is almost 20 times greater than that observed
history, laboratory findings, and renal histology. In the in Finland.
coming years, the classification will be modified by the use
of molecular genetics.
Clinical Features
CNS and early-onset NS can be classified into primary
and secondary or acquired forms (Table 25.1). CNF is con- The basic problem in CNF is severe loss of protein, begin-
sidered the prototype of CNS, and in recent years progress ning during the fetal period (7). The signs and symptoms
has been made in the knowledge of the genetics, patho- are believed to be secondary to this protein deficiency
physiology, and clinical aspects of CNF (2–4). In this chap- (7,13,14). In a recent survey, more than 80% of the chil-
ter, management of CNF is presented more thoroughly. dren were found to be born prematurely (before the thirty-
eighth week), with a mean birth weight of 2600 g (1500 to
3500 g). However, only 2 of the 46 newborns were small
PRIMARY NEPHROTIC SYNDROME
for gestational age (14). Amniotic fluid is often meconium
stained, but most neonates do not have major pulmonary
Congenital Nephrotic Syndrome of the
problems. The placenta is larger than normal and almost
Finnish Type
invariably weighs more than 25% of the baby’s birth
CNF originally referred to a severe form of CNS typically weight. The mean ratio of placental to infant weight is 0.38
seen in Finnish newborns (5,6). After the gene (named in babies with CNF, compared with 0.18 in normal babies.
NPHS1) responsible for this disorder was isolated in 1998, The reason for this is not known.
it became clear that not all cases of “typical” CNF are In typical CNF, edema and abdominal distention
caused by mutations in NPHS1 and, on the other hand, become evident soon after birth. NS was diagnosed within
mutations in NPHS1 may sometimes cause atypical (mild) the first week in 82% of the Finnish patients and within 2
forms of NS. This has led to some confusion in the nomen- months in the remaining cases (14). In contrast to most
504 V. Glomerular Disease

ogy of this manifestation is not known. Minor cardiac find-

ings, such as hypertrophy and mild functional pulmonary
stenosis, have been reported in one-fourth of the Finnish
patients (14). In a recent report form Malta, pulmonary
valve stenosis was found in three cases and a subaortic ste-
nosis in one patient (16).

Laboratory Findings
In CNF, the first urine analysis already shows proteinuria,
microscopic hematuria, and, often, some leukocyturia. The
magnitude of proteinuria depends on the serum albumin
concentration. The urinary protein concentration exceeds
FIGURE 25.1. Schematic presentation of the age at presenta- 20 g/L when the serum albumin concentration is above 15
tion for 178 children with nephrotic syndrome detected in the g/L (15) and no medication reducing filtration pressure has
first year of life. CNS, congenital nephrotic syndrome; DMS, dif-
fuse mesangial sclerosis; MCNS, minimal-change nephrotic syn- been given. This is in contrast to CNS associated with dif-
drome. (From Mauch TJ, Vernier RL, Burke BA, et al. Nephrotic fuse mesangial sclerosis (DMS), in which patients usually
syndrome in the first year of life. In: Holliday MA, Barratt TM, have less severe proteinuria (Table 25.2). In addition to
Avner ED, eds. Pediatric nephrology, 2nd ed. Baltimore: Williams
& Wilkins, 1994:788–802, with permission.) albumin, many other proteins are lost in the urine: immu-
noglobulin G (IgG), transferrin, apoproteins, lipoprotein
lipase, antithrombin III (ATIII), ceruloplasmin, vitamin
other CNS, the protein loss in CNF leads to severe hypoal- D–binding protein, and thyroid-binding globulin (17).
buminemia, and the serum albumin concentration is typi- The serum levels of these proteins and their ligands (e.g.,
cally less than 10 g/L before protein substitution (15). thyroxin) are low, leading to secondary metabolic distur-
Without albumin substitution and nutritional support, the bances. The low thyroxine concentration leads to an
classic picture of CNF develops: generalized edema, increase in thyroid-stimulating hormone (TSH) (18). Low
abdominal distention, ascites, umbilical hernias, and wid- serum albumin and postheparin plasma lipoprotein lipase
ened cranial sutures and fontanelles (5). activities and high free fatty acid concentrations lead to
Infants with CNF do not have extrarenal malforma- hypertriglyceridemia. Total and low-density lipoprotein
tions. Minor functional disorders in the central nervous cholesterol levels are high, but high-density lipoprotein
system and heart, however, are quite common during the levels are low, and the low-density lipoprotein and high-
course of the disease. Most children have muscular hypoto- density lipoprotein particles are enriched with triglycerides
nia, and computed tomography or magnetic resonance (17). These lipid abnormalities and arteriolar changes are
imaging showed mild atrophic changes in the brain in a seen already during the first year of life and may lead to an
third of the Finnish patients (14). Dystonic cerebral palsy increased risk of arteriosclerosis (19). The characteristics of
has been diagnosed in 8% of the Finnish patients; the etiol- CNF are given in Table 25.2. Today, the diagnosis can be
confirmed by the analysis of the NPHS1 gene.

TABLE 25.1. CLASSIFICATION OF CONGENITAL AND Radiologic Findings

EARLY-ONSET NEPHROTIC SYNDROME (NS)
Saraga et al. reported ultrasonographic measurements in 20
Primary NS children with CNF (20). Kidney size was normal in most
Congenital nephrosis of the Finnish type
patients during the first 2 months. The renal cortex was
Isolated diffuse mesangial sclerosis
Denys-Drash syndrome hyperechogenic, and the medullary pyramids were echolu-
Congenital NS with brain and other malformations cent in two-thirds of cases. Corticomedullary differentia-
Minimal-change NS tion was preserved. The central echo was merged with the
Focal segmental glomerulosclerosis surrounding cortical echoes in most kidneys, and the pelvic
Membranous glomerulopathy
areas were normal.
Unclassified
Secondary NS Between 2 and 12 months, the size of the kidneys was
Infections above two standard deviations in all children, but with a nor-
Congenital syphilis mal outline. The renal cortex was hyperechogenic, corti-
Toxoplasmosis, rubella, cytomegalovirus comedullary differentiation had mostly disappeared, and the
Hepatitis, human immunodeficiency virus
medullary pyramids were still echolucent. After 12 months,
Malaria
Systemic lupus erythematosus there was a decrease in kidney size, and the parenchyma was
hyperechogenic, with loss of corticomedullary differentia-
 25. Congenital Nephrotic Syndrome 505

TABLE 25.2. TYPICAL FEATURES OF CONGENITAL NEPHROSIS OF THE

FINNISH TYPE (CNF) AND DIFFUSE MESANGIAL SCLEROSIS (DMS)
Features CNF DMS

Proteinuria, onset Starts intrauterinely Can start at birth, but mostly

during first year of life
Amniotic fluid alpha-feto- Always increased Usually normal
protein
Placenta >25% of birth weight Usually normal
Proteinuria, magnitude Severe (>20 g/L with a serum Usually less severe
albumin >15 g/L)
Glomerular filtration rate Normal during the first 6–12 End-stage renal disease within
mo months after presentation
Histology Radial dilation of proximal Mesangial sclerosis contracting
tubules after 3 mo the glomerular tuft; tubular
atrophy; interstitial fibrosis
DNA analysis Mutations in the NPHS1 Mutations of the WT1 gene in
gene Denys-Drash syndrome and
some cases of isolated DMS

tion. These findings correlate with the histologic changes and a thin glomerular basement membrane (GBM), which
showing increased dilation of proximal tubules at first and is already evident in the fetal kidneys (27).
interstitial fibrosis and glomerular sclerosis later on. Biopsies taken 3 to 8 months after birth usually show
the most characteristic changes of CNF. Numerous radial
dilations of the tubules are the most obvious feature (Fig.
Pathology
25.2B). The glomeruli show moderate mesangial hypercel-
CNF kidneys are smooth surfaced, and the renal cortex is lularity, but some degenerative changes, such as shrinking
somewhat thicker than normal. In an autopsy series, the of the glomerular tuft, fibrotic thickening of the Bowman’s
mean kidney weight was almost twice that of age-matched capsule, and glomerular sclerosis, become evident (Fig.
control children (21). Older patients had smaller, “normal- 25.3A). Interstitial fibrosis and scattered lymphocyte infil-
sized” kidneys, probably because of atrophy and scarring of trates begin to develop (14). The proportion of sclerotic
the renal parenchyma. Pathologic findings are confined to and ischemic glomeruli increases. Some tubular dilatations
the renal cortex without medullary lesions. The changes are increase in size, and their epithelium becomes atrophic.
progressive with age, from occasional dilated tubules in the Interstitial fibrosis and inflammation increase.
fetal kidneys to severe tubulointerstitial and glomerular
abnormalities at the age of 1 to 2 years (21,22).
Genetics
In the fetal period, the CNF kidneys develop quite nor-
mally. At 16 to 22 gestational weeks, the glomeruli show few NPHS1 and Nephrin
changes by light microscopy, but occasional dilated tubules CNF is inherited as an autosomal recessive trait (6). The
are seen in most cases (23,24). The dilations contain bright, gene responsible for most CNF cases has been localized to
eosinophilic, colloid material, and the epithelium is low and chromosome 19q13.1 (28) and named NPHS1 (2). It has a
cuboidal. By electron microscopy, the mature glomeruli size of 26 kb and contains 29 exons. NPHS1 codes for
show effacement of podocyte foot processes and irregular nephrin, which is a 1241-residue, cell adhesion protein of
foot processes. The slit pores between the foot processes are the Ig family (Fig. 25.5). The extracellular part of nephrin
of various sizes, and the filamentous image of the slit dia- contains eight Ig-like modules and one fibronectin type III
phragm is completely missing in cases with severe NPHS1 domain. The intracellular domain has no significant
mutations (25). It is remarkable that also carriers of NPHS1 homology with other known proteins but it has nine
mutations can have these “proteinuric” changes, making the tyrosine residues, some of which may become phosphory-
pathologic diagnosis difficult (26). lated during ligand binding. The expression of nephrin was
The pathologic changes are subtle by light microscopy first reported to be restricted to glomerular podocytes (2).
during the first month. The glomeruli show a slight to Recent studies on the NPHS1 promoter have confirmed
moderate mesangial cell proliferation. Irregular microcystic this localization (29,30). The data obtained from mice,
dilatations of proximal tubules are inconstantly found on however, suggest that nephrin is also expressed in some
renal biopsy specimens (Figs. 25.2 and 25.3). Thick-walled areas of the central nervous system (e.g., cerebellum and
small arteries and fetal glomeruli are present, but they are spinal cord) and in pancreatic beta cells (31). The tissue
also seen in normal kidneys of this age group. Electron expression of nephrin in humans is not clear. Pancreatic
microscopy shows effacement of foot processes (Fig. 25.4) expression of nephrin has been reported (32), but our
506 V. Glomerular Disease

FIGURE 25.2. A: Histologic picture of the congenital nephrotic

syndrome of the Finnish-type kidney at 4 weeks of age. B: Histo-
logic picture of the congenital nephrotic syndrome of the Finnish-
type kidney at 6 months of age. Note the increase of the dilated
tubules (magnification, ×60).

results suggest that nephrin expression in humans is

restricted to the glomerulus (33).

NPHS1 Mutations
Sequencing the NPHS1 coding region has revealed two FIGURE 25.3. A: Congenital nephrotic syndrome of the Finnish
important mutations in more than 90% of the Finnish type. Histologic picture of a glomerulus showing mesangial
CNF patients (2). The first mutation, a 2–base pair deletion hypercellularity (×240). B: Diffuse mesangial sclerosis. Histologic
picture of a contracted glomerulus. Note the ribbon of
in exon 2, causes a frame shift resulting in a stop codon podocytes covering the glomerular tuft (×240).
within the same exon (Fin-major mutation). The second is a
nonsense mutation in exon 26 (Fin-minor). Fin-major leads
 25. Congenital Nephrotic Syndrome 507

FIGURE 25.5. The structure of nephrin. The protein is a trans-

membrane cell adhesion receptor with small intracellular and a
larger extracellular domain. The extracellular domain contains
eight immunoglobulin-like modules (circles) and one fibronec-
tin type III–like domain (hexagon) adjacent to the transmem-
brane domain. Nephrin is a major component on the podocyte
slit diaphragm.

Fin-minor mutations are rare outside Finland. Enrichment

of other mutations has been reported also in non-Finns. In
Mennonites, the 1481delC mutation is common and leads
to a truncated protein of 547 residues (9). On the other
hand, a homozygous nonsense mutation R1160X in exon
27 has been found in all Maltese cases (12). Importantly, 6
of the 16 cases with this mutation had an atypically mild
disease. The same mutation has been reported in six French
CNF patients, and two of them had mild disease (34).

Congenital Nephrotic Syndrome of the Finnish

Type Not Caused by NPHS1 Mutations
In the report of Lenkkeri et al. (8), no mutations in NPHS1
FIGURE 25.4. Electron microscopy of the glomerular capillary were found in 7 out of the 35 patients (20%) with classical
wall. A: Normal kidney showing the podocyte foot processes, CNF. Mutations may lie on important regulatory elements,
the glomerular basement membrane, and the endothelium. which are not known yet, but it seems that NPHS1 muta-
B: Congenital nephrotic syndrome of the Finnish-type (CNF) kid-
ney (Fin-major/Fin-major genotype) shows irregular podocyte tions are not the only cause of CNF. Koziell et al. (12) found
foot processes. C: Immunoelectron microscopy of a podocyte no NPHS1 mutations in 8 of 37 CNF cases. In two of these
pore in normal kidney. The filamentous image of the slit dia- eight patients, mutations were found in the NPHS2 gene,
phragm (SD) is seen (arrow). Antibodies against the intracellular
part of the nephrin show the localization of nephrin in the SD coding for another podocyte protein, podocin. Mutations in
(gold particles, arrowheads). D: Immunoelectron microscopy of NPHS2 normally lead to a recessive form of focal segmental
the podocyte slit pore in CNF kidney. No SD or nephrin is seen. glomerulosclerosis (FSGS) appearing later in life (35).
Koziell et al. also reported three cases of CNS patients who
had mutations both in NPHS1 and NPHS2, indicating a
to a truncated 90-residue protein, and Fin-minor leads to a functional interrelationship between the two genes (12).
truncated 1109-residue protein. In addition to Fin-major These patients showed FSGS on renal biopsy.
and Fin-minor, a few missense mutations in the Finnish
patients have been found (8). The uniform mutation pat-
Pathogenesis
tern seen in the Finnish population can be explained by the
founder effect. Normal Glomerular Filter
Several reports on NPHS1 mutations in non-Finnish Sieving of the plasma occurs in kidney glomeruli through
patients have been published (8–12). The patients come the capillary wall (glomerular filtration barrier). This bar-
from Europe, North America, North Africa, the Middle rier is composed of three layers: a fenestrated endothelium,
East, and Asia. In contrast to the Finnish patients, most the GBM, and visceral epithelial cells (podocytes) (3,36).
non-Finns have “individual mutations.” These include The podocytes enclose the capillaries in a comb-like fash-
deletions, insertions, nonsense, missense, and splicing ion, where the adjacent “spikes” (foot processes) form an
mutations spanning over the whole gene. For the moment, interrupted sheet around the capillaries. The foot processes
more than 60 mutations in NPHS1 have been identified are connected just above the GBM by an extracellular
(Fig. 25.6). Missense mutations are all located within the structure, the slit diaphragm (Fig. 25.7). This structure
extracellular part and cluster to exons coding for the Ig-like bridges the filtration pores between the adjacent podocyte
motifs two, four, and seven (8,11,12). The Fin-major and foot processes, and, by electron microscopy, it is seen as a
508 V. Glomerular Disease

FIGURE 25.6. The NPHS1 gene contains 29 exons (bars) at a size of 26 kb. For the moment, 65
different mutations have been reported in congenital nephrotic syndrome of the Finnish-type
patients. These include 32 missense mutations, 11 deletions or insertions, 14 nonsense mutations,
and eight splice-site mutations. In addition, nine amino acid changes without known phenotype
effect and nine polymorphic changes have been detected. See text for references. [■], missense;
[▼], deletion/insertion; [●], polymorphism; [↑], splice site; [★], nonsense; [♦], amino acid change
without known phenotype effect.

thin line connecting the cell membranes. The filter serves lack of CD2-associated protein causes NS in neonatal mice
both as a size-selective and a charge-selective sieve. It is (49,50). Nephrin, podocin, NEPH1, and CD2-associated
believed that the GBM restricts the passage of macromole- protein are known to interact with each other and the actin
cules especially according to the charge, whereas the slit cytoskeleton of the podocyte foot process, and defects in
diaphragm forms a size-selective filter (36,37). this interplay may cause proteinuria (37,43,51,52). Under-
Immunoelectron microscopy revealed that nephrin was
localized at the slit diaphragm area of human kidney glom-
erulus (38). Later, this localization was confirmed in mouse A Podocyte foot
and rat (39–41). A hypothetical model was presented in process
which nephrin molecules from adjacent foot processes Slit diaphragm
show a head-to-head assembly through homophilic interac-
tions and form a backbone of the slit diaphragm (3,38).
Recently, this model has been modified with other mole-
cules such as NEPH1 and FAT interacting with nephrin in
the slit diaphragm (42–44). GBM
Slit Diaphragm in Congenital Nephrotic
Syndrome of the Finnish Type
The Fin-major and Fin-minor mutations of the NPHS1
gene lead to a complete absence of nephrin in the CNF
kidney glomerulus (14). These kidneys also totally lack the B
filamentous image of podocyte slit diaphragms as studied F-actin
Nephrin
by electron microscopy (Fig. 25.4). Similarly, nephrin NEPH1
knock-out mice, dying of severe proteinuria soon after FAT
birth, lack the slit diaphragm filaments by electron micros- P-cadherin
copy (31). These findings indicate that the absence of
nephrin leads to distortion of the slit diaphragm and the
leakage of plasma proteins into urine through the “empty”
podocyte pores. Liu et al. (45) have shown that many mis-
sense NPHS1 mutations seen in non-Finnish CNF patients
lead to misfolding of the nephrin molecule and a defective GBM
intracellular nephrin transport in the podocyte. This most
probably explains why even “mild” (missense) mutations Integrin α3β1 Podocin
Dystroglycans ZO-1
cause heavy proteinuria and a typical clinical disease (12). CD2AP
Catenins
Role of Podocytes
FIGURE 25.7. A: The structure of the glomerular capillary wall.
It has become evident that podocytes play a crucial role in The podocyte foot processes are connected by slit diaphragms.
glomerular filtration (37,46,47), and mutations in genes B: A model of the podocyte foot processes showing some of the
coding for the other podocyte proteins probably lead to dis- known components of the slit diaphragm and the intracellular
attachment proteins. The interplay between the different com-
orders resembling CNF (Fig. 25.7). Mutations in the gene ponents is important in restricting the passage of plasma pro-
coding for alpha-actinin-4 cause NS in humans (48), and teins into urine. GBM, glomerular basement membrane.
 25. Congenital Nephrotic Syndrome 509

standing of the glomerular filtration barrier and its compo- plantation (58–60). With optimal therapy, growth and
nents is increasing rapidly, and it is to be expected that new development are satisfactory, and after the first months
“NS genes” will be identified in the near future. patients can spend the daytime at home and receive their
nightly albumin infusions in the hospital.
Prenatal Diagnosis
Antiproteinuric Medication
Proteinuria in CNF starts in utero, and the measurement of
alpha-fetoprotein (AFP) in the amniotic fluid and maternal A clear reduction of protein excretion with captopril and
serum has successfully been used for prenatal screening for indomethacin in CNF patients has been reported (61–63).
CNF and in families with a previous CNF diagnosis On the other hand, Birnbacher et al. found that captopril
(53,54). Fetal serum contains high levels of AFP, and leak- had no effect on protein excretion in their patients (64). In
age of fetal plasma proteins into amniotic fluid and further our experience, patients with the Fin-major and Fin-minor
to maternal serum occurs in CNF as well as in neural tube mutations do not respond to captopril or indomethacin.
defects. Elevated AFP levels and normal ultrasonographic Both of these mutations, as well as many of the missense
findings of the fetus are considered diagnostic for CNF. mutations, lead to a situation in which no nephrin is
Amniotic fluid AFP levels exceeding five multiples of the expressed on the podocyte surface (14,45). In such cases, this
normal median have traditionally been regarded as patho- therapy is probably not effective. On the other hand, if an
logic. The problem, however, is that heterozygous fetus car- NPHS1 mutation leads to a single amino acid change and
riers of NPHS1 mutations may have elevated values (up to quite normal nephrin expression, a reduction in the protein
50 multiples of the normal median) and a false-positive excretion may be observed (14). Thus, treatment with angio-
result in the AFP test (26,55). This may lead to termination tensin-converting enzyme inhibitor and indomethacin is
of pregnancy on false grounds. The temporary proteinuria worth trying in cases in which the NPHS1 mutations or their
in carrier fetuses is also associated with tubular microcysts biologic effect are not known. A kidney biopsy with immu-
and podocyte foot process effacement (26). Thus, the dif- nohistochemistry of nephrin and electron microscopic evalu-
ferentiation of affected and carrier fetuses is quite difficult. ation of the podocyte slit diaphragm may help in making the
In affected cases, the amniotic fluid AFP levels stay highly decision of the treatment strategy.
elevated, and repeated measurements of AFP levels during
the second trimester are strongly recommended before
Albumin Substitution
making the diagnosis.
Analysis of NPHS1 from placental biopsies or amniotic Proteinuria without substitution leads to protein malnutri-
fluid cells is the method of choice for a precise diagnosis of tion, reduced growth, and secondary symptoms of hypo-
CNF (2). In the Finnish population, analysis of the two proteinemia (15). Mahan et al. (56) first reported good
major mutations is feasible. This is the case also when the results in CNS with a high-energy (120 kcal/kg/day), high-
disease-causing mutations are known in a family and can be protein (3 to 4 g/kg/day), and low-sodium diet. Diuretics
specifically searched. If a fetus (without a family history of were given daily and intravenous albumin occasionally.
CNF) is suspected to have CNF based on elevated AFP val- Although this treatment substantially improved outcome,
ues, sequencing of NPHS1 is also possible, but due to the developmental delay and neurologic abnormalities were
large size of the gene (29 exons) the analysis of the whole observed in 93% of the children, and all exhibited growth
gene is time consuming and may not be done fast enough. retardation. Broyer et al. (65) demonstrated substantial
NPHS1 is mutation-rich and in almost all families, individ- catch up growth with aggressive tube feeding in CNS, but
ual mutations have been found. In a case of missense muta- their patients never reached the fourth percentile in growth.
tion, a large number of control samples must be analyzed to Since the mid-1980s, we have adopted a more aggressive
find out if the amino acid substitution is a polymorphic approach (15,17). Parenteral albumin infusions, 3 to 4 g
change or a disease-causing mutation. albumin/kg/day, are started at birth. Indwelling deep-vein
catheters are used from the age of 3 weeks. Albumin is given
as a 20% solution together with intravenous furosemide (0.5
Management of Congenital
mg/kg). It is first divided into three or four 2-hour infusions,
Nephrotic Syndrome
and after 1 month of age given as one infusion (over 6 to 8
The therapeutic decisions should usually aim at renal trans- hours) during the night. With this substitution, patients do
plantation, which today is the only “curative” therapy in not have substantial edema (15,17).
most cases (56). Immunosuppressive therapy with steroids
or cyclophosphamide is not effective (1,57). The goals are
Nutrition
to provide good nutrition, control edema, and prevent
thrombosis and infections, allowing the child to reach a Patients with CNF should receive enterally 130 kcal/kg of
weight and body size allowing a successful kidney trans- energy and 4 g/kg of protein per day (in addition to paren-
510 V. Glomerular Disease

teral albumin). Calorie intake is composed of 10 to 14% are often vague and masked by signs of focal infections
protein, 40 to 50% fat, and 40 to 50% carbohydrate. Rape- occurring at the same time. Antibiotic therapy should be
seed oil (10 to 15 mL) and fish oil (2 mL) are added to started promptly on suspicion and should cover the major
increase the ratio of monounsaturated and polyunsaturated hospital strains of bacteria. Response to treatment is usually
fatty acids and the polyunsaturated to saturated ratio of the excellent. Because of the urinary loss of IgG, no vaccina-
diet. The excess protein is given as a casein-based protein tions are given.
product and additional energy as glucose polymers. The
daily water intake is 100 to 130 mL/kg (15). Many patients
Unilateral Nephrectomy
need a nasogastric tube to guarantee their energy intake.
The children should also receive vitamin D2 (2000 IU/ Some centers have adopted a routine of performing unilat-
day), vitamin E from the rapeseed oil (2.5 to 3.0 mg/day), eral nephrectomy to reduce protein losses (73,74). This
and water-soluble vitamins, according to the recommended decreases the need and frequency of the albumin infusions
dietary allowances for healthy children of the same age. and may help in the everyday management. We propose
Supplementary magnesium (40 to 60 mg/day) and calcium bilateral nephrectomy and peritoneal dialysis at an early age
(500 mg/day for patients younger than 6 months, 750 mg/ to avoid the many problems encountered during the neph-
day for patients 6 to 12 months, and 1000 mg/day for rotic stage. Prolonged NS is also accompanied by lipid
patients older than 12 months) are also given. abnormalities, which leads to vascular changes already dur-
ing infancy (19).
Additional Medication
Dialysis and Transplantation
Patients with CNF often have low levels of serum thyroid-
binding globulin and thyroxine (66,67). McLean et al. (18) Although normal growth can be achieved with optimal
reported an increase in TSH in four of five patients with CNS therapy during the nephrotic stage, the patients are still
and a positive response to thyroxine substitution. We have had malnourished and hypoproteinemic (15,17). To opti-
a similar experience. Although serum thyroxine concentration mize treatment, we perform bilateral nephrectomy and
is always low, TSH may be normal in the beginning but commence continuous cyclical peritoneal dialysis when
increases in most patients during the first months. Thus, we the weight reaches 7 kg. The aim is to improve the child’s
have adopted a policy of routinely giving thyroxine from birth, nutritional state before kidney transplantation (56,75).
adjusting the dosage according to TSH. While receiving continuous cyclical peritoneal dialysis,
Urinary excretion of plasminogen and ATIII is accom- the patient’s general condition improves, muscle mass
panied by increased levels of macroglobulin, fibrinogen, increases, and catch up growth can be documented (75–
thromboplastin, and factors II, V, VII, X, and XII, contrib- 77). Transplantation can be performed also preemp-
uting to hypercoagulopathy (68). Thromboses and severe tively, and the results are satisfactory (78,79).
coagulation problems have been reported in children with The first report of successful kidney transplantation in
CNS (15,56,69), and the use of low-dose aspirin and dipy- CNF dates back to 1973 (80), and in 1984 Mahan et al.
ridamole therapy has been recommended (56). Our CNF reported excellent results with kidney transplantation in 17
patients are treated with sodium warfarin from 4 weeks of children with CNS (56). Two-year patient and graft survival
age, and no severe thrombotic complications have occurred was 82 and 71%, respectively. Twelve of 15 surviving chil-
since this therapy was commenced. Before surgical or vas- dren had normal school and social performances, although
cular procedures, warfarin is stopped, and ATIII (50 IU/kg) two attended special education classes. Since 1987, 56 CNF
is given to temporarily correct the ATIII deficiency. patients have received a transplant kidney in Finland. Patient
Bacterial infections are a major complication in infants survival at 5 years is more than 90%, and graft survival is
with CNF (7,56). Because of urinary losses of gamma glob- approximately 80%, with a mean glomerular filtration rate
ulin and complement factors B and D (70,71), nephrotic of 67 mL/min/1.73 m2. Mean height standard deviation
children are especially prone to infections caused by capsu- score was –1.4 at transplantation and improved to –0.9 after
lar bacteria, such as pneumococci, and prophylactic use of 5 years. Less than 20% of the patients are receiving growth
penicillin has been recommended (56). Ljungberg et al. ret- hormone therapy. A great majority (72%) of those who have
rospectively analyzed the incidence and type of infection in reached school age attend a normal class. The early patients
21 infants with CNF during the first year of life (72). The who experienced thrombotic complications have neurologic
infants had 63 verified and 62 suspected episodes of sepsis. handicaps (81–84).
The use of central venous lines had no effect on the inci-
dence, and the prophylactic use of antibiotics or immuno-
Recurrence of Proteinuria
globulins did not reduce the incidence. Thus, we do not
recommend prophylactic medication, but a high degree of In CNF patients, a special risk after kidney transplanta-
suspicion of septic infections is warranted. The symptoms tion is the recurrence of NS, which may lead to graft loss
 25. Congenital Nephrotic Syndrome 511

(85–89). Recent findings indicate that at least half of the IgM, C1q, and C3 in the mesangium or in the periphery of
patients with recurrence have circulating antinephrin the glomeruli (91,94).
antibodies, which most probably react against the glomer-
ular filter and induce protein leakage (89). Recurrence of
Clinical Features and Management
NS has occurred in 13 Finnish patients, and all of them
have a severe Fin-major/Fin-major genotype. These The pregnancy and delivery are usually normal, and the
infants produce antibodies against the “novel antigen” in size of the placenta, when recorded, has been normal with
the graft. Treatment of recurrence with steroids, cyclo- rare exceptions (101). Proteinuria can manifest in the first 3
phosphamide, and plasmapheresis often leads to remis- months of life, but it is more often detected later in infancy
sion (85,86,89). (96,101). Proteinuria is often moderate, and in contrast to
CNF, the patients develop progressive renal insufficiency
and end-stage renal disease (ESRD) within a few months or
Isolated Diffuse Mesangial Sclerosis
years (Table 25.2). A large proportion of patients develop
The term diffuse mesangial sclerosis denotes NS associ- hypertension.
ated with the histologic change of DMS of the renal glo- Steroids and other immunosuppressive drugs are ineffec-
meruli (90,91). DMS can be isolated (IDMS) or tive in IDMS. Early treatment is similar to that which has
associated with additional anomalies, especially Denys- been outlined for CNF, but with less aggressive protein
Drash syndrome (DDS). In Habib’s material, 26 substitution. Renal transplantation is the only curative
patients with DMS were isolated, and 14 were associated treatment of this disease.
with DDS (91). IDMS often occurs in females, but male
patients have also been described (92,93). An autosomal
Denys-Drash Syndrome
recessive inheritance has been suggested in some cases
(91,94,95). Mutations in the Wilms’ tumor gene (WT1) The triad of Wilms’ tumor, male pseudohermaphroditism
have been described in children with IDMS (92,96). (XY), and progressive glomerulopathy is known as DDS
Recently, Yang et al. (97) described decreased podocyte (102,103). It often presents as congenital or early-onset
expression of WT1 and strong expression of PAX2 in NS, but the onset may range from the newborn period to
most IDMS patients, suggesting that one of the genes childhood (91,104,105). The characteristic glomerular
implicated in the cascade of WT1 regulation (e.g., lesion is DMS.
PAX2) could be involved in IDMS.
Genetics
Pathology
DDS is caused by mutations in the WT1 gene, which is
The pathologic changes of DMS are characteristic (98). In located at chromosome 11p13 and encodes a transcription
the early phase, the glomeruli show increase of the mesan- factor of the zinc-finger family (106,107). Alternative splic-
gial matrix and podocyte hypertrophy. Then, the GBM ing produces four transcripts of WT1. WT1 expression is
becomes thickened, and the expanded mesangium shows a important for normal fetal differentiation of the kidney and
delicate PAS-positive mesh with embedded mesangial cells. urinary organs (108,109). Heterozygous, de novo germline
The mesangial expansion and sclerosis causes obliteration mutations in WT1 are observed in nearly all patients with
of the capillary lumens and, finally, the contraction of the DDS. More than 60 mutations have been reported. Most
glomerular tufts. There is no cellular proliferation during are missense mutations in exons 8 and 9, encoding zinc-
the progression of IDMS, but a single layer of large fingers 2 and 3 (110,111). The most prevalent is R394W
podocytes often covers the sclerotic tufts (Fig. 25.3). A cor- located at zinc-finger 3. Also, deletions, insertions, and
ticomedullary gradient of affected glomeruli is often nonsense mutations that lead to a truncated protein have
present, the deepest glomeruli being the least affected. Tub- been described in DDS patients (93,111). Frasier syn-
ulointerstitial lesions include atrophic tubules and intersti- drome, which is characterized by focal glomerular sclerosis,
tial fibrosis. Dilated tubules are also seen, but this feature is delayed kidney failure, and male pseudohermaphroditism
not as pronounced as in CNF. with complete gonadal dysgenesis, is caused by point muta-
By electron microscopy, the abundant mesangial matrix tions in the donor splice site in intron 9, resulting in the
often contains filaments and collagenlike fibrils, and the loss of the +KTS isoform (112).
GBM is thickened (98,99). Immunohistochemical analysis
of DMS glomeruli shows deposits of disorganized collagen
Clinical Features and Management
components and heparan sulfate proteoglycan, and absence
of laminin and collagen IV in the hyalinized glomerular DDS can be divided into three clinical categories: (a) geno-
segments (100). There are no significant immune deposits, typic males with all three abnormalities, (b) genotypic
although some studies have shown variable amounts of males with nephropathy and ambiguous external and/or
512 V. Glomerular Disease

internal genitalia only, and (c) genotypic females with SECONDARY NEPHROTIC SYNDROMES
nephropathy and Wilms’ tumor only (111,113). Male
pseudohermaphroditism characterized by ambiguous NS has been described secondary to some congenital and
genitalia or female phenotype with dysgenetic testes or infantile disorders. Syphilis, toxoplasmosis, some viral
streak gonads is observed in all 46,XY patients. By con- infections, and the infantile form of systemic lupus erythe-
trast, all 46,XX children reported had a normal female matosus (SLE) are the most important secondary causes of
phenotype with normal ovaries when the information congenital and infantile NS.
was available. The nephropathy may precede the tumor
(104,113,114).
Infections
The nephropathy begins with proteinuria and NS. In
contrast to CNF, renal failure is often observed at pre- Congenital syphilis has long been known to cause CNS. It
sentation (111). Progression to ESRD before the age of may cause a nephritic or nephrotic syndrome in the new-
4 years is the rule. No correlation between the genotype born (57). Proteinuria is common, but full-blown NS is
and the severity of renal disease has been observed less common. Hematuria is often present (133). NS may
(115). Kidney transplantation remains the only thera- manifest in the newborn but is more often seen between 1
peutic alternative, and favorable results without recur- and 4 months of age (57,134). Syphilis causes a combina-
rence of DMS have been reported (97). Because of the tion of glomerulonephritis and interstitial nephritis. Mem-
risk of developing Wilms’ tumor, prophylactic nephrec- branous glomerulonephritis appears to be common (135)
tomy at the time of ESRD or transplantation is usually and is a rare example of an immune complex–mediated
performed (113). glomerulonephritis in the newborn. Interstitial lymphocyte
and plasma cell infiltration usually accompanies the glo-
merulonephritis. Antimicrobial therapy, usually penicillin,
Other Primary Nephrotic Syndromes
is curative, provided that irreversible renal lesions have not
In 1968, Galloway and Mowat (116) described two siblings developed (136).
showing congenital microcephaly, NS, and hiatus hernia. Toxoplasmosis has been associated with CNS in a few
Several subsequent reports showing various brain malfor- cases (137,138). Congenital rubella has been reported to
mations and congenital or early-onset NS have been called cause CNS with membranous glomerulonephritis (139). An
Galloway-Mowat syndrome (117). The classic Galloway- association of neonatal cytomegalovirus infection and CNS
Mowat syndrome is an autosomal recessive disorder, but has also been reported (140,141). In these infections, mani-
the disease-causing gene has not been isolated (118). In festations other than NS lead to the correct diagnosis.
patients with CNS and brain malformation, the histologic Acquired immunodeficiency syndrome caused by the human
finding may be DMS (119). In several reports, the renal immunodeficiency virus is associated with nephropathy,
histology is not fully described, but pathologic changes including NS (142). This disease usually affects children
other than DMS have been reported (119–121). A wide older than 1 year, but infants with nephropathy have been
variety of additional anomalies have also been reported in reported (143). Hepatitis B virus may cause membranous
patients with CNS and brain malformation, including dys- glomerulonephritis, and infants with NS associated with
morphic facial features (122–124). Other defects include hepatitis B infection have been described (144).
ocular, limb, cardiac, and diaphragmatic anomalies
(121,125). Ocular abnormalities have also been described
Infantile Systemic Lupus Erythematosus
in siblings with DMS (93,101). Oculocerebrorenal (Lowe)
syndrome (126) and the nail-patella syndrome (osteo-ony- Although SLE is rarely diagnosed before 5 years of age, an
chodysplasia) (127) have been shown to be associated in infantile form of SLE has been reported (145–148). NS
rare cases with CNS. was the major clinical finding in five infants aged 6 weeks
Idiopathic NS in the newborn is rare but becomes more to 6 months with SLE. These patients had elevated antinu-
common during the first year of life. Minimal changes or clear antibody titers, hypocomplementemia, and diffuse
FSGS are more common (58,59,90,128) than diffuse proliferative glomerulonephritis. Response to the immuno-
mesangial hypercellularity or membranous glomerulone- suppressive therapy was poor in many cases.
phritis (90,129). Diagnosis of minimal-change nephrotic
syndrome (MCNS) in the very young infant may be diffi-
cult but is important because immunosuppressive treat- CONCLUSION
ment may be effective (130). MCNS in very young infants
often carries a worse prognosis and shows more familial Congenital and early-onset NS forms a group of severe dis-
cases than MCNS in the older age groups (131). In some eases, which so far have been mainly classified according to
reports (132), clinicopathologic classification of congenital histology. The isolation of the WTI, NPHS1, NPHS2, and
or infantile nephrosis has been difficult. other podocyte genes will most probably lead to a new
 25. Congenital Nephrotic Syndrome 513

CNS classification in the near future. If a child has pro- syndrome advocate a functional inter-relationship in glo-
teinuria from birth and no signs of renal failure or extra merular filtration. Hum Mol Genet 2002;11:379–388.
renal malformations, the diagnosis of CNF is likely and can 13. Hallman N, Norio R, Rapola J. Congenital nephrotic syn-
be confirmed by the analysis of NPHS1. If mutations in drome. Nephron 1973;11:101–110.
NPHS1 are not found, sequencing of NPHS2 may be indi- 14. Patrakka J, Kestila M, Wartiovaara J, et al. Congenital neph-
rotic syndrome (NPHS1): features resulting from different
cated. On the other hand, analysis of WT1 and NPHS2 are
mutations in Finnish patients. Kidney Int 2000;58:972–
indicated in infants with proteinuria and renal failure, espe- 980.
cially if renal histology shows DMS or FSGS. Newborns 15. Holmberg C, Antikainen M, Rönnholm K, et al. Manage-
and small infants with heavy proteinuria should have nutri- ment of congenital nephrotic syndrome of the Finnish type
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only curative treatment, and patient and graft survival, as lipoproteins and apoproteins in infants with congenital
well as the quality of life of these patients, have greatly nephrosis. Clin Nephrol 1992;38:254–263.
18. McLean RH, Kennedy TL, Rosoulpour M, et al. Hypothy-
improved.
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 26

INHERITED GLOMERULAR DISEASES

MARIE CLAIRE GUBLER
LAURENCE HEIDET
CORINNE ANTIGNAC

Inherited glomerular disease encompasses a large number STRUCTURE OF THE GLOMERULAR

of various unrelated disorders. A tentative classification may BASEMENT MEMBRANE
be suggested, taking into account clinical, morphologic,
pathogenetic, and genetic features (Table 26.1), but it is of The glomerular filtration barrier consists of three layers: the
limited value because of our incomplete knowledge of basic fenestrated vascular endothelium, the glomerular basement
defects and pathogenesis of conditions. Glomerular membrane (GBM), and the podocyte slit diaphragm. The
involvement may appear as the primary defect, either iso- GBM is a highly specialized type of basement membrane
lated, as in the congenital nephrotic syndrome of the Finn- (BM) (or basal lamina), between 310 and 380 nm in thick-
ish type, or as part of a genetically determined syndrome, as ness in the mature human kidney, with a trilamellar struc-
in Alport’s syndrome. Glomerular lesions may develop sec- ture composed of a thick lamina densa between two thin
ondarily as a consequence of inherited metabolic diseases lamina rara. It plays an important role as a selective size and
such as diabetes mellitus or familial amyloidosis. charge filtration barrier for macromolecules during the for-
The incidence of hereditary glomerulopathies is probably mation of primary urine.
under evaluated because of diagnostic difficulties. In fact, As in every BM in the body, the major components of
the differences in the observed incidence of hereditary neph- the GBM are type IV collagen, laminin, nidogen, and pro-
ropathies between groups probably depend on the extent of teoglycans (3). Type IV collagen provides the structural
family investigations. In the pediatric series of Necker- framework and is responsible for the mechanical resistance
Enfants Malades Hospital, inherited glomerulopathies are of the BM and attachment of other glycoproteins. Laminin
responsible for approximatively 6.5% of end-stage renal fail- is involved in cell attachment, cell growth, and differentia-
ure (ESRF) occurring before the age of 16 years (1). Accord- tion. Nidogen, which is always associated in equimolar pro-
ing to the European Dialysis Transplantation Association portion with laminin, seems to act as a connecting protein
registry, 15% of ESRF are due to hereditary glomerulopa- between laminin and type IV collagen. Heparan sulfate
thies. Familial occurrence of glomerulopathies was observed proteoglycans, the major proteoglycans present in the
by Waldherr in 7 to 8% of patients with biopsy-proven glo- GBM, are characterized by strongly anionic heparan sulfate
merulonephritis (2). chains and are implicated in the charge-controlled macro-
Since 1990, dramatic advances in molecular biology tech- molecular filtration through the GBM. Minor constituents
nology have led to an explosive growth in our knowledge of found in the BM are osteonectin (BM-40, secreted protein,
inherited disorders. New DNA-based diagnostic tests are pos- acidic and rich in cystein), fibronectin, and amyloid P. In
sible when the disease genes have been localized or cloned. the mesangial matrix, the major BM components are asso-
Recognition of the genetic nature of a given disease and its pre- ciated with type VI collagen, chondroitin sulfate proteogly-
cise identification have become essential in offering patients can, fibronectin, and tenascin.
and families the possibilities of accurate genetic counseling, The structure of type IV collagen, the scaffold of the
presymptomatic testing, and prenatal diagnosis. However, in GBM, is interesting because of its implication in acquired
nephrology, and especially in pediatric nephrology, the gene and hereditary glomerular diseases. Type IV collagen
defects responsible for several inherited conditions remain to belongs to the large collagen family, comprising 21 differ-
be identified. The goal is now to precisely define the orphan ent types of molecules encoded by at least 32 genes. Each
syndromes on clinical, morphologic, and genetic criteria, this collagen molecule is formed of three α-chains folded
delineation being the first and obligatory step for further together to form a triple helix. Specific features of type IV
molecular genetic investigations and continuing progress. collagen are its length, 400 nm, and the persistence at its C-
518 V. Glomerular Disease

TABLE 26.1. CLASSIFICATION OF INHERITED terminal end of a noncollagenous (NC) 26- to 28-kDa
GLOMERULOPATHIES globular domain, called NC (or NC1), which is a binding
1. Hereditary disorders of the glomerular basement membrane site between two type IV molecules. The other terminal
Alport’s syndrome and variants domain, 7S, at the N terminus is the aggregation site for
Familial benign hematuria four molecules. In addition, frequent short interruptions in
Collagen type III glomerulopathy the repeated collagen-specific Gly-X-Y sequence are present
2. Hereditary disorders of the podocyte
in the type IV collagen chains and are responsible for the
Finnish-type nephrotic syndrome
Familial steroid resistant idiopathic nephrotic syndrome flexibility of the molecule. These features are the structural
Familial focal and segmental glomerulosclerosis basis for the specific network organization of type IV col-
Diffuse mesangial sclerosis lagen (3).
Hereditary onychoosteodysplasia: the nail-patella syndrome Six type IV collagen subunit components, the chains
Epstein/Fechtner syndromes
α1–α6(IV), have been identified, and their primary struc-
3. Hereditary metabolic disorders with primary glomerular
involvement ture has been determined in human and for most of them
Adolescent-type cystinosis in mouse (4,5). COL4A1 and COL4A2 genes coding for
Fabry disease α1(IV) and α2(IV) have been cloned and mapped to the
Other lysosomal diseases with glomerular involvement long arm of chromosome 13 as a contiguous group (6).
4. Hereditary metabolic disorders with secondary glomerular
They are the most abundant, have a ubiquitous distribu-
involvement
Diabetes mellitus tion, and form heterotrimers consisting of two α1(IV)
Sickle-cell disease chains and one α2(IV) chain. Subsequently, two other
Familial amyloidosis chains, α3(IV) and α4(IV), have been identified, and their
Alpha-1 antitrypsin deficiency genes, COL4A3 and COL4A4, have been located to chro-
Various hereditary complement defects/factor H deficiency
mosome 2 (7). Their complete sequence has been deter-
Alagille syndrome
Lecithin cholesterol acyltransferase deficiency mined (8,9). The antigenic determinants responsible for
Lipoprotein glomerulopathy autoimmunization in the Goodpasture syndrome have
Type III hyperlipoproteinemia been localized to the 28-kDa NC domain of α3(IV)
Familial juvenile megaloblastic anemia (10,11). The COL4A5 gene was mapped to the Xq22
5. Other hereditary diseases associated with glomerular involve-
region, the locus previously assigned to the X-linked
ment
Charcot-Marie-Tooth disease Alport’s syndrome, making it a good candidate gene for
Cockayne syndrome Alport’s syndrome (12–14). Subsequently, the gene
Hereditary acroosteolysis COL4A6 was assigned to the same region and shown to be
Bardet-Biedl syndrome located head-to-head to COL4A5 (Fig 26.1) (15,16).
Alström syndrome
Immunohistochemical studies have shown that the α1
Familial dysautonomia
6. Hereditary glomerulopathies without extrarenal symptoms and α2(IV) chains are present in nearly all BM. By contrast,
Familial lobular glomerulopathy/familial glomerulopathy with the α3 to α6(IV) chains have a selective distribution within
giant fibrillar deposits; “fibronectin glomerulopathy” specialized BM of the kidney, eye, inner ear, choroid
Other familial glomerulopathies plexus, and lung (17). Within the kidney, mesangial areas,
7. Mitochondrial cytopathies with glomerular involvement
Bowman’s capsules, and extraglomerular BM are strongly
8. Rare and “bizarre” syndromes
Hirooka disease stained with antibodies to the α1 and α2(IV) chains,
Galloway syndrome whereas restricted labeling of the GBM and of the distal
Hunter disease tubular BM is obtained with anti-α3, -α4 and -α5(IV) anti-
Barakat disease bodies (18,19). The α6(IV) chain is absent in the GBM but
Edwards disease
is co-distributed with the α5(IV) chain in distal tubule and
Mattoo disease, etc.
collecting duct BM and in Bowman’s capsules (20,21). The

FIGURE 26.1. Schematic represen-

tation of the distribution of type IV
collagen gene on chromosomes 13,
2, and X. bp, base pair.
 26. Inherited Glomerular Diseases 519

FIGURE 26.2. Immunofluorescence microscopy. Normal distribution of the α(IV) chains in renal
and epidermal basement membranes. (See Color Plate 26.2.)

α5 and α6(IV) chains are co-distributed with the α1 and posed to explain the transmission of the disease in the large
α2(IV) chains in epidermal BM (Fig. 26.2). In the GBM, Mormon family initially reported by Perkoff. The diffi-
the α(IV) chains are organized into two separate networks, culty was essentially related to the insufficiency of the cri-
one containing α1(IV), and α2(IV) is restricted to the sub- teria accepted to identify affected subjects in the families.
endothelial side of the GBM, and the other, the major one, This problem has in large part been resolved because
consisting of α3, α4, and α5(IV) chains, is distributed hematuria is considered an indispensable diagnostic crite-
throughout the thickness of the GBM (17,18,22). rion. With this condition, it has become clear that the dis-
ease was transmitted as an X-linked dominant trait in the
“P” family (27).
HEREDITARY DISORDERS OF THE Further family analyses have demonstrated the genetic
GLOMERULAR BASEMENT MEMBRANE heterogeneity of Alport’s syndrome (24,28,29), with a par-
ticularly high frequency of dominant X-linked transmission
Alport’s Syndrome: A Hereditary Disorder (24). Autosomal dominant transmission confirmed by
of Basement Membrane Collagen father-to-son transmission has been reported in some fami-
lies with classic Alport’s syndrome (28–30) or with progres-
Classic Alport’s syndrome (23) is an inherited renal disorder
sive hematuric nephritis without deafness (31,32).
characterized by familial occurrence in successive generations
Autosomal recessive transmission has been observed, espe-
of progressive hematuric nephritis, with ultrastructural
cially in families with parental consanguinity and in which
changes of the GBM and sensorineural hearing loss. Ocular
several siblings are affected and, regardless of gender,
defects are commonly associated. Usually male patients have
present severe Alport’s syndrome, whereas parents are clini-
severe renal disease leading to ESRF before the fourth
cally unaffected (29). Last, in families having juvenile
decade, whereas most female patients have a normal life
Alport’s syndrome, the frequent absence of descendants
span. Alport’s syndrome has been reported in kindreds of all
from affected males makes it impossible to specify the exact
ethnic and geographic origins. Alport’s syndrome is consid-
mode of inheritance. Linkage studies using polymorphic
ered the cause of approximatively 0.6 to 2.3% of ESRF in
markers situated on chromosome X have confirmed the fre-
Europe and the United States (24–26). This proportion is
quency of X-linked dominant transmission and have
probably underestimated because of diagnostic difficulties.
allowed localization of the gene of the Alport’s syndrome at
The Alport’s syndrome gene frequency is estimated to be 1
Xq22 (33,34). Finally, identification of mutations in the
per 5000 to 1 per 10,000 (24).
COL4A3 and COL4A4 confirmed the genetic heterogene-
Over the last 20 years, successive observations and findings
ity of the disease (see later).
have led to better comprehension of the renal and extrarenal
manifestations of Alport’s syndrome and to the identification
of the new collagen IV genes, mutations of which are responsi- X-Linked Alport’s Syndrome
ble for the disease. Questions raised by the phenotypic hetero-
Clinical Presentation
geneity of the syndrome are partly explained by the genetic
and molecular heterogeneity of the condition. First symptoms usually occur early in life, especially in
The mode of transmission of Alport’s syndrome has males (35–38). In a group of 58 patients observed in a
long been debated, as shown by the many hypotheses pro- pediatric department, symptoms were detected before the
520 V. Glomerular Disease

age of 1 year in 14% of the children and before the age of 6 gression of hearing loss and extension to other frequencies.
years in 72% (38). In childhood, macroscopic or micro- Involvement of the conversational zones leads to hearing
scopic hematuria is the major presenting symptom (35–40) difficulties necessitating hearing aids. In female patients,
and may be observed at birth (38). However, the disease but also in some of the males affected with the “adult” type
may be first discovered in adulthood in patients presenting Alport’s syndrome, stable and mild hearing defect is
with proteinuria-microscopic hematuria, with or without detected only by audiometry. The progression of hearing
hypertension or renal failure. Alport’s syndrome may be loss roughly parallels that of renal impairment (42). Partial
revealed by routine urinalysis or by family screening in hearing improvement reported after transplantation might
mildly affected female patients. be due to the suppression of the superimposed conse-
quences of uremia on hearing.
Clinical and Laboratory Findings
Ocular Defects
Renal Disease Various types of ocular defects involving the lens, retina,
Microscopic hematuria is present in all patients and is usu- and cornea have been described (44–48). The anterior len-
ally persistent. It may be intermittent in some girls and in ticonus is a regular, conical protrusion on the anterior
younger boys (38). Single or recurrent episodes of macro- aspect of the lens. This lesion is not detected at birth and
scopic hematuria are observed in approximately one-half of develops progressively over the years, almost exclusively in
the patients, often within the 2 days after an upper respira- male subjects (38,46). With the exception of traumatic len-
tory tract infection. In our experience, these episodes are ticonus, it is specific to Alport’s syndrome. It has been
common during the first years of life and disappear after observed in 25% of patients examined in a pediatric series
the age of 10 to 15 years. The duration of macroscopic (38) and in 33% of cases in an adult series (46). Lenticonus
hematuria usually varies from 1 to 10 days but in some can be complicated by subcapsular cataract, without diag-
instances may persist for months. Proteinuria is a common nostic significance, but possibly leading to loss of visual
finding in males (36,38,41) but is not necessarily present in acuity. The microspherophakia reported by Sohar was in
younger patients. It increases progressively with age, often fact anterior lenticonus, as revealed by histologic examina-
exceeding 1 g/24 hours after 10 to 15 years, with develop- tion of the lens (49).
ment of nephrotic syndrome in approximately 40% of Retinal changes are asymptomatic but more frequent,
cases. Proteinuria is often absent, mild, or intermittent in observed in 37% of patients (46). They are always present
affected females. Leukocyturia and pyuria are incidental when there is anterior lenticonus. They consist of bilateral,
findings. densely packed whitish-yellow granulations surrounding
Long-term follow-up varies according to the gender of the foveal area. They are superficially located in the retina.
affected patients and to genetic factors. Male patients The fovea itself, whose reflection is suppressed, is dark red.
progress to terminal renal failure. They follow a similar These lesions are also specific. In the child, they can be dif-
pattern of evolution: mild and intermittent proteinuria ficult to identify because changes in pigmentation of the
during childhood, then persistent and increasing pro- macula and disappearance of the foveola reflex precede the
teinuria with possible development of nephrotic syn- appearance of perimacular granulations (38). The anterior
drome, hypertension, and renal failure. Depending on the lenticonus and macular lesions, although inconstant, are
rate of progression to ESRF, two types of the syndrome reliable diagnostic criteria for Alport’s syndrome. They are
have been distinguished: (a) a progressive or “juvenile” observed in juvenile forms of Alport’s syndrome and seem
type in which ESRF occurs around the age of 20 years, to portend poor prognosis (46).
always before 31 years according to Atkin et al. (24), and Nonspecific corneal lesions have been recently described
the course is highly stereotyped within a given family in Alport’s syndrome patients. They consist in posterior
(24,38); and (b) a “nonprogressive” (42) or “adult” type polymorphic dystrophy (47) and in recurrent corneal ulcer-
(24) in which age at ESRF is around 40 years, and the ation (48).
course is much more diverse, making individual prognosis
impossible. By contrast, most female carriers have mild
Clinical Variants of X-Linked Alport’s Syndrome
disease. Some of them progress to ESRF but usually at a
slower rate than males (26,28). Associated changes, including platelet abnormalities, esoph-
ageal leiomyomatosis, cerebral dysfunction, polyneuropa-
Hearing Defects thy, hyperprolinuria, ichthyosis, and thyroid and parathyroid
Sensorineural hearing loss is bilateral. It is never congenital. involvement, have been reported. They affect only a minor-
In our experience, the initial hearing defect, at 2000, 4000, ity of kindreds. Except for two X-linked contiguous gene
or 8000 Hz, is recognized by audiometry before the age of deletion syndromes, one resulting in esophageal leiomyo-
15 years in 85% of boys and in 18% of girls (38). Serial matosis, the other in the AMME (Alport syndrome, mental
audiologic studies of young males often demonstrate pro- retardation, midface hypoplasia, and elliptocytosis) syn-
 26. Inherited Glomerular Diseases 521

drome (50), most of these anomalies seem to be coinciden-

tal associations or complications of renal failure. On the
other hand, the syndromes characterized by the association
of platelet disorder and nephritis are not caused by type IV
collagen deficiency.

Diffuse Esophageal Leiomyomatosis and

Alport’s Syndrome
The association of esophageal, tracheobronchial, and geni-
tal leiomyomatosis (with vulvar and clitoral enlargement),
first described by Garcia-Torrès et al. (51), has now been
reported in more than 30 families (52–54). These patients
have typical X-linked Alport’s syndrome, often of juvenile
type, in which a notable factor is the frequent association of
FIGURE 26.3. Electron micrograph. Lead citrate and uranyl ace-
bilateral congenital cataract of particular severity and type. tate stain (×11,250). Alport’s syndrome. Thickened glomerular
Esophageal involvement is responsible for dysphagia and basement membrane showing splitting of the lamina densa and
may be recognized by radiologic investigations. Accidental presence of granulations.
deaths caused by tracheobronchic lesions occurring during
surgical operations have been reported. Interestingly, dif-
fuse leiomyomatosis is fully penetrant and completely The characteristic lesions of the GBM were demon-
expressed in females (52). Approximately 20% of children strated by ultrastructural studies (41,56,57). These lesions
with esophageal leiomyomatosis should be affected with associate irregular thickening of the GBM with splitting
Alport’s syndrome. and splintering of the lamina densa, thus delimiting clear
zones containing microgranulations (Fig. 26.3). The exter-
Hereditary Nephritis without Deafness nal aspect of the GBM is irregularly festooned and bor-
In some families, hematuric nephritis resembling adult- dered by hypertrophied podocytes. GBM thickening is
type Alport’s syndrome progresses to ESRF without senso- usually diffuse in the adult, whereas in the child it is seg-
rineural hearing loss (even at the audiometric level) or any mental and a second anomaly—thinning of the GBM with
other extrarenal symptom (24,55). Whether these observa- occasional ruptures—is associated and may be predomi-
tions constitute a different entity or a variant of Alport’s nant (Fig. 26.4) (37,38,58,59). Segmental thickening
syndrome has been debated. Genetic studies have provided occurs early as it has been observed in renal biopsy speci-
new criteria for classifying these syndromes. mens from children 1 to 5 years old (31,37,38,40,57).
Semiquantitative studies have shown that the percentage of
thick segments is higher in boys than in girls, and in boys it
Pathology progressively increases with age and level of proteinuria
By light microscopy, renal findings are not specific. In young (60). Thick and split GBM has been observed in patients
children, renal biopsy specimens show no abnormalities, with classical Alport’s syndrome and with Alport’s syn-
although an increased number of immature glomeruli have
been reported (35). With increasing age, the mesangium
shows a mild and irregular excess in matrix with areas of
segmental proliferation, and the capillary walls may appear
thickened and irregular. Advancing glomerular disease is
characterized by increasing mesangial enlargement and
occurrence of segmental sclerosis and hyalinosis. Tubu-
lointerstitial changes appear early. The presence of intersti-
tial foam cells has often been considered as suggestive of
Alport’s syndrome, but these cells are seen in most types of
chronic glomerulonephritis.
Conventional immunofluorescent studies of renal tissue
are usually negative. However, granular deposits of C3 or
traces of immunoglobulin M (IgM) may be observed in the
glomeruli of some patients (38). In advanced cases, deposits
FIGURE 26.4. Electron micrograph. Lead citrate and uranyl ace-
of immunoglobulins along some GBM segments and tate stain (×4800). Alport’s syndrome. Thin and regular glomeru-
deposits of IgM and C3 within segmental glomerular lar basement membrane (arrow) with attenuated lamina densa.
lesions are present. Epithelial foot processes are extensively fused.
522 V. Glomerular Disease

drome associated with leiomyomatosis (50,51). They have multilamellated in one family, but this anomaly has not
also been found in a few patients having progressive heredi- been confirmed by other investigators.
tary nephritis without deafness (31,40,56,60). In a family
of Samoyed dogs, an animal model of Alport’s syndrome,
Immunohistochemical and
the GBM is normal at birth; thickening of the GBM and
Immunochemical Anomalies
splitting of the lamina densa appear in the first weeks of life
in the males, encompassing the whole GBM around the age In 1976, McCoy described the lack of fixation of an anti-
of 3 months, whereas in the females the lesions remain seg- GBM alloantibody developed in a transplanted Alport’s syn-
mental (61). Similar GBM changes were found in another drome patient on the GBM of Alport’s syndrome patients
canine family of Alport’s syndrome (62). (68). This observation was confirmed by other investigators
The thick and split GBM lesion is specific (63). Its find- using the same type of serum or serum from Goodpasture
ing in children who have sporadic hematuria helps identify patients. The anomaly suggested the absence or the modifi-
Alport’s syndrome. However, this lesion can be absent, with cation of GBM structural antigen(s). This hypothesis was
the GBM appearing normal or presenting only nonspecific confirmed by biochemical and immunochemical studies of
lesions (42,56,60,64) or uniformly thin (39,58,59,64,65). GBM prepared from Alport’s syndrome end-stage kidneys
The GBM lesion can also appear late as reported in a family showing the absence of NC peptides that were targets for
with progressive hematuric nephritis without deafness, in anti-GBM antibodies (69–70). The Goodpasture epitopes
which lesions were observed in two brothers aged 19 and were subsequently localized to the 28-kDa NC domain of
21 years, 8 years after an initial renal biopsy that had shown the α3 chain of type IV collagen (10,11).
normal GBM (32). Last, excessive thickening of the lamina Parallel to the studies on the α3(IV) chain, the Minne-
rara interna without any change in the structure of the lam- apolis group showed that the serum of one of their trans-
ina densa has been reported in one of the families first planted Alport’s patients who had developed anti-GBM
described by Perkoff (66). antibodies recognized a 26-kDa NC peptide different from
Concordance of ultrastructural lesions within families the 28-kDa Goodpasture antigen and from the NC domain
is usual. The lesion-associating thickening of the GBM of the α(IV) collagen chains so far identified. This antigen
and fragmentation of the lamina densa is both a marker in had the same renal distribution as the α3 and α4(IV) chains
certain families and an index of severity (42,59). A few but was also present in the epidermal BM (EBM) of normal
discordances have been reported. In a study of 100 subjects (71). They suggested that this antigen would corre-
patients—88 children and 12 adults belonging to 85 fam- spond to the NC domain of a fifth type IV collagen chain,
ilies with progressive hematuric nephritis with or without α5(IV), and that anomaly in this chain would be the bio-
deafness—we found ultrastructural alterations of the chemical substrate of the Alport’s syndrome (72), a hypoth-
GBM in all of them. The GBM was thickened and split in esis further confirmed by the identification of the COL4A5
most of the patients having classic Alport’s syndrome and gene (12–14).
thin in 10 of 14 patients having progressive hematuric The distribution of α1–α6 chains of type IV collagen in
nephritis without deafness. In 18 patients with classic X-linked Alport’s syndrome has been studied by several
Alport’s syndrome, the GBM was uniformly thin (Fig. groups (19,20,43,73–77). In most, but not all male
26.3). In three of them, renal biopsy of another member patients, no expression of α3(IV), α4(IV), α5(IV), and
of the family disclosed a thick and split GBM. On the α6(IV) is detected in renal BMs, whereas the distribution of
contrary, in a large family in which seven affected mem- the chains is discontinuous in female patients, a finding
bers, aged 10 to 47 years, had been biopsied, the only consistent with the random inactivation of one X in each
GBM lesion observed was thinning. Thus, the existence female cell. In contrast, α1(IV), α2(IV), and collagen type
of uniformly thin GBM in both the child and the adult is VI are overexpressed in the GBM (78). Both α5(IV) and
not always synonymous with benign familial hematuria α6(IV) are also absent from the EBM in males and have a
and should be considered as a possible morphologic vari- segmental distribution in females (Fig. 26.5) (19,71,73,
ant of Alport’s syndrome (65). 76,78) providing a simple approach for the diagnosis of X-
In addition to GBM changes, focal thickening of Bow- linked Alport’s syndrome. In a series of 78 patients—
man’s capsules and tubular BMs are observed. They are mostly children—with Alport’s syndrome, the distribution
characterized by a marked bulging and splitting of the BMs of the α3–α5(IV) chains was abnormal in two-thirds of the
delineating clear areas containing osmiophilic granules, patients with classic Alport’s syndrome. On the contrary, in
lipid droplets, or vesicular formations. They are usually one-third of them and in eight patients who had hereditary
associated with the more severe GBM changes. nephritis without deafness, the distribution of the chains
BM of retina and cochlea have not been studied by elec- was normal in the GBM and/or the EBM. Two patients
tron microscopy. Streeten et al. (67) demonstrated thinning were studied in each of eight families, and the immunohis-
and many dehiscences of the anterior lens capsule of tochemical results were concordant (43). Abnormal distri-
patients with lenticonus. Epidermal BM was found to be bution of the α3, α4, and α5(IV) chains has been observed
 26. Inherited Glomerular Diseases 523

FIGURE 26.5. Immunofluorescent microscopy. Distribution of the α(IV) chains in renal and epi-
dermal basement membranes of male (A–C) and female (D–F) patients affected with X-linked
Alport’s syndrome. C: Absence of epidermal basement membrane labeling (arrow). F: Discontinu-
ous epidermal basement membrane labeling (arrows). (See Color Plate 26.5.)

in anterior lens capsules of one of the two patients with the absence of a few exons to the complete absence of the
Alport’s syndrome and anterior lenticonus who underwent gene, and involve various parts of the gene. They can result
capsulectomy for cataract extraction (79). The group of in total loss of expression of the gene, in a shortened
patients with abnormal distribution of the α3–α5(IV) α5(IV) chain, or in an aberrant gene product, within all
chains is of particular interest. This anomaly appears to be a cases a major effect on the organization of the collagen IV
marker of severe juvenile forms of Alport’s syndrome molecule and thus on the GBM structure.
because: (a) the thickened and split GBM indicating poor All types of small mutations, distributed throughout the
prognosis was observed in 36 of the 37 α3–α5(IV)–negative COL4A5 gene, have been observed (80,84–94). Various
patients studied, and (b) all males belonging to α3–α5(IV)– consequences at the protein level may be expected from
negative families progressed to ESRF before the age of 30 resulting changes in amino acid residues. The most com-
years (43). mon mutation found is glycine substitution by bulkier
Affected animals in both models of canine X-linked amino acid in the collagenous domain. It creates an inter-
Alport’s syndrome display the same immunohistologic ruption in the repeated collagen-specific gly-X-Y sequence
anomalies as in humans (61,62). In addition, in dogs with- and affects the formation of the triple helix. Removal of
out any clinical evidence of extrarenal involvement, anti- highly conserved cysteine in the NC domain could affect
gens were absent from the specialized membranes of the eye the correct conformation of the NC1 domain and influ-
and internal ear. ence the formation of the triple helix and of the intermolec-
ular crosslinks (84). Nonsense mutations and small
deletions or insertions resulting in a frame shift at the RNA
Mutations in the COL4A5 Gene: Molecular
level and in a premature stop codon lead to the synthesis of
Heterogeneity of X-Linked Alport’s Syndrome
a putative truncated α5(IV) chain often lacking the NC
As soon as it was identified, the COL4A5 gene, localized at domain. Splice site mutations are expected to alter the con-
Xq22, became the candidate gene for the Alport’s syn- formation of the protein or result in premature termination
drome and, a few months later, the description of the first of the translation.
mutations confirmed this hypothesis (80). Thus far, more As indicated earlier, heterogeneity in the X-linked
than 300 different mutations have been identified by differ- Alport’s syndrome male phenotype has been documented
ent groups, corresponding to a detection rate of 40 to 80% widely. They are partly explained by the molecular hetero-
according to the screening method. geneity of the disease. As shown in a European collabora-
Deletions or other major rearrangements (duplication, tive study, large deletions, nonsense mutations, or small
insertion/deletion, inversion) of COL4A5 have been mutations changing the reading frame confer to affected
detected by Southern blot analysis in 5 to 15% of cases males a 90% probability of developing ESRF and hearing
(81–83). Deletions vary in size and location, ranging from loss before 30 years of age, whereas the same risk is approx-
524 V. Glomerular Disease

FIGURE 26.6. Immunofluorescent microscopy. Distribution of the α(IV) chains in renal and epi-
dermal basement membranes of patients affected with autosomal recessive Alport’s syndrome.
(See Color Plate 26.6.)

imatively 50% for ESRF and 60% for hearing loss in from those seen in classic Alport’s syndrome. By contrast,
patients with missense mutations (91). some features suggest autosomal recessive inheritance—con-
At the protein level in most patients with a COL4A5 sanguinity, severe disease in affected females, and mild or no
mutation, there is a loss of the α5(IV) chains in BMs. A symptoms in the parents. As in X-linked Alport’s syndrome,
striking feature is the co-absence of the α3 and α4 chains of the GBM antigenicity is normal in a minority of patients and
type IV collagen. The mechanisms resulting in such a con- abnormal in most of them (98). A peculiar pattern of α3–
figuration are controversial. It is now established that α5(IV) distribution has been identified: It is characterized by
α3(IV) and α4(IV) mRNA are expressed in male patients the co-absence of the 3 chains in the GBM and the presence
affected with X-linked Alport’s syndrome, and thus of the α5(IV) chain in collecting ducts and EBM at places
COL4A5 mutations probably result in defective molecular where the α5(IV) chain is not associated with the α3(IV) and
assembly and subsequent proteolysis of the chains (95,96). α4(IV) chains (Fig 26.6) (98). Mutations of the COL4A3 or
COL4A4 genes have been identified in autosomal recessive
Alport’s syndrome (8,9,88,99–102). The status of heterozy-
Deletions of COL4A5 and COL4A6 Genes in
gote carriers is highly variable. Most of them are asymptom-
Alport’s Syndrome Associated with Diffuse
atic or have isolated microscopic hematuria with thin GBM,
Esophageal Leiomyomatosis
which are typical features of familial benign hematuria (8,9).
No isolated COL4A6 mutation has been found in Alport’s In rare cases, heterozygotes have a slowly progressive nephri-
syndrome patients. But deletions removing the 5' end of tis. Murine models of autosomal recessive Alport’s syndrome
both COL4A5 and COL4A6 genes are observed in all have been developed by knock-out of the COL4A3 gene
patients with Alport’s syndrome associated with diffuse (103,104).
esophageal leiomyomatosis (51–54). Interestingly, in all
cases, the deletion involves only the first two exons of
Autosomal Dominant Alport’s Syndrome
COL4A6, whereas its extent in COL4A5 is variable. In con-
trast, patients with COL4A5 deletions associated with a A few cases of autosomal dominant Alport’s syndrome with or
larger deletion in COL4A6 are affected with Alport’s syn- without deafness have been reported (28–32). The renal prog-
drome without diffuse esophageal leiomyomatosis (53,54). nosis in male patients is significantly better than in X-linked
Dramatic changes in the composition of myocyte extracel- Alport’s syndrome (28). In 2000, a COL4A3 splice site muta-
lular matrix was observed in the tumoral tissue. They tion was identified in a large Irish family with autosomal dom-
include the co-absence of the α5 and α6(IV) chains con- inant inheritance. This finding completes the spectrum of type
trasting with the high level of COL4A6 transcripts, raising IV collagen mutations in all genetic forms of Alport’s syn-
the question of a potential role for this RNA in the tumoral drome (105). An autosomal dominant nephropathy with
process (97). GBM changes similar to those occurring in Alport’s syndrome
has been described in bull terrier dogs (106).
Autosomal Recessive Alport’s Syndrome
Kidney Transplantation in Alport’s Syndrome
It is now recognized that 10 to 20% of Alport’s syndrome
Patients and Anti-Glomerular Basement
patients are affected with the autosomal recessive form of the
Membrane Nephritis
disease (29). Most features of the disease are identical to
those observed in X-linked families. The nephritis usually Favorable long-term results have been observed in most
progresses to early-onset ESRF, sensorineural hearing loss is Alport’s syndrome patients who have undergone kidney
often present, and ocular lesions, identical to those in X-linked transplantation (107–109). However, approximately 3%
families, may or may not be associated. Ultrastructural of transplanted males affected with Alport’s syndrome
changes of the GBM in homozygotes are not distinguishable develop anti-GBM glomerulonephritis (88,91,110). This
 26. Inherited Glomerular Diseases 525

complication occurs within the first year after transplanta- the α3, α4, and α5(IV) chains are other criteria (91). All
tion and leads to severe crescentic glomerulonephritis and hematuric patients fulfilling four criteria are actually
subsequent graft loss. Patients are usually male and are affected with Alport’s syndrome, but, from a practical point
affected with early-onset Alport’s syndrome and deafness, of view, diagnosis of Alport’s syndrome is not always that
progressing to ESRF before or around the age of 20 years. easy. It has to be considered in every child who has persis-
A few female patients, all affected with recessive Alport’s tent hematuria of glomerular origin, even in the absence of
syndrome, also developed anti-GBM nephritis (68,88,99, proven familial disease. The investigations to be performed
101,102), but this complication has not been reported are Addis counts in parents and siblings, regular audio-
in females with X-linked disease. Recurrence of anti- grams, and ocular examinations seeking incipient anomalies
GBM nephritis on a second transplant is common (extrarenal symptoms are absent in very young children).
(111–113). Before performing renal biopsy, skin biopsy showing the
Nearly all patients who developed anti-GBM nephritis absence or focal distribution of the α5(IV) chain in the
carry large deletions of the COL43 or COL4A5 genes or EBM can be used for rapid identification of X-linked
premature stop codon predicted to result in absent or trun- Alport’s syndrome (78). Renal biopsy with immunohis-
cated proteins without the NC domain of the molecule tochemical and ultrastructural studies of the renal specimen
(88,91,99,101,102,114,115). The risk for these patients of allows one to exclude glomerulonephritis and especially IgA
developing anti-GBM nephritis reaches 15% (91). nephropathy, another frequent cause of microscopic and
The production of anti-GBM antibodies after kidney macroscopic hematuria, in children. Thick and split GBM,
transplantation is induced by the exposure of GBM anti- and abnormal GBM distribution of type IV collagen chains,
gens for which the patient has not established immune tol- are specific markers of the disease. However, normal immu-
erance. These antibodies recognize the NC domain of type nohistochemistry of the GBM and/or the finding of thin
IV collagen chains (72,116). Several types of specificities BMs do not eliminate the diagnosis of Alport’s syndrome.
have been observed. Most patients with the COL4A5 muta- We never observed normal GBM ultrastructure in children
tion develop antibodies against the 26-kDa NC domain of affected with Alport’s syndrome. Transmission of the disease
α5(IV) (118), but antibodies against the α3(IV)NC domain by asymptomatic female carriers in X-linked Alport’s syn-
have also been observed (116,117,120). The serum of drome or autosomal recessive transmission may explain the
patients with the COL4A3 mutation recognizes the NC absence of evident family history. Another possibility is the
domain of the α3(IV) chain (102,118). In addition, tran- occurrence of a neo-mutation, which accounts for up to
sient linear IgG fixation along the GBM has been observed 18% of patients with the syndrome.
in approximatively 15% of transplanted patients in the Correct identification of the mode of inheritance is nec-
absence of crescentic glomerulonephritis (119). More essary for genetic counseling. Affected and unaffected sub-
recently, this abnormal fixation has been shown to be asso- jects should be identified, making it crucial to detect
ciated with the presence of circulating anti-GBM antibod- asymptomatic carriers. In X-linked dominant Alport’s syn-
ies with a wide spectrum of reactivity to the α3, α4, and drome, affected fathers will transmit the disease to all their
α5(IV) chains (120). daughters and to none of their sons. Half of the children—
Factors other than the genetic defect are probably impli- of either sex—of affected mothers will be affected. In auto-
cated in the occurrence of immunization, as suggested by somal dominant Alport’s syndrome, affected parents trans-
the absence of anti-GBM posttransplant glomerulonephri- mit the disease to 50% of their offspring; father-to-son
tis in most patients with major COL4A5 mutations transmission is characteristic for this mode of inheritance.
(43,88,91) and the intrafamilial discordance, with regard to Presymptomatic and prenatal diagnosis by linkage analy-
the occurrence of GBM alloimmunization, observed in one sis or by direct COL4A5 study are now possible in previ-
family with a COL4A5 deletion (82,113). ously tested and informative families. In these families,
DNA testing may allow the precise identification of asymp-
tomatic female carriers, which is important in view of pos-
Diagnosis and Genetic Counseling
sible kidney donation and for genetic counseling.
Diagnosis of Alport’s syndrome is based on the finding of
hematuria with or without proteinuria, hypertension, and
Prognosis and Treatment
renal failure in association with the following features:
(a) familial hematuria, (b) sensorineural hearing loss present The prognosis of Alport’s syndrome nephropathy is poor:
either in the proband or in one of the affected relatives, and All males progress to ESRF. Hearing impairment, ocular
(c) progression to renal failure occurring in at least one changes, and family history of juvenile-type Alport’s syn-
affected subject. Flinter et al. require one additional crite- drome have an ominous significance. In an individual male
rion—ocular changes or ultrastructural lesions of the or female patient, regular increase in proteinuria is the most
GBM—for the diagnosis of Alport’s syndrome (121). Dif- accurate clinical symptom indicating a poor prognosis. The
fuse esophageal leiomyomatosis and abnormal expression of presence of glomerular, tubular, and interstitial changes by
526 V. Glomerular Disease

light microscopy; the degree of GBM thickening and split- tubular lumens. No specific deposits are detected by immu-
ting; and the defect in GBM antigenicity are also indicative nofluorescence; small granular C3 deposits may be seen on the
of severe disease. Severe COL4A5 rearrangements are asso- flocculus or the arteriolar wall. Diffuse and extreme attenua-
ciated with juvenile-type nephritis. tion of the GBM was first described by Rogers et al. in a
The renal disease is progressive. However, recently, it has well-documented family; it was the only lesion detected at
been shown that cyclosporin A could delay progression of the ultrastructural level in the five biopsied patients aged from
ESRF (122). Angiotensin-converting enzyme inhibition 19 to 51 years (132). This finding has been confirmed by
decreases proteinuria and could also slow the rate of progres- further observations (2,58,65,126–128,130,131), but, in
sion (123,124). Prospective controlled trials based on the some patients belonging to families affected with FBEH, the
precise clinical and genetic definition of the disease should be GBM has been found normal (2,128). Thinning of the
undertaken among larger series of patients to allow definitive GBM is usually regular and diffuse, but focal attenuation,
conclusions. Also, therapeutic trials on animal models are involving at least 50% of capillary walls, may be observed.
now possible. Regular dialysis does not raise specific prob- The structure and contours of the GBM are normal.
lems in Alport’s syndrome patients. Most results indicate that There is a wide discrepancy among investigators con-
posttransplant survival and renal function are as good as, or cerning the mode of identification of “thin” GBM. It may
better than, in other groups of patients. The question of the be based on subjective evaluation of GBM thickness fol-
selection of heterozygotes—asymptomatic or having isolated lowed by measurements of representative GBM segments
hematuria—as possible kidney donors is strongly debated or on systematic, numerous, random GBM measurements
(110). The development of anti-GBM glomerulonephritis in leading to the establishment of average values. The defini-
the transplanted kidney remains a rare possibility, but anti- tion of thin GBM also varies among groups. In the first
GBM antibodies have to be sought in cases of rapid deterio- study by Rogers et al., the average thickness of the GBM
ration of kidney function. was 150 nm (132). GBM thickness was between 100 and
Hearing loss has to be detected early and treated by 200 nm in most pediatric series (65,127,128). In adult
hearing aid for a better comfort of the child and for pre- series, the mean GBM thickness values leading to the diag-
venting further degradation of the auditory center. nosis of thin GBM varied from 206 to 319 nm. Overlaps
between “abnormal” values and normal values measured in
controls are observed in some series. Immunohistochemical
Familial Benign Essential Hematuria
studies of the GBM gave normal results in all patients
Familial benign essential hematuria (FBEH) is defined by tested (46,65,130,134).
familial occurrence of persistent hematuria without pro-
teinuria and without progression to renal failure or hearing
Genetic Transmission
defect. Diffuse attenuation of the GBM is usually considered
the hallmark of the condition. However, thin GBM is not FBEH is usually considered to be transmitted as an autoso-
pathognomonic for familial benign hematuria. The inci- mal dominant trait. In the family extensively studied by Rog-
dence of this hematuric disorder has not been clearly evalu- ers et al., in which eight members with persistent hematuria
ated. Recent data have shown that, at least in some families, have been identified in four generations, the disease is clearly
type IV collagen is involved in the pathogenesis of the disor- inherited as a dominant trait, but the autosomal dominant
der (8,9,125). inheritance proposed by the authors is not demonstrated in
the absence of father-to-son transmission (132). Male-to-
male transmission was observed in two out of ten families in
Clinical and Laboratory Findings
our series and in some kindreds in the series recently reported
Hematuria is often detected in childhood by routine urinal- (130,135). Recent data have demonstrated that some
ysis (58,59,126–128), but it may also be discovered in patients with “familial benign hematuria” have COL4A3 or
adults (129–131). It is usually microscopic and persistent. COL4A4 mutations and consequently are heterozygotes for
Episodes of gross hematuria have been reported, but we autosomal recessive Alport’s syndrome (8,9,136,137). How-
have never observed them in our series of patients. Clinical ever, genetic linkage to these genes has been excluded in
and laboratory investigations fail to disclose any other some kindreds (138,139).
symptom: proteinuria is absent, there is no progression to Sporadic cases of benign hematuria with thin GBM have
renal failure, and no extrarenal manifestation. been reported by different groups. Further analyses are still
necessary to elucidate their relationship to FBEH.
Pathology
Diagnosis and Prognosis
Few renal biopsies have been performed in this benign dis-
ease. By light microscopy, the renal tissue appears normal, FBEH, when diagnosed in hematuric children, implicates a
except for the occasional presence of red blood cells within favorable short-term and long-term prognosis. Diagnosis is
 26. Inherited Glomerular Diseases 527

based on a series of negative symptoms (absence of pro- in kindreds from all geographic origins. This curious associ-
teinuria and of extrarenal involvement), on the results of ation and the presence of fibrillar collagen within the GBM
family investigations demonstrating the absence of progres- have suggested that HOOD could be an inherited connec-
sion of the disease in adult males, and if necessary, on renal tive tissue disorder. However, recently, LMX1B, a transcrip-
biopsy findings showing thin GBM with normal antigenic- tion factor involved in limb and renal development has
ity. However, the distinction between FBEH and Alport’s been shown to be the NPS protein.
syndrome may be difficult, especially in young children. In
both conditions, hematuria may be the only symptom.
Dysmorphic Syndrome
Thinning of the GBM is often prominent in children
affected with Alport’s syndrome and may constitute the The dysmorphic features were first described in 1897 by
only lesion, even in adulthood. Normal antigenicity of the Little, who reported on a family of 18 members in whom
GBM is observed in approximately 30% of Alport’s syn- nails and patellae were absent (140). Several reports fol-
drome patients. It is impossible to determine whether the lowed, and the typical, bilateral, and symmetric involve-
course of the disease will be progressive or not when only ment of the nails, knees, elbows, and pelvis were put forth
adult females are affected in a kindred. In the absence of a by Mino et al. (141).
pathognomonic marker, diagnosis of FBEH should be Nail involvement is almost constant: It has been found
made with caution and reconsidered if proteinuria or extra- absent in only 6 of 147 patients, despite the presence of
renal symptoms are detected in the proband or in family other elements of the tetrad (142). It predominates on the
members during the follow-up. hands, where the thumb and the index finger are more
severely affected. Lesions range from hemidysplasia to total
nail aplasia. Common features are longitudinal pterygium
Thin Basement Membrane Nephropathy
and absence or triangular appearance of the lunulae. Nail
Thin GBM is frequently found in patients with FBEH, but dysplasia is usually visible from birth.
the lesion that seems to represent approximately 11% of The knees are abnormal in 95% of cases. The patellas are
nontransplant renal biopsies in some groups (130) is not hypoplastic or even absent. They have a tendency to dislo-
specific and is not the guarantee of benign disease. How- cate laterally, producing difficulties in learning to walk in
ever, in recent years, various terms based on the ultrastruc- children and in climbing stairs in adults (143). In fact, the
tural finding of thin GBM have been used to describe, functional disability is usually limited, and the most fre-
mostly in adults, the heterogeneous conditions character- quent complications are knee pains, recurring patella dislo-
ized by hematuria and attenuation of the GBM cations, and early onset of arthrosis. The dysplasia may also
(130,131,133) sometimes associated with proteinuria and involve other structures of the knee.
progression to renal failure. The terms thin glomerular base- Elbow dysplasia was mentioned in 94% of cases. The
ment membrane disease, thin basement membrane nephropa- radial head may be small and dislocated in a posterolateral
thy, thin membrane nephropathy, and familial thin basement position. Associated anomalies include deformities of the
membrane nephropathy are ambiguous because they suggest distal humeral extremity, hypoplasia of the olecranon, and
that the finding of thin GBM is a marker for a specific and elongation of the radial neck. These anomalies may lead to
common disorder with a uniformly good prognosis. In our mild to severe limitation of extension, pronation, and supi-
opinion, thin GBM has to be viewed as a lesion and not a nation of the forearm, especially in cases of webbing of the
clinicopathologic entity. In children with this lesion, the skin at the anterior aspect of the elbow joint.
diagnosis of Alport’s syndrome has to be considered before The presence of iliac horns, usually discovered on plain
any other possibility. The same ultrastructural lesion found film or intravenous pyelogram, was reported in 70.7% of
in late adulthood in association with isolated microscopic cases. They are asymptomatic, bilateral, triangular, and
hematuria probably has an overall favorable prognostic sig- symmetric formations arising from the posterior aspect of
nificance. However, substantial proteinuria and progression the ilium (143). In some cases, they may be detected by
to ESRF have been reported in adult patients with thin palpation through the buttock muscles.
GBM (131). Various other bone abnormalities have been described
but are not contributive to diagnosis. According to the
severity of the dysmorphic syndrome, HOOD may be dis-
Hereditary Osteo-Onychodysplasia (Nail-
covered at various ages, but the notion of family history
Patella Syndrome)
strongly contributes to its early recognition. Some severely
Hereditary osteo-onychodysplasia (HOOD), or nail-patella affected neonates with clubbed feet are misdiagnosed as
syndrome (NPS), is a rare autosomal dominant disorder arthrogryposis. Mild forms of the disease may remain
defined by the association of nail dysplasia, bone abnormal- undetected over decades.
ities, and renal involvement. Since the first description of Abnormal pigmentation of the inner margin of the iris is
the disease (140), more than 500 cases have been reported seen in approximately 50% of patients, and recently open-
528 V. Glomerular Disease

angle glaucoma has been recognized as an additional fea-

ture of the disorder (144).

Renal Involvement
Renal involvement was first reported in 1950 (145,146).
Curiously enough, renal symptoms are not found in all
patients or in all families. They seem to develop in approxi-
mately 30 to 40% of cases (142,147), but this evaluation is
perhaps overestimated. Both genders are equally affected.
The most common presenting feature is proteinuria with
occasional nephrotic syndrome sometimes associated with
microscopic hematuria.
Progression to ESRF occurs in approximatively 30% of
FIGURE 26.7. Electron microscopy. Phosphotungstic acid stain
patients with renal symptoms. Cases of rapid evolution to (×10,500). Nail patella syndrome. Irregular distribution of fibril-
renal failure have been observed in the pediatric popula- lar collagen within the glomerular basement membrane. Inset
tion. In fact, we lack precise knowledge of the long-term shows the typical periodicity of interstitial collagen (×48,000).
renal outcome in HOOD. A surprising evolution has
been observed in a neonate with typical deformities
belonging to a severely affected family: Nephrotic syn- infantile nephrotic syndrome. The lesions were evident on
drome was present at birth, but proteinuria disappeared the second renal specimen obtained 3 years later. By contrast,
within 2 weeks and was still absent 8 months later (147). in the 2-year-old child reported by Browning et al. (154),
A striking feature of the disease is the total unpredictabil- diffuse thickening of the GBM was associated with focal sub-
ity of renal prognosis, even within a given family. It is endothelial accumulation of fibrillar collagen. No tubular
illustrated by a personal observation in which a child BM involvement has been described.
developed ESRF at 15 years of age, whereas his monozy- There is a curious lack of correlation between the sever-
gote twin had only intermittent proteinuria at 27 years. ity and the extension of ultrastructural GBM lesions on one
This suggests that superimposed factors, which remain to hand, and the patient’s age, the severity of proteinuria, or
be identified, may be implicated in the rapid progression even the degree of renal failure on the other. Typical lesions
of the glomerulopathy in some patients. Goodpasture have been found in patients who were not proteinuric
syndrome, membranous nephropathy, necrotizing angii- (162,164).
tis, and IgA nephropathy have been observed in HOOD Immunohistochemical studies of the GBM using antibod-
patients (142). ies against the NC domain of α3(IV) gave conflicting
results—normal labeling in three patients and no GBM
labeling in two others (119). We detected type III collagen
Pathology with an irregular mesangiocapillary localization and observed
Light microscopy is poorly contributive. The lesions of focal- abnormal distribution of type VI collagen in glomeruli from
segmental sclerosis, when present, are nonspecific and several patients confirming the abnormal biochemical com-
correlate with the severity of renal insufficiency. Immuno- position of the glomerular extracellular matrix.
fluorescence may show focal deposits of IgM and C3.
The specific glomerular lesion has been identified by elec- Genetic Transmission: Mutations in the
tron microscopy (148). It is characterized by the presence of Gene LMX1B
clusters of fibrillar collagen irregularly distributed within
thick GBM segments and within the mesangial matrix. With HOOD is transmitted as an autosomal dominant trait. The
standard staining techniques, the GBM and the mesangial gene located at 9q34 is closely linked to those coding for
matrix often have a “moth-eaten” appearance (149). Staining ABO blood groups and for adenylate kinase (155,156). Tak-
with phosphotungstic acid often is necessary to disclose the ing into account the absence of nephropathy in some kin-
collagen bundles (Fig. 26.7). They have a fibrillar structure dreds, Lommen et al. suggested that there are two allelic
with a major periodicity of 40 to 60 nm, but, in some mutations of the HOOD locus, one causing HOOD with-
instances, no periodicity can be detected. The width of indi- out nephropathy and one associated with the possible occur-
vidual fibrils varies considerably. These lesions have been rence of nephropathy (157). The risks for affected parents of
found within the GBM in all patients studied, mostly within having a child with nephropathy, eventually leading to ESRF,
the lamina densa (148,150–154). However, we could not should be different in these two types of families.
detect any GBM collagen fiber in the first biopsy specimen In 1998, LMX1B was shown to be the NPS gene by two
from a child affected with HOOD and presenting with groups using two different approaches. Positional cloning,
 26. Inherited Glomerular Diseases 529

candidate gene approach, and identification of mutations unassociated with extrarenal symptoms (169–177). The
were used by the first group (158). For the second group, the glomerulopathy has been termed primary glomerular fibrosis
resemblance between the phenotypes in mice inactivated for (172), collagenofibrotic glomerulonephropathy (174,177), or
the Lmx1b gene and in NPS patients suggested that LMX1B collagen type III glomerulopathy (173,175,176). The glomer-
might be the NPS gene (159). Further studies confirmed this ular involvement was initially considered as being an
finding, and more than 100 LMX1B mutations have now incomplete, purely renal form of HOOD (169). However,
been reported in NPS patients (160–164). LMX1B is a analysis of morphologic, clinical, and genetic data strongly
transcription factor belonging to the LIM homeodomain suggests that they represent a new type (or new types) of
protein family playing an important role during develop- glomerulopathy.
ment. It establishes dorsoventral patterning of the limb in
which it is expressed in a temporally and spatially restricted
Pathology
manner (165). Mice with a homozygous deletion of the gene
exhibit nail and skeletal defects similar to those observed in By light microscopy, glomerular lesions are peculiar, contrary
patients and GBM abnormalities and die within 24 hours to the lesions observed in most patients affected with
after birth (165). The previously known function of LMX1B HOOD. The glomerular tuft is significantly enlarged
provides a satisfactory explanation for the occurrence of nail because of both expansion of the mesangial matrix and
and skeletal anomalies in NPS patients (159). On the other thickening of the capillary walls owing to accumulation of
hand, the recent demonstration of the early and persistent poorly stained subendothelial material. There is no signifi-
expression of lmx1b in the podocyte, and the role of the pro- cant increase in the number of mesangial cells. Conven-
tein in the regulation of the expression of type IV collagen tional immunohistologic studies are negative or show
α4 chain and other podocyte proteins opened new avenues segmental and focal deposits of various immunoglobulins
for the understanding of the NPS nephropathy (166–168). and complement factors. Expansion of the mesangial
The expression of the α3 and α4 chains of type IV collagen matrix and of the subendothelial aspect of the GBM by a
is strongly reduced in the GBM of Lmx1b–/– mice heterogeneous electron-lucent material is confirmed by
(166,168). Similarly, decreased expression of CD2AP and electron microscopic studies. The most characteristic finding
podocin—two markers of podocyte differentiation, responsi- is the detection (after selective stainings) of bundles of col-
ble when mutated of proteinuria and nephrotic syndrome— lagen fibers accumulated within the expanded mesangium
was observed in Lmx1b null mice (167). Moreover, binding and the subendothelial space of the GBM. The lamina
sites for Lmx1b were identified in both podocin and Cd2ap densa is usually normal. Thus, changes in the glomerular
promoters and in an enhancer sequence in COL4A4 intron 1 structure and distribution of the collagen fibers are differ-
(166–168). These findings strongly suggest that defects in ent from those observed in HOOD. Antibodies to type III
GBM type IV collagen and changes in podocyte phenotype collagen are strongly positive on the glomerular tuft.
contribute to glomerular disease in NPS. However, no renal
symptom and no defects in the expression of these proteins
Clinical Data
were found in heterozygous mice.
The disease has been described in both genders and in
patients aged from 1 to 70 years. Persistent proteinuria with
Diagnosis and Treatment
or without nephrotic syndrome is observed in all cases and
As seen earlier, clinical diagnosis of nail-patella syndrome is is often the presenting symptom. Microscopic hematuria
easy and based on identification of the elements of the clas- and hypertension are inconstant. Anemia has been noted in
sic tetrad. At the ultrastructural level, the finding of col- several patients (174–177).
lagen fibers within the GBM of nonsclerotic glomeruli is Concerning the progression of the disease, a striking dif-
quite specific for the condition. One exception is the detec- ference appears between adult and pediatric series. Stable
tion of similar lesions in a few patients without extrarenal renal function or slow progression to renal failure is
anomalies of the HOOD series (see Collagen Type III observed in most adult patients (174,175), whereas a pro-
Glomerulopathy). tracted course to ESRF is frequently described in children
Evolution of renal symptoms is unpredictable. No spe- (171,179).
cific treatment is available. No recurrence of GBM lesions
and no anti-GBM glomerulonephritis have so far been
Genetic Data
reported after kidney transplantation.
Family investigations were negative in most patients
reported to date. In two adult patients (170,171), the fam-
Collagen Type III Glomerulopathy
ily history of renal disease is consistent with dominant
Massive accumulation of collagen fibers within the glomer- transmission of the condition (170). By contrast, autoso-
ular tuft has been reported in 22 patients with proteinuria mal recessive transmission of the disease was suggested in
530 V. Glomerular Disease

the pediatric series on the finding of parental consanguin- GBM lesions observed in Alport’s syndrome have been
ity, involvement of several siblings of both genders, and reported. However, GBM alteration in Fechtner and Epstein
absence of renal and extrarenal symptoms in parents syndrome have mostly a focal distribution and, except from
(171,175). rare cases (180), are not specific of Alport’s syndrome.
Further studies are necessary to clarify the actual inci- In 1999, the May-Hegglin anomaly, the Sebastian syn-
dence, the long-term evolution, and the basic mechanisms drome, and the Fechtner syndrome were shown to be geneti-
responsible for the massive overproduction of type III col- cally linked to chromosome 22q (182,183) suggesting that
lagen. Of interest, in one young boy, collagen type III these three dominant MTCPs were allelic. Indeed, mutations
glomerulopathy was associated with autosomal recessive in the MYH9 gene, encoding the nonmuscle myosin heavy
factor H (FH) deficiency (176), an inherited disorder possi- chain IIA, were subsequently identified in patients affected
bly associated with the occurrence of glomerular lesions with one of these three diseases as well as in Epstein syn-
(see Factor H Deficiency). From a practical point of view, drome (184–186). This gene is expressed in the kidney from
the typical ultrastructural and immunohistochemical the early stages of nephrogenesis to adulthood, mainly in the
anomalies have to be sought in proteinuric patients with podocytes and peritubular capillaries (186).
atypical glomerular lesions that could mimic thrombotic
microangiopathy of the glomerular type. Evaluation of C3
HEREDITARY METABOLIC DISORDERS WITH
and factor-serum levels has to be systematically performed.
PRIMARY GLOMERULAR INVOLVEMENT

Hereditary Nephritis with Fabry Disease

Thrombopenia and Giant Platelets:
Anderson-Fabry disease is a rare X-linked recessive disorder
Epstein/Fechtner Syndromes
of glycosphingolipid metabolism resulting from deficiency
The association of hereditary nephritis with macrothrom- of the lysosomal hydrolase, α-galactosidase A (187). Clini-
bocytopenia (MTCP), first reported by Epstein et al. (178), cal aspects of this disease are discussed in Chapter 50.
has long been regarded as an Alport’s syndrome variant. Characteristic glycolipid accumulation within every glo-
These authors studied two families with MTCP, deafness, merular, vascular, and interstitial cell and within distal
and nephritis, a syndrome that appeared to be autosomal tubular cells has been observed in renal tissue from all hem-
dominant. In Fechtner syndrome, patients are affected with izygous patients irrespective of their age at renal biopsy. In
nephritis and MTCP as well. However, they also display heterozygous females, a peculiar feature is the presence of
cataracts and small, pale blue cytoplasmic inclusions within two intermingled cell populations, one normal and one
the neutrophils and eosinophils that were not described in massively involved by the storage disease. Degenerative
Epstein families (179). Leukocyte inclusions are also renal changes develop with age. They first affect vessels and
observed in two other autosomal dominant MTCPs with- are characterized by the presence of round fibrinoid depos-
out renal, ocular, or hearing defects: Sebastian syndrome its resulting from necrosis of smooth muscle cells. They are
and May-Hegglin anomaly. secondarily associated with nonspecific vascular, glomeru-
The Epstein and Fechtner syndromes appear heteroge- lar, and tubulointerstitial lesions (188). Of interest, the
neous because of the variable severity of hematologic, renal, reported beneficial effect of enzyme replacement therapy
and auditory symptoms. Thrombopenia may be asymp- seems to be linked to decrease glycolipid accumulation in
tomatic or responsible for hemorrhagic complications. Pro- the vascular endothelium (189).
gression to ESRF is variable and does not generally occur
before the fourth or fifth decade of life. It is absent in some
Other Glomerular Lipidoses
families. On the contrary, rapid progression to ESRF dur-
ing childhood has been reported (180). Early occurrence of Nephrosialidosis is a rare and severe form of oligosaccharido-
deafness, particularly in females, is commonly observed. In sis, or glucoproteinosis (previously classified as mucolipidosis
addition, dissociation between the renal disease and the type I). The condition is inherited as an autosomal recessive
thrombopenia has been reported. Some males, both throm- trait and is caused by neuraminidase deficiency. The clinical
bopenic and deaf, who are obligatory transmitters of and radiologic features of the disease include dysmorphic
Alport’s syndrome have no renal impairment (181), which facies, visceral storage disease, severe mental retardation, skel-
is never observed in classic Alport’s syndrome etal abnormalities, foam cells in the marrow, and cherry red
Renal histology shows variable and nonspecific abnormal- spot on funduscopy. A specific feature is the early occurrence
ities, including variable degrees of mesangial cell proliferation of progressive glomerular symptoms leading to ESRF in the
and mesangial matrix expansion and some tubular atrophy. first years of life (190). Podocytes and proximal tubular cells
Electron microscopy shows mesangial alterations, focal or show severe vacuolization. At the electron microscopic level,
diffuse effacement of podocyte foot processes, and alterations membrane-bound vacuoles look empty because of the loss of
of the GBM. GBM lesions reminiscent of ultrastructural the stored material during fixation.
 26. Inherited Glomerular Diseases 531

Glomerular symptoms are rare in other lipidoses (191). Alpha-1 Antitrypsin Deficiency
They usually are mild or absent in patients affected with
Alpha-1 antitrypsin (α1AT) is a major serine protease inhibi-
Gaucher disease, an autosomal recessive condition in which
tor synthesized by the liver. The gene responsible for α1AT
the accumulation of glucosylceramide within monocytes-
deficiency has been localized on chromosome 14. α1AT defi-
macrophages results from deficient activity of the lysosomal
ciency, phenotype ZZ, may result in emphysema in adults
enzyme glycosylceramidase. Severe symptoms, proteinuria,
(review in 197). Renal complications are rare in these
and progressive renal failure have been described in only a
patients. Conversely, approximately 25 cases of glomerulone-
few adult patients, most often after splenectomy. Large
phritis have been described in α1AT-deficient children with
“Gaucher” cells with abundant, pale, faintly fibrillar cyto-
severe chronic liver disease (198–200), another possible and
plasm have been observed in endothelial and mesangial
early complication of this autosomal dominant biochemical
position within glomeruli and also in the interstitium.
defect. Diffuse or focal segmental type I membranoprolifera-
In Niemann-Pick disease, I-cell disease, and GM1 gangli-
tive glomerulonephritis was present in most cases, but diffuse
osidosis, silent accumulation of glycolipids or mucopolysac-
endocapillary glomerulonephritis has also been observed.
charides within glomerular cells has been detected by
Immunofluorescence revealed the presence of immunoglob-
examination of autopsy material (191).
ulins, often including IgA and complement components.
The co-localization of PiZ protein with immunoproteins has
HEREDITARY METABOLIC DISORDERS WITH been observed (200,201), but is not a constant finding
SECONDARY GLOMERULAR INVOLVEMENT (199). Expression of glomerular involvement varies from no
disease to proteinuria, hypertension, and renal failure. Its
Familial Amyloidosis severity roughly parallels that of renal lesions. A direct patho-
genic link with α1AT deficiency has been suggested based on
Hereditary amyloidosis encompasses a group of autosomal
the presence of granular α1AT or PiZ subendothelial depos-
dominant disorders characterized by the extracellular accu-
its and on the increased incidence of glomerular lesions in
mulation of protein fibrils having β-pleated sheet confor-
α1AT-deficient patients compared with a controlled cirrhotic
mation. They are now classified according to the type of
population (200). Regression of nephrotic syndrome and
protein composing amyloid fibrils and for some of them
glomerular lesions has been observed after liver transplanta-
according to the type of mutation detected in the corre-
tion (201). In adults, association of α1AT deficiency and sys-
sponding gene. Transthyretin variants have been found in
temic vasculitis, especially Wegener syndrome, has been
most affected families, but systemic hereditary amyloidosis
described, suggesting involvement of the gene in the patho-
may be associated with variants of cystatin C, gelsolin, apo-
genesis of these diseases (202).
lipoprotein A1, fibrinogen, or lysozyme (192). In heredi-
tary amyloidosis, symptoms, usually of the neurologic
Factor H Deficiency
series, develop during adulthood. Except for a few families
with apolipoprotein A1, fibrinogen, or lysozyme variant, Homozygous deficit in FH, a 150-kDa glycoprotein control-
renal symptoms are not a prominent and early feature, but ling the alternative pathway of complement activation, is
severe renal involvement may be observed in all types of usually responsible for increased susceptibility to infections.
familial amyloidoses and has an ominous significance. In several families, renal involvement consisting of atypical
Familial Mediterranean fever, an autosomal recessive dis- dense deposit disease, hemolytic uremic syndrome, or col-
order common in Mediterranean and Middle Eastern pop- lagen type III glomerulopathy has been reported (203).
ulations, especially in Sephardic Jews and Armenians, is Dense deposit disease, observed in two brothers, was char-
characterized by recurrent episodes of fever, abdominal acterized by the early onset of glomerular symptoms, with
pain, joint pain, and less frequently, pleuritis or pericarditis. recurrent macroscopic hematuria and the abundance of
The prognosis of the disease depends on the development mesangial and parietal granular C3 deposits (204). FH
of amyloidosis of the AA type with prominent renal deficiency has been shown in piglets with autosomal reces-
involvement. The gene involved in familial Mediterranean sive dense deposit disease. The disease develops early after
fever, designated MEFV, has been identified by positional birth and progresses rapidly to ESRF. Transfusion of nor-
cloning (193,194). Colchicine administration has been mal porcine plasma or injection of FH into affected pigs
shown to reduce the number of attacks and to prevent amy- increases survival, demonstrating the role of FH deficiency
loidosis (195). and alternative complement activation in the pathogenesis
Development of amyloid of the AA type occurs in other of the disease (205).
types of inflammatory disorders also characterized by recur- Low C3 level has been described in several atypical,
rent attacks of fever with visceral, synovial, muscular, or recurrent, and/or familial cases of hemolytic uremic syn-
cutaneous inflammation—the Muckle-Well syndrome and drome and, recently, the role of FH deficiency and the
the TRAPS syndrome both transmitted on the autosomal identification of mutations of the FH gene have been
dominant mode (196). reported by several groups (206–213). It appears now
532 V. Glomerular Disease

that FH deficiency is one major cause of familial Hereditary Lecithin-Cholesterol

hemolytic uremic syndrome associated with a high risk Acyltransferase Deficiency
of recurrence in renal allografts. Within families, a strik-
Hereditary lecithin-cholesterol acyltransferase (L-CAT)
ing feature is the absence of correlation between the
deficiency, first described in Norway, is a rare autosomal
degree of FH deficiency and the presence and severity of
recessive disorder characterized by an inability to esterify
renal disease.
plasma cholesterol and by abnormal deposition of unesteri-
Extensive deposition of type III collagen has been identi-
fied cholesterol in tissues, including the kidney (217,218).
fied in a young patient with progressive glomerulopathy
Progression to ESRF usually occurs in the fourth or fifth
and FH deficiency; the relationship between the two
decade. Glomerular lesions resemble those seen in Alagille
abnormalities is still unclear (181).
syndrome. They include accumulation of foam cells of
endothelial and mesangial origin and massive deposition of
Alagille Syndrome lipids within the mesangial matrix and along the subendo-
thelial aspect of the GBM (217). The L-CAT gene has been
Alagille syndrome, or syndromic paucity of interlobular ducts,
localized on chromosome 16, and different point muta-
is an autosomal dominant disorder with variable pene-
tions spread in all exons of the gene have been described in
trance, recognized as a common cause of severe cholestase
families of various geographic origins (218).
is in children. Liver abnormalities are associated with a
peculiar facial appearance, cardiovascular malformations,
butterfly-like vertebral arch defect, embryotoxon, hypogo- Lipoprotein Glomerulopathy
nadism, growth retardation, and high-pitched voice. The
Lipoprotein glomerulopathy is a new type of renal disease
disease is caused by mutations in Jagged1, which encodes a
characterized by intraglomerular lipoprotein thrombosis and
ligand for the Notch1 receptor, implicated in cell differenti-
high plasma concentration of apolipoprotein E. Since the
ation (214,215). Glomerular lesions of variable severity
first publication by Saito et al. in 1989 (219), numerous
have been observed in some patients (216). They are
cases have been observed mostly, but not exclusively, in Japan
characterized by the presence of clear vacuoles containing
(220–224). The disease, observed in both genders, usually is
osmiophilic material within the mesangial matrix and some-
detected in adulthood; however, in some cases, first symp-
times within the GBM. Mesangial foam cells may be asso-
toms occurred in childhood (224). All patients have persis-
ciated (Fig. 26.8). The extent of mesangio-lipidosis is
tent proteinuria resulting in nephrotic syndrome in most of
related to the degree of cholestasis and is probably a conse-
them. Some have mild to moderate proteinuria for a long
quence of the resulting abnormal lipid metabolism. A strik-
period, whereas progression to ESRF has been observed.
ing feature is the early occurrence of these lesions during
Transplantation has been followed by the recurrence of
the first years of life in some patients contrasting with the
glomerular lesions (221,223). Curiously, systemic manifesta-
paucity of renal symptoms. Progression to renal failure in
tions of lipidosis are consistently absent. The familial occur-
adulthood may be a major complication for patients with
rence observed in some kindreds suggested a genetic basis for
this syndrome.
the disease, involving perhaps the isoform E2 of apolipopro-
tein E. Actually, a variant of apolipoprotein E, apolipopro-
tein E Sendai, characterized by a missense mutation resulting
in arginine-145 to proline substitution appears to be the
most frequent mutation in the patients (222), but other
mutations have been described (223).
Glomerular lipidosis has also been observed in patients
with familial hypercholesterolemia resulting from defect in
the receptor for low-density lipoproteins, in type III hyper-
lipoproteinemia, and in the rare cases of cholesterolic polyco-
ria. In these diseases, clinical symptoms usually develop in
adulthood.

Familial Juvenile Megaloblastic Anemia

Familial juvenile megaloblastic anemia, or Imerslund-Gräsbeck
syndrome, is a rare autosomal recessive disorder caused by
FIGURE 26.8. Electron microscopy. Lead citrate and uranyl ace- primary and selective vitamin B12 malabsorption. Anemia
tate stain (×8600). Alagille syndrome. Massive accumulation of appearing in infancy or early childhood is nearly always
lipid vacuoles within mesangial cells and matrix. Irregular distri-
bution of lipid vacuoles within the glomerular basement mem- associated with mild and nonprogressive proteinuria, usu-
brane. ally considered of glomerular origin. Minor and nonspecific
 26. Inherited Glomerular Diseases 533

glomerular changes have been described by electron and tarsal bones. Inherited, dominant or recessive, and spo-
microscopy (225). radic cases have been reported. Hypertension, proteinuria,
and progressive renal failure may occur in some patients
(237). Arteriolar thickening and sclerosis and focal glomer-
OTHER HEREDITARY DISEASES WITH ulosclerosis are the usual histopathologic findings.
GLOMERULAR INVOLVEMENT
Other Syndromes with Renal Involvement
Charcot-Marie-Tooth Disease
Renal involvement is nearly constant in Biedl-Bardet syn-
Charcot-Marie-Tooth (CMT) disease is a familial periph-
drome (BBS), an autosomal recessive disorder, the cardinal
eral neuropathy resulting in progressive symmetric atrophy
features of which are obesity, polydactylia, mental retarda-
and weakness of distal muscles and sensory loss. Various
tion, retinal dystrophy, and hypogonadism (238). The dis-
clinical forms of the disease have been described according
ease is genetically heterogenous. Six genes have been
to nerve morphology and nerve conduction velocity, age at
localized, and the first one has been identified (239). BBS is
onset of symptoms, and presence or absence of associated
responsible for severe renal disease in 15 to 55% of patients,
symptoms. This clinical heterogeneity is explained by the
according to different series. Glomerular symptoms, such as
genetic heterogeneity of the condition: Autosomal domi-
proteinuria, as well as questionable glomerular changes, have
nant, X-linked dominant, and X-linked recessive modes of
been described in only a few patients. Actually, tubular dys-
inheritance have been described (226,227). In 1993, muta-
function and abnormal kidney structure detected by urogra-
tions in the myelin protein zero gene on chromosome 1,
phy are the main renal symptoms in BBS. Cystic kidney
peripheral myelin protein 22 gene on chromosome 17 and
dysplasia and/or tubulointerstitial lesions seem to be the
connexin 32 on chromosome X were identified (228–230).
most consistent lesions in this disease (240,241).
Renal symptoms with focal glomerular sclerosis have been
Alström syndrome is a rare autosomal recessive disorder, the
reported in a few patients and seem to segregate with the
gene of which has been recently identified. It is characterized
neuropathy in some families (231). They consist of long-
by the association of profound childhood blindness, nerve
standing proteinuria occurring in childhood or in adoles-
deafness, obesity, and diabetes mellitus and differs from BBS
cence, followed by rapid progression to ESRF. Because
by the absence of mental retardation and polydactyly. It is
some of these patients are also deaf, CMT disease with
considered a classic cause of proteinuria. However, primary
renal involvement could have been considered a variant of
glomerular involvement has not been demonstrated, and
Alport’s syndrome. Deafness is a possible manifestation of
tubular dysfunction and tubulointerstitial lesions have been
peripheral involvement in CMT disease, and, except in one
observed in the few patients studied (242).
family with co-segregation of AS and CMT disease (232),
In familial dysautonomia, an autosomal recessive neurop-
no specific ultrastructural changes of the GBM have been
athy that occurs in the Ashkenazi Jewish population, renal
observed in patients with CMT disease.
involvement seems to be primarily vascular with secondary
development of glomerulosclerosis (243).
Cockayne Syndrome
Cockayne syndrome is an unusual autosomal recessive disor-
HEREDITARY GLOMERULOPATHIES WITHOUT
der. Characteristic symptoms are poor growth, neurologic
EXTRARENAL SYMPTOMS
abnormalities, premature aging, senile face, sensorineural
hearing loss, cataracts, pigmentary retinopathy, sun sensitiv-
Familial Lobular Glomerulonephritis/
ity, and dental caries, as reported in a comprehensive review
Fibronectin Glomerulopathy: A
of 140 patients by Nance and Berry (233). Death occurs in
Heterogeneous Entity?
most cases during the first or second decade. Increased cell
sensitivity to the killing effect of ultraviolet radiation has Several familial cases of atypical lobular glomerulopathies
been observed, caused by a specific defect in the DNA repair have been reported (244–247). They were characterized by
system. Renal symptoms consisting of hypertension, mild extensive subendothelial and mesangial deposits in which
proteinuria, or renal insufficiency develop in approximately immunoglobulins and complement components were
10% of patients. The basic lesion seems to be diffuse and inconstant or lacking. Patients had persistent proteinuria,
homogeneous thickening of the GBM (234,235). The syn- hematuria, and hypertension. Slow progression to ESRF or
drome is clinically and genetically heterogeneous (236). severe cerebral vascular complications (in one family)
occurred around the third decade of life. The disease is
transmitted as an autosomal dominant trait. Recently,
Hereditary Acro-Osteolysis with Nephropathy
strong reactivity of deposits to fibronectin derived from the
Hereditary acro-osteolysis is a rare disorder characterized by plasma was shown in patients previously reported and in
arthritis-like episodes and progressive resorption of carpal additional families (248–252). The names “fibronectin
534 V. Glomerular Disease

glomerulopathy” or “familial glomerulonephritis with Glomerular involvement has been initially described in
fibronectin deposits” were suggested for this glomerulopa- three children who developed nephrotic syndrome with
thy (220). However, the pathogenic significance of focal segmental glomerulosclerosis before the age of 5 years
fibronectin deposition remains to be confirmed. It has been (258,259). These patients had mitochondrial cytopathies
suggested that genetically altered fibronectin is trapped manifested by the association of myopathy, ophthalmople-
within the glomerular tuft, but fibronectin was excluded as gia, and deafness or pigmentary retinitis and hypoparathy-
a causative gene by linkage analysis in a large family (250). roidism.
Linkage analysis of a large pedigree led to the localization of Since 1994, approximately 40 cases of glomerulopa-
a gene at 1q32 (253). thies, initially isolated or associated with extrarenal symp-
The homogeneity of this new entity is not demon- toms, have been reported. The clinical presentation of the
strated. Clinical evolution varies between families with renal disease is nonspecific: occurrence of proteinuria at
regard to the occurrence of cerebral vascular complications, various ages between 1.6 and 35.0 years, progressive
progression to ESRF, evolution after transplantation, and increase with age with eventual development of nephrotic
ultrastructural appearance of deposits. One child developed syndrome, and variable rate of progression to ESRF. An
nephrotic syndrome at 3 years of age (252). In some kin- important feature is the usual absence of hematuria. Ster-
dreds, deposits are homogeneous and granular with a oid therapy is ineffective. Proteinuria was the revealing
restricted subendothelial and mesangial distribution (245, symptom of the disease in 12 patients aged 10 to 32 years
248), whereas in others they are fibrillar (246,247,249) and who secondarily developed extrarenal symptoms. Pro-
also observed in subepithelial location (246). Of four teinuria/nephrotic syndrome was also found in patients
patients from different families who were reported to have aged 17 to 34 years who have maternally inherited diabe-
undergone kidney transplants, the disease recurred in one tes and/or deafness or, less frequently, complete or incom-
only (248,250). plete MELAS (mitochondrial encephalomyopathy with
lactic acidosis and stroke-like episodes) syndrome. This
observation led to the identification of the mitochondrial
Other Familial Glomerulopathies
DNA MELAS mutation in most patients with mitochon-
A curious form of hereditary nephropathy has been drial glomerulopathy.
described by Grottum et al. in 11 members of a large fam- Focal and segmental glomerulosclerosis was observed in
ily (254). Persistent moderate proteinuria was abruptly nearly all renal biopsies. An increased number of abnormal
followed by rapidly progressive renal failure, malignant mitochondria of various shapes and sizes was found in
hypertension, and microangiopathic anemia and throm- podocytes by Hotta et al. (271), but in most cases, no mito-
bocytopenia occurring at the ages of 24 to 31 years (254). chondrial lesion was seen. Mitochondrial abnormalities
Diffuse vascular damages and ischemic glomeruli with appear to be more frequent and prominent in proximal
IgM and C3 deposits were observed in all cases. A glomer- tubular cells despite the absence of tubular symptoms.
ular disease with a similar clinical course has been Arteriolar deposits have been described in the five biopsies
observed in another family. The presence of diffuse round examined by Doleris et al. (269). They could result from
mesangial deposits of C3 was the characteristic feature in individual myocyte necrosis and participate in the progres-
this family (255). In both families, the disease appears to sion of glomerular lesions.
be autosomal dominant.

RARE AND “BIZARRE” SYNDROMES

MITOCHONDRIAL CYTOPATHIES
Several curious syndromes with a glomerular component
Mitochondrial cytopathies are a heterogeneous group of have been described in single or few families (274–280).
diseases. They are due to genetic defects of one or several Their basic defect and final classification have not been
mitochondrial enzyme complexes that play a major role in determined. According to recent data on mitochondrial
oxidative phosphorylation and energy production. They cytopathies, some of these disorders could be caused by
have long been regarded as neuromuscular diseases only, mitochondrial defects.
but any organs dependent on mitochondrial energy supply
may be affected by these disorders (256). For a long time,
renal involvement was regarded as being rare, the most fre- ACKNOWLEDGMENTS
quent manifestation, usually seen in children, being proxi-
mal tubulopathy manifested by a de Toni Debré Fanconi A. Beziau, Y. Deris, L. Guicharnaud, M. Lacoste, M. Sich,
syndrome (257). More recently, several cases of glomeru- technical assistance. D. Bronner, B. Coupé, secretarial sup-
lopathy have been reported (257–273), and this expression port. Institut National de la Santé et de la Recherche Scienti-
is probably more frequent than initially thought. fique (INSERM), Assistance Publique des Hôpitaux de Paris.
 26. Inherited Glomerular Diseases 535

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 27

STEROID-SENSITIVE IDIOPATHIC
NEPHROTIC SYNDROME IN CHILDREN
PATRICK NIAUDET

In children, the most common cause of nephrotic syndrome more dependent on the initial response to corticosteroids
is idiopathic nephrotic syndrome, also called nephrosis. Idio- than on the results of renal biopsy.
pathic nephrotic syndrome is defined by the association of a
nephrotic syndrome and minimal changes on renal biopsy by
light microscopy with foot process fusion of epithelial cells EPIDEMIOLOGY
on electron microscopy. No immunoglobulin (Ig) or com-
plement fraction deposit is seen on immunofluorescence The incidence of idiopathic nephrotic syndrome varies
examination. Patients with minimal change disease most with age, race, and geography. The annual incidence in
often respond to corticosteroids. As renal biopsy is not usu- children in the United States has been estimated to be 2.0
ally performed when the patient responds to corticosteroids, to 2.7 per 100,000 (2), with a cumulative prevalence of 16
the term minimal change disease has become synonymous per 100,000. Geographic or ethnic differences are well
with steroid-sensitive nephrotic syndrome. known. In the United Kingdom, for example, the incidence
However, in some patients who respond to corticoste- of idiopathic nephrotic syndrome is sixfold greater in Asian
roids, histologic examination shows diffuse mesangial prolif- than in European children (3); this is also true for Indians
eration or focal and segmental glomerular sclerosis (FSGS). (4), Japanese, and Southwest Asians. Idiopathic nephrotic
In addition, IgM deposits may be found. Several authors syndrome is rare in Africa, where most cases of nephrotic
believe that minimal change disease is a distinct disease and syndrome seem to be related to structural glomerular
that diffuse mesangial proliferation, FSGS, and IgM nephrop- lesions unresponsive to steroids (5). Such differences under-
athy are also distinct entities. There is no doubt that patients line the role of genetic as well as environmental factors in
with diffuse mesangial proliferation or FSGS are more fre- the pathogenesis of this disease.
quently resistant to corticosteroids and have a significant Although idiopathic nephrotic syndrome accounts for
propensity for progression to renal failure. Conversely, only 25% of adult cases (6), it is by far the most common
patients with minimal change disease rarely progress to end- cause of nephrotic syndrome in children. Almost all neph-
stage renal failure. However, there is an overlap between rotic children between 1 and 6 years of age in Western
these histologic variants, and a significant proportion of countries have idiopathic nephrotic syndrome. The Inter-
patients with FSGS responds to corticosteroids, whereas national Study of Kidney Disease in Children (ISKDC)
some patients with minimal change disease are resistant to found minimal change disease in 76.6% of children with
corticosteroids. Moreover, in the early stages, FSGS and min- primary nephrotic syndrome (7).
imal change disease are indistinguishable (1). There is male preponderance in children, with a male to
Experience has shown that response to steroid therapy female ratio of 2:1 (7,8), but both sexes are similarly affected
carries a greater prognostic weight than the histologic fea- in adolescents. The familial occurrence of idiopathic neph-
tures seen on the initial renal biopsy. Therefore, two types rotic syndrome is well known. In a European survey, White
of idiopathic nephrotic syndrome can be described accord- found that 3.3% of 1877 patients with idiopathic nephrotic
ing to the response to corticosteroids: steroid-responsive syndrome (excluding congenital nephrotic syndrome) had
idiopathic nephrotic syndrome, in which proteinuria rap- affected family members, mainly siblings (9). Nineteen of
idly resolves, and steroid-resistant idiopathic nephrotic syn- the 63 children had minimal change disease, and 18 of them
drome (see Chapter 29), in which the nephrotic syndrome had affected siblings, whereas the remaining child’s father
persists despite the treatment. This distinction is also more was affected with the disease. This incidence was greater than
helpful for the clinician, as the therapeutic options are among the general population. Idiopathic nephrotic syn-
544 V. Glomerular Disease

drome was also reported in identical twins (10). In one series milk or egg) may be responsible for relapses of steroid-sensi-
of familial cases (11), the disease tended to develop in sib- tive nephrotic syndrome. Laurent et al. evaluated the effect
lings at the same age, with the same renal histology and the of an oligo-antigenic diet given for 10 to 15 days to 13
same outcome. Another study reported 34 patients in 15 patients. This diet coincided with improvement of pro-
families; the response to corticosteroids was identical within teinuria in nine, including complete remission in five (15).
members of individual families (12). The age and sex distri- The association between minimal change disease and
bution, the relapsing course, and the absence of renal failure malignancies mainly concerns lymphomatous disorders:
in 15 steroid-responsive familial cases were comparable to the Hodgkin’s disease and non-Hodgkin’s lymphomas (16). The
sporadic cases. nephrotic syndrome may be the presenting feature of the dis-
ease. It usually disappears after successful treatment of the
malignancy. Other types of neoplasias that have been associ-
ASSOCIATED DISORDERS ated with idiopathic nephrotic syndrome include colon carci-
noma, bronchogenic neoplasia, and small cell carcinoma (17).
Idiopathic nephrotic syndrome is, by definition, a primary Eosinophilic lymphoid granuloma in Asians (Kimura’s
disease. Nevertheless, in a number of cases, an upper respi- disease) has also been reported in association with steroid-
ratory tract infection, an allergic reaction, or another factor responsive idiopathic nephrotic syndrome. Several cases of
may immediately precede the development or relapse of the minimal change disease have been reported in association
disease. with the onset of insulin-dependent diabetes mellitus. The
Many agents or conditions have been reported to be disease is usually responsive to corticosteroids and follows a
associated with idiopathic nephrotic syndrome (e.g., infec- relapsing course.
tious diseases, drugs, allergy, vaccinations, and malignan-
cies) (Table 27.1). The question remains whether these
factors are real causes, a simple coincidence, or precipitat- HISTOCOMPATIBILITY ANTIGENS AND
ing agents. IDIOPATHIC NEPHROTIC SYNDROME
Allergy is associated with up to 30% of cases (13,14).
Among a list of anecdotal cases, the allergens reported A three- to fourfold increased incidence of HLA-DR7 in chil-
include fungi, poison ivy, ragweed pollen, house dust, jelly- dren with idiopathic nephrotic syndrome has been reported
fish stings, bee stings, and cat fur. A food allergen (i.e., cow’s (18,19). Clark et al. found a strong association between HLA-
DR7 and the DQB1 gene of HLA-DQW2 and steroid-sensi-
tive nephrosis and suggested that the β-chains of DR7 and
TABLE 27.1. CONDITIONS ASSOCIATED WITH DQW2 contribute to disease susceptibility (20).
IDIOPATHIC NEPHROTIC SYNDROME An association with HLA-B8 was reported in Europe.
Allergy
Children with atopy and HLA-B12 have a 13-fold increased
Pollen risk of developing idiopathic nephrotic syndrome.
Fungi
Cow’s milk
House dust CLINICAL FEATURES
Bee stings
Cat fur
Poison ivy The disease is characterized by a sudden onset, with edema
Drugs being the most frequent presenting symptom. Edema
Nonsteroidal antiinflammatory drugs increases gradually and becomes clinically detectable when
Ampicillin fluid retention exceeds 3 to 5% of body weight. It is often
Gold
Lithium
initially apparent around the eyes and misdiagnosed as an
Mercury allergy. Edema is gravity dependent. During the day, peri-
Trimethadione orbital edema decreases while it localizes to the lower
Malignancies extremities. In the reclining position, it localizes to the
Hodgkin’s disease back. It is white, soft, and pitting. Edema of the scrotum
Non-Hodgkin’s lymphoma
Colon carcinoma
and penis, or labia, may also be observed. Anasarca may
Bronchogenic carcinoma develop. Blood pressure is usually normal but sometimes
Others elevated. The abdomen may bulge with umbilical or
Viral infection inguinal hernias. When ascites build up rapidly, the child
Kimura’s disease complains of abdominal pain and malaise. Abdominal pain
Diabetes mellitus
Myasthenia gravis
may also result from severe hypovolemia, peritonitis, pan-
Immunization creatitis, thrombosis, or steroid-induced gastritis. Shock is
not unusual after a sudden fall of plasma albumin, with
 27. Steroid-Sensitive Idiopathic Nephrotic Syndrome in Children 545

abdominal pain and peripheral circulatory failure. Emer- tein cholesterol ratio. Patients with severe hypoalbumin-
gency treatment is needed. emia have increased triglycerides and very-low-density
The nephrotic syndrome is occasionally discovered dur- lipoprotein. Apoproteins and apolipoproteins B, CII, and
ing routine urine analysis. The disease may also be revealed CIII are also elevated. The levels of lipoprotein(a) are ele-
by a complication. Peritonitis due to Streptococcus pneumo- vated in nephrotic patients.
niae is a classical mode of onset. Deep vein or arterial Serum sodium is often reduced due in part to hyper-
thromboses and pulmonary embolism may also occur dur- lipemia and in part to the dilution from renal retention of
ing the first attack or during a relapse. water due to hypovolemia and inappropriate antidiuretic
hormone secretion. Hyperkalemia may be observed in cases
of renal insufficiency. Hypocalcemia is related to hypoalbu-
LABORATORY FINDINGS
minemia, and the level of ionized calcium is usually nor-
mal. Plasma creatinine is elevated in one-third of cases and
Urine Analysis
returns to normal when remission occurs.
Nephrotic range proteinuria is defined as urinary protein Hemoglobin levels and hematocrit are increased in
excretion greater than 50 mg/kg/day or 40 mg/m2/hr. It is patients with plasma volume contraction. Thrombocytosis
higher at onset and decreases as plasma albumin concentra- is common and may reach 5 × 108/L or 109/L. Fibrinogen
tion falls. In young children, it may be difficult to obtain a and factors V, VII, VIII, and X are increased, whereas anti-
24-hour urine collection, and urinary protein to creatinine thrombin III, the heparin cofactor, and factors XI and XII
ratio or albumin to creatinine ratio in untimed urine speci- are decreased. These abnormalities contribute to a hyperco-
mens is useful. For these two indices, the nephrotic range is agulable state.
200 to 400 mg/mmol. In most cases, proteinuria is highly
selective, consisting of albumin and lower-molecular-
COMPLICATIONS
weight proteins. The selectivity of proteinuria may be
appreciated by polyacrylamide gel electrophoresis or by the
Acute Renal Failure
evaluation of the Cameron index that is the ratio of IgG to
transferrin clearances. A favorable index would be below Some patients have a reduction of the glomerular filtration
0.05 to 0.10; a poor index is above 0.15 or 0.20. A poor rate (GFR) attributed to hypovolemia, with complete return
Cameron index is often associated with FSGS. However, to normal after remission. A reduced GFR may be found
there is a considerable overlap in results, and the test has despite normal effective plasma flow (21,22). Bohman et al.
limited value. Microscopic hematuria is present in 20% of showed a close relationship between the degree of foot-process
cases and has no influence on the response to steroid ther- effacement and both the GFR and the filtration fraction, sug-
apy. Conversely, macroscopic hematuria is exceptional and, gesting that foot-process effacement leads to a reduction of the
in the context of idiopathic nephrotic syndrome, may be glomerular filtering area or of permeability to water and small
related to renal vein thrombosis. The urine sediment often solutes (23). This reduction is transitory, with a rapid return
contains fat bodies. Hyaline casts are also usually found in to normal after remission.
patients with massive proteinuria, but granular casts are not Marked oliguria may occur in children (24). Oliguric
present unless there is associated acute renal failure and renal failure may be the presenting symptom. Renal failure
acute tubular necrosis. Urinary sodium is low, 1 to 2 mmol/ may be secondary to bilateral renal vein thrombosis that
day, resulting in sodium retention and edema. can be diagnosed by sonography. Acute renal failure has
also been reported with interstitial nephritis. Skin rash and
eosinophilia are suggestive of this diagnosis, which is often
Blood
associated with furosemide or other medication.
Plasma protein levels are markedly reduced, less than 50 g/ Acute renal failure is usually reversible, often with intra-
L, due to hypoalbuminemia. Plasma albumin level is usu- venous albumin and high-dose furosemide-induced diuresis
ally lower than 25 g/L and may be less than 10 g/L. Elec- (25). In some cases in which glomerular structure is normal
trophoresis shows a typical pattern with low albumin, on initial histology, renal failure may persist for as long as 1
increased α2-globulins, and, to a lesser extent, β-globulins, year (21) and rarely be irreversible (26).
whereas γ-globulins are decreased. IgG is considerably
decreased, IgA slightly decreased, and IgM is increased.
Infections
Lipid abnormalities include high levels of cholesterol,
triglyceride, and lipoproteins. Total cholesterol and low- Bacterial infections are frequent in nephrotic children (Table
density lipoprotein cholesterol are elevated, whereas high- 27.2). Sepsis may occur at the onset of the disease. The most
density lipoprotein cholesterol remains unchanged or low, common infection is peritonitis, often with S. pneumoniae.
particularly high-density lipoprotein 2, leading to an Other organisms may be responsible: Escherichia coli, Strepto-
increased low-density lipoprotein to high-density lipopro- coccus bovis, Haemophilus influenzae, and other gram-negative
546 V. Glomerular Disease

TABLE 27.2. INFECTIONS IN NEPHROTIC SYNDROME TABLE 27.4. NEPHROTIC HYPOVOLEMIA

Clinical syndrome Risk factors Clinical features Precipitating factors

Pneumococcal peritonitis Low immunoglobulin G Abdominal pain Severe relapse

Haemophilus infection Low factor B Hypotension Infection
Gram-negative sepsis Edematous tissue Sluggish circulation Diuretics
Staphylococcus cellulitis Impaired lymphocyte function Relative polycythemia Paracentesis
Corticosteroids Acute tubular necrosis Diarrhea
Immunosuppressive drugs Thrombosis

organisms. Apart from peritonitis, children may develop symptoms and may be confirmed by angiography or angio-
meningitis, pneumonitis, or cellulitis. Several factors may scintigraphy. Renal vein thrombosis should be suspected in
explain the propensity of nephrotic children to develop bac- patients with nephrotic syndrome who develop sudden macro-
terial infections: low IgG levels due to an impaired synthesis, scopic hematuria or acute renal failure. In such cases, Doppler
urinary loss of factor B, and impaired T-lymphocyte func- ultrasonography shows an increase in kidney size and the
tion. Factor B is a cofactor of C3b in the alternative pathway absence of blood flow in the renal vein. Thrombosis may also
of complement, which has an important role in opsonization affect the arteries (e.g., pulmonary arteries or other deep veins).
of bacteria such as S. pneumoniae.
Viral infections may be observed in patients receiving cor-
Hypovolemia
ticosteroids or immunosuppressive agents. Varicella is often
observed in these young children and may be life-threatening Hypovolemia is common and typically observed early dur-
if acyclovir therapy is not promptly initiated. Of interest, ing a relapse (Table 27.4). Sepsis, diarrhea, or diuretics may
measles infection may induce long-lasting remissions. precipitate hypovolemia. Hypovolemic children often have
abdominal pain, low blood pressure, and cold extremities.
Hemoconcentration with a raised hematocrit accompanies
Thrombosis
hypovolemia.
Nephrotic patients are at risk of developing thromboembolic
complications (Table 27.3). Several factors contribute to this
RENAL BIOPSY
increased risk of thrombosis: a hypercoagulable state, hypo-
volemia, immobilization, and infection. A number of hemo-
Indications
static abnormalities have been described in nephrotic patients:
an increase in platelet aggregability; an increase in fibrinogen Renal biopsy is not indicated at onset in a child 1 to 8 years
and factors V, VII, VIII, X, and XIII, whereas the levels of anti- of age with typical symptoms of idiopathic nephrotic syn-
thrombin III (heparin cofactor), proteins C and S, and factors drome. Complete remission induced by corticosteroid ther-
XI and XII are decreased; and an increase in fibrinolytic system apy strongly supports the diagnosis. A renal biopsy is
components (e.g., tissue plasminogen activator and plasmino- indicated at disease onset in clinical settings suggesting
gen activator inhibitor-1) (27). The incidence of thromboem- another type of glomerular disease. These include moderate
bolic complications in nephrotic children is reported to be nephrotic syndrome, a long previous course of mild pro-
approximately 3%. However, this percentage may underesti- teinuria, macroscopic hematuria, marked hypertension,
mate the true incidence. In one series, systematic evaluation by and persistent renal insufficiency. A decreased plasma C3
ventilation-perfusion scans showed defects consistent with pul- fraction is also an indication for renal biopsy. Age younger
monary embolism in 28% of all patients with steroid-depen- than 12 months and older than 11 years is another indica-
dent minimal change disease (28). Pulmonary embolism tion, even in patients with a typical clinical picture.
should be suspected in cases with pulmonary or cardiovascular In steroid-dependent patients, the therapeutic approach
is not influenced by histology. Therefore, a renal biopsy is
TABLE 27.3. THROMBOSIS IN
not necessary before initiating a course of alkylating agents
NEPHROTIC SYNDROME but is recommended before starting cyclosporine treatment
to allow assessment of nephrotoxicity on a later biopsy.
Clinical syndrome Risk factors

Pulmonary emboli Hypovolemia

Pulmonary artery thrombosis Hyperviscosity
Pathology
Cerebral venous thrombosis Low antithrombin III Light microscopy shows minimal changes in the majority of
Renal vein thrombosis High fibrinogen
Peripheral venous and artery Platelet hyperaggregability
children with steroid-responsive nephrotic syndrome. FSGS
thrombosis Hyperlipemia occurs in only 5 to 10% of such patients (Fig. 27.1) (29).
Mesangial proliferation is present in a small number of
 27. Steroid-Sensitive Idiopathic Nephrotic Syndrome in Children 547

edly reduced (33). They postulated a reduced sialic acid

content in the GBM, as sialic acid residues are responsible for
glomerular negative charges (34).
Van den Born et al. produced a mouse monoclonal anti-
body to partially purified heparan sulfate proteoglycan isolated
from rat glomeruli (35). By indirect immunofluorescence, the
monoclonal antibody bound to the GBM on rat kidney sec-
tions. By electron microscopy, a diffuse staining of the GBM
was observed. After intravenous injection, the monoclonal
antibody was localized along the GBM with a granular stain-
ing and 1 day later in the mesangium with a concomitant
decrease in staining along the GBM. By electron microscopy, 1
FIGURE 27.1. Responses to steroids in children with idiopathic hour after injection, the antibody was bound mainly to the
nephrotic syndrome according to histology/responders (check-
ered bars) and nonresponders (dotted bars). DMP, diffuse
inner side of the GBM. Intravenous injection of this antibody
mesangial proliferation; FSGS, focal and segmental glomerular in rats resulted in selective proteinuria. This model shows that
sclerosis; MCD, minimal change disease. neutralization of heparan sulfate anionic charges may contrib-
ute to albuminuria.
Levin et al. and Boulton-Jones et al. presented data indi-
patients. Immunofluorescence generally reveals no immune cating that loss of negative charges was not restricted to the
staining, but mesangial deposits of IgM and, more rarely, glomeruli in patients with idiopathic nephrotic syndrome
IgA, IgG, and C3 may be observed. but was also found on erythrocyte and platelet membranes,
A complete description of the histopathology of mini- as shown by reduced binding of Alcian blue, a cationic dye
mal change disease, diffuse mesangial proliferation, focal (36,37). A cationic protein is found in the plasma and the
and global sclerosis, FSGS, and IgM-associated nephropa- urine of patients in relapse.
thy is presented in Chapter 29.
IMMUNE SYSTEM IN STEROID-RESPONSIVE
IDIOPATHIC NEPHROTIC SYNDROME
PATHOPHYSIOLOGY:
MECHANISMS OF PROTEINURIA
Cellular Immunity
In normal individuals, the clearance of albumin is approxi- In 1974, Shalhoub postulated that idiopathic nephrotic syn-
mately 1% of that of neutral proteins with similar molecular drome might be secondary to a disorder of T-lymphocyte
weight (e.g., polyvinylpyrrolidone or dextran). Similarly, the function (38). He hypothesized that clonal expansion of a T-
clearance of neutral dextran is higher than that of anionic lymphocyte subpopulation might result in the production of
sulfate dextran with similar molecular weight. These data lymphokines, which increased the permeability of the glomer-
indicate that the permeability of the glomerular basement ular filtration barrier to proteins. Data supporting this hypoth-
membrane (GBM) is determined not only by the size but esis were the response of the disease to corticosteroids and
also by the charge of the protein. It is believed that the alkylating agents; the remission occurring in association with
anionic charge of the GBM is responsible for the charge measles, which depresses cell-mediated immunity; the suscep-
selectivity of filtration. The anionic (negative) charges of the tibility of patients to pneumococcal infections; and the occur-
GBM repulse negatively charged albumin molecules, which rence of minimal change nephrotic syndrome in patients with
have an isoelectric point of 4.6. Hodgkin’s disease.
The mechanism of proteinuria in the absence of histologic Peripheral blood T-lymphocyte subpopulations have
alterations on light microscopy has suggested an electrochem- been shown to be altered in children during a relapse, with
ical disorder of the GBM. Indeed, it was shown that the glo- an increase in memory T-cell subsets (CD45RO+CD4+ T
merular Kf is diminished despite increased permeability to cells and CD45RO+CD8+ T cells) (39). Increased expres-
serum albumin. Using polyvinylpyrrolidone (30) or dextrans sion of the interleukin (IL)-2 receptor on the T-lymphocyte
(31) with Einstein-Stokes radii between 2.0 and 4.8 nm as surface is found in patients with minimal change disease
test macromolecules, the pore size of the GBM was shown to during relapse but not during remission (40).
be reduced contrasting with massive albuminuria. This sug- Cell-mediated immunity is depressed in patients with
gested a loss of glomerular negative charges. Kitano et al., idiopathic nephrotic syndrome and returns to normal with
using polyethylamine as a cationic probe, reported a decrease remission. Their lymphocytes have an impaired response to
in the anionic charges of the GBM in minimal change disease mitogens (41,42). This decreased response is partly reversible
(32). Carrie et al. studied renal biopsy sections stained by col- when normal human serum is added to the culture medium
loidal iron and showed that its glomerular uptake was mark- instead of autologous serum, suggesting both intrinsic
548 V. Glomerular Disease

defects of the cells and the presence of inhibitory factors in Koyama et al. found a glomerular permeability factor in
the serum (43). Macrophages may be responsible for the the supernatant of a T-cell hybridoma derived from peripheral
impaired response of mononuclear cells to mitogens. The T lymphocytes from a patient with minimal change disease
proliferative response of these cells returns to normal during (53). The supernatant induced proteinuria when injected in
remission. Therefore, a soluble factor may inhibit the prolif- the rat. Histology showed partial fusion of glomerular epithe-
erative response of T lymphocytes. Other abnormalities of lial cell foot processes without immune deposits.
cell-mediated immunity have been described in minimal Several studies have shown increased in vitro production
change nephrotic syndrome. Skin reactivity to common anti- of IL-2, IL-4, and interferon-γ by lymphocytes and high
gens and recall response to common antigens are decreased plasma and urine concentrations of the soluble IL-2 receptor
during relapses and return to normal during remission (44). in patients during a relapse. The soluble IL-2 receptor sup-
Sahali et al. used a subtracted complementary DNA presses lymphocyte proliferation. Patients in relapse have
library screening to identify genes that might be differentially increased CD4+ and CD8+ IL-13 mRNA expression as com-
expressed in patients in relapse compared to those in remis- pared to patients in remission or controls (54). The IL-13
sion (45). They found several upregulated genes, some of and IL-4 cytokines are produced by stimulated Th2 lympho-
which are closely involved in T-cell activation. Relapsing cytes. Activation of Th2 lymphocytes is known to play a key
patients display persistently high levels of nuclear factor-κB role in atopy. Glomerular epithelial cells express IL-4 and IL-
DNA-binding activity and downregulation of IκBα messen- 13 receptors, and both cytokines increase transcellular ion
ger (mRNA), which may account for increased production of transport in cultured cells as well as basolateral secretion of
several cytokines. In contrast, nuclear factor-κB binding lysosomal proteinases (55,56). Such effects of IL-4 and IL-13
activity returns to normal during remission with concomitant may explain an alteration of glomerular permeability.
upregulation of IκBα and downregulation of most of the Increased IL-2 concentrations have been found in superna-
cytokines. An upregulation of c-maf expression and a down- tants of lymphocyte cultures from patients with idiopathic
regulation of IL-12 recepter during relapse may suggest that nephrotic syndrome, and IL-2 can induce proteinuria and a
T-cell activation in patients with minimal change disease reduction of anionic sites of the GBM when injected into the
evolves toward a T-helper-2 phenotype (46). rat kidney. Nephrotic syndrome has been observed in patients
treated with recombinant IL-2 and interferon-α (57,58).
Humoral Immunity Garin et al. reported an increased serum concentration of IL-8
and the presence of its mRNA in mononuclear cells from
Patients with minimal change disease have depressed serum patients with minimal change disease during relapses (59).
IgG levels. This is more pronounced during relapses but per- They also found that IL-8 may affect the metabolism of the
sists during remission (47). Conversely, serum IgM is ele- GBM and postulated that it might alter glomerular permeabil-
vated. Altered serum levels of IgG and IgM may be ity. Serum concentrations of tumor necrosis factor-α and its
secondary to abnormal T-cell regulation of Ig synthesis (48). mRNA in peripheral mononuclear cells from patients with
Factors B and D are decreased during relapses but return to idiopathic nephrotic syndrome are increased in comparison
normal during remission. Specific antibodies to pneumococ- with controls or patients in remission (60).
cal or streptococcal antigens are reduced in these patients up Schnaper and Aune (61–63) reported the presence of a lym-
to 20 years after remission. This suggests that patients with phokine, the soluble immune response suppressor, in the urine
minimal change nephrotic syndrome have a defect of the and serum from patients with steroid-responsive nephrotic syn-
immune response that is not directly related to the nephrotic drome, including minimal change nephrotic syndrome and
state. However, it does not imply that it is pathogenic. other histopathologic forms of nephrotic syndrome. The solu-
ble immune response suppressor is produced by regulatory T
Circulating Factors lymphocytes and inhibits both delayed hypersensitivity reac-
tions and antibody responses. The soluble immune response
Increased production of several lymphokines by activated T
suppressor may contribute to the decreased immune respon-
lymphocytes may alter glomerular permeability to albumin.
siveness of patients with minimal change disease.
Lagrue et al. described a vascular permeability factor, a lym-
phokine found in the supernatant of concanavalin A–acti-
vated lymphocytes from patients with minimal change TREATMENT
disease that enhances vascular permeability when injected in
guinea pigs (49,50). The vascular permeability factor is pro- Symptomatic Treatment
duced by T lymphocytes and is distinct from IL-2.
Diet
Cyclosporine suppresses in vitro vascular permeability factor
production. (51). Tanaka et al. found that supernatants of Diet includes a protein intake of 130 to 140% of the normal
peripheral mononuclear cells stimulated with concanavalin daily allowance according to statural age. Salt restriction is
injected in the renal artery of rats induced proteinuria along necessary for the prevention and treatment of edema. A very
with reduction of the GBM anionic charges (52). low salt diet is necessary in the case of edema. Fluid restric-
 27. Steroid-Sensitive Idiopathic Nephrotic Syndrome in Children 549

tion is recommended for moderate to severe hyponatremia Infections and Immunizations

(plasma sodium concentration less than 125 mmol/L). A
Prophylaxis of S. pneumoniae with oral penicillin is often pre-
reduction of saturated fat is advisable. Carbohydrates are
scribed to children during initial corticosteroid treatment.
given preferentially as starch or dextrin-maltose, avoiding
Although antibody response to pneumococcal vaccine is
sucrose, which increases lipid disturbances.
blunted in children with steroid-responsive nephrotic syn-
drome, vaccination with the conjugated pneumococcal vaccine
Hypovolemia (7vPCV) is recommended (67). In cases of peritonitis, antibi-
otics against both S. pneumoniae and gram-negative organisms
Hypovolemia, a consequence of rapid loss of serum albu-
are started after peritoneal fluid sampling. Varicella is a serious
min, may be aggravated by diuretics. When symptomatic,
disease in patients receiving immunosuppressive treatment or
this complication requires emergency treatment by rapid
daily corticosteroids. Varicella immunity status should therefore
infusion of plasma (20 mL/kg) or albumin 20% (1 g/kg)
be assessed. In the case of exposure of an at-risk patient, acyclo-
administered with monitoring of heart rate, respiratory
vir therapy should be initiated. Immunization with live virus
rate, and blood pressure.
vaccines is contraindicated in immunosuppressed patients.

Diuretics
Specific Treatment
Diuretics should only be used in cases of severe edema, after
hypovolemia has been corrected. Patients with anasarca may Corticosteroid treatment should be initiated in all cases of
be treated with furosemide (1 to 2 mg/kg) or, if necessary, idiopathic nephrotic syndrome regardless of histopathology.
furosemide and salt-poor albumin (1 g/kg infused over 4 The majority of patients is steroid responsive: 89% of chil-
hours) (25). This approach is effective immediately, but it is dren studied by White et al. (68) and 98% of the patients
not long-lasting. Moreover, respiratory distress with conges- with minimal changes on histology. Steroid responders may
tive heart failure has been observed in some patients (64). relapse, but the majority still responds to steroids throughout
Spironolactone (5 to 10 mg/kg) may be effective to minimize the subsequent course. Only 1 to 3% of patients initially ste-
hypokalemia in patients with normal renal function. Diuret- roid sensitive subsequently become steroid resistant and are
ics must be used with caution. They may induce intravascu- defined as late nonresponders (69).
lar volume depletion with a risk of thromboemboli and acute
renal failure as well as severe electrolyte imbalance. Refrac- Initial Treatment
tory edema with serous effusions may require drainage of
ascites or pleural effusions. Head-out immersion has been Steroids should not be started immediately, as spontaneous
reported to be helpful in some cases (65). remission occurs in 5% of cases within the first 8 to 15
days. Some of these early spontaneous remissions are defin-
itive. Infection must be treated before starting steroids, not
Thromboemboli only to prevent the risk of overwhelming sepsis during
Patients with severe hypoalbuminemia are at risk for treatment, but also because occult infection may be respon-
thromboembolic complications. Prevention includes mobi- sible for steroid resistance (3). Patients should have a tuber-
lization, avoiding hemoconcentration, and treating early culin test (i.e., purified protein derivative) before initiation
sepsis or volume depletion. Prophylactic warfarin may be of immunosuppressive therapy.
given to patients with a plasma albumin concentration Glucocorticoid therapy is started when the diagnosis of
below 20 g/L, a fibrinogen level greater than 6 g/L, or an idiopathic nephrotic syndrome is most likely in a child or
antithrombin III level less than 70% of normal. Patients at after renal biopsy has been performed. Prednisone remains
risk may alternatively be treated with low-dose aspirin and the cornerstone of therapy. Prednisolone has the advantage of
dipyridamole, although no controlled trials have been per- being soluble in water, making treatment easier in young
formed to demonstrate their efficacy in preventing throm- children, but it may fail to induce remission in some patients
bosis (66). Heparin is given initially if thrombosis occurs who respond quickly to the same dosage of prednisone. The
alone or with thrombolytic agents. The heparin dose neces- differences in intestinal absorption and drug interactions
sary to obtain a therapeutic effect is often greater than nor- (e.g., with aluminium gels) may explain decreased efficacy in
mal due to decreased antithrombin III levels. some children.
The ISKDC regimen consists of prednisone, 60 mg/m2/
day, with a maximum of 80 mg/day in divided doses for 4
Antihypertensive Drugs
weeks, followed by 40 mg/m2/day with a maximum of 60
Hypertension is treated using beta-blockers or calcium-chan- mg/day in divided doses on 3 consecutive days per week for
nel blockers during acute episodes. In cases of persistent 4 weeks (70). The Arbeitsgemeinschaft für Pädiatrische
hypertension, an angiotensin-converting enzyme inhibitor or Nephrologie showed that an alternate-day regimen (40 mg/
an inhibitor of angiotensin receptor 2 is preferred. m2 every other day for 4 weeks) after initial daily therapy
550 V. Glomerular Disease

resulted in a significantly lower number of patients with Persistent remission for 18 to 24 months after stopping
relapses and fewer relapses per patient (71). It also showed treatment is likely to reflect definitive cure, and the risk of
that on alternate days, prednisone could be given in a single later relapses is low. Ten percent to 20% of patients relapse
dose rather than in divided doses. several months after stopping treatment and are most often
A response occurs in most cases within 10 to 15 days cured after three or four episodes, which respond to a stan-
(median, 11 days). According to the ISKDC data, approxi- dard course of corticosteroids. The remaining 50 to 60%
mately 90% of responders enter remission within 4 weeks experience relapses as soon as steroid therapy is stopped or
after starting glucocorticoids, whereas less than 10% go into when dosage is decreased. In some cases, exacerbation of
remission after 2 to 4 more weeks of daily medication (70). A proteinuria is only transient, and spontaneous remissions
small number of patients go into remission after 8 to 12 are observed (82). The risk of relapse is greater in children
weeks of daily steroids (6,72), but prolongation of daily ste- aged younger than 5 years at onset and in male patients.
roid treatment beyond 4 or 5 weeks increases the risk of side These steroid-dependent patients often raise difficult thera-
effects. An alternative for patients who are not in remission peutic problems.
after 4 weeks is to administer three to four pulses of methyl- Steroid-dependent patients may be treated with repeated
prednisolone (1 g/1.73 m2). This additional regimen seems courses of prednisone, 60 mg/m2/day, continued 3 days
to be associated with fewer side effects than prolongation of after the urine has become protein free, followed by alter-
daily high-dose steroids and probably produces remission nate-day prednisone, 40 mg/m2, for 4 weeks as proposed by
more rapidly in the few patients who would have entered the the ISKDC (70). Another option consists of treating
second month of daily therapy (73). relapses with daily prednisone, 40 to 60 mg/m2, until pro-
The duration of initial steroid therapy influences the risk of teinuria has disappeared for 4 to 5 days. Thereafter, predni-
relapse. The Arbeitsgemeinschaft für Pädiatrische Nephrologie sone is switched to alternate days, and the dosage is tapered
compared a standard regimen of 4-week daily prednisone and 4 to 15 to 20 mg/m2 every other day, according to the steroid
weeks of alternate-day prednisone with a shorter course com- threshold (i.e., the dosage at which the relapse has
prising prednisone given at a dose of 60 mg/m2/day until the occurred). Treatment is then continued for 12 to 18
urine was protein free, followed by alternate-day prednisone months. The first approach allows better definition in
until serum albumin returned to normal (74). Treatment lasted terms of number of relapses but is associated with more
approximately 1 month in children receiving the short course. relapses. The latter regimen is associated with fewer steroid
However, these children had twice the relapse rate so that, at the side effects as the cumulative dosage is lower. Prolonged
end of the trial, they had received an amount of prednisone sig- courses of alternate-day steroid therapy are often well toler-
nificantly greater than had the standard treatment group. ated by young children, and growth velocity is not affected.
After an 8-week steroid regimen, 50 to 70% of children However, prednisone dosage must be as low as possible to
experienced relapses. Several controlled studies have com- minimize side effects. In adolescents, steroid therapy is
pared the 8-week regimen with longer duration of steroid often accompanied by decreased growth velocity.
regimen (3 to 7 months), including 4 to 8 weeks of daily A controlled trial has shown that deflazacort reduces the
prednisone followed by alternate-day prednisone (75–79). risk of relapse in comparison with equivalent doses of pred-
With a follow-up of 2 years, a significant reduction of 25 to nisone without additional side effects (83). However,
30% in the relapse rate was observed with a prednisone regi- deflazacort is not available in all countries.
men of 3 months or more. The role of upper respiratory tract infections in exacerbat-
The number of children with frequent relapses is also ing nephrotic syndrome has been highlighted in all series:
decreased with a longer initial course of prednisone. Longer 71% of relapses were preceded by such an event in a prospec-
duration is more important than the cumulative dose of tive study, although only 45% of respiratory infections were
prednisone in reducing the risk of relapse. This relative risk followed by an exacerbation of proteinuria (84). Mattoo et
decreases by 0.133 (13%) for every additional month of al. found that the risk of relapse was decreased during upper
treatment up to 7 months (80). There are no data showing respiratory tract infections when prednisone was given daily
that treating for more than 7 months is beneficial. However, for 5 days rather than on alternate days (85).
an alternate-day regimen over 1 year did not reduce the rate Leisti et al. suggested a role for postcorticosteroid adre-
of relapse compared to a 5-month alternate-day regimen nal suppression in triggering relapses (86), and some clini-
(81). Although the studies were not designed to analyze the cians have suggested possible prevention by low-dose
side effects of glucocorticoids, the authors did not report maintenance hydrocortisone (87,88) (Fig. 27.2).
increased toxicity with longer duration of treatment.
Alternative Treatments
Treatment of Relapses
Alternative treatment is required in children who relapse on
Approximately 30% of children experience only one attack alternate-day prednisone therapy and experience severe glu-
and are definitively cured after a single course of steroids. cocorticoid side effects (e.g., growth retardation, behavior
 27. Steroid-Sensitive Idiopathic Nephrotic Syndrome in Children 551

FIGURE 27.2. Treatment of idiopathic nephrotic syndrome regardless of histopathology.

disturbances, cushingoid features, hypertension, cataracts, Levamisole given for 6 months was compared with
or osteopenia). Alternative treatment is also indicated in cyclophosphamide given for 8 to 12 weeks in a retrospec-
children at risk of toxicity (e.g., diabetes or during tive study involving 51 children with steroid-dependent
puberty), in children with severe relapses accompanied by nephrotic syndrome (96). The relapse rate and the cumula-
thrombotic complications, or severe hypovolemia and in tive dose of prednisone were reduced to the same extent
those with poor compliance. Alternative treatments include with both drugs.
levamisole, which has a weak steroid sparing effect, and Side effects include neutropenia, agranulocytosis, vomit-
alkylating agents (e.g., cyclophosphamide, chlorambucil, or ing, cutaneous rash, and neurologic symptoms, including
cyclosporine). insomnia, hyperactivity, and seizures. However, levamisole
is well tolerated in most children.
Levamisole
Alkylating Agents
The beneficial effect of levamisole was first described by
Tanphaichitr et al. (89). Levamisole was subsequently Alkylating agents have been used for more than 40 years to
reported to reduce the risk of relapse in steroid-dependent achieve long-lasting remission in patients with idiopathic
patients (90–93). A significant steroid-sparing effect at a nephrotic syndrome.
dose of 2.5 mg/kg every other day was demonstrated in a
prospective controlled trial of the British Association for Cyclophosphamide
Paediatric Nephrology (94). Another controlled study con- The efficacy of cyclophosphamide for preventing relapses
firmed the efficacy of levamisole for preventing relapses of idiopathic nephrotic syndrome was reported more
(95). However, the beneficial effect of levamisole is not sus- than 20 years ago (97) and confirmed in a prospective
tained after stopping treatment. study by Barratt and Soothill, who compared an 8-week
552 V. Glomerular Disease

course of cyclophosphamide to prednisone alone (98). to 3 weeks. Severe microbial and viral infections have
An ISKDC trial found a 48% relapse rate after a mean been reported, including malignant hepatitis and mea-
follow-up of 22 months in children treated with a com- sles encephalitis.
bination of cyclophosphamide and prednisone com- Long-term toxic effects include the risk of developing
pared to a 88% relapse rate in patients on prednisone malignancy, which has been reported in patients with pro-
alone (99). longed therapeutic courses. Gonadal toxicity, as with cyclo-
Several studies have addressed the relationship between phosphamide, is more common in male patients. Azoospermia
dose, duration of treatment, and therapeutic efficacy. Treat- is total and probably irreversible at cumulative doses above 10
ment for 12 weeks at a daily dose of 2 mg/kg was found to to 20 mg/kg. No case of azoospermia was reported in patients
be more effective than an 8-week course, with 67% as com- given less than 8 mg/kg.
pared to 22% remaining in remission after 2 years (100).
However, a randomized trial showed that prolonging the Cyclosporine
course of cyclophosphamide from 8 to 12 weeks did not In a number of uncontrolled studies, cyclosporine has been
further reduce the proportion of children experiencing shown to reduce the incidence of relapses in 75 to 90% of
relapses (101). patients with steroid-dependent idiopathic nephrotic syn-
Cyclophosphamide is less effective in patients with steroid drome (113). However, most patients experience relapses
dependency compared to patients with frequent relapses when the dosage is tapered or when cyclosporine is with-
(102). The incidence of relapse after cyclophosphamide is drawn. Relapse rate usually returns to the pretreatment
significantly higher in patients with FSGS (73%) or mesan- rate. Hulton et al. found that patients in whom cyclospor-
gial proliferation compared to 22% in children with minimal ine had been discontinued and later restarted had more
change disease (103). relapses, requiring steroids in addition to cyclosporine to
Cyclophosphamide toxicity includes bone marrow depres- maintain remission (114).
sion, hemorrhagic cystitis, gastrointestinal disturbances, alope- The effects of cyclosporine have been evaluated in two
cia, and infection. Leukopenia is frequently observed, but comparative trials in steroid-sensitive patients. Cyclospo-
weekly hematologic monitoring may limit its severity, and rine at a dosage of 6 mg/kg/day for 3 months then tapered
concomitant steroids help blunt marrow depression. Hemor- over 3 months was compared with chlorambucil given for
rhagic cystitis rarely occurs. Alopecia, which is variably pro- 2 months. At 12 months, 30% of patients who had received
nounced, remits a few weeks after stopping treatment. Viral chlorambucil and only 5% of those who received cyclo-
infections can be overwhelming if cyclophosphamide is not sporine were still in remission (115). A multicenter ran-
stopped in due time. domized controlled trial compared cyclosporine for 9
Long-term toxicity includes malignancy, pulmonary fibro- months and then tapered over 3 months with oral cyclo-
sis, ovarian fibrosis, and sterility. The risk of sterility is greater phosphamide for 2 months (116). After 2 years, 25% of
in boys than in girls. The cumulative threshold dose above the patients (50% of adults and 20% of children) who
which the risk of oligo- or azoospermia increases is between had received cyclosporine had not relapsed, whereas 63%
150 and 250 mg/kg (104–106). Azoospermia is reversible in of those treated with cyclophosphamide (40% of adults
some patients (107). In girls, the cumulative dose associated and 68% of children) were still in remission. During the
with sterility is greater but not well defined. Pregnancies have year after treatment, the relapse rate (1.8 vs. 0.7) and the
been reported after treatment courses of longer than 18 steroid dosage required (109 vs. 23 mg/kg/yr) were signif-
months (108). icantly higher in children who had received cyclosporine.
Tejani et al. performed a randomized controlled trial
Chlorambucil comparing low-dose prednisone and cyclosporine versus
Beneficial results have also been achieved with chloram- high-dose prednisone for 8 weeks as initial treatment in
bucil in steroid-responsive idiopathic nephrotic syndrome. 28 children (117). Thirteen of the 14 children receiving
In 1973, Grupe demonstrated the efficacy of chlorambucil the combined treatment went into remission compared to
given for 2.5 to 12.0 weeks, with a relapse rate of only 13% only 8 of the 14 receiving prednisone alone (p <.05). The
(109). A controlled trial of chlorambucil for 6 to 12 weeks duration of remission after ending treatment was compa-
showed prolonged remissions of 1 to 3 years (110). Balu- rable in both groups. Ingulli and Tejani reported that
arte et al. obtained similar results in relapsing steroid- severe hypercholesterolemia may inhibit cyclosporine effi-
responsive nephrosis (111). Williams et al. showed that low cacy and that patients with severe hyperlipidemia require
daily doses are preferable: 91% of patients on a dose of 0.3 higher doses for maximal effectiveness (118).
mg/kg and 80% of those on 3 mg/kg were still in remission Considering the high rate of cyclosporine dependency as
4 years later (112). well as its nephrotoxicity, steroid-dependent patients should
Acute toxic effects are less frequent with chloram- first be treated with alkylating agents before resorting to
bucil than with cyclophosphamide. Leukopenia and cyclosporine (113,119). In patients being treated with cyclo-
thrombocytopenia may occur and are reversible within 1 sporine, it is often difficult to determine if any observed
 27. Steroid-Sensitive Idiopathic Nephrotic Syndrome in Children 553

changes in renal function are transient or due to the nephro- Fakhouri et al. recently reported on the outcome in
toxic effect of the drug. In these cases, it is advisable to adulthood of 102 patients born between 1970 and 1975
reduce dosage or even stop cyclosporine treatment. Lesions (129). Forty-two percent presented at least one relapse in
of chronic cyclosporine-induced nephrotoxicity can develop adulthood. A young age at onset and a high number of
without any appreciable decline of the GFR (120,121). As it relapses during childhood were associated with a risk of
is often necessary to use long therapeutic courses of cyclo- relapse in adulthood.
sporine, repeat renal biopsies are advised to detect lesions.
They most often consist of tubulointerstitial injury, charac-
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syndrome. J Pediatr 1973;82:598–604. 1982;57:544–548.
110. Grupe WE, Makker SP, Ingelfinger JR. Chlorambucil treat- 129. Fakhouri F, Bocquet N, Taupin P, et al. Steroid sensitive
ment of frequently relapsing nephrotic syndrome. N Engl J nephrotic syndrome: from childhood to adulthood. Am J
Med 1976;295:746–749. Kidney Dis 2003;41:550–557.
 28

STEROID-RESISTANT IDIOPATHIC
NEPHROTIC SYNDROME IN CHILDREN
PATRICK NIAUDET

Idiopathic nephrotic syndrome is defined by the combination tologic variants of the idiopathic nephrotic syndrome carry
of nephrotic syndrome (proteinuria, hypoalbuminemia, prognostic significance, they cannot at present be considered
hyperlipidemia, and edema) with nonspecific histologic as separate entities (13).
abnormalities of the kidney, including minimal changes, The term MCD has become synonymous with steroid-
focal segmental glomerulosclerosis (FSGS), and diffuse sensitive idiopathic nephrotic syndrome, although renal biopsy
mesangial proliferation. Glomeruli show a fusion of epithe- is usually not performed in patients who respond to steroid
lial cell foot processes on electron microscopy and no sig- therapy. Indeed, in many centers, renal biopsy is recom-
nificant deposits of immunoglobulins (Igs) or complement mended only for those patients who do not respond to ster-
on immunofluorescence. oids. Consequently, renal biopsy findings in recent published
Many authors consider minimal change disease (MCD), series are not representative of the true incidence of various his-
diffuse mesangial proliferation, and FSGS as separate diseases topathologic categories seen in idiopathic nephrotic syndrome.
because of differences in response to corticosteroids and sub- It is, therefore, more appropriate to classify the patients accord-
sequent clinical course (1). Indeed, these various pathologic ing to their response to steroid therapy. Response to glucocor-
features carry prognostic significance. Patients with FSGS ticoid therapy has greater prognostic value than the histologic
and those with diffuse mesangial proliferation more fre- features seen on initial renal biopsy. Thus, two types of neph-
quently have hematuria, are often resistant to corticosteroid rosis can be defined: steroid-responsive (see Chapter 27) and
treatment, progress more often to renal failure, and may have steroid-resistant nephrotic syndrome (SRNS).
a recurrence of the nephrotic syndrome soon after transplan- In this chapter, we focus on the 10% of children with idio-
tation. Recent data demonstrate differences between the pathic nephrotic syndrome who do not respond to corticoste-
basic pathophysiology of FSGS and MCD. FSGS appears to roids and are at risk for extrarenal complications of nephrosis
be a podocyte disease (2). The notion of podocyte dysregula- as well as the development of end-stage renal disease (ESRD).
tion (3–5), the different expression of cyclin-dependent Steroid-resistant idiopathic nephrotic syndrome accounts for
kinase inhibitors in MCD and in FSGS, the role of these cell more than 10% of children who progress to ESRD.
cycle disturbances leading to podocyte proliferation and mat- SRNS is a heterogeneous entity. In some patients, particu-
uration (6), and the identification of parvovirus B19 in glo- larly those who experience a recurrence after renal transplan-
meruli of patients with FSGS (7,8) support the separation of tation, the disease is probably immunologically mediated by
minimal change nephrotic syndrome and FSGS as distinct a cytokine that increases the permeability of the glomerular
entities. Moreover, Streulau et al. found transforming growth basement membrane (GBM) to proteins. In other cases (e.g.,
factor β1 (TGF-β1) gene expression in 18 of 20 patients familial cases), the disease is related to a primary defect of the
with steroid-resistant FSGS and in only 3 of 14 steroid-sensi- filtration barrier. Recent studies have highlighted the roles of
tive patients (9). These data support a sequence of specific newly identified podocyte proteins and demonstrated that
immunologically mediated events that contribute to progres- mutations of corresponding genes may be responsible for
sive renal damage in children with FSGS. SRNS (14).
In the early stages, FSGS and minimal change nephropa-
thy are indistinguishable (10). A significant number of
patients with FSGS respond favorably to glucocorticoid ther- CLINICAL AND BIOLOGIC FEATURES
apy (11), whereas some steroid-resistant patients have no
sclerotic changes on systematic histopathologic evaluation SRNS may occur during the first year of life, but it usually
(11,12). Therefore, some authors believe that, although his- starts between 2 and 7 years of age, with a male to female
558 V. Glomerular Disease

ratio of 2:1 (15). The disease is characterized by sudden The urine sediment of patients with idiopathic neph-
onset, with edema being the major presenting symptom. rotic syndrome often contains fat bodies. Hyaline casts are
Edema becomes clinically detectable when fluid retention also usually found in patients with massive proteinuria, but
exceeds 3 to 5% of body weight. Edema is gravity depen- granular casts are not present unless there is associated
dent and localized to the lower extremities in the upright acute renal failure and acute tubular necrosis. Macroscopic
position and to the dorsal part of the body in the reclining hematuria is rare, occurring in 3% of patients. Microscopic
position. Edema is white, soft, and pitting, retaining the hematuria is far more common and may be observed in up
marks of clothes or finger pressure. Anasarca may develop to two-thirds of patients, particularly those with FSGS.
with ascites and pleural and pericardial effusions. Although Urinary sodium excretion is low (less than 5 mmol per
there may also be abdominal distention, dyspnea is rare. 24 hours), associated with sodium retention and edema.
Periorbital edema may limit eye opening, and edema of the Kaliuresis is usually higher than natriuresis but may be
scrotum and penis or labia may be seen. Blood pressure is reduced in oliguric patients.
usually normal but is sometimes elevated. Rapid formation
of ascites is often associated with abdominal pain and mal-
Blood Chemistry
aise, but these symptoms may also be related to concomi-
tant hypovolemia. Abdominal pain is occasionally due to a Serum proteins are markedly reduced, and serum lipid usu-
complication such as peritonitis, thrombosis, or, rarely, ally increased. Plasma protein levels are below 50 g/L in
pancreatitis. Cardiovascular shock is not unusual, secon- 80% of patients and below 40 g/L in 40% (17). Albumin
dary to the sudden fall of plasma albumin, with abdominal concentration usually falls below 20 g/L and may be less
pain and symptoms of peripheral circulatory failure with than 10 g/L. Electrophoresis shows not only low albumin
cold extremities and hypotension. levels but also increased α2-globulins and, to a lesser extent,
The nephrotic syndrome is occasionally discovered dur- β-globulins, whereas γ-globulins are decreased. IgG is mark-
ing a routine urine analysis. Macroscopic hematuria is edly decreased, IgA is slightly reduced, IgM is increased,
observed in a few cases. SRNS may present with an infec- while IgE is normal or increased. Among other proteins,
tious or thrombotic complication. fibrinogen and β-lipoproteins are increased and antithrom-
bin III is decreased.
Hyperlipidemia is a consequence of (a) an increased
Proteinuria during the Urinalysis
hepatic synthesis of cholesterol, triglycerides, and lipo-
Nephrotic range proteinuria is defined as urinary protein excre- proteins; (b) a decreased catabolism of lipoproteins due to
tion greater than 50 mg/kg/day or 40 mg/m2/hr mean value. a decreased activity of lipoprotein lipase that normally
Proteinuria during the first days may be higher, as the urinary transforms very-low-density lipoprotein to low-density
concentration of proteins also depends on the plasma albu- lipoprotein (LDL) via intermediate-density lipoprotein;
min concentration. In young children, it may be difficult to and (c) decreased LDL receptor activity and an increased
perform accurate 24-hour urine collections, and urinary pro- urinary loss of high-density lipoprotein (HDL) (18).
tein to creatinine ratio or urine albumin to urine creatinine Total cholesterol and LDL cholesterol are elevated, whereas
ratio in untimed urine specimens is useful. For these two indi- HDL cholesterol remains unchanged or low, particularly
ces, the nephrotic range is 200 to 400 mg/mmol (16). HDL2, leading to an increased LDL to HDL cholesterol
The amount of protein excreted in the urine does not ratio. Patients with severe hypoalbuminemia have increased
reflect the quantity of protein crossing the GBM because a triglycerides and very-low-density lipoprotein. Apopro-
significant amount is reabsorbed in the proximal tubule. teins and apolipoproteins B, CII, and CIII are also ele-
Typically, in severe nephrotic syndrome with glomerular vated. The levels of lipoprotein(a) are elevated in nephrotic
lesions and resistance to steroid treatment, the urine con- patients, which further contributes to an increased risk of
tains not only albumin but also higher-molecular-weight cardiovascular and thrombotic complications.
proteins. This can be seen on polyacrylamide gel electro- Serum electrolytes are usually within the normal range.
phoresis and can also be quantified by means of the selec- A low sodium level may be related to dilution from inap-
tivity index. The selectivity index is the ratio of IgG propriate renal retention of water due to hypovolemia and
(molecular weight, 150 kDa) to albumin (70 kDa) or trans- inappropriate antidiuretic hormone secretion. A mild
ferrin (80 kDa) clearances. A selectivity index above 0.15 or reduction of plasma sodium concentration may be an arti-
0.20 is frequently observed in SRNS. However, the test is fact related to hyperlipidemia depending on reference labo-
of limited clinical value because of its poor specificity. Some ratory methodology. Serum potassium may be high in
children with SRNS and tubulointerstitial lesions have oliguric patients. Serum calcium is consistently low as a
both glomerular and tubular proteinuria with an increased result of hypoproteinemia. Ionized calcium is usually nor-
excretion of β2-microglobulin, retinol-binding protein, and mal but may be decreased due to urinary loss of 25-hydroxy-
lysozyme due to impaired protein reabsorption in the prox- vitamin D3 (19) and normal but inappropriate levels of
imal tubule. calcitriol (20). Blood urea nitrogen and creatinine concen-
 28. Steroid-Resistant Idiopathic Nephrotic Syndrome in Children 559

trations are usually within the normal range or slightly Pediatric Nephrology Study Group (27) reported 75 chil-
increased, reflecting a modest reduction in glomerular fil- dren with FSGS followed for periods of 7 to 217 months.
tration rate (GFR). Twenty-one percent had progressed to ESRF, 23% had
A few patients with FSGS and a poor subsequent out- decreased GFR, 37% had a persistent nephrotic syndrome,
come present with a Fanconi syndrome: glycosuria, ami- and 11% were in remission.
noaciduria, urinary bicarbonate loss, and hypokalemia (21). Progression to ESRF has been reported to be more rapid
A defect in urinary acidification has also been reported (22). in patients of African or Hispanic descent when compared
with whites. Ingulli and Tejani found that among 57 black
and Mexican American children, 50% of them reached
Hematology
ESRF in 3 years, and 95% reached this stage after 6 years
Hemoglobin levels and hematocrit are increased in patients (28). In addition, among the children with idiopathic
with plasma volume contraction. Anemia with microcytosis nephrotic syndrome, the proportion of those with steroid-
may be observed and is probably related to urinary loss of resistant FSGS was increased in black and Mexican Ameri-
siderophilin. Thrombocytosis is common and may reach 5 × can children.
108 or 109/L.
Growth
COMPLICATIONS
Growth may be severely affected in children with persistent
nephrotic syndrome. Hypothyroidism related to urinary
Acute Renal Failure
loss of iodinated proteins has been observed and may be
Renal function is usually within normal limits at presenta- treated. Low plasma IgF1 and IgF2 levels associated with
tion. A reduction of the GFR, secondary to hypovolemia, urinary loss of carrier proteins have also been reported (29).
is frequent (23). A reduced GFR may be found in patients The response of such patients to growth hormone therapy
with normal effective plasma flow (24,25). Bohman et al. has not been studied.
showed a close relationship between the degree of foot-
process fusion and both GFR and filtration fraction, suggest-
Infections
ing that fusion of foot processes could lead to a reduction
of glomerular filtering area or of permeability to water Bacterial infections are frequent in nephrotic children. Sepsis
and small solutes (26). may occur at the onset of the disease. The most common
Acute renal failure may be secondary to bilateral renal vein infection is peritonitis, often with Streptococcus pneumoniae
thrombosis. Interstitial nephritis is another possible cause of (30). Other organisms may be responsible: Escherichia coli,
acute renal failure, especially in association with furosemide Streptococcus bovis, Haemophilus influenzae, and other gram-
administration. negative organisms. Apart from peritonitis, children may
develop meningitis, pneumonitis, and cellulitis. Several fac-
tors may explain the propensity of nephrotic children to
Chronic Renal Failure
develop bacterial infections: low IgG levels due to an
Patients with SRNS are at major risk of developing end-stage impaired synthesis, urinary loss of factor B, and impaired T-
renal failure (ESRF), which is seen in less than 3% of lymphocyte function. Factor B is a cofactor of C3b of the
patients with idiopathic nephritic syndrome who respond to alternative pathway of a complement that has an important
glucocorticoid therapy. ESRD develops in at least 50% of role in opsonization of bacteria, such as S. pneumoniae.
patients with SRNS. As patients develop ESRD, all features
of their nephritic syndrome may improve because a decrease
Thrombosis
in urinary protein excretion parallels their decreased GFR.
We have retrospectively analyzed in the Enfants Malades Nephrotic patients are at risk of developing thromboembolic
series the outcome of 181 children with SRNS who have complications. Several factors contribute to the increased risk
been followed for at least 5 years. Eighty-five percent were of thrombosis: hypercoagulability state, hypovolemia, immo-
primary nonresponders, 15% were late nonresponders, and bilization, and infection. A number of hemostatic abnormali-
13% had a sibling affected by the same disease. Initial renal ties have been described in nephrotic patients: (a) increased
biopsy had shown MCD in 62 cases and FSGS in 119 platelet aggregability; fibrinogen; and factors V, VII, VIII, X,
cases. Renal survival rates were 65% at 5 years, 50% at 10 and XIII; and (b) decreased levels of antithrombin III, heparin
years, and 34% at 15 years. It is interesting to note that the cofactor, proteins C and S, and factors XI and XII. Further-
rate of progression to ESRF was similar in patients with more, fibrinolytic system components (e.g., tissue plasmino-
minimal changes or FSGS on initial biopsy. gen activator, plasminogen activator inhibitor-1) are increased
Data reported in other series are difficult to compare, as (31). The incidence of thromboembolic complications in
most of them deal with patients with FSGS. The Southwest nephrotic children is close to 3%. However, this percentage
560 V. Glomerular Disease

may be underestimated as shown by systematic ventilation-

perfusion scans, which have demonstrated defects consistent
with pulmonary embolism in 28% of patients with steroid-
dependent idiopathic nephrotic syndrome (32). Pulmonary
embolism should be suspected in patients with SRNS who
present with acute pulmonary or cardiovascular symptoms.
The diagnosis may be confirmed by angiography or angioscin-
tigraphy. Renal vein thrombosis should be suspected in cases
with sudden macroscopic hematuria or acute renal failure.
Doppler ultrasonography shows an increase in kidney size and
the absence of blood flow in the renal vein. Thrombosis may
affect the arteries (e.g., pulmonary arteries) or other deep veins.

RENAL BIOPSY

Light microscopic analysis of kidneys from patients with FIGURE 28.1. Diffuse mesangial proliferation without sclerosis.
SRNS demonstrates three major morphologic patterns: min- Light microscopy with trichrome light green (×400).
imal changes, diffuse mesangial proliferation, and FSGS.

Minimal Change Nephropathy Focal and Segmental Glomerular Sclerosis

On light microscopy, glomeruli may be normal with nor- The glomerular lesions affect a variable proportion of
mal capillary walls and normal cellularity. Swelling and vac- glomeruli (11,17,34). The focal changes are limited to a
uolation of epithelial cells and a slight increase in mesangial part of the tuft, with the other capillary loops showing no
matrix are often observed. Mild mesangial hypercellularity modification. The lesions always predominate at the cor-
may be noted (17,33,34) as well as scattered foci of tubular ticomedullary junction (39). The segmental lesion affects
lesions and interstitial fibrosis. a few capillary loops that stick together either at the
Ultrastructural changes are always present, involving hilum or at the periphery of the tuft (Fig. 28.2) (40,41).
podocytes and mesangial stalks. Podocyte foot-process The clinical course has been found to be more benign
fusion is generalized and constant; its extent is closely when the location of these sclerotic lesions is peripheral
related to the degree of proteinuria (35). Other epithelial (the tip lesion), although such findings have not been
changes consist of microvillus formation and the pres- confirmed by other authors (42,43). Hyaline material is
ence of numerous protein reabsorption droplets. The often present within the sclerotic lesions. A clear halo
GBMs are normal, with no parietal deposits. The endo- zone is observed at the periphery of the sclerotic seg-
thelial cells are often swollen (36). Mesangial alterations ments. The segmental lesion has a different aspect
include mesangial cell hyperactivity, increased mesangial depending on whether it affects a group of capillary loops
matrix, and occasionally finely granular, osmiophilic free in Bowman’s space or is adherent to Bowman’s cap-
deposits located along the internal side of the basement
membrane. These ultrastructural alterations are nonspe-
cific and are probably related to massive proteinuria.

Diffuse Mesangial Proliferation

Some patients with steroid-resistant idiopathic nephrotic
syndrome show a marked increase in mesangial matrix
associated with hypercellularity (Fig. 28.1) (17,23,34,
37). However, peripheral capillary walls are normal, and
immunofluorescence microscopy is negative. Electron micros-
copy shows foot-process fusion similar to the changes
observed in MCD. The presence of mesangial hypercel-
lularity has been reported to have prognostic significance
with a higher rate of progression to renal failure (37),
FIGURE 28.2. Focal and segmental glomerular sclerosis. Oblit-
but these findings have not been confirmed by other eration of capillary lumens by a combination of sclerosis and
authors (27,38). hyalinosis. Light microscopy with trichrome light green (×280).
 28. Steroid-Resistant Idiopathic Nephrotic Syndrome in Children 561

ment of epithelial cells from basement membranes, with

filling of the resulting space by cell debris and new mem-
branes (49).
A subgroup of patients has collapsing focal segmental
glomerular sclerosis characterized by a global collapse of
the glomerular capillaries with marked hypertrophy of
epithelial cells (Fig. 28.4) (50). These patients rapidly
progress to renal failure. Such features closely resemble
those observed in human immunodeficiency virus–associ-
ated nephropathy (51). Many authors consider collapsing
glomerulopathy to be a distinct form of FSGS that may
be idiopathic, observed in renal transplants of patients with
recurrent nephrotic syndrome, or associated with human
immunodeficiency virus or parvovirus infection (52,53).
FIGURE 28.3. Focal and segmental glomerulosclerosis. Segmen- Idiopathic collapsing glomerulopathy predominates in
tal lesion of the tuft is characterized by the obliteration of capil- blacks and has a poor prognosis (54).
lary lumens by granular and lipid deposits. Multilayered
basement membrane material is seen around the lesion. Note Recent studies on collapsing glomerulopathy and on
the presence of focal detachment of podocytes along the adja- patients with recurrence of nephrotic syndrome after renal
cent capillary lumens. Electron microscopy with silver impregna- transplantation have shown important changes in the
tion (×3900).
podocytes with major cell cycle derangement (4,5). The
normal, mature podocyte does not divide and does not
express proliferative markers (e.g., proliferating cell nuclear
sule. The free sclerotic segments are always surrounded antigen and Ki-67). The podocyte expresses several cell-
by a crown of flat or hypertrophied podocytes. The surface proteins (e.g., WT-1, C3b receptor, glomerular
podocytes form a continuous layer overlying the dam- epithelial protein-1, podocalyxin, synaptopodin, and vimen-
aged areas of the tuft and in close apposition to the clear tin). The first stages of FSGS are characterized by the loss
halo. When the sclerotic lesion is adherent to Bowman’s of cell surface proteins and the expression of macrophage
capsule, there is a direct synechia between the collapsed markers and cytokeratin. Proliferation markers (prolifer-
capillary loops and Bowman’s basement membrane. The ating cell nuclear antigen and Ki-67) are expressed. This
rest of the tuft and the nonsclerotic glomeruli show “podocyte dysregulation” is accompanied by podocyte
either minimal changes or diffuse mesangial prolifera- detachment from the GBM.
tion, both with foot-process fusion. Glomerular hyper- FSGS is an irreversible scarring process in the glomer-
trophy is common in FSGS; when such hypertrophy is uli, as shown by the analysis of repeat biopsies (47,48,55).
found in MCD, it is somewhat predictive of further Studies in experimental animals (56) as well as in neph-
development to FSGS (44,45). rotic patients have shown that proteinuria precedes the
Tubular atrophy and interstitial fibrosis are often present development of focal sclerotic lesions. The same sequence
and apparently proportional to the glomerular damage was reported in patients with recurrence of the disease
(11,46). Focal glomerular lesions should therefore be sus-
pected when focal tubular and interstitial changes are
found associated with minimal glomerular changes. On
immunofluorescence, the segmental lesions show strong
staining with anti-IgM and anti-C3 antisera.
By electron microscopy, the lesion is characterized by
the presence of paramesangial and subendothelial, finely
granular, osmiophilic deposits (46–48), with either dis-
appearance or swelling of endothelial cells, and an
increase in mesangial matrix material (Fig. 28.3). Fatty
vacuoles may be seen in the middle of the abnormal
deposit or in the cytoplasm of endothelial and mesangial
cells. The peripheral synechia, located between podocytes
and basement membrane, is formed by the apposition of
acellular material in which thin and irregular layers of
newly formed basement membranes are visible. Modifi-
FIGURE 28.4. Collapsing glomerulopathy. Early glomerular
cations of the podocytes consist of focal cytoplasmic lesion characterized by the irregular collapse of capillary loops.
degeneration, breakdown of cell membranes, and detach- Light microscopy with periodic acid-Schiff (×320).
562 V. Glomerular Disease

three main histologic patterns. Some patients with clear

evidence of minimal changes on initial biopsy have FSGS
on a second biopsy. A high proportion of these patients
are steroid resistant. Tejani found that FSGS lesions had
developed in 60% of 48 patients with steroid-resistant
MCD, in association with aggravation of symptoms (61).
The progression to sclerosis may occur from minimal
change or from mesangial proliferation (37,62). Con-
versely, some patients who show diffuse mesangial prolif-
eration, regardless of whether it is associated with focal
sclerosis on initial biopsy, may demonstrate minimal
change or FSGS on repeat biopsy (27).
In conclusion, it may be considered that, at least in chil-
FIGURE 28.5. Idiopathic nephrotic syndrome with minimal dren, MCD, FSGS, and diffuse mesangial proliferation
changes. Diffuse mesangial deposits of immunoglobulin M.
Immunofluorescence (×300).
represent histologic variations of idiopathic nephrotic syn-
drome that may be found alone or in any combination on
sequential biopsies in the same patient.
after transplantation. Within weeks after recurrence of
proteinuria, podocytes appear swollen and vacuolated by
CLINICOPATHOLOGIC CORRELATIONS
electron microscopy. The podocytes exhibit strong mitotic
activity, with multinucleation and expression of the pro-
The relative frequencies of the three histologic patterns
liferating cell nuclear antigen and Ki-67 proliferation
differ in steroid-sensitive and steroid-resistant patients.
markers.
A report of the International Study of Kidney Disease in
FSGS is not a specific histopathologic lesion: Similar
Children showed that among 354 patients with idio-
alterations may be seen in persistent idiopathic proteinuria;
pathic nephrotic syndrome who had an initial response
heroin-associated nephropathy; and, independently, in asso-
to prednisone, 95.5% had MCD, 3.0% had FSGS, and
ciation with acquired immunodeficiency syndrome, Alport’s
1.5% had diffuse mesangial proliferation. Conversely,
syndrome, hypertension, pyelonephritis, and obesity. FSGS
among 55 patients who did not respond to prednisone,
has also been reported in renal hypoplasia with oligomega-
45.5% had MCD, 47.5% had FSGS, and 7.0% had dif-
nephronia, after partial nephrectomy, and in other condi-
fuse mesangial proliferation (63). This study also ana-
tions associated with a reduction in nephron number,
lyzed the numbers of responders and nonresponders
including reflux nephropathy and obstructive uropathy.
within each histologic category. Among the 363 patients
with minimal change, 193.1% were responders and
Immunoglobulin M–Associated 6.9% nonresponders, whereas among the 37 patients
Nephropathy with FSGS, 29.7% responded to prednisone and 70.3%
did not. Waldherr et al. found that only 2 out of 36
Immunofluorescence microscopy is usually negative (57,58).
patients with diffuse mesangial proliferation responded
However, mesangial deposits of IgM, IgG, C3, and, more
to corticosteroids (37).
rarely, IgA have been reported (Fig. 28.5). Cohen et al. found
that patients with IgM deposits in the mesangium had a
poor response to corticosteroids (59). Other studies have not PATHOPHYSIOLOGY
confirmed these findings. Habib et al. reported on immuno-
fluorescence studies in a series of 222 children with idio- In 1974, Shalhoub postulated that lymphokines may be
pathic nephrotic syndrome (60). Although IgM was the Ig responsible for the increased permeability of the GBM
most frequently present in the glomeruli (54 of 222), there (64). Lagrue et al. first described the vascular permeability
was no correlation between IgM deposits, the initial response factor (VPF), a lymphokine found in the supernatant of
to steroid therapy, or the final outcome. IgM deposits have concanavalin A–activated lymphocytes from patients with
also been described in association with diffuse mesangial pro- MCD that enhances vascular permeability when injected
liferation and with FSGS. intradermally into the guinea pigs (65,66). Heslan et al.
showed that VPF was produced by T lymphocytes and
was distinct from interleukin (IL)-2 (67,68). Maruyama
Relationship between the Different
et al. showed that cyclosporine at concentrations ranging
Histologic Patterns
from 100 to 250 ng/mL was able to suppress the in vitro
Repeat renal biopsies in patients with idiopathic nephrotic production of VPF by mononuclear cells from patients
syndrome have shown morphologic transition between the with MCD (69). VPF was also found in other diseases,
 28. Steroid-Resistant Idiopathic Nephrotic Syndrome in Children 563

such as IgA nephropathy. Tanaka et al. found that the et al. found that the nephrotic syndrome recurs when
supernatants of concanavalin A–activated lymphocytes Buffalo/Mna rats receive a kidney from a healthy
from patients with MCD or FSGS induced a marked pro- LEW.1W rat (80). Conversely, proteinuria and renal
teinuria when injected in the renal artery of rats together lesions regress when kidneys from a Buffalo/Mna rat are
with a reduction of the anionic charges of the GBM (70). transplanted into normal LEW.1W rats. Although recur-
Boulton-Jones et al. infused lymphocyte culture superna- rence of proteinuria is not immediate after transplanta-
tants into the renal artery of rats and found reduced col- tion, this model may provide new insights into the
loidal iron staining in the kidneys, suggesting a loss of pathogenesis of FSGS and the mechanisms of recurrence
negative charges of the GBM (71). Similarly, Wilkinson et after transplantation in humans.
al. showed that the infusion of plasma from nephrotic Two additional observations indicate that circulating
patients to rabbits induced proteinuria and a reduction in factors, rather than innate kidney defects, render the
the number of anionic sites on the GBM (72). GBM more permeable to proteins: Lagrue et al. reported
Koyama et al. described a glomerular permeability the case of a woman with steroid-resistant FSGS who gave
factor in the supernatant of T-cell hybridoma derived birth on two occasions to children who were proteinuric
from the fusion between peripheral T lymphocytes of a and hypoalbuminemic at birth. In both cases, proteinuria
patient with MCD and a T-cell line, CCRF-HSB2 (73). and nephrotic syndrome disappeared within 2 and 3
The glomerular permeability factor was identified by the weeks, respectively (81). The second observation concerns
ability of the supernatant to induce a proteinuria when the report of a 20-year-old man with MCD who died of
injected intravenously (IV) in the rat. In addition, rats cerebral hemorrhage and whose kidneys were transplanted
injected with the supernatants show a partial fusion of into two recipients whose primary renal disease was not
foot processes of glomerular epithelial cells and no idiopathic nephrotic syndrome. In both cases, proteinuria
immune deposits. Glomerular permeability factor is dif- and all features of MCD disappeared within 6 weeks of
ferent from the other known lymphokines. Its molecular transplantation (82).
weight is between 60,000 and 160,000. The pathogenesis of glomerulosclerosis is still unknown
Other lymphokines may play a pathogenic role in (83). Several factors, including hemodynamic factors,
SRNS (74). Increased IL-2 levels have been found in lym- cytokines and growth factors, hyperlipidemia, and plate-
phocyte culture supernatants from patients with idio- let activation, lead to an increase of mesangial matrix
pathic nephrosis, and IL-2 can induce proteinuria and a production by resident cells. Patients with lesions of
reduction of the anionic sites of the GBM when injected FSGS have a significant glomerular hypertrophy. Experi-
into the rat kidney (68,75). A nephrotic syndrome has mental and clinical data demonstrate that abnormal glo-
been described in several patients treated with recombi- merular growth is associated with glomerular sclerosis.
nant IL-2 and interferon-α. Growth factors involved in the process include platelet-
The rapid recurrence of proteinuria after renal trans- derived growth factor, TFG-β, angiotensin II, throm-
plantation in 30% of patients with SRNS is a strong boxane A2, macromolecule deposition, coagulation factors,
argument for the presence of a circulating factor that and lipids (84–86). It is interesting to note that angio-
increases the permeability of the GBM. Savin et al. iden- tensin regulates the expression of platelet-derived growth
tified a serum factor in some patients with FSGS that factor and TFG-β. This may explain the fact that angio-
increases the albumin permeability of isolated rat glo- tensin-converting enzyme inhibitors may prevent the
meruli. The presence of the factor was strongly predic- decline of renal function in nephrotic patients.
tive of the recurrence of proteinuria after renal
transplantation (76). A VPF, which induces proteinuria
Genetic Factors
when injected into the renal artery of rats, was found in
the serum from a transplanted patient who had recur- In recent years, there have been several reports on the
rence of the nephrotic syndrome (77). However, subse- molecular basis of familial cases of idiopathic nephrotic
quent attempts to isolate such a factor have failed. syndrome with FSGS. Moreover, the discovery of
Dantal et al. treated patients who had recurrent neph- molecular defects leading to FSGS has provided impor-
rotic syndrome with plasma protein A adsorption (78). tant insight in the critical role of newly identified
The administration to rats of material eluted from the podocyte proteins in the glomerular filtration barrier to
protein A columns increased urinary albumin excretion. proteins.
The active fraction had a molecular weight below Mathis et al. performed linkage analysis in a large
100,000. The factor or factors that may be responsible family in which FSGS followed an autosomal dominant
for recurrent nephrotic syndrome after transplantation mode of inheritance (87). Some patients developed
appear to be bound to an Ig (79). severe proteinuria and ESRD by the fourth decade,
The Buffalo/Mna strain of rats spontaneously devel- whereas others had only mild proteinuria. The responsi-
ops proteinuria with FSGS at 2 months of age. Le Berre ble gene, located on chromosome 19q13, encodes α-
564 V. Glomerular Disease

actinin-4, an actin-filament cross-linking protein (88). by exon 5 and the other to three amino acids [lysine-threo-
Mutations in this gene were detected in affected patients nine-serine (KTS)] encoded by the 3' end of exon 9—lead
(88). In vitro, mutant α-actinin-4 binds filamentous to the synthesis of four isoforms with definite and stable
actin more strongly than does wild-type α-actinin-4. proportions and different functions. The target genes
Regulation of the actin cytoskeleton of glomerular potentially regulated, most often negatively, by WT-1
podocytes may be altered in this subpopulation of FSGS include genes coding for transcription factors (e.g., PAX2,
patients. PAX8, NovH, and WT-1) and for growth factors or their
Another family with autosomal dominant FSGS was receptors (insulin-like growth factor-2, insulin-like growth
studied by linkage analysis (89,90). The disease was diag- factor-R, platelet-derived growth factor-A, TGF-β, and
nosed during the third decade of life, with high-grade EGFR) (98). WT-1 is strongly expressed during embryo-
proteinuria progressing to ESRD in a relatively high per- fetal life (99). In the mature kidney, WT-1 expression per-
centage of cases. The authors detected a linkage to chro- sists only in podocytes and epithelial cells of Bowman’s cap-
mosome 11q21-q22 with a maximum lod score of 9.89. sule. WT-1 gene disruption in mice results in the absence
Winn et al. described another large family with autosomal of kidneys and gonads, suggesting a key role of WT-1 in the
dominant FSGS but no linkage to chromosome 11q21 or maturation of the genitourinary tract.
19q13 (90). Frasier syndrome is characterized by male pseudoher-
Fuchshuber et al. mapped a locus to chromosome 1q25- maphroditism and progressive glomerulopathy (100). Pro-
q31 for autosomal recessive SRNS characterized by an teinuria is discovered during childhood, usually between 2
early-onset steroid resistance, a rapid progression to renal and 6 years of age or later. It increases progressively with
failure, and the absence of recurrence after transplantation time and does not respond to corticosteroids or to immu-
(91). Using a positional cloning approach, Boute et al. nosuppressive agents. The disease runs a slow, progressive
identified the causative gene, NPHS2, which is expressed course to ESRD. Renal biopsy shows FSGS. No recurrence
only on podocytes and encodes an integral membrane pro- is observed after transplantation. Patients have female
tein, podocin (92). By immunoelectron microscopy, podo- external genitalia, and it is often the evaluation of primary
cin is located at the foot processes, opposite the slit amenorrhea in nephrotic females that leads to the diagno-
diaphragm (93). This 42-kDa protein is structurally related sis of 46,XY gonadal dysgenesis, which is frequently com-
to human stomatin, an adapter protein that links mecha- plicated by gonadoblastoma.
nosensitive channels to the cytoskeleton on the cell surface. Barbaux et al. detected point mutations in the donor
Podocin may link membrane proteins (e.g., nephrin) to the splice site in intron 9 of the WT-1 gene (101). These muta-
cytoskeleton. Ten different NPHS2 mutations comprising tions were heterozygous, and appeared de novo in the two
nonsense, frameshift, and missense mutations were found patients whose parents were studied. The mutations result
to segregate with the disease. Podocin mutations have also in loss of the +KTS isoform. Similar intronic mutations
been identified in 15 to 30% of patients with sporadic have been described by other authors (102,103). This indi-
SRNS (94,95). cates that donor splice-site mutations in WT-1 intron 9 are
CD2-associated protein (CD2AP), which anchors CD2 constant in Frasier syndrome. The former definition of
receptors of T lymphocytes to the cytoskeleton, is also Frasier syndrome included only 46,XY patients with a
expressed in the podocyte. Shih et al. observed that mutant female phenotype. However, Frasier syndrome mutations
mice lacking CD2AP develop proteinuria and renal failure may be responsible for isolated persistent glomerulopathy
(96). They showed that CD2AP and nephrin interact with focal sclerosis in genetically female patients and in
directly, suggesting an important role of this protein in the patients with Denys-Drash syndrome (104,105).
anchoring of nephrin to the slit diaphragm or into the sig- Nephrotic syndrome with FSGS has been reported in
naling pathways (97). No mutations in the CD2AP gene patients with mitochondrial cytopathies (106–108). An A
associated with SRNS have been reported to date. to G transition at position 3243 in mitochondrial DNA
The Wilms’ tumor suppressor gene, WT-1, encodes a was reported in some of these patients who presented with
transcription factor presumed to regulate the expression of isolated nephrotic syndrome or with nephrotic syndrome
numerous target genes through DNA binding. It plays a in association with mitochondrial myopathy, encephalopa-
key role in renal and gonadal maturation. Mutations in thy, lactic acidosis, stroke-like episodes, or progressive
WT-1 have been associated with nephroblastoma or glo- external ophthalmoplegia. Other patients develop diabetes
merular diseases (Denys-Drash syndrome and Frasier syn- mellitus or hearing loss.
drome). The WT-1 gene contains ten exons covering Some familial cases of minimal change nephrotic syn-
approximately 50 kb of genomic DNA. Exons 1 to 6 drome are associated with other abnormalities (e.g., micro-
encode a proline/glutamine-rich transcriptional regulatory cephaly and hiatus hernia), as in the Galloway-Mowat
region, whereas exons 7 to 10 encode the four zinc-fingers syndrome (109). Spondyloepiphyseal dysplasia was found
of the DNA-binding domain. Two alternative splicing in association with FSGS (110). Other syndromes, includ-
regions—one corresponding to the 17 amino acids encoded ing microcephaly or disturbances of neuronal migration,
 28. Steroid-Resistant Idiopathic Nephrotic Syndrome in Children 565

have also been reported in association with early-onset been more often used than chlorambucil. The rate of full or
nephrotic syndrome (111). partial remission is higher in patients with partial steroid
resistance, those with late steroid resistance, or those in
whom initial renal biopsy has shown MCD, by comparison
TREATMENT
with those showing initial resistance to corticosteroids or
FSGS. The International Study of Kidney Disease in Chil-
Symptomatic Treatment
dren recently reported on 60 children with steroid-resistant
Symptomatic treatment—including dietary recommenda- FSGS who were randomly allocated to receive (a) predni-
tions; use of diuretics; prevention and therapy of infectious sone, 40 mg/m2 on alternate days for 12 months (control
and thromboembolic complications; and treatment of hypo- group) or (b) cyclophosphamide, 2.5 mg/kg body weight for
volemia, hypertension, and hyperlipidemia—is identical to 3 months plus prednisone, 40 mg/m2, on alternate days for
that recommended for the glucocorticoid-responsive patient 12 months (117). Complete remissions were observed in
with idiopathic nephrotic syndrome (see Chapter 27). 28% of children in the control group and in 25% of children
who received cyclophosphamide. The authors concluded
that there was no beneficial effect of cyclophosphamide in
Immunosuppressive Therapy
these patients. Geary et al. reported full or partial response to
By definition, patients with SRNS have not responded to cyclophosphamide in 12 of 29 steroid-resistant patients with
standard glucocorticoid regimens (see Chapter 27). FSGS (118). Renal failure developed less frequently in partial
responders (one of nine) than in those who did not respond
at all (seven of eight). Siegel et al. observed complete remis-
Pulse Methylprednisolone
sions in six steroid-resistant patients with MCD, three of
Methylprednisolone pulse therapy has been advocated for whom relapsed but became steroid responsive (119). Simi-
patients with SRNS. The protocol proposed by Mendoza et larly, Bergstrand et al. reported that some patients with
al. consists of methylprednisolone (30 mg/kg IV) adminis- steroid-resistant nephrosis treated with cyclophosphamide
tered every other day for 2 weeks; weekly for 8 weeks; every had become steroid responsive (120). Conversely, White and
other week for 8 weeks; monthly for 9 months; and then Glasgow observed no improvement after cyclophosphamide
every other month for 6 months in association with oral treatment in 15 steroid-resistant children with focal sclerosis
prednisone and, if necessary, cyclophosphamide or chloram- (121). Cameron et al. reported only 1 responder out of 13
bucil (112). At an average of over 6 years of follow-up, 21 of children with steroid-resistant nephrosis and FSGS who
32 children were in complete remission, and the 5-year inci- received cyclophosphamide (122). Similarly, Tejani et al.
dence of ESRD was approximately 5% versus 40% in his- reported no remission with cyclophosphamide in ten steroid-
toric controls (113). Side effects included nausea during the resistant children (123). In a controlled trial involving 13
infusion of pulse methylprednisolone in almost all, slowed children with steroid-resistant minimal change nephrotic
growth in four patients, small cataracts that did not interfere syndrome, IV pulse cyclophosphamide was shown to be ben-
with vision in five patients, and infections in two patients. eficial when compared to oral cyclophosphamide (124). Ren-
There were no cases of abdominal striae, diabetes mellitus, or nert et al. treated ten children with steroid-resistant FSGS
aseptic necrosis of bone. with IV cyclophosphamide. Only two of the five patients
Initial enthusiasm for usage of pulse methylprednisolone who were initial nonresponders went into remission, whereas
in patients with SRNS has been decreased by subsequent all five late nonresponders achieved complete reemission
studies that have failed to produce similar results. A prelimi- (125).
nary multicenter report of 15 children with FSGS was The side effects of cyclophosphamide include bone mar-
unable to confirm the efficacy of pulse corticosteroids (114). row depression, hemorrhagic cystitis, gastrointestinal tract
A mean of 15 pulses were given, and eight patients also symptoms, alopecia, and infection. Hemorrhagic cystitis
received an alkylating agent. At the end of the study, only occurs infrequently at the doses used in these patients.
four patients maintained a complete remission, whereas five Long-term toxic effects include the risk of cancer, pul-
patients had a poor outcome with progression to ESRD or monary fibrosis, ovarian fibrosis, and sterility. Gonadal tox-
death. Waldo et al. did not observe complete remission in ten icity is well established, and the risk of sterility is greater in
children who were treated with this protocol (115), whereas boys than in girls. The cumulative threshold dose above
Hari et al. reported a 65% response rate (116). which oligo- or azoospermia may occur is above 250 mg/kg
(126,127). Azoospermia is reversible in some patients
(128). The risk of cancer is difficult to define with the short
Alkylating Agents
courses used to treat idiopathic nephrotic syndrome.
Although alkylating agents have little therapeutic effect in Chlorambucil has been used in a few studies. Baluarte et al.
patients with SRNS, they are still widely used either alone or described remissions in 10 of 17 cases (129). Williams et al.
in combination with corticosteroids. Cyclophosphamide has treated six children who all went into remission with a follow-
566 V. Glomerular Disease

up of 1.3 to 9.4 years (130). We treated 74 steroid-resistant not respond to the combined treatment. Complete remission
children with chlorambucil, 0.2 mg/kg, for 2 to 6 months, and occurred in more than one-half of the patients within the first
only 14 (19%) achieved complete or partial remission. 2 months of this treatment, which makes it likely that the
Acute toxic effects are less frequent with chlorambucil treatment was responsible for the remission, although sponta-
than with cyclophosphamide. Leukopenia and thrombocyto- neous remission cannot be excluded. It is interesting to note
penia may occur and are reversible within 1 to 3 weeks. that eight patients who relapsed after cyclosporine treatment
Severe viral infections have been reported. Long-term toxic subsequently responded to steroids and experienced a steroid-
effects include an increased risk of malignancy (131). dependent course. Progression to renal failure was observed
Gonadal toxicity, which predominantly affects male patients, only in patients who had not responded (12 patients) or had
is probably irreversible (azoospermia) at cumulative doses only a partial response (1 patient) to the combined treatment.
above 10 mg/kg. Ingulli et al. reported that prolonged cyclosporine treat-
ment in children with steroid-resistant FSGS reduced pro-
teinuria and slowed progression to ESRF (146). The dose
Combined Immunosuppression
of cyclosporine (4 to 20 mg/kg/day) was titrated to the
Trompeter proposed aggressive immunosuppression for a serum cholesterol level to achieve a remission. In this study,
subgroup of patients with FSGS, refractory nephrotic syn- only 5 of the 21 treated patients (24%) progressed to ESRF
drome, or rapid progression to renal failure (132). The reg- compared to 42 of 54 patients from a historical control
imen included vincristine, 1.5 mg/m2/wk in eight weekly group who had not received this treatment.
IV doses; cyclophosphamide, 3 mg/kg/day for 8 weeks; and Ponticelli et al. compared cyclosporine to supportive
prednisolone, 2 mg/kg/day initially, with progressive taper- therapy in a randomized trial (147). Seven of the 22 treated
ing after 2 weeks. Lasting remissions were observed in 7 of patients went into complete remission, six experienced par-
21 children, starting 6 months to 3 years from the onset of tial remission, and nine did not respond. Only 38% of the
therapy. Almeida et al. observed a complete and stable patients who responded had sustained remissions. In the
remission in only two of the seven children who received control group, only 3 of the 19 patients achieved partial
this regimen (133). remission. Tejani and Liberman compared cyclosporine and
placebo in 25 children with steroid-resistant FSGS (148).
All 12 patients who were treated demonstrated a decrease
Azathioprine
in proteinuria compared to two in placebo-treated patients.
Azathioprine was considered to be ineffective in steroid- Gregory et al. treated 15 children with SRNS cyclospor-
resistant patients after the report of Abramowicz et al., who ine combined with prednisone. They observed a remission
found no difference between a 3-month course of azathio- in 13 children after a mean duration of treatment of 2
prine and placebo (134). However, Cade et al. reported months (149).
complete remission in 13 adult patients with steroid- Singh et al. recently reported the effect of cyclosporine
resistant idiopathic nephrotic syndrome (135). in 42 children with steroid-resistant FSGS (150). The
mean proteinuria decreased from 7.1 to 1.8 g/day, whereas
the serum albumin increased from 2.1 to 3.5 g/dL. The
Cyclosporine
mean serum creatinine increased from 0.85 to 1.26 mg/dL.
Cyclosporine has been used to treat patients with SRNS. Twenty-five patients responded with a complete remission.
Initial reports showed that only 20% of 60 steroid-resistant Patients who respond to cyclosporine often relapse when
children achieved complete remission (136–142). A partial the dose is tapered or discontinued (151). Many reports
response was observed in 13% of cases, but it was usually indicate that the prolonged use of cyclosporine is associated
transient (143). with chronic nephrotoxicity. The most prominent histo-
The Collaborative Study of Sandimmune in Nephrotic logic feature of chronic cyclosporine nephrotoxicity is the
Syndrome analyzed the data from different clinical studies, presence of tubulointerstitial lesions, characterized by
including 226 steroid-resistant patients, adults, and chil- stripes of interstitial fibrosis containing groups of atrophic
dren (144). The analysis demonstrated that the rate of tubules. Cyclosporine-associated arteriolopathy is rarely
complete remission was significantly higher when cyclo- observed. Classen et al. did not find significant changes on
sporine was given in combination with steroids: 24% com- the renal biopsies of five patients treated for a mean period
pared to 14%. of 10 months (152). Habib and Niaudet performed repeat
The French Society of Pediatric Nephrology reported the renal biopsies in 42 patients (153). There were no signifi-
results of a prospective trial, including 65 children treated with cant changes in 18 patients, several foci of atrophic tubules
cyclosporine, 150 to 200 mg/m2, and prednisone, 30 mg/m2/ with thickened basement membranes within stripes of
day for 1 month and on alternate days for 5 months thereafter interstitial fibrosis in 15 patients, and extensive lesions of
(145). Twenty-seven patients (42%) went into complete remis- interstitial fibrosis with atrophic tubules in nine patients. In
sion and four (6%) partial remission, whereas 34 (52%) did this study, 9 of the 15 patients with several foci of atrophic
 28. Steroid-Resistant Idiopathic Nephrotic Syndrome in Children 567

tubules and all nine patients with severe interstitial fibrosis trolled trials in adult patients have shown that lipid-
had MCD on pre- and posttherapy biopsies without FSGS, lowering agents can prevent the decline of renal function in
suggesting that the lesions were secondary to cyclosporine a variety of chronic nephropathics. No such studies have
and not to the natural course of the disease. At the time of been performed in children.
most recent biopsy, all children, including the nine patients
with the most severe tubulointerstitial lesions, had normal
renal function. Thus, morphology is a more sensitive indi- RECURRENCE OF IDIOPATHIC NEPHROTIC
cator of chronic cyclosporine nephrotoxicity than GFR. SYNDROME IN TRANSPLANTED KIDNEYS
Other side effects give less cause for concern and respond to
standard therapy: elevation of blood pressure, hyperkalemia, A major problem in patients with idiopathic nephrotic syn-
hypertrichosis, gum hypertrophy, and hypomagnesemia. drome who progress to ESRF and subsequently undergo
renal transplantation is the risk of recurrent disease in the
graft. The overall risk of recurrence is estimated at 25%. The
FK506
risk appears to be different in children and adults. Senggutu-
A few patients have been treated with the immunophilin van et al. reported recurrence in 8 of 16 children compared
modulator FK506 with poor results (154). to 3 of 27 adults (163). In children, recurrence is more fre-
quent when the disease is diagnosed after 6 years of age
(164,165). Similarly, a rapid progression of the disease to
Nonsteroidal Antiinflammatory Drugs
ESRF was a major factor associated with recurrence: In most
Nonsteroidal antiinflammatory drugs may decrease pro- series, when the duration of disease has been shorter than 3
teinuria. Several authors have used indomethacin in the years, the nephrotic syndrome recurs in one-half of the
treatment of idiopathic nephrotic syndrome with variable patients (166,167). The histopathologic pattern observed on
results. Donker et al. found a reduction of proteinuria in the first biopsy during the course of the disease is also an
patients with focal sclerosis associated with a simulta- important predictive factor (163,165,166,168,169). Recur-
neous reduction of GFR (155). Velosa et al. also reported rence occurs in 50 to 80% of patients in whom initial biopsy
a significant reduction in proteinuria in patients treated showed diffuse mesangial proliferation but in only 25% of
with meclofenamate (156). patients with MCD on initial biopsy. Most patients who
The detrimental effect of nonsteroidal antiinflamma- experience a recurrence in a first graft show recurrence in a
tory drugs on renal function is well established, and patients second graft (170–173). Conversely, when a first graft has
with renal disease seem more vulnerable (157,158). Positive been lost due to rejection without recurrence, a second graft
sodium balance, increased edema, and risk of arterial can be performed safely, as no recurrence has been described
hypertension are recognized complications. The decreased in this setting.
GFR observed with nonsteroidal antiinflammatory drugs is In children, recurrence of proteinuria occurs in most cases
usually reversible and marked only in salt-sodium– within the first hours or days after transplantation. A high
depleted patients. However, irreversible renal failure was proportion of patients with immediate recurrence show
reported with a high incidence in a prospective study in delayed graft function (164,170,174). In some patients, pro-
children with nonresponsive nephrotic syndrome and teinuria recurs several months later. Proteinuria is most often
focal sclerosis (159). associated with a nephrotic syndrome. Transplant biopsy,
when performed early, usually shows minimal glomerular
changes with foot-process fusion (175–177). Lesions of
Angiotensin-Converting Enzyme Inhibitors
FSGS appear after several days or weeks (176).
The angiotensin-converting enzyme inhibitor captopril was Graft failure occurs in approximately 60% of patients
reported to dramatically decrease nephrotic range proteinuria with recurrence versus 23% of those without recurrence
secondary to renovascular hypertension and secondary FSGS (163,164,170,178). Some patients may maintain adequate
(160). A decrease in proteinuria and even complete remission renal function for several years despite persistent nephrotic
have also been observed in patients with chronic glomerulopa- syndrome (164,167).
thies with or without hypertension (161). Reduction of pro- The role of cyclosporine in recurrent SRNS remains
teinuria by 50% without a concomitant decrease in GFR was unclear. There is no evidence that cyclosporine can prevent
reported in children with SRNS (162). Further studies are the recurrence of nephrotic syndrome after transplantation
needed to confirm these observations. (179–184). After the introduction of cyclosporine, the inci-
dence of recurrence did not change, but graft survival
improved (164,179,180,183). In patients who have recur-
Lipid-Lowering Agents
rent disease, high doses of cyclosporine may be effective.
Hyperlipidemia has been shown to accelerate the progres- Mowry et al. reported on 11 children who received 12 renal
sion of glomerular sclerosis in experimental animals. Con- transplants and who had been treated with high-dose cyclo-
568 V. Glomerular Disease

sporine, plasma exchanges, or a combination of both (185). entities or a spectrum of disease. In: Robinson RR, ed.
Remission was observed in 10 of the 12 recipients. Ingulli Nephrology: proceedings of the IXth International Congress of
and Tejani reported two children with recurrent nephrotic Nephrology, Los Angeles: Springer-Verlag, 1984:634–644.
syndrome who both achieved remission when cyclosporine 2. Kriz W, Elger M, Nagata M, et al. The role of podocytes in
dosage was gradually increased from 15 mg/kg/day to 27 the development of glomerular sclerosis. Kidney Int Suppl
1994;45:S64–S72.
and 35 mg/kg/day (186). A similar experience was reported
3. Bariety J, Nochy D, Jacquot C, et al. Diversity and unity of
by Srivastava et al. (187). In our group, 17 children with focal and segmental glomerular sclerosis. Adv Nephrol Necker
recurrence were treated with IV cyclosporine at an initial Hosp 1998;28:1–42.
dose of 3 mg/kg/day, which was then adjusted to maintain 4. Bariety J, Bruneval P, Hill G, et al. Posttransplantation
whole blood levels between 250 and 350 ng/mL. In 14 of relapse of FSGS is characterized by glomerular epithelial cell
the 17 cases (82%), proteinuria completely disappeared transdifferentiation. J Am Soc Nephrol 2001;12:261–274.
after 20.8 ± 8.4 days (range, 12 to 40 days). The treatment 5. Barisoni L, Mokrzycki M, Sablay L, et al. Podocyte cell
was ineffective in the remaining three patients. Plasma cycle regulation and proliferation in collapsing glomerulop-
exchanges were performed in four patients during the first athies. Kidney Int 2000;58:137–143.
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after transplantation (189). thy. Kidney Int 2001;59:2126–2133.
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patients and, in some cases, combined with increased renal transcription of CTL-effectors and TGF-beta1 in chil-
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tion 1990;50:594–596. 195. Solomon LR, Cairns SA, Lawler W, et al. Reduction of post-
181. Pirson Y, Squifflet JP, Marbaix E, et al. Recurrence of focal transplant proteinuria due to recurrent mesangial proliferative
glomerulosclerosis despite cyclosporin treatment after renal (IgM) glomerulonephritis following plasma exchange. Clin
transplantation. BMJ 1986;292:1336. Nephrol 1981;16:44–50.
182. Schwarz A, Krause PH, Offermann G, Keller F. Recurrent 196. Zimmerman SW. Plasmapheresis and dipyridamole for
and de novo renal disease after renal transplantation with or recurrent focal glomerular sclerosis. Nephron 1985;40:241–
without cyclosporine. Am J Kidney Dis 1991;17:524–531. 245.
183. Vincenti F, Biava C, Tomalanovitch S, et al. Inability of 197. Cameron JS. The enigma of focal segmental glomeruloscle-
cyclosporine to completely prevent the recurrence of focal rosis. Kidney Int 1996;50:S119–S131.
 29

IMMUNE MECHANISMS OF
GLOMERULAR INJURY
ALLISON A. EDDY

OVERVIEW dent glomerular cells and include cytokines and growth

factors, reactive oxygen metabolites, bioactive lipids [platelet-
Immunologic pathways initiate and propagate glomerular activating factor (PAF) and eicosanoids], proteases, and vaso-
injury. Over the past decade, major advances in cellular, active substances [endothelin (ET) and nitric oxide (NO)]. It
molecular, genetic, and immunologic sciences have provided is hoped that by understanding the immunopathogenesis of
significant new insights into the pathogenesis of glomerulo- glomerulonephritis, new therapeutic strategies will emerge.
nephritis. For the purpose of review, the immunopathogenic Furthermore, future genetic studies may find that genotype-
mechanisms can be divided into two basic phases. Primary dependent expression and/or activity levels of certain key
mechanisms of glomerulonephritis focus on those events that mediators predict risk and severity of glomerulonephritis.
initiate glomerular injury. These initial events alone rarely
produce significant and sustained damage. The role of the
humoral immune system, dependent on B lymphocyte acti- STRUCTURE OF THE GLOMERULAR
vation and antibody production, has been recognized for CAPILLARY WALL AND MESANGIUM
nearly a century (Fig. 29.1). Although two distinct antibody-
mediated pathways have been proposed, current evidence A basic understanding of the architecture of the glomerulus
suggests that antibody interaction with antigens in situ is necessary to appreciate the basis of the dynamic interac-
within the glomerulus (which may or may not lead to the tions between intrinsic components of the glomerular cap-
formation of immune complexes) predominates, whereas illary wall and mesangium and the blood-borne mediators
glomerular trapping of immune complexes preformed within of glomerular injury. The glomerular basement membrane
the circulation occurs rarely, if ever. The cellular immune sys- (GBM) is a specialized extracellular matrix lined by the
tem, dependent on T-lymphocyte activation, occasionally endothelial and visceral epithelial cells. It acts as a filtration
triggers glomerular injury. However, for many glomerular barrier with selective permeability, allowing passage of
diseases, separation of B-cell and T-cell pathways is rather small plasma molecules but almost completely restricting
artificial as the cellular and humoral effector limbs of the filtration of molecules larger than albumin. It also plays a
immune system often collaborate. more dynamic role regulating the adhesion and behavior of
The secondary immune mechanisms of glomerular injury adherent cells. Chemical analyses have shown the GBM to
implicate cascades of inflammatory mediators that are be 80% collagenous proteins, primarily collagen IV (α3,
recruited to propagate renal damage after the primary glo- α4, and α5 chains). A number of the other components
merular attack. Some of these mediators play essential roles; have been identified such as laminin-11 and entactin/
others may modify disease severity, but their participation is nidogen (1). The GBM also contains proteoglycans, the
not essential. The secondary mediators are variable depend- most abundant of which is agrin. Although constituting
ing on the nature of the primary injury, but they often only 1% of the dry weight of the GBM, these anionic com-
include lymphohemopoietic cells [polymorphonuclear leu- ponents contribute significantly to the charge-selective
kocytes (PMNs), monocytes, and platelets] and activated properties of the ultrafiltration unit. The onset of pro-
components of the complement cascade. Resident glomeru- teinuria is usually characterized by a loss of charge as well as
lar endothelial cells, mesangial cells, and epithelial cells may size selectivity. At least four theories have been proposed to
be activated and further contribute to the inflammatory explain the alterations in the GBM charge: masking by
response. Most of the secondary mediators that have been immune complexes, depolymerization by oxygen radicals,
identified are products of lymphohemopoietic cells or resi- alterations in proteoglycan synthesis, and proteolytic diges-
576 V. Glomerular Disease

FIGURE 29.1. Immunofluorescence and electron microscopic features of three distinct patterns of
immune-mediated glomerular injury. In anti–glomerular basement membrane (GBM) nephritis,
immunoglobulin G (IgG) is deposited in a linear pattern along the GBM (A). Although dense
immune deposits are not detected by electron microscopy, there may be expanded lucent suben-
dothelial areas (B). Membranous nephropathy is characterized by granular deposits of IgG and C3
(C) along the epithelial aspect of the glomerular capillary wall, which correspond to the subepithe-
lial deposits present on electron microscopy (D). Immune complex–induced glomerulonephritis,
illustrated by the renal biopsy of a patient with lupus nephritis, is characterized by the deposition
of immunoglobulins and complement (E) along the glomerular capillary wall and within the
mesangium. Electron microscopy reveals electron-dense deposits in similar locations (F).

tion (2). On the other hand, exactly how erythrocytes abundant component. During the filtration process, some
traverse the GBM remains enigmatic. A recent ultrastruc- plasma enters the mesangial zone, gaining access through
tural study suggests that they may traverse through gaps in the mesangial “waist” or “angle,” bypassing the GBM. Cir-
the GBM (3). culating immune complexes commonly become lodged in
The mesangium, the region occupying the centrolobular this area.
position between the mesangial cells and the perimesangial The intrinsic glomerular cells—specialized epithelial
basement membrane, is similar in composition but not cells (podocytes), mesangial cells, and endothelial cells—
identical to the peripheral GBM. It consists of a dense net- play an active role in the regulation of both normal physio-
work of microfibrils; fibronectin appears to be the most logic processes and pathologic states. Mesangial cells have
 29. Immune Mechanisms of Glomerular Injury 577

TABLE 29.1. GLOMERULAR MATRIX ADHESION MOLECULES

Glomerular expression

Matrix ligand Mesangial Epithelial Endothelial

β1 integrins (VLA family of lymphocytes antigens)

α1 Laminin, collagens I and IV + – +
α2 Laminin, collagens I and IV + + +
α3 Fibronectin, laminin, collagen, entactin + + +
α5 Fibronectin, RGD peptide + – +
α8 Fibronectin, vitronectin, tenascin + – –
β3 integrins
αv Vitronectin, fibronectin, fibrinogen + – +
β5 integrins
αv Vitronectin, fibronectin + + –

+, present; –, absent; RGD, arginine, glycine, aspartate, amino acid sequence; VLA, very late antigen.

smooth muscle cell–like characteristics and attach to the eral cell-cycle proteins that promote mitosis (9). Cell-cycle
GBM at several sites. Consequently, mesangial cell contrac- inhibitory agents such as E2F decoy oligonucleotides and
tion modifies glomerular filtration. In response to injury, cyclin-dependent kinase inhibitors have been used experi-
these cells may change phenotypically, and presumably mentally to prevent mesangial cell proliferation after
functionally, by expression of additional smooth muscle cell immunologic injury, with beneficial outcomes (10,11).
proteins such as α smooth muscle actin, desmin, and calpo- Mesangial cells may produce several products that may
nin and matrix-binding integrin receptors (Table 29.1) contribute to acute and chronic glomerular damage such as
(4,5). Mesangial cells express receptors that may actively vasoactive hormones (renin, angiotensin II, vasopressin, ET),
engage in antibody-mediated glomerular injury: receptors bioactive lipids (PGs, leukotrienes, PAF), peptide growth
for the Fc fragment of immunoglobulin G (IgG) (FCγRIII factors and cytokines (PDGF, IL-1α, IL-1β, insulin-like
or CD16) and IgA. growth factor-1, IL-8), matrix proteins (mainly collagen I
Mesangial cell proliferation, a common feature of glomer- but also types III, IV, and V; laminin; and fibronectin), gly-
ulonephritis, may replenish cells depleted by mesangiolysis, cosaminoglycans, thrombospondin, procoagulants, fibrinoly-
but proliferation may also have pathologic consequences by tic factors (plasminogen activators), proteinases, and reactive
enhancing the production of proinflammatory and profi- oxygen species (ROS). Megsin is a recently identified serine
brotic substances. Most proliferating mesangial cells are resi- protease inhibitor that is produced primarily by mesangial
dent glomerular cells, but precursor cells may also migrate in
from the extraglomerular mesangium within the juxtaglom-
erular apparatus and from the bone marrow (6–8). Given the
close association between mesangial hypercellularity and glo-
merular injury, understanding the mechanisms that regulate
their number is important. Proliferation is controlled at mul-
tiple levels: mitogens and their receptors, intracellular signal-
ing pathways, and intranuclear events. Many proteins are
mesangial cell mitogens including platelet-derived growth
factor (PDGF)-B and -C, insulin-like growth factor-1, epi-
dermal growth factor (EGF), transforming growth factor α
(TGFα), low-dose TGFβ, basic fibroblast growth factor FIGURE 29.2. Cell-cycle regulatory proteins in glomerular cell
(FGF), chemokines, insulin, interleukin-1 (IL-1), tumor proliferation. Cellular proliferation is characterized by the orderly
necrosis factor (TNF), ET, serotonin, bradykinin, vaso- progression of cells through sequential phases from the quies-
cence (G0), resting (G1), DNA synthesis (S), resting (G2), and mitosis
pressin, thrombin, fibronectin, and prostaglandin (PG)F2α. (M). Each step is regulated by cell-cycle proteins (cyclins) that bind
Recent studies have identified several nuclear proteins and activate cyclin-dependent kinases (CDK). Each step may be
that tightly regulate the mesangial cell cycle at multiple blocked by negative cell-cycle proteins, CDK inhibitors. Recent
studies indicate that the several CDK inhibitors are expressed in
levels (Fig. 29.2). Their actions ultimately determine normal glomeruli and play a major role in the regulation of the
whether the retinoblastoma protein, a negative cell-cycle glomerular cell proliferative responses to mitogens produced dur-
regulator, will be inactivated by phosphorylation. With ret- ing injury. CDK inhibition has been shown to decrease mesangial
cell proliferation, decrease matrix expansion, and improve renal
inoblastoma protein inactivation, transcription factors of function in rats with Thy 1 nephritis (11). (Courtesy of Dr. Stuart
the E2F family are released to promote transcription of sev- Shankland, University of Washington).
578 V. Glomerular Disease

FIGURE 29.3. Molecular anatomy of the glomerular podocyte. The basal sole plate of the
podocyte approximates the glomerular basement membrane (GBM) and is directly attached to it in
areas of focal contact via α3β1 integrin receptors and the α,β dystroglycan (α-, β-DG) complex. Adja-
cent podocytes are joined laterally by a modified adherens junction of “slit diaphragms.” Genetic
mutations in several slit diaphragm–associated proteins cause inherited nephrotic syndrome,
including nephrin, CD2AP, podocin, and the nephrin homologue neph-1. The apical membrane
extending from the slit diaphragm into Bowman’s space is covered with an anionic glycoprotein
coat made predominantly of podocalyxin. Each of these domains is connected to the intracellular
actin cytoskeleton that functions to maintain podocyte structural integrity. The complexity of the
contractile apparatus has become appreciated with the identification of several key subcompo-
nents that interact with actin, including myosin II, α-actinin-4 (α-act-4), talin (T), vinculin (V), and
synaptopodin (synpo). MAGI-1, membrane-associated guanylate kinase inverted-1; NHERF2, NA +/
H+ exchanger regulatory factor 2; P, paxillin; P-Cad, P-cadherin. (From Mundel M, Shankland SL.
Podocyte biology and response to injury. J Am Soc Nephrol 2002;13:3005–3015, with permission.)

cells and is associated with mesangial proliferation and can be rapidly induced in animals by injection of antibodies
matrix expansion when overproduced (12). to slit diaphragm proteins such as nephrin (15). A parallel
Glomerular podocytes serve several functions such as process has been observed in some congenital nephrotic syn-
pinocytosis of proteins that have leaked through the GBM, drome patients who develop nephrotic syndrome after renal
synthesis of GBM matrix proteins, and restriction of passage transplantation in association with antinephrin antibody
of proteins at the level of the slit diaphragm. Epithelial cells production. In many glomerular diseases, podocytes com-
synthesize many biologically active products, including extra- pletely detach from the GBM and are shed into the urinary
cellular matrix proteins [type IV collagen, heparan sulfate space (16). Unlike mesangial cells, glomerular podocytes are
proteoglycan (HSPG), fibronectin], cyclooxygenase (COX), terminally differentiated cells that, with a few exceptions,
and lipoxygenase products; urokinase-type plasminogen acti- fail to proliferate in response to injury. The reasons for this
vator and plasminogen activator inhibitor-1; and antioxidant are not yet entirely clear. Highly differentiated podocytes
enzymes, IL-1, and angiopoietin-1. Podocytes express C3b/ express high levels of cyclin-dependent kinase inhibitors,
C4b and Fc receptors that may interact directly with p21, p27, and p57 (Fig. 29.2). A proliferative phenotype
immune complexes. The outer podocyte cell membrane is can be restored by genetic deficiency of p21 or p27 (17,18).
differentiated into three structurally and functionally distinct However, the complete answer to this question is likely to be
regions that are illustrated in Figure 29.3 (13). more complex with evidence that injured podocytes can
The functional hallmark of glomerular injury is pro- enter the cell cycle but arrest at the G2/M phase. This fail-
teinuria. The filtration barrier of the glomerular capillary ure to proliferate and self-replenish is problematic, as
wall is regulated at the level of the GBM and the slit dia- podocyte loss is now recognized as a harbinger of progressive
phragm. Perturbations of podocyte slit diaphragm function glomerular sclerosis (19).
underlie most if not all types of glomerulonephritis and are Glomerular endothelial cells line the capillary wall. These
often associated with significant structural changes as well, cells assume a flattened profile and are densely perforated by
namely, foot-process retraction or effacement. The cellular transendothelial pores or fenestrae, water channels that may
processes that cause foot-process effacement remain to be permit direct size-dependent access of blood-borne factors to
elucidated, but they may recapitulate embryonic develop- the basement membrane (20). Endothelial cells can proliferate
ment in reverse and an uncoupling of the actin cytoskeleton (21). Their integrity after renal injury is partially maintained
from membrane functional domains (13,14). Proteinuria by vascular endothelial growth factor (VEGF) and angiopoi-
 29. Immune Mechanisms of Glomerular Injury 579

TABLE 29.2. ENDOTHELIAL LEUKOCYTE ADHESION MOLECULES IN EXPERIMENTAL GLOMERULONEPHRITIS

Family Member Primary cellular distribution Ligand

Selectins L-selectin (leukocyte) PMN, Mφ, lymphocytes Mucins (e.g., addressin, CD34,
Gly-CAM)
P-selectin (platelet) Platelets, activated endothelium Mucins (e.g., sLe x)
E-selectin (endothelial) Activated endothelium Mucins (e.g., sLe x)
β1 integrins (VLA α4β1 (VLA-4) Mφ, lymphocytes, eosinophils ± PMN VCAM-1, fibronectin
proteins)
β2 (leukocyte) inte- CD11a/CD18 (LFA-1) All leukocytes ICAM-1, 2, 3
grins
CD11b/CD18 (Mac-1) PMN, Mφ ICAM-1, C3bi, fibrinogen, factor
X
CD11c/CD18 (p150, 95) PMN, Mφ ICAM-1
CD11d/CD18 Mφ subset ?
IgG-like ICAM-1 (CD54) Endothelium, some leukocytes, mesangial β2 integrins
cells
ICAM-2 Endothelium, some leukocytes CD11a/CD18
ICAM-3 (CD50) All leukocytes ?
VCAM-1 Activated endothelium, glomerular pari- α4β1 (VLA-4)
etal epithelium, ± mesangial cells

Gly-CAM, glycosylation-dependent cell adhesion mocecule-1; ICAM, intercellular adhesion molecule; LFA, lymphocyte function-associated antigen; Mφ,
monocytes/macrophages; PMN, polymorphonuclear leukocytes; sLe, sialyl Lewis; VCAM, vascular cell adhesion molecule; VLA, very late antigen.

etin-1 that bind to their endothelial receptors, Flk-1 and Tie-2, acid (AA) metabolites. The identification of anti–β1 integrin
respectively (22). Angiopoietin-2 also binds to the Tie-2 recep- antibodies in polyclonal antisera against the tubular brush-
tor, but it antagonizes and destabilizes capillary integrity (23). border fraction Fx1A and GBM, which induce proteinuria
Glomerular endothelial cells appear unique in many respects in experimental animals, suggests that integrins play an
compared to endothelial cells of other origin. Although less important role in maintaining the permselectivity of the glo-
extensively studied than other glomerular cells, they also merular capillary wall. Recent studies suggest the α1β1 may
undergo specific alterations in structure, function, and metab- also play an important role in extracellular matrix remodel-
olism during glomerular injury, making them active partici- ing during progressive glomerulosclerosis (24,25).
pants in injurious and reparative processes. Endothelial cells
synthesize several coagulation proteins, growth factors, and
cytokines; extracellular matrix proteins; and vasoactive mole- HUMORAL IMMUNE MECHANISMS OF
cules. The surface of endothelial cells expresses angiotensin- INDUCTION OF GLOMERULAR INJURY
converting enzyme (ACE) and ET-converting enzymes that
catalyze the formation of the vasoconstrictors angiotensin II Antibody-mediated glomerular injury is the result of a B-
and mature ET, respectively. They may express several different lymphocyte–driven immune response. B-lymphocyte activa-
adhesion molecules that promote interactions with blood- tion requires T-lymphocyte participation. The fact that helper
borne leukocytes, platelets, and the third component of com- T lymphocytes recognize antigen in the context of major his-
plement (Table 29.2). Other functions include synthesis of tocompatibility complex (MHC) class II antigens (HLA DR
reactive oxygen metabolites and the potential to serve as anti- in humans) has led to speculation that the risk of developing
gen-presenting cells. immune-mediated glomerulonephritis may be linked to the
Cellular interactions with the GBM and extracellular inheritance of certain MHC class II genes. Although beyond
matrix play an essential role in maintaining glomerular integ- the scope of this review, several studies have suggested an asso-
rity and function. Among the numerous cell-matrix adhesion ciation between various DR antigens and the risk of develop-
molecules, those of the β1 integrin family of membrane gly- ing anti-GBM nephritis, IgA nephropathy, membranous
coproteins (also known as very late antigens) have been most nephropathy, and lupus nephritis (26–28).
extensively studied in the glomerulus (Table 29.1). The β1
chain associates with one of 12 different α chains, the most
Interaction of Antibodies with Intrinsic
abundant glomerular isoform being α3β1. In addition to
Matrix Components of the Glomerular
maintaining glomerular capillary wall integrity, integrins can
Extracellular Matrix
function as classical signal-transducing receptors to initiate a
variety of cellular responses that may play a role in the glo- Normal structural components of the GBM may be the tar-
merular response to injury: mitogenesis and synthesis of get of an autoantibody attack leading to the development of
matrix proteins, reactive oxygen metabolites, and arachidonic anti-GBM nephritis, which in humans may occur with
580 V. Glomerular Disease

(Goodpasture syndrome) or without pulmonary involve- electron-dense deposit formation, although histologic changes
ment. Experimentally, anti-GBM nephritis is induced by and proteinuria do not develop. A glycoprotein [matrix met-
passive administration of heterologous antibodies (nephro- alloproteinase (MMP)-50/100] has been identified that is
toxic serum nephritis) or by active immunization with heter- unique to rat glomerular mesangium, the expression of which
ologous GBM in an immunologic adjuvant (experimental is increased in several models of nephritis. A similar unique
autoimmune glomerulonephritis). human mesangial antigen has been identified (32).

Glomerular Basement Membrane Antibody Interaction

The GBM target antigen(s) in the classic experimental model After injection of heterologous anti-GBM antiserum (or
of nephrotoxic serum (or Masugi) nephritis has not been nephrotoxic antiserum), the antibody binds to the GBM
completely elucidated and is likely multiple. In contrast, the within minutes, gaining free access through the open glo-
dominant human Goodpasture antigen is known to be part merular endothelial fenestrae. By immunofluorescence
of the collagen IV molecule, and it has been further localized microscopy, the antibody is observed in a linear pattern
to a conformation-dependent epitope residing within the corresponding to the distribution of the GBM. Formation
amino-terminal noncollagenous NC-1 globular domain of of electron-dense deposits along the glomerular capillary
α3(IV) collagen (29,30). The gene encoding α3(IV) col- wall does not ensue (Fig. 29.1). Alterations in glomerular
lagen is located in the q35-37 region of chromosome 2. structure and function occur as a direct consequence of
Immunization with antibodies to α3(IV) collagen or dimers antibody binding to the GBM, leading to proteinuria and a
of α3(IV) induces mild nephritis in animals. Less common, decline in glomerular filtration rate (GFR). Ultrastructural
other components of the GBM may also serve as antibody changes include a rearrangement of GBM laminin, foot-
targets. In general, immunization with collagen IV, laminin, process fusion, loss of GBM anionic–binding sites, and evi-
fibronectin, and HSPG or administration of their specific dence of cellular injury. Some of these changes may be
antisera alone produces minor morphologic changes without induced by the interaction of the antiserum with glomeru-
proteinuria, although more severe disease has occasionally lar epithelial cell antigens such as the β1 integrins and ami-
been reported. nopeptidase A (33).
Anti-GBM antibodies may play a role in other forms of
glomerulonephritis. For example, patients with poststrepto-
Secondary Mediators
coccal glomerulonephritis may develop circulating antibodies
to type IV collagen, laminin, entactin/nidogen, and HSPG. Although antibody binding alone causes some alterations
Anti-DNA antibodies isolated from the sera of patients with in glomerular structure and function, additional mediators
systemic lupus erythematosus (SLE) cross-react with HSPG are needed for the full expression of the injury, otherwise
and type IV collagen, probably via antibody-bound DNA- the initial damage subsides after 24 hours. This subsequent
histone complexes. Humans with X-linked dominant heredi- period of glomerular inflammation is associated with the infil-
tary nephritis lack the α5 chain of collagen IV, a gene that is tration of PMNs, chemoattracted by complement proteins
encoded in the Xq region of the X chromosome. This genetic C5a and C3b generated by interactions with the anti-GBM
defect results in a secondary failure of α3 and α4 collagen IV antibody. Depletion of PMNs or complement prevents
chains to be integrated into the GBM. Primary mutations in proteinuria. There is some evidence that the membrane
the genes that encode the α3 and α4 chains of collagen IV are attack complex (MAC) of complement also plays a role in
responsible for autosomal recessive hereditary nephritis. After mediating cellular damage. In experimental models of anti-
renal transplantation, approximately 2 to 4% of the Alport GBM nephritis induced by heterologous antiserum, a sec-
syndrome recipients develop anti-GBM disease in the normal ond phase of glomerular inflammation develops 5 to 7 days
renal allograft due to the formation of anti-α3, -α4, and/or later as a result of the production of antibodies by the
-α5 collagen IV antibodies (31). immunized animal (autologous antibodies) to the heterolo-
gous antibody, which is now acting as a foreign antigen
planted along the GBM. In addition to antibody comple-
Mesangial Matrix
ment–induced damage, other mechanisms of injury are
The antigenic composition of the mesangial matrix is similar now recognized. T cells sensitized to GBM antigens play an
to that of the GBM, yet it appears to be largely protected important role and may even cause severe glomerulone-
from immune attack in anti-GBM nephritis, presumably phritis in the absence of nephritogenic antibodies (34). The
because the nephrotoxic antibodies bind avidly to the GBM complement-binding domain of IgG (as opposed to the
before they gain access to the mesangium. It is possible that antigen-binding domain) may also interact directly with
unique mesangial matrix antigens exist and are the target of mesangial cells, podocytes, and inflammatory cells through
immune attack. Injection of a monoclonal antibody to a rat interactions with Fc receptors. Studies in mice demon-
mesangial matrix component (81 kDa) induces mesangial strated that deficiency of the γ chain (shared by Fc receptors
 29. Immune Mechanisms of Glomerular Injury 581

I, II, and III) was associated with significantly milder anti- with megalin and may function in a regulatory role to control
GBM nephritis (35). megalin-ligand interactions. Purified antibodies to either
megalin or RAP induce subepithelial immune complex forma-
tion, but neither antibody alone produces the full renal disease
Interaction of Antibodies with Glomerular
spectrum that was first observed by Edgington and colleagues
Cell–Surface Antigens
when they injected polyclonal anti-Fx1A antiserum into rats
Experimental models involving the binding of antibodies (36a). This has led to the notion that other antigen-antibody
to cell-surface antigens have now been described for the interactions may also be involved. Candidates include anti-
three cell types intrinsic to the glomerulus. Antibody attack bodies to dipeptidyl peptidase IV, β1 integrin, laminin, and
is directed to the epithelial cells in Heymann nephritis and antibodies to complement regulatory proteins, Crry and
in glomerular diseases associated with the anti–slit dia- CD59. The nephritogenic antigen(s) in idiopathic human
phragm antibodies, to mesangial cells in anti–Thy 1 dis- membranous nephropathy is still unknown; megalin is not
ease, and to endothelial cells when animals are injected expressed by human podocytes. There is still speculation that
with antibodies to ACE and possibly with antineutrophil circulating immune complexes involving exogenous antigens
cytoplasmic antibodies (ANCA). These interactions may may also play a role in human membranous nephropathy (37).
produce glomerular injury associated with the formation of
immune complexes (in situ) or associated with perturba- Antibody Interaction
tions in normal cellular function but without the formation The primary interaction that initiates the formation of sub-
of immune complexes. Knowledge gained from investiga- epithelial immune deposits in passive Heymann nephritis is
tion of these experimental models has revolutionized think- the binding in situ of antibodies to megalin on the foot pro-
ing about the immunopathogenesis of glomerular injury. In cesses of glomerular podocytes. In vitro studies suggest that
situ mechanisms now appear to play the dominant role in cross linking of the antibodies redistributes the antigens
most forms of human glomerulonephritis. into progressively larger aggregates that are subsequently
shed into the subepithelial space. These complexes may
then be modified by the addition of further megalin and/or
Podocytes
RAP molecules (36).
The model of Heymann nephritis has been extensively
studied because it resembles human membranous nephrop- Secondary Mediators
athy (Fig. 29.1). Heymann nephritis can be induced in sus- Induction of renal injury and proteinuria depends on the
ceptible strains of rats by active immunization with the activation of complement, but inflammatory cells are not
antigen. Proteinuria develops by 8 to 10 weeks in 30 to required. Immunofluorescence studies reveal early and prom-
80% of the immunized animals. A similar lesion can also be inent participation of the complement cascade (C3 and
induced passively by injection of the appropriate antibody, neoantigens of the MAC of complement) in the subepithelial
producing proteinuria within 5 days. deposits. Rats depleted of complement fail to develop pro-
teinuria despite antibody deposition, although perturbations
Nephritogenic Antigen in glomerular flow can be detected by micropuncture studies.
Extensive work by several investigators has determined that the Neutralization of Crry and CD59, complement regulatory
primary target antigen is a large 517-kDa glycoprotein named proteins expressed on the surface of podocytes (by antibodies
megalin (previously called glycoprotein gp330 and gp600) (36). naturally present in anti-Fx1A antiserum), promotes pro-
Megalin, a member of the low-density lipoprotein receptor teinuria (38). Sublethal podocyte attack by C5b-9 has several
gene family, is an endocytosis receptor for multiple ligands consequences, including the production of eicosanoids and
such as plasminogen, plasminogen activator complexes, apo- ROS and disruption of focal adhesions that link the actin
lipoprotein E–enriched β very-low-density lipoprotein, lipo- cytoskeleton to extracellular matrix (39,40). Disruption of
protein lipase, clusterin/apolipoprotein J, retinol-binding slit diaphragms and reduction of the glomerular heparan sul-
protein, insulin, β2 microglobulin, thyroglobulin, and certain fate agrin are other structural consequences associated with
polybasic drugs, to name a few. Megalin is also present on the development of proteinuria (41). Once glomerular dam-
other epithelial cells, including the brush border of proximal age is established, ongoing complement activation appears to
tubules where it serves an important role in albumin reabsorp- be unnecessary because complement depletion at this time
tion, pneumocytes, and ependymal cells in the brain. Mice does not abolish proteinuria. After transplantation of a kid-
that lack megalin develop abnormalities in the kidneys, lungs, ney with active Heymann nephritis into a normal syngenic
and central nervous system, and most die of respiratory failure rat, complement deposits begin to disappear within 2 to 4
shortly after birth. A second antigen is involved, a 44-kDa pro- weeks, but proteinuria persists much longer. These studies
tein known as RAP (receptor-associated protein). This protein suggest that structural alterations to the glomerular filter are
is homologous to human low-density lipoprotein receptor– long-standing once damaged by the antibody complement-
related protein/α2-macroglobulin receptor. RAP associates dependent pathway.
582 V. Glomerular Disease

Mesangial Cells genic, although the specifics of the connection between the
antibodies to the granulocyte proteins (proteinase 3 and
The rat Thy 1 model of glomerulonephritis has proven to be an
myeloperoxidase) and endothelial injury are still unravel-
important investigative model as so many glomerular diseases
ing. Classical “immune complexes” are not formed, but
are characterized by mesangial cell proliferation. Thy 1 is an 18-
recent data support a direct link between antibody-mediated
kDa membrane glycoprotein most notably expressed in the
activation of neutrophils and monocytes and glomerular
thymus and brain. It is also expressed on the surface of normal
endothelial injury (45). Endothelial cells may also express
rat mesangial cells; circulating rat lymphocytes are negative.
and/or internalize proteinase 3 and become directly involved
Mesangial cell injury is induced by the injection of heterolo-
in the pathogenetic process—both ANCA-dependent and
gous antithymocyte serum or anti–Thy 1.1 monoclonal anti-
-independent (46,47). Recent studies in animal models
body. Anti–Thy 1 nephritis is a biphasic model of glomerular
provide convincing evidence that antimyeloperoxidase anti-
disease, the first phase characterized by mesangiolysis (1 hour to
bodies cause glomerular damage (48).
2 days) and the second phase by mesangial cell proliferation
ACE present on endothelial cells has been used as an
and glomerular microaneurysm formation (4 to 14 days).
experimental target for antibody attack to induce epimem-
Despite histologic and glomerular micropuncture evidence of
branous nephropathy in rabbits, but whether there is a
significant glomerular injury, proteinuria is often mild. Mesan-
human counterpart to this model is unknown. The anti-
giolysis is the consequence of antibody-directed, complement-
ACE antibody binds to and redistributes endothelial cell-
mediated injury to mesangial cells and can be abrogated by
surface ACE, followed by shedding of antigen-antibody
complement depletion but not by the elimination of leuko-
complexes from the cell membrane in a sequence of events
cytes. Deposition of components of the terminal complement
reminiscent of that proposed for Heymann nephritis. The
cascade within the mesangium suggests that cell lysis is medi-
reason that these complexes move from a subendothelial
ated by the MAC of complement. The hypercellular phase is
(days 1 to 2) to a subepithelial position (days 3 to 24) con-
mainly due to the proliferation of the smooth muscle–type
centrated at filtration slits is not entirely clear, but it may be
mesangial cells. It does not occur if the mesangiolytic phase is
related to hydrodynamic factors as the plasma filtrate
prevented by complement depletion. Lymphohemopoietic
crosses the GBM. The glomerular lesion is characterized
cells—PMNs, monocytes, and platelets—also invade the glom-
histologically by complement deposition and the presence
erulus during the initial phase of mesangial injury. Several
of PMNs, but their respective roles in the pathogenesis of
mesangial cell mitogens have been identified, and their effects
the glomerular disease remain to be defined.
on mesangial cell cycle regulatory proteins are becoming clari-
fied and offer the possibility of future therapeutic targeting (Fig.
29.2). Integrin-dependent interactions between mesangial cells Interaction of Antibodies with Exogenous
and their matrix also modulate their proliferative response (25). Antigens Trapped or Planted within
Platelets and PDGF-B appear to play an important pro-mitotic the Glomerulus
role in the Thy 1 model. Although antimesangial cell antibod-
Exogenous macromolecules trapped or planted within the
ies appear to be uncommon in humans, they have been identi-
glomerulus may become antigenic targets for antibodies,
fied in a few patients with IgA nephropathy and lupus nephritis
resulting in the in situ formation of immune complexes.
(42,43). Nonetheless, mesangial cells respond to and elicit a
Although still unproven, this pathway may account for
large variety of pathophysiologic events, whether initiated by an
some forms of immune complex nephritis occurring in
antibody or another form of injury.
humans after exposure to certain drugs, toxins, or microor-
ganisms or in association with malignancy. Endogenous
antigens originating at extrarenal sites may also induce
Endothelial Cells
renal injury by a similar mechanism.
The ability of antiendothelial antibodies to cause glomerular
damage is best exemplified by renal allograft rejection. Several
Antigen Candidates
antigen families may be targeted, especially MHC class I and
II antigens. Antiendothelial antibodies have also been demon- Several experimental manipulations have been used to trap
strated in patients with SLE, systemic vasculitis, and throm- antigens in the glomerulus with glomerular injury ensuing as
botic microangiopathy, although the relationship of these circulating antibodies bind in situ to the planted antigen. Sev-
antibodies to the pathophysiology of the disease remains eral factors might promote antigen entrapment. Low (subne-
unclear. An animal model of antibody-induced renal throm- phritic) concentrations of antiglomerular antibodies may
botic microangiopathy has recently been developed and some initially bind and serve as a magnet for further antibody
of the mediators of injury identified, but the target antigen(s) recruitment (e.g., antiidiotypic antibodies, antirheumatoid
have yet to be determined (44). factor antibodies or antiheterologous antibodies as occurs in
There is increasing evidence that ANCA associated with animals injected with nephrotoxic serum produced in another
pauciimmune necrotizing glomerulonephritis are patho- animal). Other possibilities include electrostatic interactions
 29. Immune Mechanisms of Glomerular Injury 583

(cationic antigens binding to GBM anionic sites such as strep- to-antibody ratio, has a major influence on its rate of clear-
tococcal proteinase) (49), glomerular carbohydrate interac- ance from the circulation (faster rates being associated with
tions with lectins (e.g., lectin-like domains on microbial a lower risk of nephritis) and on the pattern of deposition
pathogens), glomerular matrix interactions (e.g., DNA- within glomeruli. Large-latticed complexes formed near
histone complexes binding glomerular heparan sulfate and antigen:antibody equivalence are efficiently eliminated by
fibronectin), or mesangial entrapment of a molecule of colloi- the mononuclear phagocyte system and are minimally
dal size as they percolate through normal mesangial channels. entrapped by glomeruli, mainly in the mesangium. The
smallest immune complexes pass freely through the glomer-
ulus without being trapped. It is the intermediate-size
Glomerular Deposition of Circulating
immune complexes (e.g., Ag2:Ab2) that are retained within
Immune Complexes
glomeruli. In primates, soluble-circulating immune com-
Since the recognition of the in situ mechanism of glomerular plexes are eliminated via a complement-dependent “eryth-
immune complex formation, the concept of the entrapment rocyte shuttle” within the mononuclear phagocyte system.
of immune complexes preformed in the circulation has been Within minutes of formation, circulating immune com-
increasingly challenged. It is now believed that most immune plexes bind complement and attach to the erythrocyte
complexes form in situ even in such classical diseases as lupus complement receptor 1 (CR1). This interaction protects
nephritis. Nonetheless, the “circulating immune complex” peripheral organs such as the kidney from immune-complex
pathway likely contributes to some extent in the pathogene- deposition and injury. Erythrocytes bearing immune com-
sis of human glomerulonephritis, and nearly 75 years of plexes circulate to the liver, where the complexes are trans-
work using animal models of immune-complex glomerulo- ferred to Kupffer cell Fc receptors and catabolized.
nephritis have taught us a great deal about the immuno- Physicochemical properties such as net charge are
pathogenesis of glomerular injury. With rare exception, the important. Given the net negative charge of the glomerular
nature of the nephritogenic antigens in human glomerulone- capillary wall, cationic antigens are more nephrotoxic than
phritis are unknown. Putative antigens include foreign serum neutral or anionic ones. Both size and charge also influence
proteins, drugs, food antigens, infectious organisms (bacte- the location of trapping. Small highly cationic complexes
ria, parasites, viruses, fungi, and mycoplasma), and certain (isoelectric point, 9.5 to 11.5) deposit along the glomerular
endogenous antigens (nucleic acids, thyroid antigens, tumor capillary wall, whereas large cationic complexes tend to
antigens, nuclear non-DNA antigens, erythrocyte antigens, lodge in the mesangium. The high renal blood flow rates
and renal tubular antigens). and transcapillary hydraulic pressure gradients are thought
to explain why the kidney is particularly vulnerable to
immune complex–mediated tissue injury. Alterations in
Animal Models of Immune Complex
glomerular hemodynamics may affect the severity of injury.
Glomerulonephritis
For example, serum sickness is worse in hypertensive rab-
The classic model of immune complex–induced tissue bits, a reduction in GFR decreases immune-complex depo-
injury is acute serum sickness, induced in rabbits by inject- sition, and renal artery stenosis protects the kidney from
ing heterologous plasma proteins such as albumin or oval- immune-complex disease.
bumin. The clinicopathologic features resemble those of
acute poststreptococcal glomerulonephritis. Glomerular
In Situ Modification and Damage
injury begins abruptly during the phase of antibody-depen-
dent immune elimination of the antigen coincident with Trapped glomerular immune complexes are free to escape
the appearance of circulating immune complexes. Injury unless a series of events occur in situ to stabilize them.
resolves again in 2 to 3 weeks. The most striking and exten- Intraglomerular immune complexes enlarge over time, the
sively studied spontaneous models of immune complex morphologic counterpart of the in situ interactions that
occur in certain strains of mice and resemble lupus nephri- rearrange, enlarge, and stabilize the lattice structure. In fact,
tis (NZB X NZW)F1, BXSB, and MRL/lpr/lpr mice. the immune complex is in a state of dynamic equilibrium
with its component parts in the circulation. The immune
response typically involves polyclonal B-cell activation,
Glomerular Immune Complex
involving a heterogeneous group of antibodies. The addi-
Deposition and Injury
tion of antibodies of increasing affinity may provide further
Multiple factors influence the capacity of a circulating stability to the complex. The entire immune complex may
immune complex to remain soluble, lodge within, and ini- act as a planted antigen, recruiting additional antibody sys-
tiate glomeruli injury. First, the antigen must be immuno- tems such as rheumatoid factors (antibodies reactive with
genic and persist in the circulation long enough to allow the Fc and occasionally Fab fragment of IgG), antiidiotypic
antibody generation and binding. The size of the immune antibodies binding to unique epitopes of the Fab antibody
complex that is formed, determined primarily by the antigen- fragment, and immunoconglutinins, which are IgM anti-
584 V. Glomerular Disease

bodies that react with sites on activated fragments of C3 of IgA alone is insufficient to cause glomerular injury. IgA
and C4. At other times, subsequent events dissolve the may also bind to mesangial cells and trigger subsequent
complexes. The addition of nonprecipitating antibodies, effects, although the specific receptor involved remains to
excess antigen, and/or complement proteins may decrease be identified—it appears to be distinct from the known
and ultimately dissolve glomerular immune deposits. Pro- receptors IgA FcαR1 (CD89), asialoglycoprotein receptor,
teolytic enzymes may also accelerate complex removal. and the polymeric Ig receptor (55).
Once nephritogenic immune complexes stabilize within Whereas C3 is almost always present in association with
the glomerulus, they trigger a sequence of events that IgA immune deposits, IgA is an inefficient activator of the
induce glomerular inflammation and proteinuria. Marrow- alternative pathway. The recent recognition that IgA can
derived monocytes are the key mediators of proteinuria in activate complement by the third or mannan-binding lectin
experimental models of acute serum sickness. pathway may be relevant to IgA glomerular injury (56).
Alternatively it may be the co-deposited IgG or IgM mole-
cules that activate complement and mediate renal injury. In
Unique Features of Immunoglobulin
most experimental IgA models, hematuria is C3 depen-
A Nephropathy
dent, but the terminal components C5b-9 are not required.
New insights into the pathogenesis of experimental IgA
nephropathy have been made in recent years and are fur-
ther discussed in Chapter 31. Only a few unique factors CELLULAR IMMUNE MECHANISMS OF
will be mentioned here. It appears that many antigens may INDUCTION OF GLOMERULAR INJURY
be involved, although this remains speculation. One recent
study identified Haemophilus parainfluenzae antigens in the T lymphocytes play an intricate role in the immune
mesangium of 31% with IgA nephropathy and 4% with responses that cause glomerulonephritis, usually culminat-
other renal diseases, illustrating one example of a possible ing in the generation of nephritogenic antibodies. Consid-
link between microbial pathogens in this disease (50). The erably less common, it has long been suspected that T cells
IgA molecule is unique in that 10 to 20% circulates in a may occasionally cause glomerular injury by recruiting
polymeric form, with monomers linked by J chain. monocytes in the absence of antibodies. Evidence that T
Humans produce two isotypes—IgA1 and IgA2—but it cells alone, working in the absence of antibodies, can
appears that most of the glomerular deposits are mono- induce glomerular injury, has been convincingly demon-
meric IgA1. Despite a strong clinical correlation between strated in animal models of autoimmune glomerulonephri-
acute mucosal infections (respiratory and gastrointestinal), tis. Transfer of GBM-sensitized CD4+ T cells to normal
and perhaps abnormal mucosal immunity, and nephritis animals can induce glomerulonephritis (34,57). In a related
exacerbations, most of the data point to the bone marrow model based on the injection of subnephritic doses of anti-
and not the mucosal immune system as the origin of the GBM antibodies (that serves as the “planted” antigen for
glomerular IgA1 deposits. Bone marrow transplantation subsequent immunologic attack by the host immune
data, both human anecdotes and studies in the spontaneous system), crescentic glomerulonephritis develops even in
ddY mouse model, suggest that IgA nephropathy is a pri- antibody-deficient mice, whereas disease is attenuated in T-
mary stem cell disorder (51). cell–depleted animals (58).
Several recent studies have focused on the biochemical An alternative T-cell–dependent antibody-independent
structure of the IgA molecule and have consistently mechanism that may cause glomerular dysfunction involves T-
reported differences in the carbohydrate structure between cell activation and release of a “lymphokine” that alters the glo-
patients and controls (52,53). The number of O-linked merular permselectivity barrier, causing proteinuria in the
galactose sugars in the hinge region of IgA1 is significantly absence of glomerular inflammation (59). Such a mechanism,
reduced in the patients. Although the metabolic basis of first proposed Shalhoub in 1974 (59a), is still the favored
this galactose deficiency is not yet known, it is thought to immunopathogenetic paradigm for idiopathic minimal
be of primary pathogenetic significance. Relevant observa- change nephrotic syndrome and focal segmental glomerulo-
tions include its delayed rate of clearance from the circula- sclerosis (FSGS) despite the lack of definitive evidence. The
tion, increased binding to mesangial cells, and recognition nature of the putative lymphocyte-derived glomerular perme-
as an antigenic determinant by naturally occurring IgA1 ability factor remains elusive despite several proposed candi-
and IgG antibodies. dates [reviewed in (60)]. Savin et al. identified a factor in the
The ability of IgA to bind to fibronectin and certain lec- plasma of FSGS patients that exerts permeability changes in a
tins may facilitate glomerular uptake. After the observation rat glomerular bioassay and predicts the risk of allograft-recur-
that mice lacking uteroglobin, an antiinflammatory protein rent FSGS in some patients (60a). The factor itself has not
with high affinity for fibronectin, spontaneously develop been isolated. Immunoabsorption to protein A suggests that it
IgA nephropathy, additional studies have yet to confirm its may circulate in association with IgG. Similar permeability
role in human IgA nephropathy (54). Mesangial deposition activity has also been identified in the plasma of patients with
 29. Immune Mechanisms of Glomerular Injury 585

FIGURE 29.4. Antigen-dependent T-cell activation and functional polarization. When T cells
encounter antigen-presenting cells (APC), at least two signals must be transmitted before an
immune response is initiated. The first signal is emitted by ligation of the T-cell receptor with
antigenic peptides bound to major histocompatibility complex (MHC) molecules on the APC. T-
helper cells (CD4+) interact with MHC class II, and T-cytotoxic cells (CD8 +) interact with MHC class
I molecules. A second signal is required for full activation. It may be provided by CD28 that acti-
vates T cells after binding to its ligand, B7-1 or B7-2, on APCs. Once activated, T cells begin to
express a CD28 homologue (CTLA-4) that binds B7-1 and B7-2 with higher affinity and inhibits T-
cell activation. Therapeutic administration of CTLA-4 can decrease the severity of autoimmune
nephritis. Two new members of the CD28 superfamily are ICOS (inducible costimulator) and PD-1
(programmed death-1) that interact with their respective APC ligands, B7RP-1, and PD-L1 or PD-
L2. The second major costimulatory family is the tumor necrosis factor (TNF)–TNF-R family, which
includes CD154 expressed by activated T cells and CD40 expressed by APCs, including B cells. The
CD154-CD40 system appears to be particularly important in antibody-mediated diseases such as
lupus nephritis. Several new TNF-TNF-R superfamily members have been identified and await
investigation in glomerulonephritis (GN): 4-IBB/4-IBBL, CD134/CD134L, and CD27/CD70. Resident
glomerular mesangial and epithelial cells may function as nonprofessional APCs to activate T
cells, but it is unlikely that they can initiate a primary immune response. T-helper (Th) cells acti-
vated by antigen can be divided into two distinct subsets based on the cytokines they produce.
Th1 cells synthesize interleukin-2 (IL-2), interferon-gamma (IFNγ), TNF, and granulocyte-macro-
phage colony stimulating factor (GM-CSF) and mediate delayed-type hypersensitivity reactions
with monocyte and cytotoxic T-cell activation and immunoglobulin class switching to favor the
production of complement-fixing immunoglobulin G (IgG) isotypes. The Th1 pathway predomi-
nates in hypercellular/proliferative forms of glomerulonephritis. In contrast, Th2 cells are charac-
terized by the production of IL-4, -5, -10, and -13 and mediation of allergic, immediate-type
hypersensitivity reactions associated with mast cells and production of IgE and the weaker com-
plement-fixing IgG1. The Th2 cytokine profile is commonly associated with nonproliferative glo-
merular diseases such as minimal change nephrotic syndrome and membranous nephropathy.
Cytokine therapy has been used experimentally to attenuate Th1-dependent proliferative dis-
eases: either administration of Th2 cytokines or inhibition of Th1 cytokines/receptors. (Modified
from Kitching AR. Cytokines, T cells and proliferative glomerulonephritis. Nephrology
2002;7:244–249.)
586 V. Glomerular Disease

podocin mutations, indicating that this “factor” is not unique glomerulonephritis. Although most of these cells migrate into
to idiopathic FSGS (61). Alternatively, it has been suggested glomeruli from the circulation, they may also proliferate in situ
that proteinuria may be the consequence of the loss of an (67). Beginning with landmark studies in the late 1970s, the
inhibitory substance, perhaps a lipoprotein, which serves in pivotal role played by Mφ in the genesis of primary glomerulo-
normal individuals to block the permeability factor (62,63). nephritis has clearly been established by numerous studies. Mφ
Two fundamental aspects of T-lymphocyte biology have been may also be recruited during a secondary wave of glomerular
extensively investigated in the past decade, yielding insights injury, associated with disease progression, as demonstrated in
that bear relevance to the basic immunology and possible ther- the remnant kidney model and in lipid-induced glomerular
apeutics of glomerulonephritis. The first is the identification of disease. Although unraveling the effector pathways of mono-
pairs of costimulatory molecules involved with MHC mole- cyte-dependent glomerular damage is challenging owing
cules in antigen recognition, and the second is the identifica- to their multifunctionality, several activities appear to be
tion of functionally distinct subsets of T cells (64–66). These involved: (a) phagocytosis, which may eliminate immune reac-
functions are reviewed in Figure 29.4. tants but may also activate pathways that promote damage;
(b) antigen presentation to T cells after induction of
MHC class II surface molecules; and (c) biosynthesis of
SECONDARY MEDIATORS OF
numerous proinflammatory products: cytokines, procoagulant
GLOMERULAR INJURY
and fibrinolytic proteases and proteins, matrix-degrading
enzymes, ROS, vasoactive substances, and cationic proteins.
After the primary immunopathogenic event that initiates glo-
Although Mφ express CRs (CR1 and CR3) and are chemoat-
merular injury, a series of secondary mediators are recruited
tracted to C5a in vitro, glomerular Mφ recruitment is usually
and activated to execute the inflammatory response. Definitive
complement-independent in experimental models. Mφ also
proof that these pathways are involved in renal damage is usu-
express receptors for antibody Fc fragments that may partici-
ally based on the results of selective inhibition or depletion of
pate in their recruitment and activation (68). It is also quite
the effector system under investigation and by studies in
clear that activated Mφ and PMN represent a highly heteroge-
knock-out mice.
neous population of cells, and even in glomerulonephritis
some of these cells are programmed to be antiinflammatory
Polymorphonuclear Leukocytes and to terminate ongoing injury.
PMNs are present in several human and experimental glomeru-
lonephritides in which they contribute to inflammation-associ- Glomerular Leukocyte
ated injury. Depletion of PMNs during the heterologous phase Recruitment Mechanisms
of anti-GBM nephritis and in the Con A-anti–Con A model of
immune-complex nephritis prevents proteinuria. In PMN- To reach a site of inflammation, leukocytes must first adhere
dependent lesions, the cells appear abruptly, and several factors to and subsequently emigrate through retracted endothelial
may play a role in their recruitment, including chemoattraction junctions and the basement membrane out into the sur-
to C3a, C5a, or alpha (C-X-C) chemokines; immune adher- rounding tissue [reviewed in (69)]. This is accomplished by a
ence to CR1 or CR3; and Fc receptor–dependent binding to multistep process involving a series of leukocyte surface
antibodies. Among numerous toxic PMN secretory products, adhesion molecules that bind to cognate receptors on the
ROS and proteolytic enzymes (e.g., GBM-degrading serine endothelium (Fig. 29.5; Table 29.2). Leukocyte migration to
proteases, neutral proteinases such as elastase and cathepsin G sites of inflammation is directionally regulated by chemoat-
and matrix-degrading metalloproteinases) have been most fre- tractant molecules, especially chemokines (chemotactic cyto-
quently implicated in glomerular damage. Other PMN activi- kines). These small proteins (8 to 10 kDa) are subdivided
ties that may contribute include proteolytic activation of into four major families (already more than 50 ligands and
plasma protein cascades, such as the coagulation pathway; 17 different receptors identified) based on the position of the
release of cationic proteins that may bind and neutralize GBM first two cysteine residues (Table 29.3) (70). Only two of the
anionic charge sites; and the synthesis of phospholipid metabo- families have been extensively investigated. The α-chemo-
lites [PGs, thromboxanes (Tx), leukotrienes, and PAF], hydro- kines share a common CXC structure (X denotes any inter-
gen peroxide, and vasoactive substances (histamines). Products vening amino acid), and many are involved in the
derived from PMN granules that are targeted by ANCA have recruitment of PMN. They bind to G-protein–coupled
been implicated in the genesis of endothelial cell damage and CXC receptors and activate intracellular phosphatases. Each
focal necrotizing glomerulonephritis. receptor typically binds more than one chemokine making
for great pleiotropy and redundancy in the system. PMN are
known to express CXCR1 and CXCR2. It has recently been
Monocytes and Macrophages
reported that glomerular podocytes also express CXCR1, -3,
Monocytes and macrophages (Mφ) are present in many glo- -4, and -5, although their role here has yet to be determined
merular diseases, particularly in crescentic and postinfectious (71). IL-8 (that binds to CXCR1 and 2) has been implicated
 29. Immune Mechanisms of Glomerular Injury 587

binds to monocyte CCR1 and CCR5, and its inhibition has

also been protective in experimental proliferative nephritis.
However, neither MCP-1 nor RANTES blockade achieves
sustained reversal of monocyte infiltration and proteinuria in
any model. It has become evident that several of the chemo-
kines have other effects, including cellular activation and gen-
eration of Th1 or Th2 responses. For example, inhibition of
macrophage inflammatory proteins (MIP)-1α and -1β (CC
chemokines) decreased proteinuria without affecting mono-
cyte numbers. Rather surprising, despite the beneficial effect
reported in experimental nephritis with either antireceptor
FIGURE 29.5. Mechanisms of glomerular leukocyte recruitment. antibody therapy or inhibition of MCP-1, genetic deletion of
The earliest and relatively transient adhesions, which cause leuko- CCR1 and CCR2 has not been protective in the same disease
cytes to “roll” along the endothelium, are mediated by surface
receptors with lectin-like domains (selectins) that bind to specific model (73–76). In fact, both knock-out mice develop worse
glycoprotein ligands (mucins). Not expressed by normal glomeruli, anti-GBM nephritis, perhaps due to a greater Th1 polarity
P- and E-selectins have been detected in some diseased human and more aggressive crescent formation.
glomeruli. Their only ligand in normal glomeruli is CD34. Selectin
blocking and neutralization studies have not been shown to pre- Fractalkine is the only known membrane-bound chemo-
vent leukocyte recruitment or proteinuria. In fact, in a study of P- kine. It belongs to a third chemokine class, CXXXC, and
selectin–deficient mice, anti–glomerular basement membrane binds to its own unique receptor (CX3CR1) that is expressed
(GBM) nephritis was more severe compared to wild-type mice
(149). Once activated, leukocytes soon become firmly attached to on natural killer cells, T cells, and mesangial cells (77,78).
the endothelium by engaging leukocyte integrins (α1β1 and β2) Chemokine expression is primarily induced by proinflamma-
with their endothelial ligands, which are members of the immuno- tory cytokines such as IL-1 and TNF-α. The transcription
globulin (Ig) gene superfamily: intercellular adhesion molecules
(ICAM) and vascular cell adhesion molecules (VCAM) (reviewed in factor NF-κB is frequently involved, and NF-κB inhibition
Table 29.2). Low-level glomerular ICAM-1 and ICAM-2 expression may be an effective strategy to reduce glomerular inflamma-
has been observed in some studies, whereas VCAM-1 appears lim- tion (79). In addition to lymphohematopoietic cells, mesan-
ited to parietal epithelial cells of Bowman’s capsule. Thus far, inter-
ventions designed to prevent leukocyte recruitment in acute gial cells may be an important source of chemokines in
glomerular disease by selectively blocking a single leukocyte inte- glomerulonephritis. Resident glomerular cells may also
grin ligand have generally been ineffective. Whether this is due to respond to chemokines via specific receptors. Mesangial cells
overlapping functions of other receptors or whether other reac-
tions promote leukocyte adherence to the glomerular endothe- express CCR1 and CX3CR and proliferate in response to IL-
lium remains to be determined. Galectins, osteopontin, and CD44 10 via an unknown receptor.
may be involved, for example. In addition to recruitment, leuko-
cyte integrin-ligand interactions may activate several intracellular
signaling pathways and alter leukocyte functions. These effects Platelets and Platelet-Activating Factors
may partially explain why mice with genetic ICAM-1 deficiency and
nephrotoxic serum nephritis develop milder chronic renal damage Intraglomerular platelets or their degradation products are
(150). Directed by chemokines, leukocytes ultimately migrate
between endothelial cells to the site of injury. commonly observed in diseases such as membranoprolifer-
ative glomerulonephritis and lupus nephritis. Evaluation of
circulating platelets isolated from such patients often shows
in PMN recruitment in some inflammatory glomerular dis- evidence that they have been activated in vivo. Although
eases. Additional CXC chemokines are chemoattractants for antiplatelet therapy has been advocated in the diseases dis-
other cells but not PMN. These include interferon gamma cussed earlier, its efficacy has not been clearly established.
(IFN-γ)–inducible protein of 10 kDa (IP-10) and monokine The role of platelets as inflammatory mediators in experi-
induced by IFN-γ (Mig); both have been detected in mental glomerulonephritis has been investigated for several
inflamed glomeruli (72). decades now, but results have been inconsistent because of
The role of the second chemokine family in glomerulone- the difficulties inherent in platelet depletion and nonspe-
phritis, the CC or β chemokines, has been more extensively cific drug inhibition studies.
investigated. In general, the CC chemokines recruit mono- Mechanisms of glomerular platelet recruitment to the
cytes and other mononuclear cell lineages but not PMN. At kidney are poorly characterized. Complement activation is
least 10 CC receptors are known; monocytes express CCR1, critical in a model of in situ immune-complex nephritis
-2, and -5. Monocyte chemoattractant protein-1 (MCP-1) (Con A-anti–Con A) and in anti–Thy 1 nephritis, but
has been most extensively investigated; MCP-2, -3, -4, and -5 whether this is a result of immune adherence to CR1 or of
also exist. MCP-1 binds to CCR2 and its expression is often chemoattraction to C5a is unknown. Platelets may adhere
detected in proliferative and crescentic glomerulonephritis. to and become activated by the GBM in a reaction reminis-
MCP-1 deficiency or neutralization usually decreases glomer- cent of the in vitro effects of collagen. Other factors that
ular monocyte numbers and disease severity. RANTES (regu- have been implicated in intraglomerular adherence or acti-
lated upon activation, normal T cell expressed and secreted) vation of platelets include immune adherence to Fc recep-
588 V. Glomerular Disease

TABLE 29.3. CHEMOKINES IN GLOMERULAR DISEASE

Family Members Abbreviation Receptors Primary target cells

CXC (alpha) Growth-related onco- GRO-α CXCL1 CXCR2 PMN

gene alpha
Platelet factor 4 PF4 CXCL4 ? PMN, eosinophils
Interleukin-8 IL-8 CXCL8 CXCR1, CXCR2 PMN
Monokine induced by Mig CXCL9 CXCR3 T cells
interferon gamma
Interferon gamma– IP-10 CXCL10 CXCR3 Th1 cells, NK cells
induced protein
CC (beta) Monocyte chemoattrac- MCP-1 CCL2 CCR2 Mφ, dendritic cells, T cells, NK
tant protein-1 cells
Macrophage inflamma- MIP-1α CCL3 CCR1, CCR5 Mφ, dendritic cells, Th1 cells,
tory protein-1α NK cells
Macrophage inflamma- MIP-1β CCL4 CCR5 Mφ, dendritic cells, Th1 cells,
tory protein-1β NK cells
Regulated on activa- RANTES CCL5 CCR1, CCR3, CCR5 Mφ, dendritic cells, Th1 cells,
tion, normal T cell NK cells
expressed and
secreted
Eotaxin CL11 CCR3 Eosinophils, basophils, Th2
cells, dendritic cells
CX3C Fractalkine FKN CX3CL1 CX3CR1 T cells, NK cells
C Lymphotactin LTN XCL1 XCR1 T cells

Mφ, monocytes/macrophages; NK, natural killer; PMN, polymorphonuclear leukocyte; Th, T helper.
Note: This is a partial listing, highlighting chemokines most frequently investigated in glomerular disease. CXCR4 is also a T-cell coreceptor for
human immunodeficiency virus (HIV)-1; CCR5 is a coreceptor for HIV-1 strains that infect macrophages and activated T cells.

tors, activation of the coagulation cascade, TxA2, and PAF. by specific surface receptors to initiate a variety of cellular
The role of platelet adhesion molecules, including P-selec- responses (Table 29.4). Most of these factors can be produced
tin, glycoprotein Ib, platelet-endothelial cell adhesion mol- by native glomerular cells as well as inflammatory cells.
ecule (PECAM-1), and β3 integrins (e.g., glycoprotein IIb-
IIIa) in glomerular disease has not yet been determined.
Epidermal Growth Factor Family
Several platelet products may be injurious to glomeruli
such as bioactive lipids (PAF and TxA2), platelet factor 4 (a One of the first circulating growth factors to be recognized,
cationic protein that participates in the coagulation cascade EGF was originally isolated from mouse submandibular
and is a CXC chemokine for leukocytes), and growth fac- glands and human urine. Most studies have failed to iden-
tors (PDGF, TGF-β1). PAF has a multitude of biologic tify immunoreactive EGF in diseased glomeruli. In vitro,
effects that may be mediated by the phospholipid itself EGF stimulates mesangial cell contraction and PG synthe-
(leukocyte chemoattraction, altered vascular permeability) sis; intrarenal EGF infusion decreases GFR and renal blood
or indirectly by stimulating release of substances such as flow. TGFα shares 30% sequence homology with EGF and
Tx, PGs, leukotrienes, lysosomal enzymes, superoxide, IL- exerts its biologic action by binding to the EGF receptor. A
1, MCP-1, and TNF. PAF is not only produced by platelets newer family member, heparin-binding EGF-like growth
but also in turn triggers platelet aggregation, activation, and factor, a mesangial cell mitogen, is expressed in disease
degranulation. Antagonists to PAF or its receptor have models associated with mesangial cell proliferation (80).
decreased disease severity in several experimental glomeru- Heparin-binding EGF also has direct glomerular hemody-
lonephritis models. Platelets may further modulate glomer- namic effects (81).
ular injury by activating C3 and C5 of the complement
cascade, activating the intrinsic coagulation cascade, or by
Fibroblast Growth Factor
synthesis of β-thromboglobulin (inhibits endothelial release
of prostaglandin I2) or vasoactive amines such as histamine FGF-2 or basic FGF (bFGF) is a potent angiogenic factor
and serotonin. that also regulates matrix synthesis and is a mitogen for all
intrinsic glomerular cells. FGF-2 has affinity for heparin and
HSPG and may bind to basement membranes/extracellular
Soluble Secreted Peptides
matrix in vivo. In the Thy 1 nephritis model, production of
Growth factors and cytokines are small polypeptide messen- bFGF increases during the phase of mesangial cell prolifera-
gers (most approximately 6 to 30 kDa) that bind to target cells tion. Disease severity is enhanced by exogenous FGF and
 29. Immune Mechanisms of Glomerular Injury 589

TABLE 29.4. SOLUBLE SECRETED PEPTIDES IN ACUTE GLOMERULAR INJURY

Promotes injury Prevents injury Either (model-dependent)

Fibroblast growth factor-2 Interleukin-4 Granulocyte-colony stimulating factor

Granulocyte-macrophage colony stimulating factor Interleukin-11 Interleukin-6
Heparin-binding epidermal growth factor–like growth factor Interleukin-13 Interleukin-10
Interferon-gamma Vascular endothelial growth Interleukin-12
Interleukin-1 factor Transforming growth factor β
Interleukin-8
Interleukin-18
Macrophage colony stimulating factor
Macrophage migration inhibition factor
Platelet-derived growth factor
Tumor necrosis factor-α

attenuated by FGF-2 neutralization/antagonism (82). FGF diseases. IL-1 induces mesangial cell proliferation and
may also contribute to the genesis of glomerulosclerosis as promotes synthesis of a variety of substances: eicosanoids,
chronic administration causes podocyte damage in rats, and oxygen radicals, collagenase, cytokines, chemokines, and
it enhances cytokine-induced endothelial cell apoptosis (83). adhesion molecules. It also enhances collagen synthesis by
glomerular epithelial cells. Renal IL-1 mRNA levels are
increased in several glomerular diseases, and neutraliza-
Colony-Stimulating Factors
tion studies with a soluble IL-1 receptor antagonist
Colony-stimulating factors (CSF) are a family of glyco- diminish inflammation and proliferation in rats with
proteins that not only regulate the development of hema- anti–Thy 1 or nephrotoxic serum nephritis (88). IL-8 is
topoietic progenitor cells but also promote the an important mediator of granulocyte chemoattraction
inflammatory response of mature cells such as macro- and activation. Mesangial cells produce IL-8 when stimu-
phages. Increased CSF expression has been reported in lated with cytokines such as IL-1 and TNF-α. IL-8 neu-
several proliferative glomerular diseases. Mice deficient in tralization prevents neutrophil infiltration in a model of
granulocyte-macrophage (GM)–CSF develop less inflam- immune complex glomerulonephritis. In humans, urinary
mation (PMN and Mφ) after injection of nephrotoxic IL-8 levels are higher in patients with glomerulonephritis
antiserum and membranoproliferative glomerulonephritis associated with leukocyte infiltration. IL-18 treatment has
has been reported in a patient exposed to chronic GM- been reported to exacerbate SLE nephritis and cell-medi-
CSF treatment (84,85). The effects of G-CSF may be ated crescentic nephritis (89,90).
dose-dependent based on the observation that low-dose ILs with antiinflammatory effects that have been shown
G-CSF promotes a Th2 response and worse nephritis in to attenuate glomerulonephritis include IL-4, IL-10, and
MRL-lpr/lpr mice, whereas high-dose therapy downregu- IL-11. Promotion of a Th2 response and inhibition the
lated expression of the Fcγ receptor III and attenuated proinflammatory effects of macrophages are effects of IL-4.
nephritis (86). Increased M-CSF has also been observed Both mesangial cells and podocytes can produce IL-4. The
in proliferative nephritis. Macrophage migration inhibi- protective role of IL-4 has been documented in the anti-
tory factor is another proinflammatory protein shown to GBM nephritis model, both by studies in IL-4–deficient
be upregulated in glomerular diseases associated with a mice that develop worse disease and by IL-4 therapy that
leukocyte infiltrate. Anti–migration inhibitory factor attenuates disease (91–93). In this model, IL-11 therapy
antibodies partially reverse glomerular injury induced has also been protective (94). IL-10 suppresses the nephri-
with anti-GBM antiserum (87). togenic Th1 immune responses; IL-10–/– mice develop
worse nephrotoxic serum nephritis than IL-10+/+ mice
(95). In the Thy 1 nephritis and anti-GBM nephritis
Interleukins
model, IL-10 therapy is protective (95,96).
ILs are a large family of cytokines (at least 25) that play a Some ILs may have either pro- or antiinflammatory
central role in inflammatory and immunologic responses. effects depending on the type of disease evaluated. For
The expression and function of several members have example, IL-6 is a cytokine produced by several blood-
been examined in glomerulonephritis. In addition to the borne cells as well as glomerular endothelial and mesangial
role played by ILs in the polarization and function of cells. IL-6 may be detectable in glomeruli and the urine in
Th1/Th2 cells, several ILs are produced by and/or regu- several proliferative glomerular diseases in animals and
late the behavior of resident glomerular cells. IL-1, IL-8, humans. Neutralizing anti–IL-6 antibodies and IL-6 recep-
and IL-18 have proinflammatory roles in most glomerular tor blockade decreased the severity of lupus nephritis,
590 V. Glomerular Disease

whereas IL-6 transgenic mice spontaneously develop glo- effects that may prove to be relevant to initiation of immune-
merulonephritis (97). Yet, IL-6 recombinant protein therapy mediated glomerular injury. Although TGFβ therapy has
reduced disease severity in rats with anti-GBM nephritis been reported to attenuate experimental autoimmune dis-
(98). Anti-GBM nephritis is less severe in IL-12–deficient eases such as arthritis and encephalitis, mice overexpressing
mice, whereas IL-12 DNA plasmid therapy reduces the Smad7, an inhibitor of TGFβ signaling, were found to have
severity of immune complex nephritis associated with graft- less severe anti-GBM nephritis (107).
versus-host disease (99,100).
Tumor Necrosis Factor
Interferon Gamma
TNF-α is a proinflammatory cytokine primarily produced
IFN-γ is a member of a family of glycoproteins (α, β, and γ) by monocytes and macrophages, although mesangial cells
with immunomodulatory, antiviral, and antineoplastic activ- can also synthesize TNF-α. It is particularly important in the
ity. IFN-γ has been shown to play a key role in several genesis of septic shock and the associated renal endothelial
autoimmune diseases including lupus nephritis. Deletion of damage. TNF-α is also thought to play a role in proliferative
IFN-γ or its receptor or treatment with a neutralizing anti- glomerular diseases including anti-GBM nephritis, murine
body or IFN-γR-Fc chimeric protein reduces renal disease in lupus nephritis, and serum sickness. Anti-GBM nephritis is
several SLE mouse models (101,102). Macrophages have less severe in TNF-α knock-out mice (108). TNF-α can
been identified as an important source of IFN-γ, and IFN-γ induce mesangial cell contraction and synthesis of a variety
expression regulates glomerular monocyte migration (103). of products such as IL-1, IL-8, GM-CSF, MCP-1, PGs, pro-
coagulant, NO, and ROS.
Platelet-Derived Growth Factor
Vascular Endothelial Growth Factor
PDGF is a dimeric glycoprotein of four known isoforms—
PDGF-A, -B, -C, and -D. It has diverse biologic activities, VEGF, also known as vascular permeability factor, promotes
including chemoattraction and activation of inflammatory capillary repair and angiogenesis through endothelial cell
cells, mitogenesis, and promotion of tissue repair. It also has mitogenesis. Four isoforms have been identified: VEGF121
vasoconstrictor properties. The mitogenic effect of PDGF on and VEGF165 (the most abundant) are soluble secreted forms,
mesangial cells is especially remarkable, but it may also trig- whereas the 189 and 206 isoforms are usually cell-bound.
ger a chemotactic and contractile response. Mesangial cells Within glomeruli, podocytes and activated mesangial cells
may also produce PDGF. During the mesangial proliferative produce VEGF, whereas its receptors, flt-1 and flk-1, are
phase of Thy 1 nephritis, PDGF neutralization by antibody, expressed on endothelial cells. Inhibition of VEGF exacerbates
aptamers, PDGF receptor-IgG, or a PDGF receptor tyrosine Thy 1 nephritis and results in the development of microaneu-
kinase selective inhibitor is effective in blocking proliferation rysms, whereas treatment with VEGF165 promotes recovery
(104). Enhanced expression of PDGF and the PDGF-B from thrombotic microangiopathy (22,44,109).
receptor has been observed on human glomerulonephritis.
The role of the more recently discovered PDGF-C and
PDGF-D remains to be determined, although it has already Coagulation Cascade
been shown that PDGF-C can be made by podocytes and The normal glomerulus is remarkably protected from the
induce mesangial cell proliferation (105,106). formation of thrombi, but fibrin deposition is frequently
observed as a feature of three pathologic states: thrombotic
microangiopathy, proliferative and necrotizing glomerulo-
Transforming Growth Factor
nephritis, and crescent formation. Earlier studies using anti-
TGFβ is a family of polypeptide growth factors with at least coagulation with warfarin, heparin, or hirudin (a thrombin
three distinct mammalian isoforms. TGFβ has diverse bio- antagonist) yielded conflicting data on the potential for this
logic actions that are relevant to glomerular disease, includ- intervention to diminish the severity of crescentic glomeru-
ing its ability to modulate (sometimes in a dose-dependent lonephritis. Studies with fibrinolytic agents (ancrod, strep-
manner) inflammatory and immunologic responses, cellular tokinase or tissue plasminogen activator) have generally
proliferation, apoptosis and differentiation, angiogenesis, been more convincing with attenuated disease reported in
and extracellular matrix metabolism. TGFβ appears to be the models of anti-GBM nephritis, serum sickness, and lupus
primary mediator of glomerulosclerosis. TGFβ production nephritis. The most compelling data that fibrin plays a sig-
by resident and/or inflammatory cells may occur early in the nificant role are based on a study of anti-GBM nephritis in
course of glomerular injury, but its pathogenetic role in the fibrinogen-deficient mice (110). The number of intraglo-
acute phase of glomerulonephritis is still not clear. It is most merular monocytes, crescent formation, and serum creatinine
commonly reported to have antiproliferative and proapop- levels were significantly reduced in the fibrinogen–/– mice.
totic effects. TGFβ also has striking immunosuppressive In proliferative glomerular diseases, fibrin’s primary role may
 29. Immune Mechanisms of Glomerular Injury 591

be monocyte recruitment to the glomerular tuft and to Complement Cascade

Bowman’s space to the site of crescent formation. The path-
Since the first description of complement proteins in diseased
ways that regulate glomerular fibrin deposition and removal
human glomeruli in 1956, the role of complement has been
are reviewed in Figure 29.6.
extensively investigated in experimental glomerular disease
models. It is now evident that the complement cascade is mul-
tifunctional, somewhat paradoxically functioning systemically
to prevent the formation and deposition of immune com-
plexes but at a local level contributing to the inflammatory
response through its intraglomerular effects. Most of the solu-
ble complement components are made in the liver, but glo-
meruli may contribute to local production, especially during
inflammation (111). The three pathways of complement acti-
vation are reviewed in Figure 29.7.
Complement-mediated glomerular injury may be a con-
sequence of leukocyte recruitment, or complement itself may
directly attack target cells. Complement activation generates
the small chemoattractant peptides C3a and C5a that are
especially effective in recruiting and activating PMN express-
ing C3a and C5a receptors. The latter fragment is unique for
its chemotactic activity, which leads to the recruitment and
activation of PMNs. Observations during the heterologous
phase of anti-GBM nephritis provide the most compelling
evidence for the complement PMN–dependent mechanism
of glomerular injury. The MAC of complement may also
FIGURE 29.6. Regulation of glomerular fibrin deposition and activate the transcription factor NF-κB leading to further
removal. Fibrin deposition may be the consequence of intraglom- inflammation due to IL-8 and MCP-1 production (112).
erular activation of prothrombotic cascades and/or decreased
endogenous fibrinolytic activity, primarily intraglomerular tissue The importance of this pathway is illustrated by studies in
plasminogen activator (tPA). Most of the fibrin associated with complement-depleted or -neutralized animals that are pro-
immunologic glomerular diseases is generated by activation of the tected from the early injury induced by anti-GBM antibod-
extrinsic coagulation pathway that begins with the activation of
factor VII by the tissue factor (TF) procoagulant to generate VIIa. TF ies. Despite the presence of C3a and C5a receptors on several
can be produced by resident glomerular cells or monocytes/mac- other cells, including monocytes, the glomerular recruitment
rophages in response to several inflammatory stimuli. In addition, of monocytes is usually complement independent. Mesangial
normal renal vessels constitutively express a TF inhibitor, tissue fac-
tor pathway inhibitor (TFPI), which is downregulated in many cres- cells also express the C5a receptor that may lead to direct cel-
centic glomerular diseases. The importance of the TF pathway has lular activation in the absence of PMN (113).
been confirmed in experiments showing worse disease with TFPI Based on important observations in the models of passive
neutralization and milder disease with recombinant TFPI therapy
or TF inhibition (151). Several other factors contribute to the anti- Heymann nephritis and anti–Thy 1 nephritis, it was recog-
thrombotic environment within normal glomerular such as PGI2, nized that the terminal complement components C5b-C9
nitric oxide, antithrombin III, and thrombomodulin (152,153). It is that form the MAC can directly induce glomerular injury
becoming apparent that certain coagulation proteins may them-
selves have proinflammatory effects that may contribute to glom- without inflammatory cells. Many subsequent studies using
erular injury. For example, mesangial cells express and can be different strategies to inhibit or deplete complement or
activated by a thrombin receptor, protease-activated receptor-1 experiments in rodents with genetic complement deficiencies
(PAR-1). The receptor is activated by the protease TRAP. Mice
genetically PAR-1–deficient develop less severe anti-GBM nephritis, have clearly established the requirement for C5b-9 formation
whereas PAR-1 activation by recombinant TRAP therapy causes within glomeruli as the key event that initiates glomerular
worse disease but only in PAR-1–expressing mice (154). Mesangial cell damage. The nature of this injury is still an area of active
cells proliferate after exposure to factor Xa, a VIIa cofactor for
thrombin generation (155). Fibrin itself may also bind to cell sur- investigation, but several possibilities have been recognized.
faces through several potential mechanisms, especially by binding Multiple C9 molecules of the MAC polymerize to form ring-
to αvβ3 integrin (156). Glomeruli produce plasminogen activators, like structures that insert into the lipid bilayer of cell mem-
mainly tPA, which normally protect the kidney from the damaging
effects of fibrin accumulation. Mice with a genetic deficiency of branes, forming channels. Insertion into erythrocyte cell
tPA or plasminogen develop worse anti-GBM nephritis (157). Pro- membranes leads to hemolysis, but nucleated cells are more
duction of PA inhibitors, especially plasminogen activator inhibitor- resilient. C5b-9 can induce cell death by apoptosis, and such
1 (PAI-1) and perhaps protease nexin-1, may depress endogenous
plasmin activity and promote glomerular fibrin deposition. In addi- a mechanism may explain the early phase of complement-
tion to its role in acute glomerular injury, it is now evident that the dependent mesangiolysis in rats with Thy 1 nephritis and
glomerular tPA/PAI-1 balance is involved in the chronic phase of glomerular endothelial cell loss in antibody complement–
injury and glomerulosclerosis through activities that regulate accu-
mulation of not only fibrin but also extracellular matrix proteins dependent thrombotic microangiopathy (114). “Sublytic”
[reviewed in (158)]. uPA, urokinase-type plasminogen activator. levels of C5b-9 have been shown to significantly modify the
592 V. Glomerular Disease

1, ROS, and PG; and endothelial cells produce MCP-1 and

IL-8 (112,115). At least some of these products are coupled
to alterations in the glomerular capillary permselectivity bar-
rier that lead to proteinuria. What remains perplexing is the
mechanism by which MAC causes glomerular injury and
proteinuria in immunologic diseases associated with noncel-
lular antigens. Although the GBM lacks a lipid bilayer, C5b-
9 does bind to it. In factor H–deficient Yorkshire piglets that
spontaneously develop membranoproliferative glomerulone-
phritis type II (dense intramembranous deposit disease in the
absence of immune complex deposition) and factor H
knock-out mice with MPGN, deposits of C5b-9 can be seen
along the GBM early in the course of the disease (116).
Glomerular cells are normally protected from complement-
mediated injury due to a cell-surface defense mechanism that
inhibits complement activation and/or function (117). In
human glomeruli, three such molecules have been well charac-
terized, but additional molecules exist. Decay accelerating fac-
tor (DAF or CD55) and membrane cofactor protein (MCP or
CD46) regulate C3 and C5 activation. CD59 inhibits at the
level of C8 activation, preventing MAC formation (Fig. 29.7).
In glomeruli, MCP and CD59 are expressed by all three types
FIGURE 29.7. The complement cascade. The complement cas- of intrinsic cells, whereas DAF expression is minimal, some-
cade is composed of at least 35 plasma proteins and several cell times found on podocytes. Studies based on human biopsies
membrane receptors. The cascade is divided into three activation
pathways: the classical, alternative, and mannan-lectin recogni- show that MCP and CD59 expression is increased, and DAF
tion pathways, which activate different early components, and a is expressed de novo in the mesangium in various forms of glo-
common terminal unit leading to the formation of C5b-9 and the merulonephritis. Crry is the rodent homolog with activities
membrane attack complex (MAC) of multiple C9 molecules. In
humans, the classical pathway is activated by the binding of Clq similar to DAF and MCP. Not only does blockade of comple-
to the Fc region of immunoglobulin G1 (IgG1), IgG2, IgG3, or IgM. ment regulatory proteins such as DC59 or Crry exacerbate
The alternative pathway is usually triggered by immunoglobulin- complement-dependent glomerulonephritis, but also soluble
independent interactions with substances such as damaged tis-
sues, polysaccharides, or microorganisms. IgA may activate the Crry and CD59 can effectively inhibit complement and atten-
alternative pathway, but it does not capture C3 molecules effi- uate renal injury (118,119).
ciently. GBM can also activate the alternative pathway. The Two additional complement regulatory proteins are of
recently identified lectin complement pathway leads to comple-
ment activation when the plasma protein mannan-binding lectin interest for their potential to attenuate glomerular injury. CR1
(MBL), which has a structure similar to C1q, binds to specific car- (CD35) preferentially binds to C3b and C4b and is expressed
bohydrate structures. MBL normally circulates as a complex with on glomerular podocytes. CR1 also functions as a regulatory
a family of serine protease (MBL-associated serine proteases-1, -2,
-3) that becomes activated as a consequence of MBL-carbohy- molecule to protect cells from injury, and treatment with solu-
drate interactions. These proteases subsequently cleave C3 and ble CR1 has been shown to attenuate glomerular injury. Clus-
C4. C3 nephritic factor (C3NeF) is an IgG autoantibody that reacts terin is a plasma protein that is frequently found within
with the alternative pathway C3 convertase (C3bBb), an interac-
tion that enhances enzyme activity leading to C3 consumption. glomerular immune deposits in association with vitronectin
The membrane attack complex of polymerized C9 molecules and soluble C5b-9. Clusterin depletion enhances immune
forms ring-like structures that may insert into cellular and base- complex glomerulonephritis in mice (120).
ment membranes to cause dysfunction (illustrated within renal
basement membrane in the lower-right immunoelectron photo- Systemically, the primary function of the complement is
micrograph). At least four cell surface proteins, highlighted in one of defense and protection against infectious and autoim-
black boxes at their sites of action, inhibit complement activation mune disease. This role is powerfully illustrated by the obser-
and protect glomerular cells from complement-mediated dam-
age: decay accelerating factor (DAF), membrane cofactor protein vation that individuals with genetic complement deficiencies
(MCP), complement receptor 1 (CR1), and CD59. (The MAC photo- have an increased incidence of infectious diseases and SLE
micrograph is from Falk et al. Ultrastructural localization of the that is often associated with glomerulonephritis. The risk is
membrane attack complex of complement in human renal tissues.
Am J Kidney Dis 1987;9:121–128, with permission.) greatest with deficiencies of the early components of the clas-
sical pathway.
The complement cascade plays a role in the normal pro-
behavior of the targeted cell. For example, several intracellu- cessing and elimination of immune complexes before they
lar signaling pathways are activated in glomerular epithelial can deposit in tissues such as glomeruli. In vitro studies
cells, leading to increased synthesis of eicosanoids and matrix have shown that components of the classical pathway help
protein; mesangial cells respond by increased synthesis of IL- to maintain circulating immune complexes soluble by
 29. Immune Mechanisms of Glomerular Injury 593

interfering with their aggregation so that they can be elimi- teinuria. In addition to its potential role as an antigenic tar-
nated by the mononuclear phagocytic system. In primates, get in ANCA-associated vasculitis, myeloperoxidase catalyzes
the opsonization of immune complexes by complement the formation of another toxic product by reaction of H2O2
facilitates hepatic elimination by the “erythrocyte shuttle.” with a halide, usually to produce hypochlorous acid. The role
In the lupus-prone mouse, deficiency of C3, C4, and factor of highly toxic hydroxyl radicals in glomerular injury is less
B causes worse renal disease (121–123). The alternative clearly defined. Both H2O2 and O2– may be converted to
complement pathway is able to dissolve antigen-antibody OH⋅ via the Haber-Weiss reaction, and OH⋅ scavenger ther-
complexes by intercalating C3 molecules into their lattice apy has been beneficial in several experimental models, but
structure. This activity may help to clear immune complex in general the agents used may have additional effects unre-
deposits from glomeruli. Recent observations in C1q-defi- lated to OH⋅ depletion. Analogous to atherosclerosis, lipid
cient mice, which like deficient humans develop a lupus- peroxidation products may also deposit in glomeruli with
like syndrome, suggest that complement plays an impor- proinflammatory effects. Glomeruli are normally equipped
tant role in the clearance of apoptotic cells (a potential with effective antioxidant defenses—catalase, superoxidase
source of nuclear autoantigens) (124). dismutase, and glutathione peroxidase. The response of these
endogenous systems has not been extensively investigated in
glomerulonephritis, but a recent study reported a decline in
Reactive Oxygen Species
glomerular antioxidant activity during the phase of ROS
ROS are partially reduced oxygen molecules containing an generation in rats with Thy 1 nephritis (126).
unpaired electron that are generated during oxygen metabo-
lism and are capable of an independent existence. Oxygen
Eicosanoids
normally accepts four electrons to be converted directly into
water. Acceptance of one electron generates superoxide Eicosanoids (eicos is the Greek word for 20 indicating their
anion (O2–), two electrons produce hydrogen peroxide number of carbon atoms) are a family of biologically
(H2O2), and three electrons generate the hydroxyl radical important autocoids derived from AA that are released
(OH⋅). Although H2O2 itself is not an oxygen radical, it is from plasma membranes by cytosolic phospholipases (rate-
an important precursor. ROS may be generated by intrinsic limiting step). Two major pathways generate oxidized AA
glomerular cells (endothelial, mesangial, epithelial) or infil- products of interest in glomerular disease: the COX and
trating leukocytes by a variety of stimuli, and they can cause lipoxygenases (Fig. 29.8). Each pathway has its own recep-
glomerular damage. In experimental models of glomerular tors; some are expressed on glomerular cells. Whereas both
disease, alterations in glomerular permselectivity and pro- resident glomerular cells and inflammatory cells are active
teinuria have been linked to ROS generation. Other effects in AA oxidation, most often resident cells produce COX
relevant to the glomerulus include the ability of ROS to
influence the susceptibility of the GBM to degradation
directly or indirectly via the activation of latent proteases
(collagenase, gelatinase) or by inactivation of collagenolytic
enzyme inhibitors (α1-protease inhibitor), halogenation and
damage to glomerular structures via interactions with hypo-
halous acid derivatives (derived from PMN), mediation of
cellular toxicity, vasodilation of the microcirculation, and
altered metabolic activity of intrinsic glomerular cells. ROS
may also be involved in the initiation phase of glomerular
damage, for example, by altering the hexameric structure of
collagen IV to expose the normally “hidden” Goodpasture
antigen (125).
Several different ROS may cause glomerular injury.
Superoxide anions are the most abundant species produced
by the respiratory burst of PMNs, but they are poorly reac-
tive in aqueous solution and are unlikely to mediate tissue
injury. It is more likely that the toxic glomerular effects of
O2– relate to its conversion to hydroxyl radicals via the FIGURE 29.8. Glomerular effects of eicosanoids. Arachidonic
acid released from plasma membranes by phospholipases is
Haber-Weiss reaction. Hydrogen peroxide appears to be an metabolized by cyclooxygenase and lipoxygenase enzymes to
important mediator of PMN-dependent models of glomeru- yield eicosanoids that can regulate glomerular hemodynamics
lar diseases. Infusion of H2O2 directly into the renal artery of and/or the intensity of inflammation. Some eicosanoids preserve
renal blood flow and dampen inflammation, whereas others
rats causes dose-dependent proteinuria, and depletion of compromise glomerular blood flow and enhance inflammation.
H2O2 with catalase therapy reduces PMN-dependent pro- HETE, hydroxyeicosatetraenoic acid.
594 V. Glomerular Disease

products, and leukocytes produce lipoxygenase products, nificant antiinflammatory effects, particularly because of
with some overlap. Eicosanoids have profound effects on their ability to block leukotriene-mediated PMN chemo-
renal blood flow and leukocyte recruitment/activation that taxis and β2 integrin–dependent adhesion. Within
may either preserve renal blood flow and prevent inflam- inflamed glomeruli, interaction between PMNs (a source of
mation (PGs and lipoxins) or decrease GFR and promote 5-lipoxygenase) and platelets (a source of 12-lipoxygenase)
inflammation (Tx and leukotrienes). results in the generation of lipoxins. Mφ are a rich source of
Two COX genes have been identified that encode con- 15-lipoxygenase. The antiinflammatory effects of 15-
stitutive COX-1 (and the recently identified COX-3 splice lipoxygenase were nicely demonstrated in an anti-GBM
variant) and the inducible COX-2, respectively (127). Nei- nephritis model by gene transfection (133).
ther isoform is easily detected in normal glomeruli, but sev- Two dietary manipulations that induce changes in
eral inflammatory stimuli induce COX-2 expression during eicosanoid metabolism have been shown to attenuate glo-
glomerulonephritis. Mesangial cells are the most important merulonephritis severity. First, dietary supplementation with
intrinsic glomerular source of prostaglandin E2; epithelial fish oil that contains omega-3 fatty acids (especially eicosa-
and endothelial cells produce some. Prostaglandin E2 is pentaenoic acids and docosahexaenoic acid) modifies both
often synthesized in response to acute glomerular injury in COX and lipoxygenase metabolites. Second, dietary deple-
which it serves to preserve renal blood flow by vasodilation tion of essential fatty acids impairs renal production of a spe-
and mesangial cell relaxation. It may also have an antiin- cific lipid-derived Mφ chemoattractant and eicosanoids such
flammatory role, suppressing lymphocyte function and as LTB4. In addition to alterations in eicosanoid biosynthesis,
macrophage recruitment, preserving vascular permeability, both fish oil supplementation and essential fatty acid defi-
and downregulating the synthesis of MCP-1, NO, and col- ciency have been shown to reduce macrophage production of
lagen (128,129). Prostaglandin I2 or prostacyclin may also TNF-α, ROS, and NO. Fish oil decreases mesangial cell pro-
dampen the severity of acute glomerular injury through liferation in response to mitogens such as PDGF that have
hemodynamic and antiinflammatory effects and by its abil- been implicated in the pathogenesis of mesangioproliferative
ity to inhibit platelet activation. glomerular disorders (134).
Usually Tx are the most abundant eicosanoids produced
by nephritic glomeruli. Elevated glomerular and/or urinary
Endothelin and Nitric Oxide
TxB2 (the stable TxA2 hydration product) levels have been
reported in virtually every experimental model of glomeru- ETs are a family of peptides first isolated in 1988. Three
lonephritis examined, and inhibition therapy has been ben- mammalian isoforms are known: ET-1, -2, and -3. ET, pre-
eficial, especially in lupus nephritis. A relationship between dominantly ET-1, is produced constitutively by intrinsic
TxB2 and proteinuria has been proposed, but conflicting glomerular cells, most notably by endothelial cells. Several
reports exist. A recent study reported that glomerular albu- mediators of glomerular inflammation are reported to stim-
min permeability could be increased in vitro by incubation ulate the production of ET-1 by mesangial cells in vitro,
with TxA2 (130). In addition to its ability to contract TGFβ, IL-1, TNF-α, PDGF, thrombin, and Tx, for exam-
mesangial cells and to constrict blood vessels, TxA2 also ple. Synthesis of ET-1 by mesangial cells and macrophages
induces platelet aggregation, mediates the glomerular has also been documented in vivo in several human and
effects of PAF, and may stimulate matrix protein synthesis. animal models of glomerulonephritis. Evidence of a direct
TxA2 may be synthesized by inflammatory cells (PMN, role for ET-1 in glomerular damage is based on the ability
Mφ, and platelets) as well as intrinsic glomerular cells. of ET-receptor blockade to attenuate experimental glomer-
Lipoxygenase products may also be produced during ulonephritis and the presence of glomerulosclerosis in ET-1
acute glomerulonephritis (Fig. 29.8). The 5-lipoxygenase overexpressing mice. ET may function as a mesangial cell
products (5-HETE, LTB4, and leukotrienes C4, D4, E4) mitogen and a mediator of inflammation by upregulating
appear to originate from infiltrating leukocytes, whereas adhesion molecules and by enhancing monocyte chemoat-
12-lipoxygenase predominates in platelets. Elevated levels traction and activation. ET-1 may also stimulate matrix
of LTB4 have been reported in several experimental models protein production.
and associated with worse glomerular damage due to both Several endothelium-derived factors can mediate vascular
hemodynamic effects (vasoconstriction) and enhancement relaxation, but the term endothelium-derived relaxing factor
of leukocyte recruitment and activation. Although the has become synonymous with NO. NO is a gaseous free
functional significance of all of the products of AA lipoxy- radical that is liberated from the enzymatic conversion of L-
genation has not yet been determined, studies using LTB4, arginine to citrulline by NO synthase. NO is an unstable
LTC4, and LTD4 receptor antagonists have reduced glo- molecule with a half-life of a few seconds; its stable oxidation
merular disease severity (131). products, nitrites and nitrates, or its secondary mediator,
Lipoxins are a family of more recently described cyclic guanosine monophosphate, are used to estimate rates
eicosanoids that undergo dual lipoxygenation by either 5- of NO generation. Three NO synthase isoforms have been
and 15- or 5- and 12-lipoxygenases (132). They have sig- identified: two constitutive, calcium-dependent enzymes
 29. Immune Mechanisms of Glomerular Injury 595

originally described in the brain (nNOS) and endothelium glomerulonephritis. Although a discussion of issues relevant
(eNOS), respectively, and an inducible, calcium-indepen- to the resolution of glomerular injury is beyond the scope of
dent enzyme first described in macrophages (iNOS). In nor- this chapter, a few recent observations merit presentation.
mal glomeruli low levels of eNOS and sometimes iNOS The difference between recovery and progression may be
may be present, but generally these enzymes are quiescent determined by the duration or intensity of the initial insult.
unless stimulated. The acute phase of glomerular disease is This can be shown experimentally using nephritogenic
usually characterized by increased NO production, and sev- antibodies in which the injection of a single dose induces
eral inflammatory stimuli have been shown to stimulate injury followed by full recovery, whereas multiple injections
production, endotoxin, TNF-α, IL-1, cyclic adenosine may cause progressive fibrosis. Glomerular sclerosis is char-
monophosphate, and IFN, for example. acterized by the progressive accumulation of extracellular
Despite great interest in the biologic role of NO, it matrix proteins. As damaged glomeruli repair themselves,
remains unclear whether its enhanced expression during matrix protein synthesis may be transiently increased, but
glomerulonephritis serves a primary beneficial or detrimen- in the absence of ongoing injury, this phase of mesangial
tal role because it elicits multiple effects. An emerging matrix expansion often does not progress to the destructive
theme suggests that the origin of NO—both cellular source phase of glomerulosclerosis. Matrix-degrading enzymes are
and generating enzyme isoform—may determine its fate produced by glomeruli. They include serine proteases (plas-
and function. Enhanced eNOS activity has many protec- minogen activators, elastase) and the MMPs (interstitial
tive outcomes, including vasodilation to preserve renal collagenase, gelatinases, stromelysin, and membrane-type
plasma flow, inhibition of platelet aggregation and throm- MMP). Each of these enzymes has a naturally occurring
bosis, and decreased leukocyte recruitment (135). In inhibitor(s). An increase in proteolytic enzyme secretion or
eNOS–/– mice, anti-GBM antibodies induce severe glo- a decrease in inhibitor activity could degrade early glomer-
merular damage with worse inflammation and fibrosis than ular matrix protein deposits. It is increasingly recognized
is observed in wild-type mice (136). Experimental throm- that many of these proteases/inhibitors are multifunctional
botic microangiopathy is also worsened by inhibition of proteins. For example, MMP-2 is a gelatinase that also acti-
endogenous eNOS (137). vates mesangial cells, whereas the tissue inhibitor of metal-
In other models of glomerular injury, NO is produced loproteinase-1 inhibits mesangial cell apoptosis (144,145).
by iNOS activity of inflammatory leukocytes and, less Mesangial cell proliferation is a feature of many acute
often, of mesangial cells and is often associated with glo- glomerular diseases that progress to chronic disease, but it is
merular damage. NO can be oxidized to peroxynitrite, a not clear that mesangial hypercellularity per se predicts pro-
toxic ROS (138). NO may also bind directly to iron or gression. More important is the phenotypic transformation
thiol groups on proteins and modify their function. Heme of mesangial cells into smooth muscle–like cells (expressing
oxygenase has been recognized as an important defense alpha smooth muscle actin, non–smooth muscle myosin,
against oxidative injury, and one of its activities is to down- and the intermediate filament, desmin). These cells are the
regulate iNOS expression (139). NO has been ascribed primary source of the sclerosing matrix proteins. Innate
other functions that may be relevant to glomerular injury genetic programs within mesangial precursor cells may
but these still need further in vivo investigation—mitogen- determine their phenotypic and functional fate (8). The
esis, apoptosis, vascular permeability changes, immune contribution of podocytes to the expanding pool of matrix
response modulation, and matrix synthesis (140). Pharma- proteins is unclear, although in vitro these cells synthesize
cologic blockade of iNOS using the inhibitor L-NIL has several matrix proteins. During glomerular repair, novel
been beneficial in some but not all tested models, but interstitial matrix proteins may be synthesized that further
genetic deficiency of iNOS does not alter the severity of modulate the function of glomerular cells through integrin-
anti-GBM nephritis (141,142). dependent signaling mechanisms (Table 29.1).
Apoptotic programs play a critical role (146). During
the recovery phase, apoptosis has been shown to be impor-
RESOLUTION OR PROGRESSION OF tant in the reversal of mesangial cell hypercellularity and
GLOMERULAR INJURY perhaps in the elimination of inflammatory cells. Pro-
grammed cell death may further deplete all lines of intrinsic
The immunologic events initiating glomerular injury are glomerular cell as glomeruli succumb to destruction by
often self-limited. Yet, the outcome is extremely variable, sclerosis. An important area of investigation is the study of
ranging from the regeneration of an entirely normal glomer- the molecular pathways that program glomerular cell death
ulus to global glomerulosclerosis. It is becoming evident that and whether different mechanisms are involved in the ben-
many genetic factors influence the glomerular response to eficial effects of apoptosis that terminate inflammation
injury (143). It is also clear that the events occurring simulta- compared to those that lead to acellular sclerotic glomeruli.
neously in the tubulointerstitium play a critical role in deter- During the evolution of glomerular injury, several soluble
mining the long-term outcome in patients with primary factors may be produced that help dampen or even terminate
596 V. Glomerular Disease

disease progression. These may be products of intrinsic glo- 5. Sugenoya Y, Yoshimura A, Yamamura H, et al. Smooth-
merular cells, inflammatory leukocytes, or T cells polarized muscle calponin in mesangial cells: regulation of expression
to a Th2 phenotype. Examples include Th2 cytokines (Fig. and a role in suppressing glomerulonephritis. J Am Soc
29.4), matrix-degrading proteases, fibrinolytic activity (Fig. Nephrol 2002;13:322–331.
29.6), complement regulatory proteins (Fig. 29.7), antiin- 6. Hugo C, Shankland SJ, Bowen-Pope DF, et al. Extraglomeru-
lar origin of the mesangial cell after injury. A new role of the
flammatory eicosanoids (Fig. 29.8) protective growth factors
juxtaglomerular apparatus. J Clin Invest 1997;100:786–794.
(Table 29.4), and NO. Some glycoproteins synthesized dur- 7. Imasawa T, Utsunomiya Y, Kawamura T, et al. The potential
ing the inflammatory phase may sequester and inhibit fibro- of bone marrow-derived cells to differentiate to glomerular
sis-promoting cytokines. For example, SPARC (secreted mesangial cells. J Am Soc Nephrol 2001;12:1401–1409.
protein acidic and rich in cysteine) binds PDGF, HSPG 8. Cornacchia F, Fornoni A, Plati AR, et al. Glomerulosclerosis
binds bFGF, and small proteoglycans, including decorin and is transmitted by bone marrow-derived mesangial cell pro-
biglycan, block the actions of TGF-β1. genitors. J Clin Invest 2001;108:1649–1656.
On the other hand, other products of glomerular 9. Shankland SJ. Cell cycle regulatory proteins in glomerular
inflammation have fibrogenic potential. The central role of disease. Kidney Int 1999;56:1208–1215.
TGFβ is undisputed based on data from many studies 10. Maeshima Y, Kashihara N, Yasuda T, et al. Inhibition of
(147). Many other factors contribute, some working via mesangial cell proliferation by E2F decoy oligodeoxynucle-
otide in vitro and in vivo. J Clin Invest 1998;101:2589–2597.
TGFβ interactions and other independently such as angio-
11. Pippin JW, Qu Q, Meijer L, et al. Direct in vivo inhibition of
tensin II and ET-1 [reviewed in (148)]. Numerous physical the nuclear cell cycle cascade in experimental mesangial prolifer-
features associated with glomerular injury have been impli- ative glomerulonephritis with Roscovitine, a novel cyclin-depen-
cated in the genesis of glomerulosclerosis, but how they dent kinase antagonist. J Clin Invest 1997;100:2512–2520.
function at a molecular level is not fully understood. These 12. Miyata T, Inagi R, Nangaku M, et al. Overexpression of the
include intraglomerular hypertension (which may cause serpin megsin induces progressive mesangial cell prolifera-
glomerular mechanical stretch-induced TGF-β1 synthesis), tion and expansion. J Clin Invest 2002;109:585–593.
mesangial overload, the glomerular hypertrophic response, 13. Kerjaschki D. Caught flat-footed: podocyte damage and the
podocyte protein overload, podocytopenia, hyperlipidemia, molecular bases of focal glomerulosclerosis. J Clin Invest
and secondary cellular immune responses to cryptic glo- 2001;108:1583–1587.
merular antigens exposed during injury. 14. Takeda T, McQuistan T, Orlando RA, et al. Loss of glomerular
foot processes is associated with uncoupling of podocalyxin
from the actin cytoskeleton. J Clin Invest 2001;108:289–301.
15. Topham PS, Kawachi H, Haydar SA, et al. Nephritogenic
ACKNOWLEDGMENTS mAb 5-1-6 is directed at the extracellular domain of rat
nephrin. J Clin Invest 1999;104:1559–1566.
Grants supporting the author’s research and academic 16. Mundel P, Shankland SJ. Podocyte biology and response to
efforts are greatly appreciated and acknowledged, from the injury. J Am Soc Nephrol 2002;13:3005–3015.
National Institutes of Health (DK54500 and DK44757) 17. Ophascharoensuk V, Fero ML, Hughes J, et al. The cyclin-
and the Juvenile Diabetes Foundation. Most of the refer- dependent kinase inhibitor p27Kip1 safeguards against
ences cited have been published since the writing of this inflammatory injury. Nat Med 1998;4:575–580.
chapter for the 4th edition of Pediatric Nephrology. Readers 18. Bariety J, Nochy D, Mandet C, et al. Podocytes undergo
are referred to Chapter 40 in the fourth edition and Chap- phenotypic changes and express macrophagic-associated
markers in idiopathic collapsing glomerulopathy. Kidney Int
ter 32 in the third edition for a partial listing of earlier pub-
1998;53:918–925.
lished work that is relevant to the immunopathogenesis of 19. Kriz W. Podocyte is the major culprit accounting for the
glomerular injury. progression of chronic renal disease. Microsc Res Tech 2002;
57:189–195.
20. Sorensson J, Fierlbeck W, Heider T, et al. Glomerular endo-
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 30

ACUTE PROLIFERATIVE
GLOMERULONEPHRITIS
ENDRE SULYOK

Acute proliferative glomerulonephritis (GN) includes a variety 4). Sporadic cases have been reported to occur in clusters, but
of diseases with diverse etiologies and variable morphologic endemic and epidemic outbreaks have also been recognized.
appearances clinically presenting with a nephritic syndrome. Most often, APSGN follows upper respiratory tract infec-
These include abrupt onset, gross or microscopic hematuria, tions in temperate climates or skin infections in tropic and
proteinuria varying from minimal to nephrotic range, casts on subtropic areas caused by group A streptococci or, occasion-
urinalysis, fluid overload with hypertension, and variable ally, groups C and G streptococci. Only certain streptococcal
degree of renal insufficiency with oliguria or anuria. The term strains are capable of causing GN; these are called nephritoge-
proliferative implies glomerular hypercellularity, whatever the nic streptococci. Nephritogenic strains have the cell wall M-
origin of cells. They may invade the glomeruli from the circu- protein serotypes 1, 2, 3, 4, 12, 18, 25, 49, 55, 57, and 60.
lation or may originate from glomerular mesangial, endothe- Postpharyngitis GN is usually associated with M types 1, 3,
lial, or epithelial cells. Glomerular involvement may be focal or 4, 12, 18, 25, and 49, whereas postpyoderma GN is related
diffuse, segmental or global, according to the number of glom- to specific M types 2, 49, 55, 57, and 60. The incidence of
eruli and extent of glomerular injury. GN after infection with nephritogenic streptococci is vari-
The underlying mechanism of acute proliferative GN is able and depends on the M serotype and the site of infection.
immune-mediated inflammation as evidenced by glomerular It has been reported to range from 1 to 33% of the patients,
deposits containing immune complexes and complement com- with the overall risk of approximately 15%. Clinically appar-
ponents in most cases. In pauci-immune crescentic GN, the ent GN occurs in less than 2% of children infected with var-
pathogenetic role of immune mechanisms is indicated by the ious streptococcal strains. Seasonal variation is characteristic
high rate of positivity for antineutrophil cytoplasmic antibodies. for APSGN in the temperate climate because streptococcal
It is important to emphasize that the morphologic patterns throat infections are more common in winter and early
are not specific of a distinct clinical entity, and a specific disease spring, whereas streptococcal pyoderma mostly occurs in late
may have different histologic appearances (Table 30.1). GN summer and early fall.
may be primary or secondary to a systemic process, such as The occurrence of APSGN is influenced by certain host
vasculitis (1). As the etiology, history, clinical manifestations, factors, such as age, sex, socioeconomic background, and
laboratory findings, morphologic patterns, and clinical course genetic predisposition. The disease is more frequent among
may be extremely variable, an evaluation is needed for an children between the ages of 2 and 12 years, although 5%
appropriate diagnosis and adequate therapeutic interventions. of the patients are younger than 2 years of age. Boys are
In this chapter, the acute poststreptococcal GN (APSGN) more often affected with clinically overt GN than girls,
is presented in detail because it serves as a prototype of postin- with the ratio of 2:1.
fectious proliferative GN. It is the most commonly encoun- Epidemic outbreaks tend to occur in closed communi-
tered and the best understood form of proliferative GN. The ties and in densely populated areas where poor hygiene,
terms diffuse intracapillary GN, diffuse endocapillary GN, and malnutrition, anemia, and intestinal parasites are common.
acute postinfection GN are also used in the text as synonyms. Since the 60s of the last century, marked steady declines
have been noted in the prevalence of APSGN, although
cyclic recurrences have been observed in certain communi-
ACUTE POSTSTREPTOCOCCAL
ties. The reason for the apparent decline in the prevalence
GLOMERULONEPHRITIS
of the disease is not clear and may be attributed to the
widespread use of antibiotics, improved hygienic condi-
Epidemiology
tions, changes in nephritogenic potential of streptococcus
The major aspects of the epidemiology of APSGN have been strains, or altered susceptibility of the host (5). After recov-
summarized in comprehensive reviews by several authors (2– ery from APSGN, protective immunity is acquired against
602 V. Glomerular Disease

TABLE 30.1. HISTOLOGIC CATEGORIES OF ACUTE PROLIFERATIVE

(HYPERCELLULAR) GLOMERULONEPHRITIS (GN)
Histologic pattern Causes

Diffuse intracapillary proliferative (exu- Usually postinfectious

dative) GN (with neutrophils) Occasionally related to SLE or vasculitis
Diffuse proliferative GN (without neu- Postinfectious GN
trophils) SLE
Mesangial proliferative GN Primary forms of GN
Resolving postinfectious GN
IgA nephropathy
IgM nephritis
Accompanying systemic disease
SLE
Infective endocarditis
Henoch-Schönlein purpura
Mixed connective tissue disease
Membranoproliferative (mesangiocap- Idiopathic
illary) GN Systemic immune complex diseases
Mixed cryoglobulinemia
SLE
Sjögren’s syndrome
Hereditary deficiencies of complement components
Infectious diseases
Bacterial
Infected ventriculoatrial shunts
Endocarditis
Viral
Hepatitis B and C
Human immunodeficiency virus
Protozoal: malaria, schistosomiasis
Other: mycoplasma, mycobacteria
Neoplasms
Chronic liver diseases
Miscellaneous
Diffuse extracapillary proliferative
(crescentic) GN
Diseases with crescent in majority of Anti–glomerular basement membrane antibody diseases
cases With pulmonary hemorrhage (Goodpasture’s syndrome)
Without pulmonary hemorrhage
Antineutrophil cytoplasmic antibody–associated GN
Wegener’s granulomatosis
Polyarteritis nodosa
Hypersensitivity vasculitis
Idiopathic crescentic GN
Diseases with crescent in minority of Postinfectious GN
cases SLE
IgA nephropathy
Henoch-Schönlein purpura
Essential mixed cryoglobulinemia
Membranoproliferative GN

Ig, immunoglobulin; SLE, systemic lupus erythematosus.

Adapted from Kern WF, Laszik ZG, Nadasdy T, et al. The true (hypercellular or “proliferative”) glomeru-
lonephritis. Diseases associated with the nephritic syndrome. In: Kern WF, Laszik ZG, Nadasdy T, et al.,
eds. Atlas of renal pathology. Philadelphia: WB Saunders, 1999:669–689.

the nephritogenic strains. This protective immunity is more of index cases with sporadic disease (38%) than the
likely related to the specificities and neutralizing capacity of expected attack rate of children at risk in epidemics (4.5 to
antibodies rather than to the antibody titer itself (6). 28.0%) (7). The genetic aspects of APSGN are further sup-
Family studies have suggested genetic predisposition for ported by the reports of common association of the disease
APSGN by demonstrating its higher incidence in siblings with HLA-DR1 and -DRw4, whereas patients with HLA-
 30. Acute Proliferative Glomerulonephritis 603

DRw48 and -DRw8 appear to be less susceptible to devel- There is an apparent difference in streptococcal strains
opment of primary GN (8,9). leading to APSGN through pharyngeal or skin infection.
The predominant M-protein serotypes associated with pyo-
derma and APSGN are M proteins 2, 42, 49, 56, 57, 60,
Pathogenesis
whereas M proteins 1, 4, 12, and 25 are associated with
APSGN is an immune-mediated disease associated with throat infection and APSGN. In general, the pyoderma or
throat or skin infections with certain nephritogenic strains of skin strains belong to the class II M-protein serotypes with
group A streptococci. Streptococcal constituents or products the capacity to produce opacity factor but having no reac-
that may trigger pathologic processes have not been fully tion with antibodies against C repeat region (20).
defined. Furthermore, the exact mechanism of renal injury The potential role of M proteins in inducing APSGN
remains controversial, although several theories have been has been extensively studied. Markedly elevated immuno-
proposed: (a) trapping of circulating immune complexes in globulin (Ig) G titers against the C-terminal region but not
the glomeruli (10,11); (b) molecular mimicry between strep- the N-terminal region of the M12 protein were found in
tococcal and renal antigens (i.e., normal glomerular tissue patients with APSGN as compared to patients with phar-
acting as autoantigen reacts with circulating antibody formed yngitis or chronic GN or healthy individuals. Binding of
against streptococcal antigens) (12–14); (c) in situ immune IgG to bacteria is assumed to be of importance for the
complex formation between antistreptococcal antibodies and development of APSGN. Binding of IgG and IgA aggre-
glomerular planted antigens (15,16); and (d) direct comple- gates is frequently detected among nephritogenic strepto-
ment activation by streptococcal antigens deposited in the cocci. Fc regions of IgG and IgA may bind to the surface of
glomeruli (17). certain group A streptococcus strains through M proteins
or M-like proteins. Fc receptor–positive group A strepto-
cocci isolates elicit anti-IgG response and promote deposi-
Streptococcal Factors Implicated in the
tion of IgG and C3 in the rabbit kidney immunized with
Pathogenesis of Acute Poststreptococcal
group A streptococcus. These findings suggest that Fc
Glomerulonephritis
receptors of nephritogenic streptococci may induce anti-
The group A streptococci are covered with an outer hyalu- IgG molecules and, consequently, may contribute to the
ronic acid capsule required for resistance to phagocytosis pathogenesis of APSGN by enhancing renal deposition of
and bacterial adherence to epithelial cells. Extracellular sur- immune complexes (21,22).
face molecules further consist of a group A carbohydrate The role of M proteins in inducing renal injury is further
polymer, a mucopeptide, and an M protein. The M protein supported by the well-established cross-reactivity of M pro-
extends from the cell surface as an alpha-helical coiled-coil teins with glomerular basement membrane (GBM) antigens.
dimer, which appears as hair-like projections on the surface Antiglomerular antibodies have been shown to react with
of streptococci (18). streptococcal M12 protein. A number of surface proteins
from nephritogenic streptococci have been reported to bind
M Protein and M-Like Proteins constituents of GBM (e.g., heparan sulfate, laminin, and col-
Groups A and C and human isolates of group G strepto- lagen IV). The streptococcal host factor-binding proteins
cocci possess M proteins, and more than 80 serotypes have share structural homologies with classical M proteins and are
already been identified. M proteins have the variable N- designated as M-like proteins (23).
terminal region projected outward and the highly conserved
C-terminal part anchored in the cell membrane. The genes Endostreptosin
encoding several types of M proteins, called emm genes, Endostreptosin is a 40- to 50-kDa protein derived from the
have been cloned, and their nucleotide sequences have been streptococcal cell cytoplasm. It was found to absorb anti-
established. M proteins have a common structure: the hep- glomerular antibodies from the sera of patients after
tad-repeating amino acid sequence motifs within each APSGN. Endostreptosin has been detected in the glomer-
region of the molecule. M proteins have been divided into uli in the early phase of APSGN. In the late stages of the
two classes based on their reactivity with antibodies against disease, it is not detectable, but increased serum antistrep-
the C repeat region. Class I M proteins contain a surface- tosin titers are diagnostic of APSGN. Endostreptosin
exposed epitope that reacts with these antibodies and are deposited in the GBM is considered to be an in situ activa-
opacity-factor negative, whereas class II M-protein sero- tor of the alternative pathway of the complement system
types do not contain the class I epitope, do not react with and immunologically distinct from other streptococcal
anti–C repeat region antibodies, and are opacity-factor pos- enzymes or cell wall components (24,25).
itive (19).
The nephrogenicity of group A streptococci appears to Cationic Antigens
be related to specific M-protein serotypes, but not all Cationic proteins of group A streptococci are known to have
strains of the same M-protein serotype are nephritogenic. affinity for the polyanionic GBM. Antibodies raised against
604 V. Glomerular Disease

cationic proteins from nephritogenic group A streptococcus role of neuraminidase is further supported by the demonstra-
isolates were shown to bind to renal biopsy samples from tion of free sialic acid and neuraminidase activity in the
patients with APSGN, and antibodies to these cationic anti- serum of APSGN patients and by experimental evidence that
gens were detected in APSGN patients. Cationic antigens intravenous (IV) neuraminidase administration promotes
generate in situ immune complexes and initiate glomerular renal infiltration of leukocytes and that neuraminidase-
inflammation. As C3 deposition precedes that of IgG in treated leukocytes preferentially accumulate in the kidney
APSGN, the cationic protein–related mechanisms are likely (31,32). However, neuraminidase is also produced by non-
to be involved in the later phase of the disease (26). nephritogenic streptococcus strains. It has been proposed
that the formation of autoantibody to IgG could be due to
Streptococcal Pyrogenic Exotoxin B its incorporation into a streptococcal antigen-antibody com-
Streptococci isolated from patients with nephritis produce a plex rather than to its modification by neuraminidase.
46-kDa extracellular protein not found in strains from
patients without acute GN. This nephritis strain–associated Nephritis-Associated Plasmin Receptor
protein was found to be a plasmin-binding protein and was A 43-kDa protein designated as nephritis-associated plasmin
later identified as streptococcal pyrogenic exotoxin B (SpeB) receptor (NAPlr) has been isolated from nephritogenic strep-
precursor, also known as the streptococcal proteinase zymogen. tococci. This antigen is present in glomeruli in the early stage
It is believed that the protease or superantigenic properties of APSGN, and antibodies to NAPlr are detected in the
of this exotoxin/protease/plasmin binding may cause an serum of 92% of patients within 3 months of the onset of
activation of the immune system by cleaving several host the disease. Soluble NAPlr could bind to the glomeruli and
factors that favor the release of active inflammatory media- attack activated plasmin, which has a central role in the pro-
tors, vascular permeability factors, and biologically active cess of local inflammation. In addition, NAPlr directly con-
fragments from the streptococcal wall (27). Antibody titers tributes to alternative complement activation (33).
to SpeB are markedly elevated in APSGN as opposed to
patients with acute rheumatic fever, scarlet fever, or healthy
Complement Cascade
subjects. SpeB is detected in the glomeruli of 67% APSGN
biopsy specimens as compared with only 16% of non- Humoral immunity initiated by nephritogenic streptococ-
APSGN cases (28). cal antigens is assumed to be mediated by in situ formation
of antigen-antibody complexes and by glomerular deposi-
Streptokinase tion of circulating immune complexes. These processes lead
Streptokinase, a 46-kDa, 414–amino acid, extracellular to the activation of the classical and the alternative comple-
protein is considered to be involved in the pathogenesis of ment pathways as evidenced by the depressed serum levels
APSGN. Nine different genotypes of streptokinase have and the glomerular localization of the components of com-
been identified, and ska-1, -2, -6, and -9 have been found plement cascade.
to be associated with APSGN. The streptokinase genes are The classical pathway is primarily involved in the early
highly conserved, and a greater than 90% homology in the stage of disease and is likely activated by immune com-
amino acid sequence is observed in nephritogenic strains as plexes when IgG has become deposited. Several observa-
compared with less than 60% homology for nonnephrito- tions suggest that the alternative pathway is operating as
genic strains (29). Streptokinase was found to be tightly well. These include (a) the glomerular presence of proper-
bound to the glomeruli, and glomerular deposits of strep- din; (b) C3 deposition precedes that of IgG, or even C3
tokinase could be detected with a highly sensitive staining may be present in the glomeruli without IgG; and (c) in the
technique. Streptokinase is known to form complexes with acute stage of APSGN serum levels of C3 and C5 are low,
plasminogen and plasmin, and these complexes have the whereas the levels of early components of the classical acti-
capacity to convert plasminogen to plasmin. Plasmin then vation pathway (e.g., C1q, C2, or C4) are normal or only
activates the local complement cascade, causes degradation slightly depressed (34–36). Other studies have revealed an
of extracellular matrix proteins, and induces the release of activation of the terminal complement pathway and the
vasoactive bradykinin. In an experimental model, the dele- generation of the terminal C5b-C9 complex, also desig-
tion of the streptokinase gene was shown to eliminate the nated as membrane attack complex, by showing its elevated
nephritogenic properties of the strain (30). plasma level and colocalization with C3 deposits in glomer-
ular capillaries and mesangium in patients with APSGN.
Neuraminidase The potential importance of this terminal complement
It has also been suggested that in APSGN, the autologous complex implies its capacity to stimulate the production of
IgG becomes autoimmunogenic after being desialized by vasoactive substances, proteolytic enzymes, and reactive
streptococcal neuraminidase. The modified antigenic IgG oxygen radicals—all known to damage the integrity of glo-
stimulates the anti-IgG rheumatoid factor response, which merular capillary membrane. C3a and C5a also appear to be
may lead to the formation of cryoglobulins. The pathogenic involved in tissue damage, as they function as chemotactic
 30. Acute Proliferative Glomerulonephritis 605

factors for infiltrating cells and cause histamine release and intraglomerular proliferating macrophages decreases with
increased permeability (4). The early phase of APSGN may time during the course of the disease in APSGN, whereas it
be associated with C3 nephritic factor and decreased remains constant in IgA nephropathy. Infiltrating cells
plasma levels of C3. C3 nephritic factor is an autoantibody undergo apoptosis, which is a prerequisite for the elimina-
against the C3 convertase that stabilizes this inherently tion of these cells and the resolution of renal injury.
labile enzyme and induces a continuous activation of alter- Decreased apoptosis has been found to be associated with a
native pathways with subsequent C3 depletion (37). progressive clinical course (42). In addition to macrophages,
During the course of GN, protective mechanisms have neutrophils and T lymphocytes are present in the glomeruli
been identified against complement-mediated injury of glo- but not B lymphocytes (43). Recently, proinflammatory
merular cells. Surface-bound proteins expressed on glomer- monocytes or macrophages expressing CD16 antigen have
ular cells are assumed to prevent complement activation, been demonstrated in the glomeruli and the urine of
and soluble complement receptors and those on the glo- patients with active proliferative GN, and the number of
merular visceral epithelial cells appear to attenuate glomer- these cells appears to correlate with the severity of acute
ular injury (38). glomerular inflammation (44). CD16+ macrophages pro-
duce high levels of proinflammatory cytokines [e.g., tumor
necrosis factor (TNF)], whereas antiinflammatory cyto-
Coagulation System
kines (e.g., IL-10) are absent or produced at a low level
Intraglomerular deposition of fibrin or fibrinogen-related (45). Increased local production of proinflammatory cyto-
antigen is a constant finding in APSGN. The activation of kines may induce the expression of adhesion molecules
the coagulation system is further indicated by the increased (intercellular adhesion molecule-1) and lymphocyte func-
levels of plasma high-molecular-weight fibrinogen com- tion–associated molecule-1 on endothelial cells, and these
plexes, the development of hypo- or hyperfibrinogenemia, molecules may facilitate the adhesion of circulating cells to
the depression of factor XIII, α2-macroglobulin, and the the renal endothelium and their subsequent invasion into
activation of factor XII. Fibrinolysis is increased as shown the parenchyma (46). In response to nephritogenic glomer-
by the elevation of serum and urinary fibrinogen or fibrin ular antigen, two subsets of fully differentiated T helper
degradation products. Urinary excretion of these products cells are activated. T helper-1 cells produce IL-2 and inter-
is believed to reflect the degradation of glomerular fibrin feron-gamma, activate macrophages, and induce expression
deposits and the beginning of the recovery phase of the dis- of MHC antigen, whereas T helper-2 cells secrete IL-4, -5,
ease. It is interesting to note that there is no relationship and -6, cofactors for B-cell proliferation, and stimulants for
between serum levels of C3 and fibrin degradation prod- Ig production (47). IL-1 has the potential to increase glo-
ucts. The possible involvement of platelet activation has merular inflammation, whereas antiinflammatory cytokines
also been proposed because of the diminished platelet sur- (e.g., IL-4, -6, -10, -13, and transforming growth factor β)
vival time and the presence of platelet-activating factor and reduce IL-1 synthesis by macrophages and suppress the
platelet-derived growth factor in APSGN (39). generation of oxygen radicals and nitrogenous intermedi-
ates, the secretion of proteolytic enzymes, and the expres-
sion of Fc receptors (48). It is of note that IL-4 ameliorates
Cellular Infiltration and Cytokine Production
experimental GN also by upregulating the glomerular gene
APSGN is characterized by diffuse glomerular hypercellu- expression of IL-1 decoy receptor (49). IL-8 is a selective
larity, primarily as a result of accumulation of polymor- activator and chemoattractant of polymorphonuclear leu-
phonuclear leukocytes and monocytes, an increase in kocytes secreted by monocytes and intrinsic glomerular
intrinsic glomerular cells, and a transient pathologic mesangial, endothelial, and epithelial cells. Glomerular
expansion of mesangial matrix. Resident endothelial and immunoreactivity for IL-8 correlates with neutrophil infil-
mesangial cells proliferate, and macrophages are believed tration and clinical activity in patients with APSGN. Trans-
to induce this proliferation. Glomerular macrophage infil- forming growth factors β1, β2, and β3, originating from
tration appears to be mediated by complement-induced infiltrating monocytes or macrophages or from mesangial
chemotaxis and by antigen-specific T-cell activation. cells, are also abundant in biopsy specimens of APSGN and
Streptococcal superantigens cause a selective increase in T- appear to be responsible for the accumulation of mesangial
cell receptor β and massive T-cell activation with the matrix components, including collagens, proteoglycans,
release of T-cell–derived lymphokines [e.g., interleukin and fibronectin (50).
(IL)-1 and IL-6] (40). It has recently been shown that selective podocyte injury
Hisano et al. found more infiltrating macrophages and the exacerbates mesangial cell proliferation and mesangial
four macrophage subclasses—early-, acute-, chronic-stage matrix expansion, which consequently contributes to the
inflammatory, and chronic-stage mature macrophages— progression of experimental proliferative GN. Podocyte
in the glomeruli of patients with APSGN compared to dysfunction is an important factor in the development of
patients with IgA nephropathy (41). The proportion of irreversible mesangial alterations. Basic fibroblast growth
606 V. Glomerular Disease

factor is also released by the podocytes, and it may further

amplify podocyte damage and mesangial alterations (51).
Mesangial injury or activation may also be controlled by
serine protease inhibitors. Megsin, a novel member of the
superfamily of serine protease inhibitors, is predominantly
expressed in mesangial cells, and its expression is upregulated
at the peak of hypercellularity and matrix accumulation dur-
ing various glomerular diseases. Imbalance between protein-
ases and their inhibitors may participate in the expansion of
mesangial extracellular matrix by modulating the turnover of
glomerular mesangial matrix compounds (52,53).
It is generally accepted that proinflammatory cytokines
and chemokines originating from infiltrating cells or from
resident glomerular cells are more expressed in proliferative FIGURE 30.1. Acute postinfectious glomerulonephritis. The
GN. Their expression is differentially regulated by the pres- glomeruli show global intracapillary hypercellularity with large
ence of immune complexes, and there is a relationship numbers of polymorphonuclear leukocytes in the glomerular
capillary lumina (hematoxylin and eosin stain, ×400). (Courtesy
between cytokine gene expression and clinical manifestations of Peter Degrell, University of Pécs Faculty of Medicine, Nephro-
(54). In patients with APSGN, plasma levels of IL-6 have logical Center, Pécs, Hungary.) (See Color Plate 30.1.)
been shown to parallel the clinical course, whereas TNF-α is
elevated in the acute phase and variable thereafter. Levels of
platelet-derived growth factor always remain normal (55). involvement of all glomeruli (Fig. 30.1). During the course
of the disease, distinct patterns of cellular reaction can be
recognized.
Free Oxygen Radicals
At the early stage of the disease, glomerular hypercellularity
Recent studies have revealed some major features of oxygen is due to an influx of blood-borne polymorphonuclear leuko-
stress and antioxidant defense mechanisms in children with cytes and monocytes, whereas in later stages, it is mainly caused
APSGN. Lipid peroxidation is enhanced, as demonstrated by proliferation of intrinsic endothelial and mesangial cells. T-
by increased red blood cell osmotic fragility, elevated levels of helper and T-suppressor lymphocytes also participate at the
malonyl dialdehyde, oxidized glutathione, and hemoglobin. onset of APSGN. In addition to mesangial proliferation,
Furthermore, the activities of major antioxidant enzymes mesangial matrix accumulation, growing mesangial fibers,
including superoxide dismutase, catalase, glutation peroxidase, mesangiolysis, and expression of molecules mediating the cross-
and glutation-S-transferase are markedly depressed in children communication between resident and invading cells, have been
with APSGN as compared with healthy control children. As a noted. Mesangial hypercellularity may persist long after appar-
result, the levels of reduced glutathione are significantly lower ent resolution of the disease. Extracapillary proliferation (i.e.,
and the reduced glutathione–oxidized glutathione redox sys- crescents) may also be present in a minority of cases. Capillary
tem is depleted. APSGN children, therefore, have diminished walls are not thickened, but the lumens are frequently
antioxidant defense capacity and may suffer from prolonged occluded. The GBMs may be irregular due to the characteristic
oxidative stress with increased susceptibility to lipid peroxida- humps observed on electron microscopy. Renal tubules are
tion and osmotic lysis of red blood cells (56,57). There is evi- usually not involved, although protein resorption droplets have
dence for a redox-sensitive regulation of transcription factors been noted in the proximal tubular cells and erythrocytes,
and gene expression of prooxidant and antioxidant mediators. sometimes mixed with eosinophilic cast-like material, and neu-
The genetic shifting of superoxide to nitric oxide–dominated trophils may be present in the tubular lumen. Tubular vacuola-
chemistry during the course of GN may alter the self-limited tion, dilatation, or atrophy is rarely observed.
inflammatory responses of glomerular mesangial and endothe- Renal arteries and arterioles are generally spared or show
lial cells to tissue injury (58). minimal changes. There have been occasional reports of
fibrinoid necrosis and necrotizing vasculitis that are usually
associated with hypertension (3). The involvement of inter-
Pathologic Changes stitium is not remarkable; interstitial edema and scattered
inflammatory cells may be present.
Microscopic Findings
Microscopic findings correspond to the infiltration with
Immunofluorescence Microscopy
professional immune cells, activation, and proliferation of
resident glomerular cells and matrix expansion. Early in the clinical course, coarse granular staining for IgG
Capillary tufts are enlarged and exhibit marked lobular- and C3 can be detected in the glomerular capillary walls;
ity and hypercellularity with global and relatively uniform IgM is found less frequently, and IgA and early complement
 30. Acute Proliferative Glomerulonephritis 607

FIGURE 30.2. Acute postinfectious glomerulonephritis by immu-

nofluorescence. A: Garland pattern. Large, subepithelial deposits
of immunoglobulin G on the outer side of the glomerular base-
ment membrane (×400). B: Starry sky pattern. There are fine and
coarse granular deposits of immunoglobulin G in the glomerular
capillary walls and in the mesangium (×400). C: Mesangial pattern.
Predominantly mesangial, coarse granular deposits of C3 (×400).
(Courtesy of Peter Degrell, University of Pécs Faculty of Medicine,
Nephrological Center, Pécs, Hungary.) (See Color Plate 30.2.)

components (i.e., C1 and C4) are usually absent. As the dis- lumen. The most consistent finding is the presence of large,
ease evolves, staining for C3 predominates, whereas IgG electron-dense, immune-type deposits on the subepithelial
staining becomes less apparent. Three distinct immunofluo- surface of the GBM, classically referred to as humps (Fig.
rescence patterns have been described and are designated as 30.3). These are most abundant during the first few weeks
garland, starry sky, and mesangial patterns (Fig. 30.2). Gar- of APSGN and tend to disappear 6 weeks after the clinical
land pattern consists of discrete, densely packed, sometimes onset of disease. The electron density of the deposits is vari-
confluent capillary wall deposits often seen in patients with able and diminishes with time, and the appearance of elec-
extracapillary proliferation and heavy proteinuria with worse tron-lucent regions in the deposit is regarded as a sign of
prognosis. Starry sky pattern displays fine, granular IgG and morphologic resolution (61,62). Subendothelial, mesan-
C3 deposits in the capillary walls and mesangium and occurs gial, and membranous deposits may also be present, but
mainly at the acute stage of the disease. The mesangial pat- they are less consistent and smaller in size. GBMs are gener-
tern is characterized by granular deposition of C3 and IgG ally normal in contour and thickness, although patchy
predominantly in the mesangium, with the capillary walls thickening and focal disruption may occasionally be identi-
being relatively spared. C3 deposits prevail over IgG, and this fied. The glomerular capillary endothelium may also be
pattern appears to be closely related to the resolution of the partially disrupted, with neutrophils directly adjacent to
disease. It is of note that although the distinct patterns of the denuded GBM.
immunofluorescence staining and their clinical correlates are It has been suggested that several morphologic alter-
well established, combinations of these patterns and transi- ations—including glomerular hypercellularity, crescent for-
tional forms are often observed (59,60). mation, the degree of interstitial volume, the number of
glomerular humps, and the intensity of immunofluorescent
staining for IgG and C3—may influence the initial presenta-
Electron Microscopy
tion, clinical course, and long-term outcome of APSGN. The
Ultrastructural examination shows a swelling of glomerular combination of these morphologic features is likely to have
endothelial and mesangial cells with closure of the capillary greater prognostic value than any single finding alone (3).
608 V. Glomerular Disease

TABLE 30.2. CLINICAL MANIFESTATIONS OF ACUTE

POSTSTREPTOCOCCAL GLOMERULONEPHRITIS IN
CHILDREN AND ELDERLY ADULTS
Symptom Children (%) Elderly patients (%)

Hematuria 100 100

Proteinuria 80 92
Edema 90 75
Hypertension 60–80 83
Oliguria 10–50 58
Dyspnea, heart failure <5 43
Nephrotic proteinuria 4 20
Azotemia 25–40 83
Early mortality <1 25

From Rodriguez-Iturbe B. Acute endocapillary glomerulonephritis.

FIGURE 30.3. Acute postinfectious glomerulonephritis on In: Davison AM, Cameron JS, Grunfeld J-P, et al. Oxford textbook of
electron micrograph. Hump-like, electron-dense subepithelial clinical nephrology. Oxford, UK: Oxford University Press, 1998:613–
623, with permission.
deposits (arrows) are seen in the glomerular capillary wall, with
polymorphonuclear leukocytes in the capillary lumina (×6000).
(Courtesy of Peter Degrell, University of Pécs Faculty of Medi-
cine, Nephrological Center, Pécs, Hungary.)
Edema and vascular congestion results from salt and
water retention. Edema may be mild and confined to the
periorbital area or severe with pleural effusion, ascites, and
Clinical Presentation
hypertension. Periorbital edema may be apparent in the
Typical APSGN is characterized by the abrupt onset of morning, but as the day progresses, edema localizes in the
hematuria and proteinuria often in association with edema, abdomen or lower extremities. In younger children, edema
hypertension, and mild to moderate renal functional impair- is usually generalized, whereas in adolescents and adults, it
ment. The disease is usually preceded by group A β- is limited to the face and legs. Congestive heart failure may
hemolytic streptococcus infection of the throat or skin. The complicate the clinical course of the acute nephritic syn-
long-term prognosis is generally favorable, but some patients drome presenting with orthopnea, dyspnea, cough, pulmo-
may have life-threatening acute complications or may slowly nary crackles, and gallop rhythm.
progress to renal failure. Hypertension is observed in more than 80% patients. It is
Acute nephritic syndrome follows throat or skin infec- volume dependent in addition to the increased peripheral
tion after a latent period of 1 to 2 weeks or 3 to 6 weeks, vascular resistance; plasma volume and cardiac output are
respectively. During the latent period, microscopic hema- invariably elevated. Hypertension can be associated with
turia may be detected. Some patients remain asymptom- headaches, somnolence, changes in mental status, anorexia,
atic, and the ratio of subclinical to clinically overt disease is nausea, and convulsion. Some patients present with hyperten-
estimated to be 4 to 5:1 (7,63,64). sive emergency, which is defined as blood pressure greater
The most common presenting symptoms are hematuria, than 30% of normal for age and sex or any elevation with
edema, proteinuria, and hypertension. Gross hematuria is evidence of encephalopathy, heart failure, or pulmonary
present in 24 to 40% of children with APSGN. The urine edema. Central nervous system involvement during the
is smoky or coke-colored, and the patients may have dys- course of APSGN may be secondary to hypertensive enceph-
uria, frequency, and abdominal discomfort. Macroscopic alopathy, but it can also be attributed to cerebral vasculitis. In
hematuria may be present for up to 2 weeks, and micro- normotensive children presenting with severe neurologic
scopic hematuria may remain evident for months after ill- symptoms, magnetic resonance imaging may show multiple
ness resolution. Glomerular hematuria is characterized by bilateral supratentorial lesions in the white and gray mat-
dysmorphic red blood cells and red blood cell casts best ter—a finding consistent with vasculitic infarct (64–66).
seen in freshly prepared urine by phase-contrast micros- Other nonspecific symptoms include nausea, vomiting,
copy. Mild to moderate proteinuria is common in APSGN, malaise, anorexia, weakness, lumbar pain, and abdominal
whereas proteinuria of nephrotic range is rare in children. discomfort. Clinical manifestations of APSGN in chil-
Transient oliguria occurs in approximately 50% of dren are summarized and compared to those seen in
children with APSGN, but anuria is rare. Decreased urine adults in Table 30.2 (4).
output is the result of markedly decreased filtration frac-
tion and glomerular filtration rate (GFR), diminished dis-
Diagnostic Evaluation
tal delivery of the filtrate, and maintained or even
enhanced sodium and fluid reabsorption in the distal Diagnostic work-up includes identification of nephritic
nephron. syndrome, antecedent streptococcal infection, serologic
 30. Acute Proliferative Glomerulonephritis 609

markers of immune-mediated inflammation, and, when Because APSGN has a self-limited course with a good
needed, renal histology. prognosis, renal biopsy is rarely indicated. It should be con-
Urine analysis reveals distorted, also termed dysmorphic or sidered, however, for patients with atypical history or presen-
crenated, red blood cells and red blood cell casts indicative of tation, including nephrotic-range proteinuria in the acute
glomerular hematuria (67,68). Proteinuria, usually moder- stage, normal serum complement, progressively increasing
ate, reaches the nephrotic range in 5 to 10% of patients with serum creatinine, prolonged hypocomplementemia for more
APSGN. It lasts for approximately 6 months (69,70). Leuko- than 3 months, ongoing macroscopic hematuria, or long-
cyte, hyaline, and granular casts are also frequently seen (7). lasting proteinuria (73).
There is a transient elevation of blood urea nitrogen and Considering differential diagnosis, Figure 30.4 gives a
serum creatinine due to a decreased GFR with normal or low simplified diagnostic work-up. Determination of serum
renal plasma flow and markedly depressed filtration fraction. complement levels has a critical role in the initial evaluation
Tubular function is usually preserved or mildly reduced (3). of acute nephritic syndrome and may discriminate between
Recent streptococcal infection is confirmed by the dem- diseases with low or normal complement levels (80).
onstration of increased titers of antibodies against antigens
of cell wall and extracellular products of group A strepto-
Treatment
cocci. In clinical practice, repeated measurements of anti-
streptolysin, anti–deoxyribonuclease B, antihyaluronidase, The acute nephritic syndrome is treated with restricted salt
antistreptokinase, and anti–nicotinamide adenine dinucle- and fluid intake. When significant edema or hypertension is
otidase are used to demonstrate streptococcal infection. present, furosemide or other loop diuretics should be given
The antistreptolysin and anti–nicotinamide adenine dinu- to initiate prompt diuresis and to correct fluid overload, vol-
cleotidase titers are elevated in 80% of patients with post- ume-dependent hypertension, and cardiovascular conges-
pharyngitis nephritis, whereas antihyaluronidase and anti– tion. In some patients, hypertensive emergencies may occur
deoxyribonuclease B titers are elevated in 80 to 90% of and antihypertensive agents are needed. Initial management
patients after skin infections. Antibody titers are elevated 1 of severe hypertension includes calcium-channel blockers
to 5 weeks after infections and return to their initial levels (e.g., IV nicardipine), IV labetalol or dihydralazine followed
after several months (72). Antibiotic treatment may attenu- by maintenance antihypertensive therapy with loop diuretics,
ate antibody response (73,74). IgG antibodies against the C and calcium-channel blockers (e.g., nifedipine). Administra-
region of streptococcal M protein have been claimed to be a tion of angiotensin-converting enzyme inhibitors is not rec-
more reliable diagnostic marker for APSGN because they ommended because they can cause hyperkalemia.
remain significantly elevated long after the other strepto- Occasionally, pulmonary edema may develop, which needs
coccal antibodies have normalized (21). aggressive diuretic treatment, oxygen, and morphine adminis-
In patients with APSGN, the complement system is tration. Hyperkalemia can be controlled by restriction of
activated as assessed by measurement of the total hemolytic potassium intake, administration of potassium-binding resin,
complement (CH 50), C3, and C4. More than 90% of and, in more severe cases, IV salbutamol. Dialysis is indicated
APSGN patients have low levels of C3 and CH 50 and in cases of persistent electrolyte abnormalities, fluid overload,
normal or mildly depressed C4 levels, indicating a preferen- or severe azotemia.
tial activation of the alternative pathways (4,71,75). In Streptococcal infection has usually resolved before presenta-
most cases, C3 returns to normal within 8 weeks, although tion, but patients with positive throat or skin cultures or clini-
a prolonged decrease of C3 has been reported (76). cal evidence of streptococcal pharyngitis should receive
Serum levels of IgG and IgM are elevated in 90% of antibiotic therapy (e.g., penicillin or erythromycin). Preventive
patients (3). Circulating immune complexes can be detected antimicrobial treatment is also indicated for the immediate
in 58% of cases as compared to 4% in normal individuals family members and for those individuals who are in close
(10), and 66% of patients have cryoglobulins (77). Serum contact with the affected persons. The spread of infection can
IgG levels and glomerular IgG deposits are not directly cor- also be limited if personal hygiene measures are taken (2,4,81).
related. A group of APSGN patients with elevated serum In a subset of patients with progressive clinical course or
IgG was identified as being unable to form glomerular IgG with histologic evidence of extensive crescent formation,
deposits; in these cases, the severity of disease appeared to be aggressive treatment with pulse methylprednisolone, with or
independent of serum IgG levels (78). Increased circulating without cytotoxic drugs, should be considered (82,83). The
levels and urinary excretion of some cytokines during the beneficial effect of this therapeutic intervention has been sug-
acute phase of APSGN are found to be correlated with the gested by case reports.
clinical course (54,55). Of interest, antineutrophil cytoplas-
matic autoantibodies were detected in 9% of APSGN
Prognosis and Clinical Outcome
patients, and their presence was significantly associated with
a more severe glomerular disease as assessed by serum creati- APSGN in children is a benign disease with an early mortal-
nine and crescent formation (79). ity rate of less than 1%. Most children undergo complete
610 V. Glomerular Disease

FIGURE 30.4. Diagnostic work-up and etiologic classification of acute glomerulonephritis. Number
in parentheses indicates the frequency of low complement levels. CGN, crescent-forming glomerulo-
nephritis; C3NF, C3 nephritis factor; GBM, glomerular basement membrane; GN, glomerulonephritis.
(Adapted from Yoshizawa N. Acute glomerulonephritis. Intern Med 2000;39:687–694; and Lang MM,
Fowers C. Identifying poststreptococcal glomerulonephritis. Nurse Pract 2001;34:37–47.)
 30. Acute Proliferative Glomerulonephritis 611

remission; hypertension and gross hematuria usually resolve second attack of APSGN in most patients. When the disease
over several weeks, proteinuria may last for several months, recurred, antibodies against NAPlr were not present in the
and microscopic hematuria may persist for years. Less than sera, indicating that failure to induce this specific immune
2% of the patients have been reported to progress to end- response may contribute to recurrence of APSGN (89).
stage renal failure.
Attempts have been made to identify clinical features and
morphologic markers that may serve as prognostic indicators. ACKNOWLEDGMENT
Nephrotic range proteinuria, persistent oliguria, and impaired
renal function may have an adverse effect on clinical outcome. This work was supported by the Hungarian National
Similarly, morphologic changes (e.g., extensive crescent forma- Research Foundation, grant number OTKA T 030673.
tion, atypical humps on electron microscopy, garland pattern
on immunofluorescence or glomerular necroses, adhesions,
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the subsequent antibody response. Clin Nephrol 1983;19:3– in lupus nephritis. Nephrol Dial Transplant 1997;12:273–280.
10. 43. Parra G, Platt JL, Falk RJ, et al. Cell population and mem-
25. Yoshizawa N, Oshima S, Sagel I, et al. Role of a streptococcal brane attack complex in glomeruli of patients with post-strep-
antigen in the pathogenesis of acute poststreptococcal glomeru- tococcal glomerulonephritis: identification using monoclonal
lonephritis. Characterization of the antigen and a proposed antibodies by indirect immunofluorescence. Clin Immunol
mechanism for the disease. J Immunol 1992;148:3110–3116. Immunopathol 1984;33:324–332.
26. Vogt A, Batsford S, Rodriguez-Iturbe B, et al. Cationic anti- 44. Hotta O, Ysua N, Ooyama M, et al. Detection of urinary
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1983;20:271–279. novel marker of acute inflammatory glomerular injury. Kid-
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Clin Microbiol Rev 1995;8:411–426. 45. Frankenberger M, Sternsdorf T, Pechumer H, et al. Differ-
28. Cu GA, Mezzano S, Bannan JD, et al. Immunohistochemi- ential cytokine expression in human blood monocyte sub-
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Kidney Int 1998;54:819–826. 46. Parra G, Romero M, Henriquez-La Roche C, et al. Expres-
29. Johnston KH, Chaiban JE, Wheeler RC. Analysis of the sion of adhesion molecules in poststreptococcal glomerulo-
variable domain of the streptokinase gene from streptococci nephritis. Nephrol Dial Transplant 1994;9:1412–1417.
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Orefici G, ed. New perspectives on streptococci and streptococ- body secretion by subsets of antigen-specific helper T cells.
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51. Morioka Y, Koike H, Ikezumi Y, et al. Podocyte injuries 71. Berry PL, Brewer ED. Glomerulonephritis and nephrotic
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Nephrol 1997;47:6–12. hypocomplementemia in poststreptococcal acute glomeru-
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60. Sorger K, Balun J, Hübner FK, et al. The garland type of acute penicillin to control outbreaks of acute poststreptococcal
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63. Dodge WF, Spargo BF, Travis LB. Occurrence of acute glo- glomerulonephritis in children: comparison of quintuple
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acute glomerulonephritis. Pediatrics 1967;40:1028–1030. 84. Roy S, Pitcock JA, Ettledorf JN. Prognosis of acute poststrep-
64. Glassock RJ, Adler SG, Ward HJ, Cohen AH. Primary glo- tococcal glomerulonephritis in childhood: prospective study
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65. Rovang RD, Zawada ET, Santella RN, et al. Cerebral vascu- childhood acute poststreptococcal glomerulonephritis. Pedi-
litis associated with acute post-streptococcal glomerulone- atr Nephrol 1999;13:907–911.
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66. Soylu A, Kavukcu S, Turkmen M, et al. Posterior leukoen- reserve after acute poststreptococcal glomerulonephritis.
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67. Birch DF, Fairley KF, Whitworth JA, et al. Urinary erythro- patients with epidemic poststreptococcal glomerulonephri-
cyte morphology in the diagnosis of glomerular hematuria. tis. Am J Kidney Dis 2001;38:249–255.
Clin Nephrol 1983;20:78–84. 88. Roy S, Wall HP, Etteldorf JN. Second attacks of acute glo-
68. Van Iseghem P, Hauglustaine D, Bollens W, et al. Urinary merulonephritis. J Pediatr 1969;75:758–767.
erythrocyte morphology in acute glomerulonephritis. BMJ 89. Watanabe T, Yoshizawa N. Recurrence of acute poststreptococ-
1983;287:1183. cal glomerulonephritis. Pediatr Nephrol 2001;16:598–600.
 31

IMMUNOGLOBULIN A NEPHROPATHY
NORISHIGE YOSHIKAWA

Immunoglobulin (Ig) A nephropathy was first described in Hospitals, detected in approximately 30% of biopsy speci-
1968 by Berger and Hinglais (1) and is now recognized as a mens obtained.
distinct clinicopathologic entity with a higher frequency
worldwide than any other primary glomerulopathy (2). It
was initially considered a benign condition, but extended ETIOLOGY
follow-up of patients indicates that 20 to 50% of adults
ultimately progress to end-stage renal failure (2,3). Like- Because of the frequent association between upper respira-
wise, the favorable prognosis initially attributed to children tory tract or gastrointestinal infection and the onset of mac-
with IgA nephropathy must be questioned in the light of roscopic hematuria, it has been suggested that certain viral
more recent studies (4–8). or bacterial infections may lead to IgA nephropathy. Con-
siderable effort has been directed toward the search for anti-
gens and for the antibody specificity of the mesangial IgA,
EPIDEMIOLOGY but it has met with limited success. Many antigens, includ-
ing herpes simplex virus, cytomegalovirus, Epstein-Barr
IgA nephropathy has been diagnosed all over the world, but virus nuclear antigen, adenovirus, and milk antigen, have
its prevalence varies widely from one country to another. In been identified. The observation of numerous antigenic
the Pacific Rim (e.g., Japan, Singapore, Australia, and New substances in the glomeruli indicates that the antigenic
Zealand), IgA nephropathy accounts for as many as one- materials in IgA nephropathy may be heterogeneous. The
half of cases of primary glomerulonephritis. In Europe, it presence of Haemophilus parainfluenzae antigens in a dif-
accounts for between 20 and 30% of all primary glomeru- fuse and global distribution in the glomerular mesangium
lonephritis, whereas in North America, it is responsible for and the presence of IgA antibody against H. parainfluenzae
only 2 to 10%. The explanation for this apparent variability in sera of Japanese patients with IgA nephropathy have
in incidence is uncertain, but it may be due to a racial dif- been demonstrated (12,13).
ference in the incidence of IgA nephropathy or to differ-
ences in biopsy selection practices (9).
Predisposing Genetic Factors
Genetic factors and environmental influences could
contribute to geographic differences in prevalence. A lower Predisposing genetic factors have been suggested as impor-
prevalence among blacks than whites has been reported in tant in the development of IgA nephropathy (14). More-
the United States. However, in American children, similar over, it has been suggested that genetic factors may not only
incidences of IgA nephropathy in white and black children determine susceptibility to glomerulonephritis but also
from Shelby County, Tennessee, have been reported (10). influence the pathologic severity and natural course of IgA
In Australia (11), where the population is heterogeneous nephropathy (15,16).
and includes many immigrants from Third World coun- Evidence for genetic factors being important in IgA
tries, all racial groups seem to be affected equally. nephropathy is provided by family studies (15–19). Ram-
The high incidence of IgA nephropathy in certain coun- bausek et al. reported that 9.6% of patients with mesangial
tries may reflect the practice of routine urinalysis. In Japan, IgA nephropathy in Germany had one or more siblings
all children between the ages of 6 and 18 years are screened with glomerulonephritis (15). Julian et al. described kin-
annually, and those found to have urinary abnormalities are dred from eastern Kentucky in which six patients with IgA
referred for further investigation. Thus, IgA nephropathy is nephropathy descended from one ancestor and eight other
the most common primary glomerulopathy in children patients belonged to potentially related pedigrees (17).
seen in Kobe University and Wakayama Medical University Moreover, they indicated that at least 48 (60%) of 80 IgA
616 V. Glomerular Disease

nephropathy patients who were born in same region were plasma cells in the gastrointestinal and respiratory tracts,
related to at least one other patient (18). Scolarim et al. where it is synthesized as monomers and then secreted as
reported that 26 (14%) of 185 patients with IgA nephropa- dimers linked by the J-chain, which is also produced
thy investigated in Italy were related to at least one other within the plasma cells. During the passage of dimeric IgA
patient with the disease (19). These family studies suggest molecules through the mucosal epithelium toward the
that familial predisposition is a very common finding and external lumen, the secretory component is attached
genetic factors are influenced in the pathogenesis of IgA through specific noncovalent interactions; this component
nephropathy. Recently, Gharavi et al. (20) demonstrated appears to protect the dimeric IgA from the proteolytic
linkage of IgA nephropathy to 6q22-23 under a dominant enzymes present in the external secretions. IgA has two sub-
model of transmission with incomplete penetrance. classes, IgA1 and IgA2. Approximately 90% of serum IgA is
Genetic factors are implicated in both disease suscepti- composed of IgA1 mostly produced in the bone marrow,
bility and disease progression (21). Polymorphisms of Ig whereas IgA2 is mostly derived from the local mucosa of
heavy-chain switch region gene (22), Iα1 germ-line trans- the gastrointestinal and respiratory tracts. Both IgA1 and
cript regulatory region gene (23), genes of the renin angio- IgA2 are produced in the mucosa.
tensin system (24,25), and platelet activating factor The most prominent finding in the glomeruli of renal
acetylhydrolase gene (26) have been reported. Although biopsy specimens from patients with IgA nephropathy is
some associations have emerged, they have been inconsis- mesangial IgA deposition. The majority of investigators has
tent (27). Some of the discrepancies may be caused by dif- indicated that IgA1 is the predominant subclass present in
ferent sample sizes and different geographic regions of the the glomeruli (35). The J-chain has also been identified in
patients included in the studies. the mesangium in patients with IgA nephropathy (36).
Secretory component is not present in the mesangial depos-
its, but immunofluorescence studies of renal biopsy sec-
PATHOGENESIS tions from patients with IgA nephropathy have indicated
that it binds to the mesangial areas in vitro (37). These
Although the pathogenesis of IgA nephropathy remains observations suggest that the mesangial IgA deposits are
uncertain, there is substantial evidence that it is an immune polymeric, a hypothesis further supported by the immu-
complex disease (2,28). Granular electron-dense deposits are nochemical characterization of IgA eluted from renal
observed in the glomerular mesangial areas by electron biopsy sections (38). Assessment of polymeric IgA1 pro-
microscopy and confirmed as containing IgA and C3 by duction by in situ hybridization for J-chain messenger
immunofluorescence microscopy. Circulating IgA immune RNA in IgA plasma cells shows downregulation in the
complexes have been detected by several different assays mucosa (39) and upregulation in the bone marrow (40).
often associated with IgG immune complex. Many immuno- Impaired mucosal IgA responses allowing enhanced antigen
logic abnormalities that may lead to the formation of IgA challenge to the marrow shown by de Fijter et al. (41) could
immune complex have been reported in patients with IgA be the primary abnormality in IgA nephropathy, although
nephropathy. Recurrence of IgA nephropathy frequently this remains unproven (42).
occurs in allografts (29), and a rapid disappearance of glo- A number of studies have suggested that the alternative
merular IgA deposits is observed when kidneys with mesan- complement pathway has a pathogenetic role in IgA nephrop-
gial IgA deposits are transplanted to patients without IgA athy. This hypothesis is consistent with the typical immu-
nephropathy. Although much of this work was performed in nohistologic demonstration of C3 and properdin in a
adults, there is no evidence to suggest that the findings can- pattern and distribution similar to that of IgA in the glo-
not be extrapolated to children. Moreover, glomerular IgA meruli, in the absence of C1q and C4. The detection of the
deposits associated with histologic lesions similar to those of membrane attack complex of complement further supports
human IgA nephropathy can be induced in laboratory ani- the pathogenetic role of complement activation in this dis-
mals by passive administration of preformed IgA immune ease (43). Certain types of IgA aggregates or IgA from
complex or by active immunization (30–34). patients with myeloma have been shown to activate com-
plement in vitro (44). IgA has been reported to activate the
complement system via the mannan-binding lectin path-
Nature of Mesangial Immunoglobulin
way (45). However, there is no direct evidence that comple-
A Deposits
ment activation is mediated by IgA deposition in the
IgA contributes to immunity at the level of the external glomeruli. Activation of C3 is observed in the majority of
secretory system. IgA exists in monomeric and polymeric adult and pediatric patients with IgA nephropathy, but the
forms. Monomeric IgA represents approximately 90% of mediator as well as the pathophysiologic significance of this
the serum IgA and is produced mainly by the circulating complement activation remains to be determined. C3 is
lymphocytes and plasma cells in the spleen and bone mar- deposited in the kidney but is also produced by mesangial
row. Polymeric IgA is produced mostly by lymphocytes and cells in IgA nephropathy (46).
 31. Immunoglobulin A Nephropathy 617

With regard to antibody specificity, IgA eluted from cry- deficiency or structural modification of β1,3-galactosyl-
ostat sections of IgA nephropathy biopsies has been reported transferase, the enzyme responsible for the terminal galac-
to react with mesangial areas of its own and other IgA tosylation of GalNAc on O-linked glycans (54). This
nephropathy patients’ biopsies, but not with normal kidney structural or functional deficiency may be genetically deter-
(47). Such eluates have also been shown to contain antibod- mined. The recent sequence of β1,3-galactosyltransferase
ies that react with tonsillar cells and cultured fibroblasts may help us to understand the genetic basis of these abnor-
obtained from patients with IgA nephropathy (48). malities (55,56). Circulating IgA1 has reduced terminal
In summary, the immunochemical nature of the mesangial galactose on O-linked hinge-region sugars in IgA nephrop-
deposits in IgA nephropathy is consistent with antigen-poly- athy (57), apparently because of a B-cell defect in β1,3-
meric IgA complexes predominantly of A1 subclass and, per- galactosyltransferase, the enzyme responsible for placing
haps, multispecific for ubiquitous mucosally derived antigens. terminal galactose on O-linked sugars (58). The IgA1 O-
glycan chains are truncated in IgA nephropathy (59). Cir-
culating immune complexes in IgA nephropathy consist of
Immunoglobulin A Glycosylation
IgA1 with a galactose-deficient hinge region, and the defi-
IgA glycosylation has received recent attention as a putative ciency of galactose may result in the generation of antigenic
nonimmune feature of IgA, which may explain its abnor- determinants that are recognized by naturally occurring
mal behavior and glomerular deposition in IgA nephropa- IgG and IgA1 antibodies (60). Sano et al. demonstrated
thy (49). IgA1 subclass is prominent in IgA nephropathy. that enzymatically deglycosylated human IgA1 molecules
IgA1 is unique among all Igs in its possession of a hinge accumulate and induce inflammatory cell reaction (61);
region rich in proline, serine, and threonine and character- Amore et al. showed that glycosylation of circulating IgA
ized by five O-glycosylation sites (Fig. 31.1). These O- modulates mesangial proliferation in IgA nephropathy
glycosylation sites consist of N-acetylgalactosamine O-linked (62).
to the serine residues of hinge region. Abnormal galactosy-
lated IgA1 increases affinity for glomerular fibronectin,
Immunoglobulin A Immune System
laminin, and collagen IV (50) and may lead to accumula-
tion of IgA in the mesangium (51). Preliminary data indi- There is a general agreement that serum levels of IgA are
cate that deficient galactosylation of hinge-region glycans increased in 50 to 70% of patients with IgA nephropathy,
may be detected even in family members of patients with with elevations in both monomeric and polymeric IgA.
IgA nephropathy (52). Altered amino-acid sequence of the There is an increase in polymeric IgA1-producing plasma
IgA1 hinge region is a possible mechanism to consider for cells in the bone marrow (63) and in the tonsils (64) of
abnormal galactosylation of IgA1. However, the hinge patients with IgA nephropathy. The proportion of IgA-λ in
region is a highly conserved region of the IgA1 molecule. serum IgA is also increased. Serum IgA is more anionic,
There is no evidence for any nucleotide sequence alteration owing to the increased anionicity of λ- compared with κ-
or transcriptional abnormality of the hinge region in IgA light chain (65). The binding of IgA to mesangial cells is
nephropathy (53). It has also been postulated that altered charge dependent, and anionic charge may play an impor-
galactosylated IgA1 in IgA nephropathy may be due to a tant role in IgA1 deposition in the mesangium (66). In

FIGURE 31.1. Immunoglobulin A1 (IgA1) molecule

with hinge region O-glycosylation sites. IgA1 mole-
cule with two heavy chains, each having three con-
stant region domains, CH1 to CH3, and a hinge region
between CH1 and CH2. Each serine (Ser) and threo-
nine (Thr) residue is a potential site for O-glycan side
chain. O-glycosylation sites consist of N-acetylgalac-
tosamine (GalNAc) O-linked to the Ser or Thr residues
of hinge region. Pro, proline.
618 V. Glomerular Disease

addition to the increased levels of serum IgA, various types PATHOLOGY

of autoantibodies of the IgA class have been recognized.
These IgA autoantibodies include rheumatoid factor (67), Immunohistologic Findings
antinuclear antibodies (68), and anticollagen antibodies
The diagnostic immunopathologic pattern of IgA nephrop-
(69). However, the IgA may be polyspecific, indicating a
athy is the presence of IgA in the glomerular mesangium as
polyclonal increase rather than true antigen-specific auto-
the sole or predominant Ig. IgA deposits often extend just
antibodies (70). IgA immune complexes are frequently
beyond the mesangiocapillary junctions into the adjacent
detected (71). Cultured peripheral blood lymphocytes from
capillary walls (Fig. 31.2). There are also deposits of IgG or
patients with the disease produce more IgA than do those
IgM with the same staining pattern as IgA but with lesser
of normal individuals, either spontaneously or after poly-
intensity and frequency. In our series, mesangial IgA depos-
clonal stimulation in vitro. We also demonstrated an
its were associated with IgG in 32% of patients, IgM in
increased spontaneous and pokeweed mitogen-stimulated
8%, and both IgG and IgM in 11% (86). C3 deposits were
IgA production by peripheral blood lymphocytes in chil-
observed in a similar distribution pattern in 64% of cases.
dren with IgA nephropathy (72). This increased IgA pro-
The early components of the classical complement path-
duction remained stable during the follow-up period in
way, C4 or C1q, are absent. Fibrin- or fibrinogen-related
patients with persistent urinary abnormalities but decreased
antigens are found in a diffuse mesangial distribution in 25
toward normal in patients with clinical remission.
to 70% of patients and are believed to be one of the injuri-
IgA production is T-cell dependent, and the increased
ous agents in the glomeruli (87). Although, in most
production in IgA nephropathy may indicate altered T-cell
patients, IgA is present only in the mesangial regions, in
function. An increased circulating OKT4 to OKT8 cell
approximately 10% of patients it is also observed in the
ratio, due to increased OKT4 helper T lymphocytes and
peripheral capillary walls. Such peripheral capillary wall
decreased OKT8 cytotoxic-suppressor T lymphocytes, has
deposits, whether documented by immunofluorescence or
been reported in patients (73). Increased IgA-specific
electron microscopy, have been associated with more severe
helper T-cell activity and decreased IgA-specific suppressor
clinical manifestations and a poor renal outcome (88–91).
T-cell activity have also been reported (74,75).
Defective clearance of immune complexes from the cir-
culation may also be important (76), but this seems more Light Microscopic Findings
likely to be a consequence rather than the cause of the
Various glomerular changes are observed. The most charac-
increased immune complex load.
teristic abnormality is mesangial enlargement, caused by
various combinations of hypercellularity and increase in
Mechanism of Progression matrix (Fig. 31.3). Occasionally, small eosinophilic and
PAS-positive fibrinoid mesangial deposits are also seen.
IgA alone appears to be sufficient to provoke injury in sus-
ceptible individuals (77), and deposition of polymeric IgA,
but not of monomeric IgA, can initiate glomerulonephritis
(78). There is little to suggest that the mechanisms of
mesangial proliferative glomerulonephritis, progression,
and scarring are distinct in IgA nephropathy compared
with other types of chronic glomerulonephritis. Studies in
vitro and in animal models of mesangial proliferative glo-
merulonephritis have shown the key role of cytokines and
growth factors, particularly platelet-derived growth factor
and transforming growth factor β, in the induction and
progression of mesangial injury, and there is evidence that
these are also involved in IgA nephropathy (79–81). Stud-
ies in children with IgA nephropathy suggest that mesan-
gial proliferation may in part be the result of local
production of cytokines, interleukin-1, interleukin-6,
tumor necrosis factor, platelet-derived growth factor, trans-
forming growth factor β, vascular permeability factor, and
endothelial growth factor (82–85). Although much has
been learned about the basic abnormalities of IgA in IgA
nephropathy, therapeutic interventions that may prevent
FIGURE 31.2. Immunofluorescence micrograph showing mesan-
glomerular IgA deposition or the subsequent inflammation gial immunoglobulin A deposits in a patients with immunoglobu-
and injury have to be found. lin A nephropathy.
 31. Immunoglobulin A Nephropathy 619

FIGURE 31.3. Light micrograph showing mesangial proliferation in patients with immunoglobulin
A nephropathy. Three types of mesangial changes are identified: A: Mesangial hypercellularity is
more prominent than the increase in matrix. B: The degrees of mesangial hypercellularity and matrix
increase are similar. C1,C2: The increase in matrix is more prominent than the mesangial cellularity.

Biopsies can be graded according to the amount of mesan- degree of mesangial cell proliferation varies considerably
gial cell proliferation on the basis of the World Health among glomeruli as well as segmentally within individual
Organization criteria (92). glomeruli. The proliferation is usually associated with
increased matrix. Small cellular or fibrocellular crescents are
1. Minimal glomerular lesions. The majority of glomer- frequently found but rarely affect more than 20% of the
uli appears optically normal, although a few may show a glomeruli. Capsular adhesions are frequently seen overlying
slight increase of mesangial matrix, with or without accom- lobules showing mesangial proliferation. Segmental capil-
panying hypercellularity. The number of mesangial cells per lary collapse is often observed in association with crescents.
peripheral mesangial area does not exceed three. There are A small number of glomeruli showing global sclerosis is
also small foci of tubular atrophy and interstitial lympho- often present. Tubular atrophy, interstitial fibrosis, and
cyte infiltration in some patients. interstitial lymphocyte infiltration are frequently present
2. Focal mesangial proliferation. Up to 80% of glomeruli but are not extensive.
show moderate or severe mesangial cell proliferation (i.e., 3. Diffuse mesangial proliferation. More than 80% of
more than three cells per peripheral mesangial area). The glomeruli show moderate or severe mesangial cell prolifera-
620 V. Glomerular Disease

tion, which varies in intensity in different regions of the

mesangium in a given glomerulus as well as from one glom-
erulus to another. Mesangial cell proliferation is always
accompanied by increased mesangial matrix. Cellular and
fibrocellular crescents are often found, usually affecting less
than 50% of the glomeruli, although in approximately
10% of patients, more than 50% are involved. Capsular
adhesions are frequently seen in the absence of crescents. A
small number of globally sclerosed glomeruli are often
present. Tubular atrophy, interstitial fibrosis, and interstitial
lymphocyte infiltration are frequently present and are
extensive in 10% of patients.

Three types of mesangial changes are identified in chil-

dren with IgA nephropathy (5) (Fig. 31.3): (a) Mesangial FIGURE 31.4. Electron micrograph showing numerous elec-
hypercellularity is more prominent than the increase in tron-dense deposits in the mesangium in a patient with immu-
noglobulin A nephropathy.
matrix, (b) the degrees of mesangial hypercellularity and
matrix increase are similar, and (c) the increase in matrix is
more prominent than the mesangial cellularity. to patient; in some patients, they are large and produce local-
The first type of lesion is seen in biopsies in which the ized protrusions. Peripheral glomerular capillary wall depos-
interval between onset of disease and biopsy is short. Serial its are also found in the subendothelial and subepithelial
pathologic observations reveal that prominent mesangial regions. Subendothelial deposits occur most frequently in the
hypercellularity is almost exclusively seen in initial biopsies capillary wall adjacent to the mesangium, although they are
and disappears in follow-up biopsies. These observations sug- also observed in the peripheral part of the loop. Subepithelial
gest that predominant mesangial hypercellularity is charac- deposits are reported to be unusual in adult patients but are
teristic of the early lesion of childhood IgA nephropathy and frequently found in children with IgA nephropathy. They are
may disappear within a matter of months. An increase in generally small and flat and localized to a few capillary loops;
mesangial cells, although sometimes present, is seldom strik- the humps typical of acute poststreptococcal glomerulone-
ing in adult patients (93). In contrast, biopsies with a pre- phritis are never observed. Lysis of the glomerular basement
dominant matrix increase show a long interval between onset membrane is also seen quite frequently in children (98). In
of disease and biopsy and a high percentage of glomerular affected areas of the glomerular capillary walls, the lamina
sclerosis. Serial pathologic observations reveal that this type densa is thin and irregular, and the epithelial aspect of the
of change is usually seen in follow-up biopsies. An increase in glomerular basement membrane shows irregular segments of
the amount of mesangial matrix with duration of the disease low electron density with an expanded, washed-out appear-
has also been noted in adult patients (94). These findings ance. The epithelial foot processes are generally well pre-
suggest that progression of IgA nephropathy leads to gradual served, but diffuse foot process effacement may be seen in
resolution of mesangial hypercellularity and an increase of patients with the nephrotic syndrome.
matrix associated with the development of sclerosis (95).
The severity of tubulointerstitial changes usually reflects
Repeat Renal Biopsy Findings
the severity of glomerular damage. Vascular lesions, such as
arterial or arteriolar sclerosis, are reported to be common in There have been only a few reports on the results of repeat
adults (96) but are very unusual in children with IgA neph- renal biopsies (99,100). We previously reported our results
ropathy (97). This difference may be related to the age at in children with IgA nephropathy (6). At the time of the
biopsy and the duration of disease before biopsy. second biopsy, 23 patients had shown clinical remission,
defined as complete disappearance of proteinuria and hema-
turia with normal renal function, whereas 38 had persistent
Electron Microscopy
urinary abnormalities with normal renal function. There
Electron microscopic abnormalities are mainly observed in were no differences between the two groups with regard to
the mesangium, which is variably enlarged by a combination the initial clinical findings and the pathologic findings in
of increased cytoplasm and matrix. Electron-dense deposits the initial biopsy. The second biopsy in patients who were in
in the mesangium are the most constant and prominent fea- clinical remission showed improvement of the glomerular
ture and are seen in almost all patients (Fig. 31.4). They are lesions on light microscopy, a disappearance of or a decrease
granular masses situated immediately beneath the lamina in mesangial IgA deposits, and a decreased amount of elec-
densa in the perimesangial region and expanded mesangium. tron-dense deposits. Conversely, light microscopy showed a
The size and extent of mesangial deposits varies from patient progression of histologic lesions and the persistence of both
 31. Immunoglobulin A Nephropathy 621

mesangial IgA deposits and electron-dense deposits in hematuria and is usually reversible. However, a number of
patients with persistent urinary abnormalities. Clinical investigators have documented a subset of patients with IgA
remission and histologic regression have been reported in nephropathy that is characterized by extensive crescents and
adults with IgA nephropathy (101). a rapidly progressive course (107–109).

CLINICAL FEATURES LABORATORY INVESTIGATIONS

IgA nephropathy occurs at all ages but is most common Serum IgA levels are increased in 30 to 50% of adult patients
during the second and third decades of life; it affects boys but in only 8 to 16% in children with IgA nephropathy (86).
more often than girls, with the reported male to female For this reason, it is seldom of diagnostic significance. Serum
ratio varying from less than 2:1 to 6:1 (2). In a study of Jap- complement component concentrations are usually normal,
anese children (86), the mean age at presentation was 9.3 but the C3 level should be measured routinely if the patient
years in boys and 10.3 years in girls, and the male to female has been referred for investigation after the first attack of
ratio was 3:2. The clinical presentation of IgA nephropathy hematuria to eliminate a diagnosis of postinfectious glomer-
varies. Some patients have asymptomatic microscopic ulonephritis or membranoproliferative glomerulonephritis.
hematuria with or without proteinuria. Other patients have Likewise, the antistreptococcal antibody titers should be
recurrent episodes of macroscopic hematuria. Some determined after initial hematuria. The serum creatinine
patients present with acute nephritic syndrome and, more should be measured routinely to estimate renal function; if
rarely, with acute renal failure. necessary, the glomerular filtration rate should be deter-
Sixty-two percent of our 258 Japanese children were mined. If present, proteinuria should be quantified, as pro-
found to have microscopic hematuria or asymptomatic pro- teinuria is associated with histologic lesions and a risk of
teinuria (86). Twenty-six percent presented with macroscopic progression. The plasma proteins should be measured rou-
hematuria and 12% with an acute nephritic syndrome or tinely in the presence of heavy proteinuria.
nephrotic syndrome. Several studies from Europe and the
United States reported that more than 80% of the patients
have episodes of macroscopic hematuria, and recurrent mac- DIFFERENTIAL DIAGNOSIS
roscopic hematuria is traditionally regarded as the hallmark
of childhood IgA nephropathy (102–105). However, it was The diagnosis of IgA nephropathy is based on the presence
the initial feature in only 26% of our series, presumably of IgA as the sole or predominant Ig in the glomerular
because of the school screening program that detected a high mesangium. Because diffuse mesangial IgA deposits are
prevalence of asymptomatic urinary abnormalities rather observed in a variety of other disorders (Table 31.1), the
than regional variation in the expression of IgA nephropathy. diagnosis of IgA nephropathy can be made only by exclusion.
During the observation period, 60% of our patients had one
or more episodes of macroscopic hematuria, whereas the
Relationship between Immunoglobulin
other 40% remained asymptomatic.
A Nephropathy and
Macroscopic hematuria often occurs in association with
Henoch-Schönlein Purpura
upper respiratory tract infections; less frequently, it occurs
in association with other infections involving the mucosal There is a close relationship between IgA nephropathy and
system (e.g., diarrhea and sinusitis). Episodes of macro- Henoch-Schönlein purpura (110). The morphologic and
scopic hematuria are sometimes associated with loin pain. immunopathologic features are similar in the two conditions
The interval between the precipitating infection and the (110,111), which are characterized by various degrees of focal
appearance of hematuria ranges from 1 to 2 days compared or diffuse mesangial proliferation, the diffuse deposition of
with 1 or 2 weeks in acute postinfectious glomerulonephri- IgA in the mesangium, and electron-dense deposits in the
tis. Many patients have recurrent episodes of macroscopic mesangium. Elevated serum IgA levels are found in both IgA
hematuria—each often associated with the same type of nephropathy and Henoch-Schönlein purpura nephritis, and
infection. The number of recurrences and the intervals IgA-containing circulating immune complexes have been
between different episodes are variable. The incidence of demonstrated in both conditions. Infective episodes precede
macroscopic hematuria is lower in adults (105,106). Henoch-Schönlein purpura nephritis in 30 to 50% of
Patients with a nephritic or nephrotic onset have the most patients, and the presence of H. parainfluenzae antigens in a
severe glomerular damage. The most common presenting diffuse and global distribution in the glomerular mesangium
symptom is macroscopic hematuria. Hypertension is infre- and the presence of IgA antibody against H. parainfluenzae in
quent and usually mild to moderate. Nephrotic edema is sera of Japanese children with Henoch-Schönlein purpura
reported in approximately 10% of patients. Acute renal fail- nephritis have also been demonstrated (13). The two disor-
ure is occasionally associated with episodes of macroscopic ders have been reported to coexist in different members of
622 V. Glomerular Disease

TABLE 31.1. DISEASES ASSOCIATED WITH DIFFUSE proliferation and IgA deposits are the most common find-
MESANGIAL IMMUNOGLOBULIN A (IgA) DEPOSITS ings. Most patients with chronic liver disease have clinically
IgA nephropathy (Berger’s disease) asymptomatic renal disease. The pathogenetic mechanisms
Multisystem disease that contribute to mesangial IgA deposition in chronic liver
Henoch-Schönlein purpura diseases remain unknown. Significant elevations of the
Systemic lupus erythematosus serum monomeric and polymeric IgA levels have been
Cystic fibrosis
Celiac disease
reported. Impaired hepatic clearance and increased synthe-
Crohn’s disease sis of polymeric IgA, abnormalities of IgA metabolism, and
Dermatitis herpetiformis portosystemic shunting of antigens and immune complexes
Ankylosing spondylitis have been suggested as possible causes of mesangial IgA
Neoplasms deposition (123).
Carcinomas of the lung and colon
Monoclonal IgA gammopathy
Mucosis fungoides Idiopathic Nephrotic Syndrome
Non-Hodgkin’s lymphoma
Infectious diseases A few patients with steroid-sensitive nephrotic syndrome
Mycoplasma infections show mesangial deposits of IgA on renal biopsy. They are
Leprosy
Toxoplasmosis
classified by some as IgA nephropathy, whereas others con-
Others sider that mesangial IgA in patients with minimal changes
Chronic liver disease (i.e., without cellular proliferation) is coincidental. This
Thrombocytopenia probably applies to idiopathic nephrotic syndrome associ-
Pulmonary hemosiderosis ated with mesangial IgA deposits occurring in Asians and
Mixed cryoglobulinemia
Polycythemia
explains a favorable response to steroids, which is not the
Scleritis case in true IgA nephropathy (124).

NATURAL HISTORY AND PROGNOSIS

the same family, including a pair of monozygotic twins who
developed the disorders simultaneously after a well-docu- In adult series, the incidence of renal insufficiency varies
mented adenovirus infection (112–115). Moreover, the evo- from less than 10% to as high as 45% in patients followed
lution of IgA nephropathy into Henoch-Schönlein purpura for more than 1 year. In long-term follow-up of adult
nephritis in the same patient is described in both adults and patients, 30 to 35% have been found to develop progressive
children (116–118). It has been suggested that the two con- renal insufficiency 20 years after the initial discovery of dis-
ditions are variants of the same process and that IgA nephrop- ease (2,125–127). It can be estimated that 1 to 2% of adult
athy is Henoch-Schönlein purpura nephritis without the patients will enter end-stage renal failure each year from time
rash. Although there are similarities in their pathologic and of diagnosis (128). The long-term prognosis of the 169 Japa-
immunologic features, the two conditions are clinically dif- nese children with IgA nephropathy who were followed for
ferent, and the pathogenesis is not clear. Our study suggests more than 10 years indicates that 9% of the patients had
that Henoch-Schönlein purpura nephritis is an acute disease, developed chronic renal failure by 15 years (Fig. 31.5).
with glomerular lesions nonprogressive after the onset (119).
Therefore, in most patients, the prognosis is associated with
the severity of glomerular change at the onset. In contrast,
IgA nephropathy is a chronic, slowly progressive glomerular
lesion, which may eventually lead to chronic renal failure,
whatever the presentation. A few patients with Henoch-
Schönlein purpura nephritis have recurrent episodes of mac-
roscopic hematuria and a progressive renal disease on repeat
renal biopsies. Finally, Henoch-Schönlein purpura nephritis
occurs mostly in young children and is rare in adulthood,
whereas IgA nephropathy affects mainly older children and
younger adults.

Chronic Liver Disease

FIGURE 31.5. Long-term prognosis of the 169 Japanese chil-
Glomerular IgA deposits may be observed in patients with dren with immunoglobulin A nephropathy who were followed
various types of chronic liver diseases (120–122). Mesangial for more than 10 years.
 31. Immunoglobulin A Nephropathy 623

Because of the variable rate of progression to chronic long-term studies assessing the prognosis in children have
renal failure, there have been attempts to identify features challenged earlier views that the condition represents a
present at the time of diagnosis that would predict the ulti- benign disorder. Thus, IgA nephropathy presents a thera-
mate outcome. The following clinical findings are regarded peutic challenge in both adults and children. Because of the
as poor prognostic indicators in adult patients (2): persis- variable rate of progression to renal failure, and because of
tent hypertension, persistent heavy proteinuria, and the probable multifactorial pathogenesis of the disease, the
reduced glomerular filtration rate at presentation (125– effectiveness of any treatment can only be properly evalu-
127,129–134). In children, several studies have shown ated by means of a randomized controlled trial (142). When
that the degree of proteinuria correlates with the severity considering treatment protocols, an issue of great impor-
of morphologic glomerular lesion (104,135–137) and tance is the selection of appropriate patients in whom the
heavy proteinuria at the time of biopsy predicts a poor treatment is to be evaluated. Patients with heavy proteinuria
outcome (91,138). In contrast, slight proteinuria or its at biopsy and the most severe glomerular lesions on renal
absence at the time of biopsy predicts a favorable outcome. biopsy appear to be at greatest risk of progressive renal dete-
There is general agreement that hypertension and low glo- rioration and, therefore, the most appropriate candidates for
merular filtration rate at presentation are significant factors specific therapeutic interventions. Patients with long-standing
in determining the outcome of adult patients with IgA disease and extensive, irreversible glomerular damage are
nephropathy. In children, acute renal failure at onset is unsuitable for such treatments.
usually transient and associated with macroscopic hema- Controlled double-blind trials in adult patients with IgA
turia and reversible tubular lesions. Male gender has also nephropathy (143–145) showed that treatment with fish oil
been considered an unfavorable prognostic feature by for 2 years retarded the rate at which renal function was lost,
some investigators (126), but we (91) and others (89) but a meta-analysis showed that there was only a 75% proba-
could not confirm it in a large cohort of adult and pediat- bility that fish oil was beneficial (146). Recent studies have
ric patients. Schena et al. reported an increased risk of end- indicated that angiotensin-converting enzyme inhibitors
stage renal disease in familial IgA nephropathy (139). reduce urinary protein excretion and preserve renal function
Several pathologic features are associated with a poor in adult patients with IgA nephropathy (147,148), but there
outcome: diffuse mesangial proliferation; a high propor- is no randomized controlled study demonstrating that angio-
tion of glomeruli showing sclerosis, crescents, or capsular tensin-converting enzyme inhibitors preserve renal function
adhesions; the presence of moderate or severe tubulo- in IgA nephropathy. High-dose intravenous methylpredniso-
interstitial changes; the presence of subepithelial electron- lone was shown to delay development of renal failure in a
dense deposits; and lysis of the glomerular basement randomized controlled trial in adult patients (149). How-
membrane by electron microscopy (91). Patients with dif- ever, no convincing evidence has been published to date to
fuse mesangial proliferation have been reported to have a support the use of fish oil, angiotensin-converting enzyme
significantly worse prognosis than those with focal prolif- inhibitors, or high-dose intravenous methylprednisolone for
eration or minimal lesions by light microscopy in adults the treatment of children with IgA nephropathy; at present,
(125,130,140). In many adult studies (89,125,133,141), there is no curative therapy for IgA nephropathy (150).
glomerular sclerosis and crescents have also been associ- Our recent controlled trial by the Japanese Pediatric IgA
ated with poor renal outcome. Levy and associates found Nephropathy Treatment Study Group demonstrated that
that mesangial proliferative glomerulonephritis with cres- treatment of children with severe IgA nephropathy with
cents was associated with poor prognosis in children prednisolone, azathioprine, heparin-warfarin, and dipy-
(135). Because the severity of the tubulointerstitial ridamole for 2 years early in the course of disease prevents
changes usually corresponds with the severity of the glo- immunologic renal injury and progression of the disease
merular changes, tubulointerstitial changes in IgA neph- (151). Seventy-eight children with newly diagnosed IgA
ropathy are believed to be secondary to the glomerular nephropathy showing diffuse mesangial proliferation were
injury. Vascular lesions, such as arterial or arteriolar scle- randomly assigned to receive either the combined therapy
rosis, have been reported to play an important role in the of prednisolone, azathioprine, heparin-warfarin, and dipy-
progression of IgA nephropathy in adults. However, vas- ridamole for 2 years (group 1) or the combination of hep-
cular changes are very unusual in children (5,105,108). arin-warfarin and dipyridamole for 2 years (group 2).
This difference may be related to the age at biopsy and the Urinary protein excretion was significantly reduced in
duration of disease before biopsy. group 1 patients but remained unchanged in group 2
patients. Serum IgA concentration was also significantly
reduced in group 1 patients but was unchanged in group 2
TREATMENT patients. Blood pressure and creatinine clearance were nor-
mal at the end of the trial in all but one group 2 patient
IgA nephropathy is a leading cause of chronic renal disease who developed chronic renal insufficiency. The percentage
and end-stage renal disease in adult patients, and recent of glomeruli showing sclerosis was unchanged in group 1
624 V. Glomerular Disease

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patients. The beneficial effects of prednisolone, azathio- zae antigen and antibody in renal biopsy samples and serum of
prine, heparin-warfarin, and dipyridamole treatment were patients with IgA nephropathy. Lancet 1994;343:12–16.
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majority of patients with IgA nephropathy in our series are athy. Kidney Int 2000;57:1818–1835.
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626 V. Glomerular Disease

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 32

MEMBRANOPROLIFERATIVE
GLOMERULONEPHRITIS
C. FREDERIC STRIFE
MICHAEL C. BRAUN
CLARK D. WEST

Membranoproliferative glomerulonephritis (MPGN) is char- 106 pediatric patients (8). In most series, MPGN type I has
acterized on histology by glomerular hypercellularity, increased been found to be the most common form (Table 32.2). In
mesangial matrix, thickening of the peripheral capillary walls, those studies that distinguished MPGN type I and type III,
and a splitting of the glomerular basement membranes the relative frequency of type III ranges from 14 to 37%
(GBMs) due to mesangial interposition in the capillary walls. (9–12) (Table 32.2). In our experience, type III is at least as
The term MPGN was originally used by Habib et al. (1) in common as type I (11). MPGN types I and III usually
1961 to describe the glomeruli in a group of patients with present in older children or adolescents. Occasionally, they
chronic nephritis. In 1965, this appearance was associated with have been observed in patients older than 30 years or
hypocomplementemia (2). In 1973, a second chronic nephri- younger than 5 years. The prevalence of MPGN type I has
tis, originally called dense deposit disease by Berger and Galle decreased in most series from 15 to 20% of primary glom-
(3), was classified as a variant of MPGN (4) and later desig- erulonephritides in the 1970s to less than 5%.
nated MPGN type II. The more common type, in which sub- Support for a genetic susceptibility for both MPGN
endothelial deposits predominate, was designated type I. The types I and III was provided by the observation that an
term mesangiocapillary glomerulonephritis has been used inter- extended haplotype, B8,DR3,SC01,GLO2 is significantly
changeably with membranoproliferative glomerulonephritis. A more frequent in patients with these two types than in the
nephritis morphologically similar to type I found in patients general white population (13). Also, patients with MPGN
with chronic antigenemia or malignancies makes necessary the types I and III have a similarly high frequency of inherited
additional classification of secondary as opposed to idiopathic complement deficiencies (14). Finally, there are a number
MPGN (Table 32.1). In the 1970s, several observers using of reports of familial clustering of MPGN type I (15–19).
silver-impregnated electron micrographs described a third type Reports of familial type III are rare (20). Recently, however,
of MPGN with ultrastructural features distinct from MPGN an Irish family with eight affected members in four genera-
types I or II (5–7). In MPGN type III, complex alterations of tions was reported. Significant evidence for linkage was
the GBM are revealed by the silver stain, together with deposits observed on chromosome 1q31-32 (21).
that are subendothelial and subepithelial as well as within the
GBM. Although certain aspects of MPGN types I and III are
similar, the clinical and morphologic differences between the Presentation and Clinical Manifestations
two make their separation feasible. Because this distinction is
The presentation of MPGN generally falls into three cate-
not universally accepted, this chapter considers MPGN types I
gories: nephrotic syndrome, acute nephritic syndrome, or
and III together, whereas type II is discussed separately.
asymptomatic hematuria and proteinuria discovered by
chance (Table 32.3). The specific MPGN type cannot be
distinguished by the presenting clinical features because of
MEMBRANOPROLIFERATIVE
extensive overlap (22).
GLOMERULONEPHRITIS TYPES I AND III

Epidemiology and Genetics Nephrotic Syndrome

The incidence of MPGN is difficult to ascertain; however, Edema as the presenting symptom is present in approxi-
it is roughly estimated that there are one to two cases per mately one-third of patients with either MPGN type I or
630 V. Glomerular Disease

TABLE 32.1. CLASSIFICATION OF MEMBRANO– TABLE 32.3. CLINICAL PRESENTATION OF

PROLIFERATIVE GLOMERULONEPHRITIS (MPGN) MEMBRANOPROLIFERATIVE GLOMERULONEPHRITIS
TYPES I AND III
MPGN type I
Primary or idiopathic Type I Type III
Secondary/chronic infections (%) (%)
Bacterial: endocarditis, visceral abscesses, infected ventricu-
Clinical presentationa
lar shunts, osteomyelitis
Edema 19 32
Viral: hepatitis B and C, human immunodeficiency virus
Gross hematuria 28 24
Protozoal: malaria, schistosomiasis
Asymptomatic microhematuria or pro- 22 65
Other: mycoplasma, fungal
teinuria
Immunologic (autoimmune) diseases
Additional common features at presenta-
Systemic lupus erythematosus glomerulonephritis
tion
Chronic active hepatitis
Symptoms of systemic disease 26 0
Paraprotein deposition diseases
Hypertension 60 21
Cryoglobulinemia
Low serum C3 68 86
Light chain disease
Acute infection before presentation 33 26
Neoplastic
Leukemia or lymphoma
aPatients may present with more than one clinical feature.
Neuroblastoma
MPGN type II
Idiopathic
Associated with partial lipodystrophy tration to a normal level is not occurring, as is typical for
MPGN type III resolving acute poststreptococcal glomerulonephritis.
Idiopathic

Asymptomatic Microhematuria and Proteinuria

The diagnosis of MPGN is made in approximately 50% of
III (Table 32.3). These patients have significant proteinuria patients because of the chance discovery of hematuria and
and usually microscopic hematuria with red cell and granu- proteinuria in the otherwise healthy child. This presentation
lar casts. Serum concentrations of albumin and immuno- is most common in MPGN type III patients (22). Renal
globulin (Ig) G are typically low, and approximately 70 to function and serum albumin levels are usually normal.
80% have a low serum C3 level. In the absence of effective
treatment, a nephrotic syndrome is strongly associated with
a poor prognosis. Associated Clinical Features
Braun et al. (11) observed that glomerular filtration rate esti-
Acute Nephritic Syndrome mated from serum creatinine is lower at presentation in
patients with MPGN type I when compared to those with
Gross hematuria with an acute nephritic syndrome is the MPGN type III. They speculated that the relatively greater
presenting feature in approximately 25% of patients with glomerular proliferation in MPGN type I is likely related to
MPGN. Mild hypoalbuminemia is common. Although the lower glomerular filtration rate. The higher frequency of
renal function is usually normal, a rare patient may have a hypertension at presentation in patients with MPGN type I
rapidly progressive course. Such patients usually have a low may also relate to the greater degree of glomerular prolifera-
serum level of C3, making distinction from acute poststrep- tion. Encephalopathy due to hypertension is rare at presenta-
tococcal glomerulonephritis difficult. A biopsy is often not tion but has been reported during follow-up in both treated
performed until it is apparent that the return of C3 concen- and untreated patients (10). Antecedent constitutional com-
plaints, including fatigue, lassitude, and weight loss, charac-
terize the onset in approximately 25% of patients with
TABLE 32.2. RELATIVE FREQUENCY OF
MEMBRANOPROLIFERATIVE MPGN type I but are not observed in those with MPGN
GLOMERULONEPHRITIS BY TYPE type III (22). Table 32.3 lists these clinical features.
Type I Type II Type III No. of
Series (%) (%) (%) patients Laboratory Abnormalities
Iitaka (9) 78 5 17 41 An occasional patient, especially with MPGN type I, has a
ISKDC (10) 58 19 23 73 normochromic, normocytic anemia out of proportion to the
Braun (11) 36 23 41 78
Schwertz (12) 52 34 14 50
degree of renal insufficiency. However, the distinctive feature
Totals 53 21 26 242 of the three MPGN types is hypocomplementemia (Fig.
32.1), caused in many by the presence of autoantibodies,
ISKDC, International Study of Kidney Disease in Children. called nephritic factors (NFs), directed at epitopes on comple-
 32. Membranoproliferative Glomerulonephritis 631

normal control of the complement cascade, and inhibition of

C3 synthesis mediated by circulating C3 breakdown pro-
ducts. A rare patient with hypocomplementemia as a result
of a defect in the regulatory proteins of the complement sys-
tem has an MPGN-like glomerulonephritis (23).
Of patients with MPGN type I, approximately 40% of
those with a low serum C3 level have in addition a low
serum level of C4, suggesting classical complement path-
way activation (Fig. 32.2) (22). A few also have low serum
levels of C1q and C2, but these complement components
may be low as a result of heavy proteinuria (24).
The complement perturbation in MPGN type III is dis-
tinct from that in both types I and II; C4 levels are typically
normal, and severely hypocomplementemic patients (C3 less
than or equal to 30 mg/dL) commonly have depressed levels of
C5 and properdin and of one or more of the other terminal
components, C6, C7, and C9 (25–27). Depression of the lat-
FIGURE 32.1. Activation of either the classical or alternative ter components is seen in only 10% of those with type I and is
complement pathways produces activated C3, C3b, which can not seen in type II. The NF in MPGN type III is presumably
form fluid-phase or solid-phase C3b,Bb (convertase). Normally,
both fluid-phase and solid-phase C3b,Bb are rapidly inactivated responsible for the low levels of terminal components (Fig.
by complement control proteins. Both can be stabilized by the 32.2). Known as the NF of the terminal pathway (NFt), it dif-
nephritic factor (see Fig. 32.2). C3b,Bb stabilized on a solid phase fers from that found in type II in that in vitro it converts C3
can activate C5 and form the membrane attack complex, C5-9.
(Courtesy of John Bissler, M.D.) slowly with maximum conversion in 4 hours rather than 30
minutes. The conversion is properdin dependent and activates
terminal components (28). It is believed to stabilize a conver-
ment proteins (Fig. 32.2). The serum C3 concentration is tase with the composition (C3b,Bb)n,P,NFt. It is composed of
low in nearly 80% at their initial evaluation. In all, three properdin (P) and several convertase (C3b,Bb) complexes,
mechanisms are believed to be responsible for the hypocom- which serve to activate C5.
plementemia: circulating immune complexes activating the
classical complement pathway, NFs that interfere with the
Pathology
Glomeruli in MPGN type I by light microscopy (Fig.
32.3) usually have a uniform increase in cellularity due to
an influx of leukocytes and mesangial proliferation. The
mesangial proliferation causes glomerular enlargement and
marked reduction in the number of open capillary lumens.
With silver stain, capillary walls may show a double con-
tour called tram-tracking. This is the result of new base-
ment membrane formation at the site of mesangial
interposition. With progression, the mesangial prolifera-
tion may become replaced by centrolobular hyalinization,
giving the glomeruli a progressively more lobular appear-
ance. In MPGN type III, the degree of mesangial prolifera-
tion is typically less in comparison to type I and often focal
in distribution (Fig. 32.3). Glomerular size is usually nor-
FIGURE 32.2. Fluid-phase activation of C3 in membranoprolif- mal. Double contoured capillary walls are rare, due to less
erative glomerulonephritis (MPGN). In MPGN type II, the conver- mesangial interposition. These differences, however, can-
tase stabilized by nephritic factor-a (NF a) activates only C3. The
convertase produced in type I by immune complexes and the not reliably distinguish MPGN type I from type III by
nephritic factor complex produced in type III can form a C3,C5 light microscopy.
convertase that activates both C3 and terminal components. Immunofluorescent microscopy (29) usually can distin-
This results in low C3 levels in type II, low C3 and C5 levels in
type I, and low C3,C5, and terminal component levels in type III. guish type I from types II or III (Table 32.4) in that C4 is
On a solid phase such as the renal glomerulus, it is possible that usually present in type I; C3, C4, and IgG colocalize on the
in type I, a C3,C5 convertase is deposited and nephritogenesis is periphery of the glomerular lobules, the so-called fringe
the result. Solid-phase events that are nephritogenic in types II
and III are not clear. NFt, nephritic factor of the terminal path- pattern (Fig. 32.4). In MPGN type III, C3 deposition is
way; P, properdin. (Courtesy of John Bissler, M.D.) typically in a mesangial and capillary loop pattern, C4 is
632 V. Glomerular Disease

FIGURE 32.3. Light microscopic appearance of membranoproliferative glomerulonephritis (MPGN)

types I and II. A: The glomerulus is enlarged, very cellular, and has a lobulated appearance. The
mesangium is markedly expanded. The capillary walls are thickened in appearance, and, frequently,
the capillary lumens are obscured by the mesangial expansion. In addition to the increase in mesan-
gial cells, scattered neutrophils are also identified in many of the segments. B: The type III glomerulus
is less cellular in appearance, and there is less expansion of the mesangium. Most of the capillary
loops are patent; however, thickened capillary walls are evident. (Courtesy of Dr. David Witte.) (See
Color Plate 32.3.)

rarely present, and IgG is present in small amounts in includes marked mesangial proliferation. Evidence for this
approximately 50% of cases (22). hypothesis is based on the observation that indistinguish-
Ultrastructural studies are essential to distinguish able renal pathology is present in glomerulonephritis secon-
MPGN type I from type III (Table 32.5). In MPGN dary to identified infectious, oncogenic, and other antigens
type I, uranyl-lead–stained micrographs typically dem- (Table 32.1). In addition, the pattern of complement acti-
onstrate a completely intact GBM without breaks or vation is via the classical complement pathway in at least
laminations (Fig. 32.5). There is prominent mesangial 40% of patients, favoring the presence of circulating
proliferation with extension of the mesangium at least immune complexes.
partially around the glomerular capillaries (interposi- Little is known regarding the pathogenesis of MPGN
tion), which, with the subendothelial deposits, causes type III. Normal serum C4 levels and the absence C4 in
narrowing of the capillary lumen. glomeruli suggest that immune complex formation does
In MPGN type III, uranyl-lead stains typically demon- not play a major pathogenic role. West and associates have
strate a complex thickening and irregularity of the GBM.
However, the type III lesion—characterized by a discontin-
uous fragmented, laminated appearance of the GBM (Table
32.5)—is best seen with methenamine silver stains (Fig. TABLE 32.4. COMPARISON OF
IMMUNOFLUORESCENT MICROSCOPY IN
32.5). The distribution of deposits in this lesion has been MEMBRANOPROLIFERATIVE GLOMERULONEPHRITIS
shown to vary according to the C3 level at the time of the TYPES I AND III
biopsy (Table 32.5).
Immunoglobulin G
C4 (%) C3 (%) C5 (%) (%)
Pathogenesis Type I 75 100 100 100
Idiopathic MPGN type I is presumed to be secondary to Type III 0 100 100 50
circulating immune complexes composed of IgG (anti-
Adapted from Wyatt RJ, McAdams AJ, Forristal J, et al. Glomerular
body)–unknown antigen–C3, which deposit in the suben- deposition of complement-control proteins in acute and chronic
dothelial glomerular space, giving rise to a response that glomerulonephritis. Kidney Int 1979;16:505–512.
 32. Membranoproliferative Glomerulonephritis 633

Natural History
The natural history of MPGN was described in detail by
Cameron et al. (33) in a report comparing the long-term
outcome of 69 children and adults with MPGN type I (type
III, presumably, was included in this group) to 35 with type
II (Fig. 32.6). The outcome was poor irrespective of type,
with 50% losing renal function by 10 years and 90% by 20
years. Similar results have been observed by Habib et al. (4).
Cameron et al. (33) noted that MPGN type I progressed
more slowly in children than in adults during the first 10
years of follow-up. There are no reports that compare the
courses of MPGN types I and III in the absence of effective
FIGURE 32.4. Immunofluorescence appearance of membrano- treatment. Outcome has been shown to be adversely affected
proliferative glomerulonephritis type I. The prominent periph- by the presence of nephrotic syndrome or renal insufficiency
eral distribution of immunoglobulin G deposits in the capillary
walls results in a fringe pattern. (Courtesy of Dr. David Witte.) at onset and by the presence of crescents in the initial biopsy.
(See Color Plate 32.4.)

Treatment
reported a strong association between hypocomplement- The effectiveness of treatment regimens for MPGN has to
emia and the presence of paramesangial and subendothelial be judged by the outcome of usually uncontrolled, retro-
deposits (30,31). Subendothelial deposits, in particular, spective observations on small numbers of patients. In sev-
were never found in patients normocomplementemic at eral series, the basic regimen has been supplemented in
biopsy (Table 32.5). They suggest that NFt may play a sig- some patients by other treatment modalities. Despite these
nificant role in the development and perpetuation of the shortcomings, some consensus has emerged.
ultrastructural changes that define MPGN type III.
Although this is a provocative and intriguing hypothesis,
Steroids
these findings must clearly be reconciled with other reports
that have failed to demonstrate any relationship between The treatment regimens that appear to improve outcome in
the presence of NF, the duration or severity of hypocomple- patients with MPGN types I and III have used steroids.
mentemia, and either renal survival or disease progression The Cincinnati group (11,34–36) has used high-dose (2
in MPGN type III (11,32). mg/kg to a maximum of 80 mg) alternate-day prednisone

TABLE 32.5. DIFFERENCES IN GLOMERULAR ULTRASTRUCTURE IN TYPE I AND TYPE III

MEMBRANOPROLIFERATIVE GLOMERULONEPHRITIS AS VIEWED IN URANYL-LEAD–
AND METHENAMINE-SILVER–STAINED PREPARATIONS
Type I Type III

Uranyl-lead staining
Basement membrane Intact Thickened areas with patchy dense deposits
Methenamine-silver staining
Basement membrane Intact Type III lesiona present with both ↓ [C3] and nl
[C3]
Uranyl-lead and methenamine-silver
staining
Subendothelial deposits Always present with ↓ [C3] Present in 50% with ↓ [C3]
Present in 50% with nl [C3] Never present with nl [C3]
Subepithelial paramesangial deposits Very rare; never with nl [C3]; in ~12% Present with ↓ [C3] and in those with nl [C3] if
with ↓ [C3] level has been low in past year
Subepithelial loop deposits Frequent, accompany subendothelial May be present; not clearly related to [C3]
deposits

↓ [C3], serum C3 level low at time of biopsy; nl [C3], serum C3 level normal at the time of biopsy.
Note: Correlations of deposits with serum C3 levels are taken from references (3) and (31). Italics indicate key features differentiating types I and III.
aType III lesion is a complex lesion of the GBM that appears to originate from several generations of subepithelial and subendothelial deposits

forming in conjunction with multiple interruptions of the lamina densa such that the deposits are partially confluent. Lesions develop a complex
laminated appearance because each generation of deposit is covered by new lamina densa–like material.
From Braun MC, West CD, Strife CF. Differences between membranoproliferative glomerulonephritis types I and III in long-term response to an
alternate-day prednisone regimen. Am J Kidney Dis 1999;34:1022–1032, with permission.
634 V. Glomerular Disease

FIGURE 32.5. Ultrastructural features of membranoproliferative glomerulonephritis types I and

II. A: An electron photomicrograph of a glomerulus with membranoproliferative glomerulone-
phritis type I. Note the prominent mesangial proliferation and deposits in the mesangium. The
capillary walls are thickened due to extensive interposition (arrow) in addition to prominent sub-
endothelial deposits (asterisk). In this silver-stained preparation, the lamina densa can be clearly
identified and is generally intact. B: The glomerulus with the type III lesion also shows by silver
stain extensive mesangial proliferation and deposits. The capillary walls show extensive disrup-
tions of the lamina densa, with deposits that are subepithelial and subendothelial and, at times,
completely obscure the lamina densa. (Courtesy of Dr. David Witte.)

for a minimum of 2 years. Dose reduction thereafter is quency of proteinuria and hematuria and a lower glomeru-
based on improvement of clinical parameters (e.g., urinaly- lar filtration rate.
sis, serum albumin, serum C3 level) and glomerular mor- Based on early reports of improved outcome with alter-
phology (e.g., degree of mesangial proliferation, number of nate-day prednisone, the International Study of Kidney Dis-
open capillary lumens). In the long term, prednisone dose ease in Children sponsored a randomized, double-blind,
is slowly reduced if there is no evidence of disease reactiva- placebo-controlled 5-year trial of alternate-day prednisone,
tion (e.g., increase in proteinuria or hematuria or decrease 40 mg/m2 versus placebo in 80 children with MPGN and
in serum C3 level). Because many, if not most, patients heavy proteinuria (10). The study included 42 patients
continue with at least some degree of proteinuria as a result with MPGN type I and 17 with type III. Treatment failure
of chronic glomerular damage, loss of microhematuria was defined as an increase in serum creatinine of either
appears to be the best clinical indicator of disease remission greater than or equal to 30% over baseline or greater than
(35). Most patients have continued on alternate-day ster- 0.4 mg/dL. Outcome analysis evaluating patients with
oids for at least 5 years and many for much longer periods. MPGN types I and III as a group showed that at last follow-
Reports of outcome with this regimen have been encour- up (mean 5.25 years), 33% of patients treated with pred-
aging but continue to be difficult to interpret because of nisone and 58% in the placebo group were treatment
small patient numbers, retrospective analysis of data, lack failures (p = .07). The results favored treatment with
of a control group, and use in some patients of adjunctive prednisone.
therapies. Kidney survival at 10 years was 84% and, at 20 Similar results were obtained by Mota-Hernandez et al.
years, was 54%. In the most recent report (11) using the (37), who treated eight MPGN type I patients with alter-
same alternate-day prednisone regimen, renal survival was nate-day prednisone and ten with placebo. After an average
80% at 10 years in those with type I and 70% in those follow-up of 6 years, treated patients had stable or improved
with type III. In addition to a lower renal survival, those renal function, whereas four of ten placebo-treated patients
with type III had, at 10 years, a significantly greater fre- had progressed to end-stage renal disease.
 32. Membranoproliferative Glomerulonephritis 635

ridamole (44). Uncontrolled reports have suggested no or

limited benefit from treatment with other cytotoxic agents,
antimalarials, or anticoagulants.

Disease Recurrence in Renal

Transplant Recipients
The rate of recurrence in type I MPGN ranges between 20
and 30% (48). Patients with recurrence may remain asymp-
tomatic or develop proteinuria or hematuria. Hypocomple-
mentemia is inconstant. According to Briganti et al., the
incidence of graft loss at 10 years due to recurrence is 15%
(49). No therapy has been proven to be effective. The role
of cyclosporine is uncertain, as some authors have found
that the rate of recurrence had decreased from 30 to 10%
after the introduction of cyclosporine, whereas other
authors did not find any changes. Case reports have sug-
gested that graft survival may be improved by the addition
of cyclophosphamide to immunosuppressive drugs (45,50).
There are two case reports of recurrent MPGN type III in
FIGURE 32.6. Comparison of renal survival between adults and adult cadaveric renal transplant patients (46,47).
children. (From Cameron JS, Turner DR, Heaton J, et al. Idio-
pathic mesangiocapillary glomerulonephritis. Comparison of
types I and II in children and adults and long-term prognosis. Am MPGN TYPE II (DENSE DEPOSIT DISEASE)
J Med 1983;74:175–192, with permission.)

Epidemiology and Genetics

Additional uncontrolled reports have favored the use of MPGN type II is the least frequent of the three types (Table
alternate-day prednisone (9,38–40). The observation that, 32.2). It constitutes 19 to 34% of the cases in whites and
in the absence of treatment, the disease in patients not 5% in Japanese. There is no evidence for a genetic basis for
nephrotic at presentation progresses more slowly (4,33) has the disease. We have two families in which one of a pair of
led to the recommendation that steroids be withheld in identical twins has the disease, and a similar family has
these patients (41) or used in a low dose (39,40). However, been reported (51). Onset is most often in childhood.
others have shown significantly better outcomes in patients
treated with prednisone early in the course of the disease
Presentation and Clinical Manifestations
regardless of severity (9,35).
Long-term follow-up data on patients with MPGN type Symptoms and signs at presentation can include hematuria,
III have recently been published by a number of centers proteinuria, hypertension, and edema. Hematuria is always
(11,12,42). The data should be viewed with caution, as less present; in those with gross hematuria, the onset is that of
than 50% of the patients initially studied were available for an acute nephritic syndrome. Presentation with a nephrotic
long-term follow-up, and the numbers of patients studied syndrome is common and has been associated with a poor
were small. The report by Iitaka et al. (42) is by far the prognosis (52,53). Even before onset of renal failure, hyper-
most promising, with 100% renal survival after 16 years of tension can develop, but hypertensive encephalopathy is
follow-up. However, it is uncertain if the universally good rarely a presenting event.
outcome was due to early identification and aggressive ther- The clinical course is characterized by a nephrotic syn-
apy or to an unknown factor that moderates the course of drome that persists or develops and increasingly severe
the disease in the Japanese population. Finally, the outcome hypertension (52,54,55). Occasionally, patients present
reported by Braun et al. (11) and by Schwertz et al. (12) is with renal failure and have a rapidly progressive course
less encouraging, with 70% renal survival at 10 years and (54,56). Conversely, a few may have periods after onset
50% renal survival at 15 years, respectively. when their nephritis is silent (54); their urinary abnormali-
ties completely disappear and only hypocomplementemia
remains. Signs of nephritis may return, often associated
Other Therapies
with intercurrent infection, and persist.
Other controlled prospective therapy trials have shown no There is increasing evidence that MPGN type II is a sys-
long-term benefits with the antiplatelet agent dipyridamole temic disease. First, partial lipodystrophy and hypocomple-
(43) or with cyclophosphamide, warfarin, and dipy- mentemia may be present for a considerable period before
636 V. Glomerular Disease

signs of glomerulonephritis appear (57). Second, renal

transplants in almost every case quickly develop dense
deposits in the GBM, often in the absence of manifesta-
tions of glomerulonephritis (58). Third, dense deposits
similar to those in the GBM develop in the basement mem-
branes of the sinusoids of the spleen (59) and in Bruch’s
membrane in the choriocapillaris of the eye (60). The eye
lesion is associated with drusen, which resemble those seen
in adults with macular degeneration. It is of interest that
these spleen and eye membranes, like the GBM, are unique
in that they are bathed in plasma.

Laboratory Abnormalities
FIGURE 32.7. Ultrastructural features of membranoprolifera-
Distinctive features of the disease are the complement pro- tive glomerulonephritis type II. In this electron photomicrograph
file and the frequent presence of NF (Fig. 32.2). The profile of a uranyl-lead acetate–stained preparation, the lamina densa
is characterized by levels of C3, which may be markedly can be seen to be expanded by accumulation of electron-dense
material (arrow), which has a fusiforme configuration. In addi-
depressed with normal or near normal levels of other com- tion to the dense deposit material, there are also large, discrete
ponents (25). Thus, the depressed properdin and C5 levels subepithelial deposits (asterisk) located in the paramesangial
seen in the other types of MPGN, in acute glomerulone- region. (Courtesy of Dr. David Witte.)
phritis, and in the nephritis of systemic lupus are not
present. The depressed C3 is produced by activation of
native C3 by the alternative pathway convertase, C3b,Bb, ear appearance (railroad tracks). They also observed C3
which has been stabilized by the NF of the amplification deposits within the mesangium outlining circular structures
loop, NFa (Fig. 32.2). The stabilized convertase, C3b,Bb, that represent shed fragments of the basement membrane
NFa, has a half-life more than 15 times that of the native (mesangial rings). The railroad tracks are visualized with
convertase (61). The inactivated C3 it forms is quickly labeled anti-C3c and -C3d, indicating that they are com-
inactivated by factors H and I and is subsequently degraded posed of C3b, iC3b, and C3dg, whereas the paramesangial
to the ultimate C3 breakdown products, C3c and C3dg. deposits stain only with anti-C3c, indicating that they are
All three of these derivatives may be found in the circula- formed from C3c deposited from the circulation (69).
tion (62–64). Contributing heavily to the hypocomple- The diagnostic intramembranous deposits are best seen
mentemia is an inhibition of C3 synthesis attributed to a by electron microscopy of uranyl-lead–stained specimens
negative feedback produced by the circulating C3 break- (Fig. 32.7). They usually occupy only the lamina densa but
down products (65,66). occasionally may be present only in the lamina interna
(70). The deposits may be discontinuous. Similar deposits
may also be found in Bowman’s capsule and in the base-
Pathology
ment membranes of isolated groups of tubules. If searched
In most cases, by light microscopy, the glomeruli are uni- for, subepithelial deposits can be found on the parts of the
formly hypercellular with increased mesangial matrix, and basement membrane that overlay the mesangium (parame-
the number of open capillary lumens is commensurately sangial) if the patient was biopsied when hypocomplement-
reduced. In contrast to MPGN type I, the proliferation is emic (71). Occasionally, hump-like subepithelial deposits
rarely great enough to increase glomerular size. The diagno- are present on the capillary loops.
sis of type II can occasionally be made by light microscopy
of silver-stained preparations; the basement membrane is
Pathogenesis
thickened, and at sites of the intramembranous deposits,
argyrophilia is lost. The dense deposits may also be visual- The pathogenesis of the disease is not known. Many investi-
ized by light microscopy by their ability to stain with gators find that the actual measurement of NF or of its surro-
thioflavin T (67). Deposits are located along the GBM, the gate, hypocomplementemia, does not show a straightforward
Bowman’s capsule, and the tubular basement membrane. correlation with activity of the disease. A few, however, have
By immunofluorescence, IgG, IgA, IgM, and C4 are rarely found a correlation between absence of hypocomplement-
found in glomerular deposits, indicating that the classical emia or of NF and a benign course (72,73). Evidence that
pathway of complement is not activated. NF is more than an epiphenomenon is the observation that
C3 deposits are abundant. Kim et al. (68) found C3 to patients with several uncommon conditions—one of which
be present along the margin but not within the central por- is homozygous deficiency of factor H—are like those with
tion of the dense deposits in the GBM, giving a double lin- MPGN type II in that they have convertase circulating in
 32. Membranoproliferative Glomerulonephritis 637

abnormally high concentration and have an unusual fre- Disease Recurrence in Renal
quency of glomerulonephritis. How circulating convertase Transplant Recipients
produces the nephritis is not clear (74).
The rate of recurrence of dense deposits after renal trans-
It has been proposed that in consonance with the fre-
plantation is high, ranging from 50 to 100% in various
quent association of deficiency of complement components
series (83). Only a fraction of patients with recurrence
with nephritis, the deficiency of C3 produced by NF is per
develop proteinuria and, sometimes, nephrotic syndrome.
se in some way nephritogenic. Another proposal is that it is
A number of investigators have correlated disease recur-
an immune complex disease with the complexes generated
rence with persistent or recurrent hypocomplementemia
by a subclinical infection, which in turn is a consequence of
(58,80,84), whereas others could find no correlation with
the hypocomplementemia. The absence of signs of classical
either low C3 levels or the presence of NF (81,85). The
pathway activation makes this concept unlikely.
North American Pediatric Renal Transplant Cooperative
Support for a role of complement in the pathogenesis of
Study group analyzed the 2-year kidney transplant survival
MPGN is provided by the observation that not only humans
and found an increased rate of graft loss for patients with
homozygous for deficiency in factor H can develop nephritis
MPGN type II; 45% were lost to disease recurrence. In
(23), but also that this deficiency in pigs (75) and mice (76)
published reports, graft loss due to recurrent MPGN type
produces a lethal nephritis resembling MPGN type II. In
II varies widely from 0 to 100% (52,79,80–82).
mice, deposition of C3 and C9 antedating the deposition of
IgG indicated that immune complexes do not initiate the
glomerular injury. The disease was shown to be complement
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25. Verade WS, Forristal J, West CD. Patterns of complement of type III membranoproliferative glomerulonephritis in
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26. Mollnes TE, Ng YC, Peters DK, et al. Effect of nephritic on life-table analysis. Am J Kidney Dis 1989;6:445–451.
factor on C3 and on the terminal pathway of complement 44. Cattran DC, Cardella CJ, Roscoe JM, et al. Results of a
in vivo and in vitro. Clin Exp Immunol 1986;65:73–79. controlled drug trial in membranoproliferative glomerulo-
27. Clardy CW, Forristal J, Strife CF, et al. Serum terminal comple- nephritis. Kidney Int 1985;27:436–441.
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28. Clardy CW, Forristal J, Strife CF, et al. A properdin depen- lonephritis after transplantation. Am J Kidney Dis 2000;35:
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54. Habib R, Gubler M-C, Loirat C, et al. Dense deposit dis- 71. West CD, McAdams AJ. Paramesangial glomerular deposits in
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640 V. Glomerular Disease

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 33

MEMBRANOUS NEPHROPATHY
SUDESH PAUL MAKKER

Membranous nephropathy (MN) is a chronic glomerular PATHOLOGY

disease with distinct histopathologic features. The term
membranous glomerulitis was first used by Bell in 1938, Light Microscopy
reporting a light microscopy study of autopsied kidneys
Under light microscopy, the glomeruli are normal in size or
from patients with nephrotic syndrome (NS) (1). In a sub-
slightly enlarged. Their cellularity appears normal, and there is
group whose glomeruli had no proliferation, he noted that
no infiltration with inflammatory cells or proliferation of
some had essentially normal glomeruli, whereas others had
mesangial or endothelial cells. The epithelial cells may be nor-
diffuse thickening of the glomerular basement membrane
mal or enlarged and prominent (Fig. 33.1). In most patients,
(GBM). The latter cases were termed membranous glomeru-
no epithelial cell crescents are present, but occasionally such
litis. It is interesting to note that two of them were chil-
crescents are seen in association with a clinical picture of rap-
dren. Later, Bell noted that the GBM had a vacuolated
idly progressive glomerulonephritis. The mesangial matrix is
appearance (2).
not increased, and the glomerular tufts do not show excessive
In 1957, Jones introduced the periodic acid–silver methe-
lobulation. The capillary loops are usually patent but may be
namine stain to study kidney tissue of patients with NS and
occluded in advanced cases. The predominant change in MN
observed that the GBM in membranous glomerulitis had a
is in the glomerular capillary walls, which show varying grades
peculiar appearance, with the thickened GBM resolving into
of diffuse thickening involving all capillary loops and all glo-
a thinner membrane from which protruded many silver-
meruli. The thickened capillary wall is best visualized in the
positive projections (3). Between the projections, a silver-
periodic acid–methenamine silver stained sections in which
negative hyaline material was present in the form of droplets.
the GBM itself is stained black, and the subepithelial deposits
Jones’ description is now widely accepted as the pathognomic
in the GBM are not stained. Because of their location in the
feature of MN, and his stain is routinely used in the process-
subepithelial space, these unstained deposits are surrounded by
ing of renal biopsy specimens. At approximately the same
black-staining newly synthesized GBM that appears as small
time, Movat and McGregor first used electron microscopy,
black spikes projecting from the GBM toward the urinary
and Mellors et al. used immunofluorescence microscopy in
space (Fig. 33.2). In tangentially cut sections, it may give a vac-
the study of this disease and further elucidated the ultrastruc-
uolated appearance. In the advanced stages, the subepithelial
tural and immunohistologic features of MN (4,5).
deposits are incorporated into the GBM, giving it a chain-like
The characteristic histopathologic features of MN are
appearance. As the lesion progresses, the capillary walls
(a) lack of proliferation in the glomerulus and a diffusely
increase in thickness, their lumen is occluded, and eventually
thickened glomerular capillary wall exhibiting silver-positive
the glomeruli become sclerosed.
projections, or so-called spikes, with silver methenamine
In the early stages, the tubules, vessels, and interstitium
stain under light microscopy; (b) fine granular staining for
are normal. As the disease advances, there is progressive tubu-
immunoglobulin (Ig) G and complement along the
lar atrophy, accumulation of mononuclear cell infiltrate, and
periphery of the glomerular capillary wall on immunofluo-
increasing interstitial fibrosis, and the kidney eventually
rescence microscopy; and (c) electron-dense deposits located
develops the appearance of end-stage kidney disease (6,7).
exclusively in the subepithelial position along the GBM on
electron microscopy.
Although the term membranous glomerulitis was used
Immunofluorescence Microscopy
commonly in earlier medical literature, membranaus nephrop-
athy is now the preferred term. MN is a histopathologic term With immunofluorescence microscopy, all cases of MN
used to describe a unique glomerular lesion and is not itself a show bright, fine to coarse granular staining for IgG along
clinical entity. the glomerular capillary wall (Fig. 33.3). Using monoclonal
642 V. Glomerular Disease

FIGURE 33.1. Early membranous nephropathy with mild dif-

fuse thickening of capillary loop membranes (arrowhead)
(hematoxylin and eosin stain, ×100). (Courtesy of Dr. John A. FIGURE 33.3. Membranous nephropathy, immunofluorescent
Wolfe, Sutter Hospitals, Sacramento, CA.) study showing the typical brightly stained granular immune-
complex deposits along the entire glomerular basement mem-
brane (anti–immunoglobulin G, approximately ×100). (Courtesy
of Dr. John A. Wolfe, Sutter Hospitals, Sacramento, CA.)
antibodies against human IgG subclasses, it appears that
the predominant IgG staining in the granules is for IgG4
(13). No staining for IgG is seen in the mesangium in idio- Electron Microscopy
pathic MN; if present, it should arouse the suspicion of sys- The electron microscopy findings in MN are characteristic.
temic lupus erythematosus (SLE) as the underlying cause. The hallmark is the presence of electron-dense deposits,
The staining for IgA and IgM is variable and not intense located exclusively in the subepithelial space along the cap-
and is in the same location as IgG. Staining for comple- illary wall and corresponding to the granular IgG deposits
ment C3 is similar in character and distribution to that for seen on immunofluorescence microscopy (Fig. 33.4). No
IgG and is usually, but not always, of similar intensity. electron-dense deposits are seen in the subendothelial space
Staining for C1q and C4 is seen, but the intensity is much or in the mesangium, and the mesangial cellularity is nor-
less than that for C3, and intense staining for C1q should mal. An occasional mesangial deposit should arouse suspi-
arouse suspicion of SLE. Recently, staining for membrane cion of MN associated with SLE. Four morphologic stages
attack component of complement C5b-9 has been noted in of MN are recognized:
a similar location to that of IgG (14,15). Staining for fibrin
is also commonly present.

FIGURE 33.4. Electron micrograph showing the portion of glo-

merular peripheral capillary membrane with multiple, fairly evenly
FIGURE 33.2. Membranous nephropathy, silver stain showing spaced, electron-dense immune-complex deposits located on the
formation of spikes (single arrowheads) and a moth-eaten subepithelial side of the basement membrane (arrowheads). There
appearance with multiple small holes in the basement mem- is very early deposition of basement membrane between these
branes when cut en face (double arrowhead) (Jones silver stain, deposits. Notice the loss of normal epithelial cell foot process archi-
approximately ×160). (Courtesy of Dr. John A. Wolfe, Sutter Hos- tecture (double arrowheads) (×9700). (Courtesy of Dr. Richard R.
pitals, Sacramento, CA.) Wilber, and Dr. John A. Wolfe, Sutter Hospitals, Sacramento, CA.)
 33. Membranous Nephropathy 643

1. In stage I, the GBM is normal in thickness and appear- TABLE 33.1. CLASSIFICATION OF MEMBRANOUS
ance. The electron-dense deposits are small to moderate GLOMERULONEPHROPATHY IN CHILDREN
in size but discrete. The smaller deposits are generally Idiopathic or primary
located at the site of the slit diaphragm. Moderate-size Associated with other conditions—secondary
deposits cover a greater area of the subepithelial space, Infections
adjacent to where the foot processes of the visceral glo- Hepatitis B (8,18–21)a
Congenital syphilis (22,23)a
merular epithelial cells are usually fused.
Malaria? (24)
2. In stage II, the deposits are numerous, larger, and con- Filariasis (25)
fluent. Projections of GBM, corresponding to the spikes Immunologic or autoimmune diseases
seen on light microscopy staining black with silver Systemic lupus erythematosus (8,12,18,19) a
stains, are seen between the deposits. The GBM itself is Enteropathy (27)
Crohn’s disease (28, 29)
generally not thickened.
Pemphigus (30)
3. In stage III, the deposits are larger and surrounded by Drugs
the projections of GBM. The density of the deposits is O-Penicillamine (8,17)a
decreased, and the GBM appears irregularly thickened. Neoplastic
4. In stage IV, the deposits have been incorporated into Ovarian tumor (33)
Neuroblastoma (34)
GBM and have become electron lucent and, thus, are
Gonadoblastoma (35)
sometimes difficult to differentiate from the surround- Wilms’ tumor? (36)
ing GBM. The GBM is now severely altered and irreg- Miscellaneous
ularly thickened. De novo occurrence in renal transplant (37)a
Fanconi’s syndrome (8,26)
Sickle cell disease (8)
Stage II is the most common type of lesion (10,11,16,17),
Diabetes mellitus? (38)
whereas stages III and IV are considered as showing resolu- Associated with antitubular basement membrane (8,26) and
tion. Because MN is a chronic disease, probably involving antialveolar basement membrane antibodies (38)
continuous or multiple generations of deposit formation, dif- Thrombocytopenia (8) and microangiopathic anemia (39)
ferent stages of the lesion in a patient may be seen at any Juvenile cirrhosis or alpha-1 antitrypsin deficiency (40)
given time, as reported by Habib et al. in the largest series of aAccounts for majority of the cases.
children with MN (10). There is insufficient information in
children to determine the correlation between the different
histologic stages and the clinical manifestation or course of 1. The development of MN in certain patients (both chil-
the disease (9,12,16). dren and adults) with SLE.
2. The development of MN in other autoimmune disorders
[e.g., insulin-dependent diabetes mellitus in children (38)
CLASSIFICATION (S. P. Makker, unpublished observation, 1980) and adults
(47), children with autoimmune enteropathy (27),
MN in children may be divided into two categories based Crohn’s disease (28,29), multiple sclerosis (31) or pem-
on cause: (a) idiopathic or primary; and (b) secondary, in phigus (29), adults with Graves’ disease (6,48), primary
which MN develops in association with another condition, biliary cirrhosis (6,49), Sjögren’s syndrome (6), rheuma-
such as SLE, or the patient has been exposed to certain toid arthritis (6), Hashimoto’s thyroiditis (6), dermatitis
infectious agents or drugs (Table 33.1) (8,18–39). herpetiformis (6), and ankylosing spondylitis (6)].
Compared with adults, a greater percentage of cases of 3. MN can occur in association with the development of
MN in children are secondary (19). In the largest series, autoantibodies to other renal antigens [i.e., tubular
43% had an associated condition (8,19). In several parts basement membrane in children (26) and GBM in
of the world—particularly Southeast Asia (20,21), Japan adults (50)].
(40), Africa (41–43), and certain parts of Europe (44– 4. An autoimmune MN indistinguishable from idio-
46)—hepatitis B is a significant factor in the etiology of pathic MN in humans can be produced in rats by
MN. Other infections, such as congenital syphilis (22,23), immunization with rat tubular antigens (Heymann
malaria (24), and filariasis (25), are also of etiologic signif- nephritis).
icance in those areas where they are prevalent. Whether 5. Mercury is known to produce immune dysregulation
certain host factors predispose people to the development and autoimmunity including MN in certain strains of
of MN in such infected children is unclear. Medications rats (51), and mercury exposure in adult patients can
and neoplasms are rare causes of MN in children com- also produce MN (6).
pared with adults (8,19).
The cause of the idiopathic type of MN is unknown, Genetic factors are also important in idiopathic MN.
but several observations suggest immune dysregulation: Strong associations of HLA-DR3 (52,53) and DQA1 allele
644 V. Glomerular Disease

(54) have been reported with idiopathic MN in adults. bodies against at least one expressed podocyte protein, neu-
MN has been reported in two HLA identical brothers tral endopeptidase, was reported recently (73). In this case,
(both children) (55) and, in another instance, in a father antibodies against neutral endopeptidase produced by a
and son with HLA-DR3 (56). Based on the aforemen- mother who herself had a deficiency of this protein were
tioned observations, it may be hypothesized that idiopathic transferred to her fetus, producing a MN in the newborn.
MN in humans is an autoimmune disease occurring in cer- The mother apparently had been immunized against this
tain genetically predisposed individuals and precipitated by protein during an earlier pregnancy.
unknown exogenous or endogenous events. Also, based on the studies in Heymann nephritis, it
appears that (a) the subepithelial immune deposits come to
lie in this unique position after capping and shedding of
IDIOPATHIC MEMBRANOUS NEPHROPATHY
the antigen-antibody complexes formed in situ on the sur-
face of the glomerular epithelial cell (74); and (b) after
Prevalence
immune-deposit formation complement activation ensues,
Idiopathic MN may be discovered during evaluation of resulting in the generation of the terminal membrane
asymptomatic proteinuria or NS (8,19,57). Because all chil- attack component (C5b-9), which may be an important
dren with asymptomatic proteinuria do not come to the mediator of the resulting proteinuria (75,76). A critical
attention of physicians, and because renal biopsies are not threshold of the amount of the antigen-antibody complexes
performed on all patients with asymptomatic proteinuria or and the ensuing complement activation is required for the
even those with NS, the exact prevalence of MN in children development of proteinuria (77). The generated C5b-9 is
is unknown. In the International Study of Kidney Disease in inserted into the cell membrane of the glomerular epithelial
Children, idiopathic MN was found in only 4 of 400 chil- cell and then endocytosed, transported intracellularly, and
dren with NS (58). The rarity of idiopathic MN as a cause of finally excreted into the urinary space and urine (78). How
NS in children is supported by our experience and by others the activation of complement and the formation of C5b-9
who have found that only 2 to 6% of those children with NS produce proteinuria in Heymann nephritis is not estab-
who are studied by renal biopsy have idiopathic MN lished, but reactive oxygen species (79), metalloproteinases
(8,10,16,17,59). In contrast, idiopathic MN is a common (80), phospholipases, and protein kinases (81) appear to play
glomerular disease in adults; it is responsible for 25 to 40% a role. Whether a similar mechanism operates in human
of cases of idiopathic NS (60,61). MN is unclear. However, C5b-9 can be demonstrated in
the human idiopathic MN lesion (14,15) and is also
excreted in the urine (82). In situ subepithelial antigen-
Pathogenesis
antibody deposits in experimental animals can also result
The pathogenesis of idiopathic MN is unknown. Based on from the binding of antibodies to nonglomerular, cationic,
immunofluorescence and ultrastructural findings, it is exogenous antigens implanted experimentally in the lamina
clearly an immunologically mediated disease. The granular rara externa of the GBM (83).
deposits containing IgG, which correspond to the subepi- Circulating immune complexes of appropriate size and
thelial ultrastructural electron-dense deposits, are believed charge, made in vitro by artificially covalently linking an
to represent antigen-antibody complexes. The antigen(s) in exogenous antigen and antibody followed by intravenous
the deposits and the process by which they form, enlarge, infusion into an experimental animal, also localize in the
and partially resolve are not known. Whether the antigen is subepithelial region (84) and produce the subepithelial
exogenous or endogenous, renal or nonrenal, or the same deposits of MN. Whether such a mechanism operates in
or different in various cases of MN is also unclear. Based on humans is unknown, but it appears to be unlikely.
studies of Heymann nephritis in the rat, both the active In summary, human MN is an antibody-mediated dis-
(62) and passive (63,64) types, it is possible that the depos- ease of uncertain and imprecise pathogenesis. However, the
its in idiopathic human MN, like the deposits of Heymann hypotheses that it is an autoimmune disease of the kidney
nephritis, are formed in situ after binding of an autoanti- and that the subepithelial immune deposits are formed in
body to a glomerular antigen; a large (600-kDa) glycopro- situ with an endogenous glomerular antigen are attractive.
tein variously named as gp600/gp330/megalin (65,66)
presents on the surface of the glomerular epithelial cell (67)
Clinical and Laboratory Findings
(see Chapter 39). Recently, the pathogenic region on
gp600/gp330/megalin has been localized to a small area in Clinical features of idiopathic MN at onset are summarized in
the N-terminal of this protein (68). Two case reports, one Table 33.2. MN can occur at any age, including infancy.
in a child (69) and another in an adult (70), and recent data Habib et al. (10) reported two patients of 8 and 10 months of
(71,72) in a small number of patients support this concept age, and the author has seen two infants present at 5 and 11
of pathogenesis. An interesting case supporting the concept months. Sixty percent of the children are male; 40% are
that MN can develop in a human by the binding of anti- female (Table 33.2). The children may have overt features of
 33. Membranous Nephropathy 645

TABLE 33.2. CLINICAL FEATURES OF MEMBRANOUS GLOMERULONEPHRITIS

Southwest
Pediatric
Nephrology
Habib et al. Study Group Latham et Ramirez et Trainin et al. Total
Clinical features (10) (12) al. (11) al. (16) (59) Makker number Percent

No. of cases 50 54 14 22 14 9 163

Boys 38 28 9 11 6 7 99 60
Girls 12 26 5 11 8 2 64 40
Onset
Age (yr) 0.7–15.0 6.0–15.0 3.5–6.0 0.9–20.0 2.0–15.0 0.4–15.0 — —
Asymptomatic pro- 26 8 3 5 3 0 45/162 28
teinuria
Nephrotic syn- 24 45 11 17 11 9 117/162 72
drome
Hypertension 3 16 7 8 2 1 37/163 22
Macroscopic hema- 3 0 0 0 0 0 3/163 2
turia
Microscopic hema- 30/46 30 8 14 14 9 111/159 70
turia
Renal failure 0 — — 1 1 1 2/73 3
Normal C3 11/11 — — 14 14 9 45/47 98
Course
Follow-up (yr) 1.0–10.0 0.2–14.0 1.0–16.0 1.0–11.0 0.8–7.0 1.0–14.0 — —
No treatment 5 20 1 11 3 2 42 26
Treatment
Prednisone + + + + + + — —
Cytotoxic drugs + + + + – + — —
Outcome
Remission 26 13/50 4 7 6 6 62/159 39
Active disease 19 ? 7 9 4 2 41/109 38
Chronic renal failure 5 10/44? 3 6 4 1 29/153 19

+, present; –, not present.

NS (e.g., anasarca) or asymptomatic proteinuria (57); presen- serology for syphilis, negative serologic test for hepatitis B sur-
tation with NS is more common (69%). Although presenta- face antigen (HBsAg), a negative antinuclear antibody test, and
tion with macroscopic hematuria is rare, it has been reported negative tests for poststreptococcal infection]. Lack of prompt
(10); however, microscopic hematuria at presentation is com- response to corticosteroids in a nephrotic patient may also sug-
mon (69%). Presentation with isolated microscopic hematuria gest MN. However, a definitive diagnosis can only be made by
without proteinuria or NS is extremely rare (16). Hyperten- a histologic diagnosis on a renal biopsy specimen.
sion at onset is uncommon (21%). Renal failure at onset is rare
(2.3%), but the author has seen a child present with rapidly Treatment
progressive acute renal failure, whose glomerular histology
showed MN with glomerular crescents. In patients with Treatment consists of immunosuppressive drugs and reno-
edema, laboratory findings of NS (i.e., hypoproteinemia, low protective agents (e.g., angiotensin-converting enzyme
serum albumin concentration, hyperlipidemia, and heavy pro- inhibitors or angiotensin II receptor antagonists) and lipid-
teinuria) are present. Serum complement levels CH50, C3, lowering drugs [e.g., hydroxymethylglutaryl coenzyme A
and C4 are normal in most patients (Table 33.2). reductase inhibitors (statins)]. Although there have been no
reported studies employing renoprotective agents in chil-
dren with MN, there are sufficient data in adults with MN
Diagnosis
(85) and other proteinuric renal diseases that it seems rea-
No specific clinical finding or simple, routinely performed lab- sonable to use angiotensin-converting enzyme inhibitors or
oratory test is diagnostic for MN. The condition should be angiotensin II receptor antagonists in all patients with MN,
suspected when (a) features of a chronic glomerulonephrop- except those with renal failure (86). Lipid-lowering drugs
athy (i.e., proteinuria, hematuria, abnormal urine sediment, may be used in nephrotic patients (87).
with or without hypertension, or renal failure) are present; and There are no controlled clinical trials in children of
(b) the laboratory tests for more common glomerulonephri- treatment with corticosteroids or other immunosuppressive
tides are negative [i.e., normal serum complement, negative drugs. Therefore, no definitive approach to therapy is avail-
646 V. Glomerular Disease

able at this time. However, based on uncontrolled studies followed by daily oral prednisone, 0.4 mg/kg for 27 days.
(8,12,16,17,19,59,88), the following approach appears Prednisone was then discontinued and chlorambucil
rational: Children with asymptomatic proteinuria and given at a dosage of 0.2 mg/kg/day for 1 month. These
without hypertension, renal failure, or NS should be given alternating courses of prednisone and chlorambucil were
angiotensin-converting enzyme inhibitors or angiotensin II continued for 6 months. At 5 years of follow-up, there
receptor antagonists and not treated with corticosteroids or were significantly more remissions in treated than in
other immunosuppressive therapy because most are likely untreated patients (p = .026). There was significant deteri-
to go into remission and do well. oration of renal function in the untreated group (p =
How to treat all other children with idiopathic MN .0002) but not in the treated group (p value was not sig-
remains a difficult decision at present. It is unlikely that nificant). At 10 years of follow-up, these benefits were
sufficient numbers can be collected to conduct a prospec- maintained (93). The same group from Italy performed
tive, randomized, double-blind controlled study because of another controlled trial comparing the same regimen of
the low prevalence of the disease in children. In the absence chlorambucil with steroids or with steroids alone (94).
of such information, decisions have to be based on infor- The authors found that the addition of chlorambucil was
mation available from controlled studies using immuno- associated with more remissions of the NS and preserva-
suppressive drugs performed in adults with idiopathic MN tion of renal function. In another controlled trial of
(Table 33.3). selected patients with progressive MN, cyclosporine (3.5
Alternate-day corticosteroids alone have been used in mg/kg/day for 12 months) was found to be beneficial
three controlled trials (89–91). Two trials compared a (95). Significant reductions of proteinuria (p <.02) and a
100- to 150-mg alternate-day dose of prednisone for 8 slower rate of decline of renal function (p <.02) were
weeks with placebo. In the study from the United States observed during therapy (95). Recently, in a controlled
(89), the treatment was associated with more complete trial of adult steroid-resistant nephrotic patients treated
and partial remissions of the NS. However, the follow-up for 26 weeks with prednisone (0.15 mg/kg/day) plus
period was short, and the rate of deterioration of renal cyclosporine (3.5 mg/kg/day) or prednisone alone, 75%
function was unexpectedly high in the control group. of the former and 22% of the latter groups achieved par-
The other study, from the United Kingdom (90), found tial or complete remission of proteinuria (96).
no significant difference (p <.05) between the untreated The most promising results in the aforementioned studies
and treated groups in plasma creatinine concentration, were obtained in the Italian study using alternating courses
creatinine clearance, or 24-hour protein excretion. The of daily prednisone and chlorambucil. This treatment, or
third study, from Canada (91), using 45 mg/m2 predni- treatment with cyclosporine (95,96), may be tried in chil-
sone for 6 months, concluded that the therapy was of no dren who have persistent NS with or without other features
benefit (p <.2). of severe disease (i.e., renal failure or hypertension). Children
In trials with corticosteroids and other immunosup- generally tolerate corticosteroids well and are unlikely to
pressive agents, a controlled study from Italy (92) used a develop severe side effects on dosages used in the Italian pro-
6-month course of daily corticosteroids alternating with tocol, but there is a possibility of gonadal toxicity and onco-
chlorambucil (0.2 mg/kg/day) as follows: Methylpred- genic potential with chlorambucil and renal toxicity with
nisolone was first given intravenously, 1 g/day for 3 days, cyclosporine. Newer therapies are emerging. In a preliminary

TABLE 33.3. RESULTS OF CONTROLLED TRIALS OF TREATMENT OF IDIOPATHIC MEMBRANOUS

GLOMERULONEPHRITIS IN ADULTS
Study (reference) No. of patients Follow-up (mo) Treatment Conclusion

Collaborative Study Untreated—38 26–52 Prednisone, 125 mg (100–150 mg) “Less deterioration of renal func-
Group (USA) (89) Treated—34 every other d × 8 wk tion in treated patients.”
Cattran et al. (Canada) Untreated—77 48 ± 3 Prednisone, 45 mg/m2 every other “No benefit.”
(90) Treated—81 d × 6 mo
Cameron et al. (UK) Untreated—51 52 ± 6 Prednisone, 125–150 mg every “No significant difference
(91) Treated—52 other d × 8 wk between treated and untreated
groups in plasma creatinine,
creatinine clearance or 24-h
proteinuria.”
Pontelli et al. (Italy) Untreated—39 60 median Methylprednisolone, 1 mg IV × 3 d “Produces sustained remission of
(92) Treated—42 Prednisone, 0.4 mg/kg × 27 d nephrotic syndrome and helps
preserve renal function.”
Chlorambucil, 0.2 mg/kg × 30 d
Three cycles; total: 6 mo
 33. Membranous Nephropathy 647

study, pentoxifylline (1200 mg/day for 6 months), which

suppresses the tumor necrosis factor-α cytokine, reduced
proteinuria in ten adult patients from 4.6 to 27.0 g/day to
0.0 to 10.9 g/day without causing side effects (97).
Patients with NS may develop deep vein thrombosis, par-
ticularly renal vein thrombosis. Whether prophylactic antico-
agulants should be used in these patients remains unsettled.

Clinical Course
The clinical course of idiopathic MN is variable. Some
patients (particularly those with asymptomatic proteinuria)
achieve spontaneous remission, others continue with NS, and
some develop end-stage renal failure. A review of the literature
(10,12,16,59,88) (Table 33.2) shows that after a variable fol-
low-up ranging from 1.0 to 15.8 years, irrespective of treat-
ment, 18% of the children develop chronic renal failure, 41%
achieve remission, and 37% continue to have active disease
but without renal failure. These results, however, need to be
interpreted in light of the uncontrolled and retrospective
nature of the studies and the variable follow-up time, ranging
from 2.0 months to 15.8 years. It is important to note that all FIGURE 33.5. Ultrastructural findings in the transplanted Hey-
mann nephritis rat kidney at weeks 0, 28, and 38 transplanted
29 patients who went to chronic renal failure had NS at onset into a normal syngeneic Lewis rat. At 0 time, the subepithelial
or subsequently during the course. Hypertension at onset is deposits are evident. After 28 weeks, the glomerular basement
associated with a less favorable outcome (Makker SP, unpub- membrane is thickened, the subepithelial deposits have become
lucent, and the subepithelial contour is irregular. At 38 weeks, the
lished observation, 2003) (10,12,16,46,59,88). There is insuf- latter changes persist (×43,000). (Electron microscopy performed
ficient information to determine the frequency of spontaneous by Seymour Rosen, M.D., Harvard Medical School, Boston.)
remission in a child presenting with NS who is not treated or
to determine the relationship between sex, age at onset, and
renal failure at onset and the final outcome.
MEMBRANOUS GLOMERULONEPHROPATHY
ASSOCIATED WITH HEPATITIS B
Resolution
Epidemiology
Idiopathic MN is a chronic disease and, therefore, most
likely involves continuous or multiple generations of Many cases of MN associated with hepatitis B in children have
deposits. Do children achieving complete remission (nor- been reported from several parts of the world, including the
mal urine) stop forming new deposits, completely resolve United States (99), Europe (8,44,46), Africa (41,43), and the
their previous deposits, and normalize their GBMs? If these Orient (20,21,39,100). The largest series of patients have
changes do occur, how long does it take? It is difficult to come from Southeast Asia (20,21), consistent with the higher
answer these questions because sequential renal biopsies prevalence of HBsAg-carrier state. The prevalence of HBsAg
after complete remissions are not often performed. We have carriers is approximately 0.3 to 1.0% in North America, 1.0%
studied the problem in active Heymann nephritis of the rat in western Europe, 7.0% in Africa, and 10.0% in Southeast
(98). Proteinuric MN kidneys were transplanted into nor- Asia, where it is endemic (101). For instance, in Taiwan, 95%
mal, unilaterally nephrectomized syngeneic Lewis rats to of all MN, excluding SLE, is due to hepatitis B (21). In these
follow the course of the resolution of established MN in a areas, vertical transmission from infected mothers to their chil-
normal host, thus preventing any new generation of depos- dren and horizontal transmission between siblings is likely
its. Sequential renal biopsies were performed over time because 33 to 36% of siblings of patients show a positive blood
(Fig. 33.5). We found that the resolution of the process was test for HBsAg (21). Transmission in adolescents through drug
very slow. At 38 weeks, the deposits were still positively abuse or sex is also possible (102).
stained for rat IgG by immunofluorescence, and electron
microscopy showed thickened lamina densa, irregular sub-
Clinical Features
epithelial contour, and lucent deposits in the lamina densa
of GBM. This suggests that if a comparable pace of resolu- Children with MN associated with hepatitis B, like the children
tion occurs in humans, complete resolution in children will with idiopathic MN, usually have NS or are detected during
take several years, even after the cessation of new deposits. evaluation of persistent proteinuria (8,20,21,40–46,99,100).
648 V. Glomerular Disease

Microscopic hematuria is common, and even macroscopic noted in South African children suggests a role for genetic
hematuria has occasionally been reported. Rarely, a patient may factors (104). The presence of IgG in the subepithelial
have only microscopic hematuria without proteinuria (20). In deposits shows that these deposits contain immune com-
almost all large series, boys predominate overwhelmingly (75 to plexes. How the deposits are formed is unclear. Two hypoth-
80%) (8,20,21), as opposed to idiopathic MN, in which boys eses have been considered. According to the first, circulating
and girls are affected in approximately equal numbers (Table immune complexes consisting of antigen(s) of hepatitis virus
33.2). Age at onset in large series has varied from 2 to 16 years. and their corresponding antibodies (elicited as the result of
Retrospective analysis of symptomatology at onset reveals that chronic infection) are trapped in the glomerulus and come to
patients start with flu-like illness consisting of low-grade fever, lie in the subepithelial space during the formation of glomer-
poor appetite, nausea, vomiting, and malaise, but usually no ular ultrafiltrate. According to the second hypothesis, certain
jaundice. After these symptoms subside, hematuria and pro- freely circulating hepatitis B virus antigens pass through the
teinuria are noted (20). However, most come to attention GBM during the process of ultrafiltration and are implanted
because of the onset of edema as the result of MN, and evi- in the subepithelial space. Theoretically, cationic antigens are
dence for hepatitis is discovered only during evaluation. The more likely to pass through the GBM than anionic ones
liver may be enlarged in some patients at onset. because of the anionic nature of the GBM. Once implanted,
Laboratory tests on serum for a profile of hepatitis B anti- the deposits form by the binding of antibodies crossing the
gen and antibodies show a positive result for HBsAg in all GBM from the circulation.
patients, usually a negative result for hepatitis B antibody, and To prove either hypothesis, hepatitis antigen(s) must be
a positive result for hepatitis B early antigen (HBeAg) in more demonstrated in the glomerular immune deposits. Indeed,
than 90% of patients (20,21). Serum levels of complement hepatitis B antigens have been demonstrated in these
components C3 and C4 are usually low (21) at onset, but they deposits by immunofluorescence microscopy. Since the first
may return to normal at some time during the course of the study by Combes et al. (105), HBsAg, HBeAg, and HBcAg
disease. Serum levels of liver transaminase enzymes may be ele- have been detected by many investigators, but the findings
vated on presentation and, in some cases, persist chronically have been inconsistent (41). Part of the explanation for the
(8,20,40,44). Liver biopsies usually show chronic hepatitis inconsistent results is the questionable specificity of the
(8,20). However, the long-term course of the liver disease older reagents used in the earlier studies. Recent studies, as
remains to be studied. Hepatitis B–associated MN is suspected alluded to earlier, have used Fab2 portions of the highly spe-
in a child with features of a glomerular disease (i.e., pro- cific monoclonal antibodies (20,21,103) and have found
teinuria, hematuria, or NS), who during laboratory evaluation that only HBeAg (and not the other antigens) is present in
is found to have a positive test result for HBsAg in serum, and the glomerular immune deposits, suggesting the impor-
the diagnosis is established by renal biopsy. If serum comple- tance of HBeAg in pathogenesis. The molecular weight of
ment C3 levels are low, it is an additional piece of evidence in free HBeAg in circulation is approximately 100,000 dal-
favor of hepatitis B–associated MN versus idiopathic MN. On tons, and its isoelectric point is 4.8 (anionic). These physico-
the basis of clinical manifestations alone at the time of presen- chemical characteristics of HBeAg probably do not favor
tation, the two conditions may be difficult to differentiate, and the in situ hypothesis.
a high index of suspicion is necessary. To substantiate the hypothetical circulating immune
complex, complexes of the same antigen antibody specific-
ity as those detected in the glomerular immune deposits
Pathology
and of a size and charge that would localize in subepithelial
The pathology of MN associated with hepatitis B is similar space must be demonstrated in circulation. The available
to that of idiopathic MN and is indistinguishable in most information in this regard is limited and discordant (45).
cases. The two conditions may be differentiated on the basis Takekoshi et al. (106) have demonstrated circulating
of demonstration of HBeAg in the glomerular immune immune complexes of HBeAg and antibody of an approxi-
deposits by immunofluorescence microscopy. When Fab2 mate size greater than IgG in the serum of two children
portions of monoclonal antibodies to HBsAg, hepatitis B they studied. However, the problem is complicated by the
core antigen (HBcAg), and HBeAg are used for detection, fact that circulating immune complexes of hepatitis B anti-
HBeAg is present in most (90% or greater) patients with gens and antibody are present in patients with chronic hep-
hepatitis-associated MN, but it is not seen in idiopathic MN atitis B infection without MN.
(20,21,103). HBsAg and HBcAg were not detected in hepatitis-
associated MN in these studies.
Treatment and Course
The course of hepatitis B–associated MN is variable, ranging
Pathogenesis
from spontaneous recovery in some (106) to end-stage renal
The exact pathogenesis of hepatitis B–associated MN is failure in others (42,107). Retrospective analysis of six studies
unknown, but a high frequency of HLA allele DQB1*0603 (20,21,42,107–109) comprising a total of 82 children, most of
 33. Membranous Nephropathy 649

whom had been treated with corticosteroids, showed that from the histology observed in idiopathic MN. In a large
within 12 months after diagnosis, nearly 60% were in remis- series of children from the United States who had renal
sion. The remainder had persistent disease, with 7.3% having involvement secondary to SLE, MN was present in nearly
chronic renal failure and 2.4% end-stage renal failure (103). In 10% (113). In another study from the United States, 10 of
one large study of 52 patients from Taiwan (20), 38 of whom 64 patients evaluated for MN had SLE as the underlying
were treated with corticosteroids, 64% of the children were in cause (12). However, in a study from France (18), only 2 of
remission at 1 year and 92% at 7 years after follow-up, and 65 children with MN had SLE.
only one patient was in renal failure. Four children who Children with MN associated with SLE commonly have
received no treatment were in remission at 2 years. In another features of a glomerular disease associated with other extra-
series of 70 patients from South Africa (110), 60 of whom renal manifestations of SLE. These usually include hema-
were untreated, it was reported that the cumulative probability turia and proteinuria, with or without NS and with or
of remission was 64% at 4 years and 84% at 10 years; after 90 without hypertension or renal failure. However, patients
months of follow-up, three patients were still nephrotic, and may have only a glomerular disease and no other manifesta-
two had developed end-stage renal failure. The average dura- tions of SLE. Laboratory tests on serum for SLE are usually
tion of proteinuria in these patients was 30 months, with positive (i.e., antinuclear antibodies, anti–double-stranded
remission of proteinuria usually occurring 6 months after DNA antibodies, and low serum complement C3 and C4).
clearance of HBeAg from the serum. Overall, these two studies However, occasionally all clinical manifestations may be
suggest that although there can be a prolonged period of mor- absent, and the laboratory tests for SLE may be negative at
bidity, the eventual outcome in most of the children is favor- presentation and become positive only later; in some cases,
able. On the other hand, in another study from Taiwan (20) of this may be years. Patients with inherited deficiency of the
32 patients treated with corticosteroids, 70.6% had persistent second component of complement are prone to develop
disease and 12.3% progressive disease after 2 years of follow- SLE, and these patients may present initially with MN and
up, and HBsAg in serum remained positive in most cases later develop SLE. Renal biopsy findings of SLE-associated
throughout the course of the disease, even among some who MN and idiopathic MN may be identical. However, pres-
were in clinical remission (20,21). However, children whose ence of mesangial deposits and strong positive staining of
serum became negative for HBsAg were more likely to have the deposits for C1q and C4, as strong as the staining for
remission than those whose serum remained positive for C3, are suspicious of SLE. The treatment of MN associated
HBsAg (20). The treatment for hepatitis-associated MN is with SLE is similar to the treatment of other SLE patients
evolving, but at present, there is no optimal and accepted treat- with renal involvement, including the use of corticosteroids
ment for hepatitis-associated MN in children. Corticosteroids and cytotoxic drugs.
do not appear to be beneficial and probably should not be
used because they may enhance viral replication in mononu-
clear cells (111). Recently, recombinant human interferon-α MEMBRANOUS NEPHROPATHY
(subcutaneous injections three times a week) has been reported IN RENAL ALLOGRAFTS
to be beneficial (112). In this study, two groups of 20 patients
each with persistent proteinuria and NS who had previously MN in a renal allograft may develop de novo or as a result of
not shown a response to corticosteroids were either treated the recurrence of the original idiopathic MN. The recur-
with recombinant human interferon-α for 1 year or given only rence rate in adults has been reported to vary from 26.3
supportive treatment. At the end of the study, all (20 of 20) (114) to 50.0% (115). Because of the paucity of cases, the
treated patients were free of proteinuria; however, in the recurrence rate in children is unknown. Several cases of de
untreated patients, two were also free of proteinuria, 12 had novo MN in the transplanted kidney have been reported
mild proteinuria, and only six had persistent heavy pro- (37). In children, the incidence of de novo occurrence of
teinuria. Thus, without treatment, 70% of the patients showed MN in transplanted kidneys is estimated to be approxi-
improvement in proteinuria. All treated patients had flu-like mately 1 to 2% (36,116), but in one large series from
symptoms in the first 2 weeks of therapy; later, 30% of the France, 9.3% of the renal transplants in children developed
patients developed psychiatric problems. Longer follow-up of de novo MN (36). Part of the reason for the higher inci-
treated and untreated patients is needed to draw conclusions dence in this report may be the greater frequency of routine
regarding the recommendation of this therapy. renal transplant biopsies. MN was discovered in nine
patients by the latter approach, with none of them having
proteinuria, suggesting that the actual incidence of the con-
MEMBRANOUS NEPHROPATHY ASSOCIATED dition is higher. In other patients, it is discovered during
WITH SYSTEMIC LUPUS ERYTHEMATOSUS evaluation of proteinuria, NS, or a suspected rejection. The
pathogenesis of de novo MN is not understood. However,
The histopathology of the renal lesion in some patients there is a high rate of recurrence of the de novo MN in the
with SLE can have an appearance that is indistinguishable second allograft, suggesting that host factors play a role
650 V. Glomerular Disease

(36). No correlation between the development of de novo MEMBRANOUS NEPHROPATHY

MN and age or sex of the patient, the original disease pro- ASSOCIATED WITH TUMORS
ducing end-stage renal failure, and the time between the
transplant and the onset of MN has been observed. How- MN has been associated with a number of malignancies in
ever, ureteral obstruction (116,117) and a good histocom- adults (6,122)—the most common being bronchogenic
patibility match (118,119) could be possible predisposing carcinoma, gastric carcinoma, and adenocarcinoma of the
factors. The course of de novo MN is variable. Some patients colon. A few cases have also been reported in children
never even develop proteinuria, whereas others develop NS; (Table 33.1). In two cases reported in children (33,34), res-
in some, it may contribute to the deterioration of renal olution of MN was observed after excision of the tumor.
function in association with chronic rejection. In general,
the demise of the graft is more often caused by the rejection
process than the de novo MN. MEMBRANOUS NEPHROPATHY ASSOCIATED
WITH OTHER DISORDERS

MEMBRANOUS NEPHROPATHY ASSOCIATED In addition to SLE, MN has been reported with a number
WITH OTHER INFECTIONS of other autoimmune and immunologic diseases. Cases in
children have been reported with enteropathy (27), Crohn’s
MN may be associated with a number of other infections disease (28,29), pemphigus (30), multiple sclerosis (31),
(Table 33.1). Congenital syphilis should be considered in immune thrombocytopenic purpura (123), and sarcoidosis
the differential diagnosis in any infant presenting with NS. (124). In a recent report of MN associated with enteropa-
Infants with congenital syphilis also often have other clini- thy (27), autoantibodies to a 55-kDa epithelial cell protein
cal features of congenital syphilis, and most have micro- common to intestine and kidney were detected in the
scopic hematuria. The diagnosis can be confirmed by a serum of the child and were temporally related to the devel-
positive serologic test result for syphilis. Complement levels opment of enteropathy and MN.
of CH50, C3, and C4 are low at onset and return to nor- A distinct entity consisting of antitubular basement
mal after treatment with penicillin (21). Renal biopsy find- membrane–associated tubulointerstitial nephritis, Fanconi’s
ings are similar to those of idiopathic MN, except that syndrome, and MN has been recognized (26). All of the
staining to C1q is commonly present (22). Treponema reported children except for one with this disease have been
antigen has been demonstrated in the glomerular immune boys. The tubular autoantigen is a 58-kDa protein that is
deposits (120). Early treatment with penicillin leads to located in the basement membrane of the proximal tubule
complete recovery. This emphasizes that the physician (125). The glomerular autoantigen is a 600-kDa protein
needs to be aware that (a) a potentially curable condition (gp600/gp330/megalin/LRP II) (26). Isolated cases of MN
can be the cause of NS in infants, and (b) once the offend- are associated with a number of other conditions in chil-
ing agent has been eradicated, MN can resolve. dren (Table 33.1).
Other parasitic infections, such as malaria, filaria, and
hydatid disease, can produce MN and should be considered
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 34

CRESCENTIC GLOMERULONEPHRITIS
MICHAEL J. DILLON

Crescentic glomerulonephritis is the histopathologic corre- changes in the underlying glomerular tuft. In addition,
late of the clinically defined condition of rapidly progressive crescents were divided into cellular, fibrocellular, or fibrous
glomerulonephritis (RPGN) (1). It is relatively rare in child- categories according to their histopathologic appearances
hood (2–9) and is characterized by the presence of extensive and were further subcategorized according to other criteria,
crescents demonstrable on renal biopsy and by sudden and including changes in the glomerular tuft, immunochemical
progressive decline in renal function. It can accompany most staining, and clinicopathologic correlations.
forms of primary glomerulonephritis in childhood and can
be associated with various systemic disorders [e.g., systemic
lupus erythematosus (SLE)] and several forms of systemic INCIDENCE
vasculitis (2,3,7,8). The severity of the condition, the multi-
ple etiologies, and the well-recognized poor prognosis have The real incidence of crescentic glomerulonephritis in chil-
resulted in the use of multiple treatment regimens, the effi- dren is unknown. It comprises 2 to 5% of all cases of glomer-
cacy of which have been difficult to assess (10), and there has ulonephritis in adults (13); Andrassy et al. (14) calculated the
been a lack of clarity in identifying variables with therapeutic annual incidence in Germany to be 0.7 per 100,000 adults.
and prognostic implications. Miller et al. (15) reported crescentic lesions in 56 of 372 chil-
dren with glomerular pathology on renal biopsy; in 26, there
was a clinical course compatible with RPGN. These data are
DEFINITION not dissimilar to those quoted for adults, emphasizing the
relative rarity of the condition in children.
There are a number of definitions of crescentic glomerulonephri-
tis, and this varied nomenclature has created problems in data
interpretation and comparing outcomes of treated patients. CAUSES
There is general agreement that crescentic glomerulonephritis
is defined by the presence of large epithelial crescents within There are a number of ways of considering the causes of cres-
Bowman’s space, but the appearance of the crescents and the centic glomerulonephritis. There are classifications that con-
number required for the diagnosis are arguable. In childhood, sider the immunofluorescence staining patterns on renal
more than 75% (3), 50% or more (2,7), and occasionally as biopsy and subdivide disorders according to the appearances
few as 20% of glomeruli affected have been considered to rep- into (a) anti–glomerular basement membrane (GBM) anti-
resent crescentic glomerulonephritis (5). In a series described body disease; (b) no staining or pauci-immune disease; and
by Neild et al. (11) of 39 patients with RPGN and extensive (c) immune complex disease (13,16). Using these criteria,
crescent formation, ten patients younger than 14 years of age the most frequently observed pattern in childhood (in con-
had greater than 60% of glomeruli affected. However, in trast to adults) is immune complex disease, constituting 80%
another series of 13 children with RPGN, Cunningham et al. of cases, with pauci-immune and anti-GBM disease making
(4) reported the number of glomeruli affected as varying from up the remainder in a ratio of 2:1 (2,7,13).
10 to 100%. In other series with undoubtedly crescentic glo- Other classifications consider RPGN or crescentic glomer-
merulonephritis, the clinical factors have been emphasized ulonephritis under headings of disease category (e.g., anti-
irrespective of the number of crescents (12). GBM disease), primary systemic vasculitis, other disorders
Jardim et al. (7) considered that crescentic glomerulone- (e.g., SLE), primary glomerulonephritis, infection-associated
phritis was present when large epithelial crescents, filling glomerulonephritis, and miscellaneous causes (Table 34.1).
Bowman’s space, occurred in 50% or more of glomeruli In four large series of childhood patients with crescentic
present in a renal biopsy specimen, irrespective of any glomerulonephritis reported from the United States, France,
656 V. Glomerular Disease

TABLE 34.1. CAUSES OF CRESCENTIC the United Kingdom, and India, the pattern of disease was
GLOMERULONEPHRITIS IN CHILDHOOD similar but there was variation, as might be expected,
Anti-GBM antibody disease between centers (2,3,7,8).
Anti-GBM nephritis, Goodpasture’s syndrome, with perinu- The Southwest Pediatric Nephrology Study Group (2)
clear antineutrophil cytoplasmic antibody–associated reported on 50 children with histologically 50% or more glo-
microscopic polyangiopathy, posttransplantation in Alport’s meruli affected by crescents. Thirteen had nonspecified
syndrome
Primary systemic vasculitis
immune complex disease, nine had SLE, seven had idiopathic
Henoch-Schönlein purpura, microscopic polyarteritis or poly- (nonimmune complex) crescentic glomerulonephritis, six had
angiopathy, idiopathic crescentic nephritis, Wegener’s poststreptococcal glomerulonephritis, four had immunoglob-
granulomatosis, Churg-Strauss syndrome ulin (Ig) A nephropathy, three had Henoch-Schönlein pur-
Systemic disorders pura, three had vasculitis, three had possible anti-GBM
SLE, Behçet’s syndrome, Weber-Christian disease, relapsing
polychondritis, essential mixed cryoglobulinemia, mixed
disease, and two had dense deposit disease (Table 34.2).
connective tissue disease, dermatomyositis, juvenile rheu- Niaudet and Levy (3) reported the findings in 41 chil-
matoid arthritis, Sjögren’s syndrome, sarcoidosis dren with more than 75% crescents on biopsy. Eleven had
Primary glomerulonephritis Henoch-Schönlein purpura, nine had membranoprolifera-
Mesangiocapillary glomerulonephritis, IgA nephropathy, tive glomerulonephritis, five had acute glomerulonephritis,
membranous nephropathy
Infection-related glomerulonephritis
three had anti-GBM disease, three had IgA nephropathy,
Poststreptococcal glomerulonephritis, legionella, myco- three had polyarteritis, three had glomerulonephritis with-
plasma, syphilis, hepatitis B and C, tuberculosis, human out evidence of deposits, two had immune complex glo-
immunodeficiency virus, bacterial endocarditis, shunt merulonephritis, one had SLE, and one had shunt nephritis
nephritis, leprosy, visceral abscess (Table 34.2).
After medication
Penicillamine, rifampicin and hydralazine, enalapril, interleu-
Jardim et al. (7) described 30 children with 50% or
kin-2, interferon-α, phenylbutazone, propylthiouracil, iso- more crescents on biopsy. Of these, nine had Henoch-
niazid Schönlein purpura, seven had mesangiocapillary glomer-
Miscellaneous causes ulonephritis (type I in six and type III in one), five had
Malignant tumors, leukemia and lymphomas, silicosis, alpha- vasculitis (three had microscopic polyarteritis, one had pol-
1-antitrypsin deficiency, hyper-IgD syndrome
After transplantation
yarteritis nodosa, and one had Wegener’s granulomatosis),
Anti-GBM disease in Alport’s syndrome (mentioned above), four had idiopathic crescentic nephritis, two had poststrep-
recurrence of IgA nephropathy and mesangiocapillary tococcal glomerulonephritis, two had anti-GBM disease,
glomerulonephritis, anti-GBM, Henoch-Schönlein pur- and one had SLE (Table 34.2).
pura, SLE Srivastava et al. (8) reported 43 children with crescents
GBM, glomerular basement membrane; Ig, immunoglobulin; SLE,
in more than 50% of glomeruli. Of these, 11 had post-
systemic lupus erythematosus. streptococcal glomerulonephritis; six had an underlying

TABLE 34.2. CAUSES OF CRESCENTIC GLOMERULONEPHRITIS IN CHILDREN (PERCENTAGE)

Niaudet and Jardim et al.c Srivastava et al.d
SPNSGa (N = 50) Levyb (N = 41) (N = 30) (N = 43)

Nonspecified immune complex disease 26 4.8 — —

Systemic lupus erythematosus 18 2.4 3.3 2.3
Idiopathic crescentic glomerulonephritis 14 7.3 13.3 60.4
Poststreptococcal glomerulonephritis 12 12.1 6.6 25.5
Immunoglobulin A nephropathy 8 7.3 — —
Henoch-Schönlein purpura 6 26.8 30.0 6.9
Vasculitis 6 7.3 16.6 —
Anti–glomerular basement membrane disease 6 7.3 6.6 2.3
Mesangiocapillary glomerulonephritis 4 21.9 23.3 —
Shunt nephritis — 2.4 — —
Juvenile chronic arthritis — — — 2.3

N, number of patients.
aData from SPNSG (Southwest Pediatric Nephrology Study Group). A clinico-pathological study of crescentic glomerulonephritis in 50 children.

Kidney Int 1985;27:450–458.

bData from Niaudet P, Levy M. Glomerulonéphritis à croissants diffus. In: Royer P, Habib R, Mathieu H, Broyer M, eds. Néphrologie pédiatrique,

3rd ed. Paris: Flammarion, 1983:381–394.

cData from Jardim HMPF, Leake J, Risdon RA, et al. Crescentic glomerulonephritis in children. Pediatr Nephrol 1992;6:231–235.
dData from Srivastava RN, Moudgil A, Bagga A, et al. Crescentic glomerulonephritis in children: a review of 43 cases. Am J Nephrol 1992;12:

155–161.
 34. Crescentic Glomerulonephritis 657

TABLE 34.3. CLINICAL FEATURES OF CRESCENTIC

GLOMERULONEPHRITIS IN CHILDREN
Symptom Incidence (%)

Hematuria (macroscopic) 50–90

Proteinuria 72–100
Oliguria 4–100
Edema 13–90
Anemia ~70
Hypertension 17–85
Renal impairment 100

Data from references 2–4, 7, and 8.

systemic disorder consisting of Henoch-Schönlein purpura

in three, SLE in one, anti-GBM disease in one, and chronic FIGURE 34.1. Cytoplasmic staining antineutrophil cytoplasmic
rheumatoid arthritis in one; and in 26, the condition was antibody pattern in a standard indirect immunofluorescence assay.
classified as idiopathic (Table 34.2).

more impaired than might have been deduced from ran-

CLINICAL PRESENTATION dom plasma creatinine values. Anemia, which is frequently
present, is more marked than anticipated (13) and is usu-
Most children with crescentic glomerulonephritis have an ally normochromic and normocytic.
acute nephritis presentation with proteinuria, hematuria, Serologic abnormalities vary according to the underlying
volume overload, hypertension, and renal impairment. This disease state. Hypocomplementemia is not uncommon in
may evolve quickly and initially give the impression of a immune complex disease and SLE. Circulating anti-GBM
postinfectious glomerulonephritis but can be associated antibodies would be expected in anti-GBM disease (17); if this
with a more protracted clinical course that can be deceptive condition is suspected, it is essential to have a rapid result from
and may be the cause of a delay in diagnosis and introduc- the laboratory, as initiation of early treatment is critically
tion of appropriate treatment. In patients in whom the important. Circulating anti-GBM IgG antibodies can be dem-
crescentic glomerulonephritis is secondary to or associated onstrated by indirect immunofluorescence or enzyme immu-
with a systemic disorder (e.g., lupus or vasculitis), clinical noassay. Enzyme immunoassay is more sensitive and specific as
features of this may be present and helpful diagnostically. compared to indirect immunofluorescence.
Pulmonary hemorrhage might suggest anti-GBM disease or Raised anti–streptolysin O levels with or without raised
vasculitis. However, in childhood, anti-GBM glomerulone- anti–deoxyribonuclease B are features of poststreptococcal
phritis often occurs without lung involvement, and pulmo- glomerulonephritis, but they can be increased coinciden-
nary hemorrhage is the exception rather than the rule. tally in other forms of crescentic glomerulonephritis and
In the series of 30 patients reviewed by Jardim et al. (7), may, therefore, not be diagnostically helpful. Antinuclear
all had hematuria (macroscopic in 15); all had proteinuria
(nephrotic syndrome in 14); edema was present at the onset
in 24; hypertension was present in 9; oliguria requiring dialy-
sis for fluid control occurred in 15; and the glomerular filtra-
tion rate (GFR) was less than 30 mL/min/1.73 m2 at
presentation in 22 patients and between 30 and 60 mL/min
per 1.73 m2 in the remaining 8. These data are very similar
to those recorded in other reports in the literature (2–4).
However, it is important to emphasize that there is a fairly
wide spectrum of presenting clinical features (Table 34.3).

LABORATORY FINDINGS

Laboratory investigations substantiate the clinical impres-

sion of renal impairment as well as confirming the presence
FIGURE 34.2. Perinuclear staining antineutrophil cytoplasmic
of hematuria and proteinuria. The degree of renal failure antibody pattern in a standard indirect immunofluorescence
can be deceptive with GFR findings, often substantially assay.
658 V. Glomerular Disease

FIGURE 34.3. Cellular crescent. FIGURE 34.5. Fibrous crescent.

and anti–double-stranded DNA antibodies point to a diag- tis. ANCA-positive crescentic glomerulonephritis is also
nosis of SLE nephritis. associated with propyl thiouracil treatment in children (23).
Elevated levels of antineutrophil cytoplasmic antibodies
(ANCAs) may reflect an underlying vasculitic cause, such as
Wegener’s granulomatosis or microscopic polyarteritis (or HISTOPATHOLOGY
polyangiopathy) (18,19). Two neutrophil primary-granule
enzymes are the targets of the majority of ANCAs in sys- Controversy exists as to how many glomeruli have to be associ-
temic vasculitis. ANCAs directed against proteinase-3 are ated with crescents to designate the disease as crescentic glomer-
strongly associated with Wegener’s granulomatosis and pro- ulonephritis. As mentioned previously, authors vary in their
duce a cytoplasmic staining pattern in a standard indirect views, and more than 75% (3), 50% or more (2,7,8), and
immunofluorescence assay (19,20) (Fig. 34.1). ANCA occasionally even 20% of glomeruli affected have been consid-
directed against myeloperoxidase, producing a perinuclear ered to represent crescentic glomerulonephritis (5). There is, in
staining pattern, is often seen in microscopic polyangiopa- addition, a need to characterize the nature of the crescents, and
thy, but perinuclear ANCA may also be directed against three categories are recognized. Cellular crescents are those in
other epitopes and is seen in other nonvasculitic conditions which there is prominent proliferation of epithelial cells, with
(19,21) (Fig. 34.2). The presence of circulating ANCA in a some admixture of macrophages and occasionally neutrophils
child with crescentic glomerulonephritis might be helpful filling the urinary space and compressing the tuft (Fig. 34.3).
diagnostically with a cytoplasmic pattern suggestive of Fibrocellular crescents are those in which strands of membrane-
Wegener’s granulomatosis and a perinuclear pattern sugges- like material and collagen fibers are present among the cells
tive of microscopic polyangiopathy (22). In children with forming the crescent (Fig. 34.4). Fibrous crescents are those in
idiopathic disease, which is considered probably to be a which the cells of the crescent have virtually all disappeared to
form of microscopic polyarteritis, such a positive finding be replaced by collagen (7) (Fig. 34.5).
can be helpful, but negative results do not exclude Wegener’s The nature of the underlying disease causing the crescentic
granulomatosis, nor do they exclude microscopic polyangii- glomerulonephritis is based on various criteria: (a) the

FIGURE 34.4. Fibrocellular crescent. FIGURE 34.6. Mesangiocapillary glomerulonephritis.

 34. Crescentic Glomerulonephritis 659

irrecoverable renal impairment. This process leading to

glomerulosclerosis is similar in most causes of crescentic
glomerulonephritis, and at a late stage, it is usually not pos-
sible to distinguish the etiology from histopathologic
examination.

IMMUNOPATHOGENETIC MECHANISMS

In severe inflammatory glomerular injury, whatever the ini-

tial insult or injury, crescent formation may be the final
pathway. A crescent is a proliferation of extracapillary cells
within Bowman’s space. There is evidence that crescent for-
mation is initiated by cytokine-driven proliferation of the
FIGURE 34.7. Anti–glomerular basement membrane glomeru-
lonephritis with linear immunoglobulin G demonstrable by parietal glomerular epithelial cells (24–26). Associated with
immunochemical staining. this, localized breaks in the GBM of Bowman’s capsule
(27–29), mediated by activated leukocytes, are followed by
macrophage infiltration into Bowman’s space with local
associated changes in the glomerular tuft, such as those of fibrin formation (30). Current evidence suggests that cres-
mesangiocapillary glomerulonephritis (Fig. 34.6); (b) the cent formation is a feature of cell-mediated rather than
immunochemical staining, such as the presence of linear IgG humoral immune mechanisms (31).
staining along the glomerular capillary walls in anti-GBM glo-
merulonephritis (Fig. 34.7); and (c) clinicopathologic factors,
such as in polyarteritis nodosa, Wegener’s granulomatosis, and TREATMENT AND OUTCOME
microscopic polyarteritis (7). However, the identification of the
underlying glomerulonephritis is often difficult, but immuno- The heterogeneity and poor outcome of childhood crescen-
fluorescence studies can prove helpful in, for example, mesan- tic glomerulonephritis has led to multiple treatment regi-
giocapillary glomerulonephritis, anti-GBM disease, IgA mens. Steroids (oral or high-dose intravenous), cytotoxic
nephropathy, and Henoch-Schönlein purpura (Fig. 34.8). agents, anticoagulants, antiplatelet agents, and plasma
It is clear that the number of crescents is not necessarily exchange, alone or in different combinations, have all been
the most important factor in terms of subsequent outcome. described (2,3,7,8,10,11,32) (Table 34.4).
This is much more closely related to the nature of the cres- High-dose intravenous methylprednisolone, often recom-
cents, with fibrous crescents having a worse prognosis than mended in adults (33), has been used in children with cres-
cellular or fibrocellular crescents (7). It is also clear that the centic glomerulonephritis by Ferraris et al. (32), coupled with
histopathologic appearances of the crescents can change cyclophosphamide, with long-term renal recovery in five chil-
quite rapidly from cellular to fibrous, especially if thera- dren after a mean follow-up of 35 months. Methylpredniso-
peutic intervention is delayed. The implication from this lone has also been used alone in 29 children with severe
evolution is that the underlying glomeruli became seriously proliferative glomerulonephritis (12), with a favorable out-
compromised with progression to glomerulosclerosis and come (GFR greater than 80 mL/min/1.73 m2) observed in
18 (62%) after a mean follow-up of 35 months.
Combined immunosuppressive and anticoagulant ther-
apy as reported by the Southwest Pediatric Nephrology
Study Group (2) resulted in 24 of 47 (52%) patients recov-
ering renal function. The importance of anticoagulants in
this regimen has been emphasized by Cunningham et al.
(4) who reported recovery of renal function in 7 of 13
(54%) patients with crescentic glomerulonephritis. A simi-
lar regime used by Niaudet and Levy (3) was associated
with renal recovery of varying degree in 19 of 41 (46.3%)
patients (Table 34.5).
Treatment regimens used in the study of Jardim et al. (7)
included corticosteroids, cyclophosphamide, azathioprine,
anticoagulant and antiplatelet agents, and plasma exchange
FIGURE 34.8. Henoch-Schönlein purpura mesangial deposition in varying combinations. Despite these intensive therapeutic
of immunoglobulin A demonstrable by immunochemical staining. approaches, approximately 50% of patients eventually pro-
660 V. Glomerular Disease

TABLE 34.4. TREATMENT OPTIONS FOR CRESCENTIC led to its use in other disorders (e.g., immune complex–
GLOMERULONEPHRITIS IN CHILDHOOD mediated RPGN) (34). The literature remains confusing in
Induction terms of the benefits of plasma exchange in RPGN from
Prednisolone 2 mg/kg/d PO other causes, with some showing benefit (35) and others no
If fulminant disease, methyl prednisolone, 600 mg/m 2/d × 3 IV, effect (36). Pusey et al. (37) demonstrated that dialysis-
followed by PO prednisolone in above dose dependent patients were more likely to recover renal func-
Cyclophosphamide, 2 mg/kg/d PO (for 2–3 mos), or pulsed
cyclophosphamide, 500–750 mg/m 2/dose IV every 3–4 wks
tion if treated with plasma exchange. Jardim et al. (7)
×6 treated 24 children by plasma exchange; although statisti-
If fulminant or unresponsive disease or if anti-GBM disease, cally there was no significant difference between patients
plasma exchange, two volume exchanges daily × 5–10 d who were and who were not exchanged, nine of the ten
Antiplatelet therapy ± anticoagulation patients with a GFR of greater than 30 mL/min per 1.73
Treatment for underlying disease (often covered by above)
with antimicrobial therapy, removal of offending drugs,
m2 at latest follow-up received this treatment. However, the
and treatment of malignant disease majority of the patients who progressed to renal failure
Maintenance despite plasma exchange had fibrous crescents on presenta-
Alternate-day low-dose prednisolone (tapering from high tion. In the Niaudet and Levy report (3), plasma exchange
induction dose) was used in seven of 41 patients, and only the two patients
Azathioprine, 2 mg/kg/d, at least until remission maintained
for >1 yr and in practice for approximately 18 mo
not requiring dialysis on presentation recovered renal func-
Cyclosporin, methotrexate, and mycophenolate mofetil as tion. This emphasizes that the need for dialysis at onset is a
alternative maintenance drugs if azathioprine incapable of predictor of worse outcome as has been shown in several
controlling the disease series (3,7,11). Gianviti et al. (38) also showed that the
Refractory disease addition of plasma exchange to other intensive therapy in
Humanized monoclonal antibodies [e.g., anti-CD4 and anti-CD52
or anti–tumor necrosis factor agents (e.g., infliximab)]
patients with RPGN had beneficial effects.
Supportive therapy Evidence-based data on treatment are very limited and
Dialysis, diuretics, antihypertensives, and so forth unavailable in children. However, for adults, an analysis of
evidence-based recommendations was published by Jindal
in 1999 (39). In this report, early aggressive therapy was
gressed to end-stage renal failure. Four children on long-term recommended despite weak supporting evidence because of
follow-up had GFRs greater than 80 mL/min per 1.73 m2, the high risk of end-stage renal disease. Treatment for anti-
six had GFRs of 30 to 80 mL/min per 1.73 m2, and three GBM antibody-induced crescentic glomerulonephritis
had GFRs of less than 30 mL/min per 1.73 m2. Patients with should be initiated early and should include pulsed methyl-
fibrous crescents did less well, and there was evidence to sup- prednisolone, a 2-week course of plasmapheresis, and 2
port the view that the longer the interval between disease months’ treatment with corticosteroids and cyclophospha-
onset and commencement of treatment, the worse the prog- mide. Treatment for pauci-immune crescentic glomerulo-
nosis. All patients whose treatment was delayed to beyond 3 nephritis should be pulsed methylprednisolone, followed
months after disease onset went into end-stage renal failure; by oral corticosteroids and cyclophosphamide for 6 to 12
those treated 1 to 3 months after onset had variable results months. Recurrences can be managed similarly along with
(two out of five patients recovered renal function); and the appropriate supportive therapy.
12 patients treated in the first month after onset responded In terms of prognosis, it is accepted that poststreptococcal
well (7) (Table 34.5). crescentic glomerulonephritis has a better prognosis, with
The benefits of plasma exchange in anti-GBM nephritis spontaneous improvement after supportive management
have been demonstrated in adult patients (34) and have (3,40). Roy et al. (41) found no advantage from immuno-

TABLE 34.5. OUTCOME IN CHILDREN WITH CRESCENTIC GLOMERULONEPHRITIS

Niaudet and
SPNSGa (N = 50) Levyb (N = 41) Jardim et al.c (N = 30) Srivastava et al.d (N = 43)

Normal renal function 13 of 47 (27.6%) 13 (31.7%) 4 (13.3%) 6 (13.9%)

Chronic renal impairment 11 of 47 (23.4%) 6 (14.6%) 10 (33.3%) 14 (32.5%)
End-stage renal failure 23 of 47 (48.9%) 22 (53.6%) 16 (53.3%) 23 (53.4%)

N, number of patients.
aData from SPNSG (Southwest Pediatric Nephrology Study Group). A clinico-pathological study of crescentic glomerulonephritis in 50 children.

Kidney Int 1985;27:450–458.

bData from Niaudet P, Levy M. Glomerulonéphritis à croissants diffus. In: Royer P, Habib R, Mathieu H, Broyer M, eds. Néphrologie pédiatrique,

3rd ed. Paris: Flammarion, 1983;381–394.

cData from Jardim HMPF, Leake J, Risdon RA, et al. Crescentic glomerulonephritis in children. Pediatr Nephrol 1992;6:231–235.
dData from Srivastava RN, Moudgil A, Bagga A, et al. Crescentic glomerulonephritis in children: a review of 43 cases. Am J Nephrol 1992;12:155–161.
 34. Crescentic Glomerulonephritis 661

suppression and anticoagulants over conservative manage- lone pulses. Proceedings of 8th International Congress of
ment in children with poststreptococcal disease. However, Nephrology, Athens. Basel: Karger, 1981:305–311.
progression to renal failure has been reported in children (3– 13. Rees AJ, Cameron JS. Crescentic glomerulonephritis. In:
5,7,8); hence, the outlook is not uniformly good. Davison AM, Cameron JS, Grünfeld J-P, et al., eds. Oxford
Of other causes of crescentic glomerulonephritis, mesan- textbook of clinical nephrology, 2nd ed. Oxford, UK: Oxford
University Press, 1998:625–646.
giocapillary glomerulonephritis, microscopic polyangiitis,
14. Andrassy K, Kuster S, Waldherr R, et al. Rapidly progressive
anti-GBM disease, and the idiopathic form seem to have a glomerulonephritis: analysis of prevalence and clinical
worse prognosis than, for example, Henoch-Schönlein pur- course. Nephron 1991;59:206–212.
pura or SLE, which are among the more common causes of 15. Miller MN, Baumal R, Poucell S, et al. Incidence and prog-
the condition (3,7). However, the outlook for any patient is nostic importance of glomerular crescents in renal disease of
very dependent on the need for dialysis at onset and the childhood. Am J Nephrol 1984;4:244–247.
presence of fibrous crescents on biopsy (3,7,11). 16. Bidani AK, Lewis EJ. Idiopathic rapidly progressive glomer-
In conclusion, it is clear that crescentic glomerulone- ulonephritis and Goodpasture’s syndrome. In: Edelmann
phritis in childhood, as in adult practice, is caused by many CM, ed. Pediatric kidney disease, 2nd ed. Boston: Little,
different conditions with diverse pathogenetic mechanisms. Brown and Company, 1992:1223–1245.
These determine the natural history and the most appropri- 17. Levin M, Rigden SPA, Pincott JR, et al. Goodpasture’s syn-
drome: treatment with plasmapheresis, immunosuppression
ate treatment. Precise rapid diagnostic categorization and
and anticoagulation. Arch Dis Child 1983;58:697–702.
aggressive appropriate therapy are, in the majority of cases, 18. Walters MD, Savage COS, Dillon MJ, et al. Antineutrophil
indicated in light of the well-recognized poor prognosis in a cytoplasm antibody in crescentic glomerulonephritis. Arch
substantial proportion of those affected. Dis Child 1988;63:814–817.
19. Dillon MJ, Tizard EJ. Antineutrophil cytoplasmic antibod-
ies and antiendothelial cell antibodies. Pediatr Nephrol
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6. Dilma MG, Adhikari M, Coovadin HM. Rapidly progres- type of glomerular crescent cells. Am J Clin Pathol 1985;
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8. Srivastava RN, Moudgil A, Bagga A, et al. Crescentic glo- 26. Muller GA, Muller CA, Markovic-Lipkovski J, et al. Renal
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et al. Rapidly progressive glomerulonephritis in Thai chil- 27. Burkholder PM. Ultra structure demonstration of injury and
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12. Robson AM, Rose GM, Cole BR, et al. The treatment of 29. Bonsib SM. Glomerular basement membrane necrosis and
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32. Ferraris JR, Gallo GE, Ramires J, et al. “Pulse” methyl pred- antibodies. Kidney Int 1991;40:757–763.
nisolone therapy in the treatment of acute crescentic glo- 38. Gianviti A, Trompeter RS, Barratt TM, et al. Retrospective
merulonephritis. Nephron 1983;34:207–208. study of plasma exchange in patients with idiopathic rapidly
33. Bolton WK, Sturgill BC. Methyl prednisolone therapy for progressive glomerulonephritis and vasculitis. Arch Dis
acute crescentic rapidly progressive glomerulonephritis. Am Child 1996;75:186–190.
J Nephrol 1989;9:368–375. 39. Jindal KK. Management of idiopathic crescentic and diffuse
34. Lockwood CM, Pinching AJ, Sweny P, et al. Plasma proliferative glomerulonephritis: evidence-based recom-
exchange and immunosuppression in the treatment of ful- mendations. Kidney Int 1999;(Suppl)70:S33–S40.
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Lancet 1977;i:63–67. nificance of extracapillary proliferation: clinico-pathological
35. Thysell H, Bygren P, Bengstsson U, et al. Immunosuppres- review of 60 patients. Nephron 1976;16:1–19.
sion and the additive effect of plasma exchange in treatment 41. Roy S, Murphy, WM, Arant BS. Poststreptococcal crescen-
of rapidly progressive glomerulonephritis. Acta Med Scand tic glomerulonephritis in children: comparison of quintuple
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 S E C T I O N

VI

TUBULAR DISEASE
 35

NEPHRONOPHTHISIS-MEDULLARY
CYSTIC KIDNEY DISEASE
FRIEDHELM HILDEBRANDT

DEFINITION OF THE NEPHRONOPHTHISIS- tion and gene identification by positional cloning, different
MEDULLARY CYSTIC DISEASE COMPLEX disease variants can be distinguished on the basis of distinct
gene loci (Table 35.1).
The nephronophthisis-medullary cystic disease (NPH- In juvenile NPH (NPH1), a gene locus has been
MCKD) complex describes a distinct clinicopathologic entity mapped to chromosome 2q12.3 (10). This locus was fur-
of inherited diseases that lead to chronic renal failure on the ther refined (11–16), and the gene (NPHP1) responsible
pathologic basis of a chronic sclerosing tubulointerstitial for NPH1 was identified by positional cloning (17,18).
nephropathy (1). Whereas NPH is an autosomal recessive dis- Approximately 85% of patients with NPH1 carry large
ease with onset of end-stage renal disease in adolescence, the homozygous deletions of the NPHP1 gene (19,20). Sponta-
term medullary cystic kidney disease is used for autosomal neously occurring deletions of the NPHP1 locus (20) as
dominant variants with onset in adulthood. NPH represents well as specific loss-of-function point mutations of NPHP1
the most frequent genetic cause of chronic renal failure in the have been characterized (18,21,22). In a subset of patients
first two decades of life (6–8). Diseases of the NPH-MCKD with large deletions in NPHP1, there is an association with
complex are characterized clinically by a defect in urinary oculomotor apraxia type Cogan (23). Another subset shows
concentrating capacity, anemia, and progression into terminal an association with retinitis pigmentosa (22). No specific
renal failure in adolescence or young adulthood (2,3). On molecular characteristics have been detected that clearly
renal histology, NPH-MCKD exhibits a characteristic triad of identify the subgroup of NPH1 patients with extrarenal
disruption of renal tubular basement membranes (TBMs) associations. The protein encoded by NPHP1, “nephrocys-
with tubular cell atrophy, tubulo-interstitial fibrosis, and cyst tin,” contains an src-homology 3 (SH3) domain with high
formation (4,55). Extrarenal manifestations have been sequence similarity to the c-crk proto-oncogene. This sug-
described in association with recessive NPH, but are absent gests that nephrocystin may play a role as a docking protein
from MCKD. After the first descriptions of NPH by Smith in cell-cell or cell-matrix signaling processes, or both. Neph-
and Graham (2) and Fanconi et al. (3), more than 300 cases rocystin is highly conserved in evolution, including the
have been reported (4,5), and the disease complex has been nematode Caenorhabditis elegans.
extensively reviewed (1,6–8). The term nephronophthisis is Infantile NPH (NPH2) is a distinct disease entity, in
used for the recessively inherited variants of the complex, and which end-stage renal failure occurs within the first 3 years
the term medullary cystic kidney disease denotes the autosomal of life (24,25). NPH2 differs from other forms of NPH by
dominant variants. the presence of enlarged kidneys and cortical microcysts
and the absence of medullary cysts. Histologically, there is
no disruption of TBMs. A gene locus (NPHP2) for NPH2
ETIOLOGY AND MOLECULAR GENETICS was localized to 9q22-q31 in one Bedouin kindred (26).
Morphologically, the disease phenotype encompasses fea-
To date, no aberrations of chromosome number have been tures of both NPH and autosomal recessive polycystic kid-
reported in any disease of the NPH-MCKD complex, with ney disease (ARPKD). Recently, recessive mutations in the
the single exception of a partial monosomy of chromosome inversin gene have been identified as the cause of NPHP2
3 described by Sarles et al. (9). Because little insight had (26a).
been available into the pathogenesis of NPH-MCKD, a In NPH type 3 (NPH3), a third NPH gene locus
positional cloning approach was used toward the identifica- (NPHP3) for “adolescent NPH” has been localized to
tion of genes for the complex. After chromosomal localiza- chromosome 3q21-q22 (27), and several candidate genes
666 VI. Tubular Disease

TABLE 35.1. DISEASE VARIANTS, GENE LOCI, AND EXTRARENAL MANIFESTATIONS OF THE
NEPHRONOPHTHISIS-MEDULLARY CYSTIC KIDNEY DISEASE (NPH-MCKD) COMPLEX
Disease Inheritance ESRD (median in yr) Chromosome Gene (product) Extrarenal association

NPH
NPHP1 (juvenile) AR 13 2q12.3 NPHP1 (nephrocystin) SLS, OMA
NPHP2 (infantile) AR 1–3 9q22-q31 INV (inversin) Situs inversus, SLS
NPHP3 (adolescent) AR 19 3q22 ? SLS
NPHP4 AR 20 1p36 NPHP4 (nephroretinin) SLS, OMA
MCKD
MCKD1 AD 62 1q21 ? Hyperuricemia
MCKD2 AD 32 16p12 UMOD (Thamm-Horsfall Hyperuricemia
protein)

AR, autosomal recessive; AD, autosomal dominant; ESRD, end-stage renal disease; OMA, oculomotor apraxia type Cogan; SLS, Senior-Loken syndrome.

have been excluded (28,29). In addition, linkage to The histologic changes of NPH are characteristic but
NPHP3 was identified in families with Senior-Loken syn- not specific for the disease and develop postnatally. Typi-
drome (SLS) (30). cally, there is pronounced thickening and multilayering of
The disease gene for NPH type 4 (NPH4) has been iden- the TBM, which represents the most characteristic histo-
tified on chromosome 1p36 (31–33). The gene and its gene logic feature of the NPH-MCKD complex (Fig. 35.1). By
product nephroretinin are novel and highly conserved in light microscopy, there appears to be a sequence of events.
evolution. Subsets of patients with mutations in the NPHP4 TBM disruption is followed by lymphocytic and histocytic
gene exhibited SLS as well as Cogan syndrome (31,32). peritubular infiltration. Subsequently, atrophic or dilated
The locus for MCKD type 1 (MCKD1) has been mapped and tortuous tubules develop predominantly at the corti-
to 1q21 (34–36). Microscopic and macroscopic pathology of comedullary junction. In advanced stages, the picture
dominant MCKD is indistinguishable from recessive NPH.
The MCKD1 locus has been refined to 4 centi-Morgan (cM)
(37), and the gene (NPR1) for natriuretic peptide receptor-1
has been excluded as a candidate gene (38).
A second locus, MCKD type 2 (MCKD2), has been
mapped to chromosome 16q12 (39–44). The MCKD2 locus
was refined to a 1.3-megabase (Mb) interval, demonstrating
that it co-localizes with the locus for familial juvenile hyper-
uricemic nephropathy (45). The UMOD gene has very
recently been identified as causative for MCKD2. There is
evidence for existence of a third MCKD locus (46,47).

HISTOPATHOLOGY

Renal histopathology is similar in all variants of the NPH-

MCKD complex, and has been described in 27 patients with
NPH by Waldherr et al. (4). Kidney size is normal or moder-
ately reduced. There is always bilateral renal involvement.
Macroscopically, the kidney surface has a finely granular
appearance, most likely due to the protrusion of dilated corti-
cal collecting ducts. There are between 5 and approximately 50
cysts of 1 to 15 mm in diameter located preferentially at the
corticomedullary border. The cysts arise primarily from the
distal convoluted and medullary collecting tubules, as shown
by microdissection (48), but may also appear in the papilla.
Cysts are observed only in approximately 70% of autopsy cases FIGURE 35.1. Renal histology of nephronophthisis-medullary
and seem to arise late in the course of the disease (49). There- cystic kidney disease. Note the characteristic triad of tubular
basement membrane disruption, tubular cell atrophy with cysts,
fore, the presence of cysts is not a prerequisite for diagnosis. and interstitial infiltration and fibrosis. Hematoxylin-eosin stain.
No cysts are present in organs other than the kidney. (Courtesy of Prof. R. Waldherr, Heidelberg, Germany.)
 35. Nephronophthisis-Medullary Cystic Kidney Disease 667

merges into a diffuse, sclerosing, tubulo-interstitial nephrop- (62). Nephrocystin expression leads to phosphorylation of
athy. TBM changes and cyst formation are most prominent Pyk2 on tyrosine residue 402, which in turn activates the
in distal tubules, where cysts are lined with a single layer of transcription factors ERK1 and ERK2 (extracellular signal–
cuboidal or flattened epithelium (50–53). Glomeruli dem- related kinases 1 and 2). Thus, nephrocystin helps recruit
onstrate periglomerular fibrosis with splitting and thicken- Pyk2 to cell-matrix adhesions, thereby initiating Pyk2-
ing of Bowman’s capsule. Glomerular obsolescence is only dependent signaling (62). Nephroretinin, the product of the
present in nephrons that have been destroyed by tubular NPHP4 gene mutations that cause NPH4 and SLS type 4
alterations. Leakage of Tamm-Horsfall protein from dam- (32), has been shown by co-immunoprecipitation to interact
aged collecting tubules into the interstitium has been dem- with nephrocystin (33). In patients with NPH, abnormal
onstrated (54). On transmission electron microscopy, there expression of the α5 integrin fibronectin receptor has been
is thickening, splitting, attenuation, and granular disinte- demonstrated in renal TBMs (65). This leads to the hypoth-
gration of the TBM without clear stages of transition (55). esis that renal tubular cells in NPH express α5 integrin
A marked increase of microfilaments is seen at the base of fibronectin receptor within focal adhesions as a compensa-
the tubular epithelial cells. tory mechanism for defective function of the α6 integrin
molecule. This, in turn, would lead to destruction of the
TBM, which is a typical histologic finding early in the course
PATHOGENESIS
of NPH. In addition, alterations in hepatocyte growth factor
(66) and Pax2 (67) have been described in NPH.
Changes in tubular function characterize NPH-MCKD and
include decreased urinary concentration ability and ami-
noaciduria. Proteinuria is a rare finding. Histologic changes
ANIMAL MODELS
(see Histopathology) also emphasize the central role of
altered tubular structure in the pathogenesis. Cysts do not
Further support for the hypothesis that a defect in tubular
seem to be important for disease progression (56). Cohen
cell-cell or cell-matrix interaction is an important pathoge-
and Hoyer (57) have hypothesized that NPH results from a
netic mechanism in NPH comes from the finding that tar-
primary defect of TBM matrix components. Such abnormal-
geted disruption of the mouse tensin gene leads to a renal
ities lead to reduced mechanical compliance of the distal
phenotype closely resembling human NPH (68). Because
tubule with consecutive cyst development. In SLS, both
tensin is a major component of focal adhesions, defects in
renal and retinal changes are thought to be degenerative
these matricellular contact points might play a central role
rather than dysplastic (58).
in the pathogenesis of NPH. Additional mouse models
Recent data suggest that NPH results from a primary
with features of NPH include kd/kd mice (69,70), angio-
defect in tubular cell-cell contacts (adherens junctions) or
tensin-converting enzyme knock-out mice (71), and Bcl-2
tubular cell-matrix interactions, or both (59). The NPHP1
knock-out mice (72). Recently, strong synteny of the
gene product, nephrocystin, is a docking protein that binds
human NPHP3 locus has been demonstrated to the locus
through multiple domains of protein-protein interaction to
pcy mouse whose renal histopathology closely resembles
proteins of cell-cell signaling and cell-matrix signaling at
human NPH (73). Also, the RhoGDI-α-“knock-out”-mouse
adherens junctions and focal adhesions, respectively. The src-
model exhibits a renal cystic phenotype, which shares
homology-3 domain of nephrocystin binds to proline-rich
certain histologic features with human NPH (74).
peptides on the protein p130cas (c-crk–associated substrate),
Recently, nephrocystin was shown to interact with
which is known to be a major mediator of adherens junction
inversin, the gene product defective in NPH2, and both
and focal adhesion signaling (60–62). Nephrocystin, in
nephrocystin and inversin have been demonstrated to
MDCK cells, co-localizes to adherens junctions with
co-localize to single cilia on MDKC cells (26a). This
p130Cas and with the integral adherens junction protein E-
expression pattern provides a possible link between the
cadherin (61). Expression of an exogenous nephrocystin con-
pathogenesis of NPH and the pathogenesis of polycystic
struct with a defective SH3 domain in MDCK cells
kidney disease because polycystin-1 and polycystin-2 have
decreased electrical resistance (63). In addition, it has been
been shown to be expressed in cilia of renal epithelial
shown that targeting of nephrocystin to adherens junctions
cells, where they might exert a function as a mechanosen-
of MDCK cells was dependent on the nephrocystin homol-
sory renal tubular flow sensor (75).
ogy domain (NHD). The NHD represents the C-terminal
half of nephrocystin, which by secondary structure predic-
tion is featureless and exhibits no homology with any known
CLINICAL PRESENTATION
proteins. Binding of nephrocystin to the actin-organizing
proteins filamin A and B occurs via the NHD domain (63).
Nephronophthisis
Nephrocystin binds to the signaling proteins p130Cas
(64), focal adhesion kinase 2 (Fak2, alias Pyk2), and tensin, Diseases of the NPH-MCKD complex are characterized by
which are integral components of focal adhesion signaling the insidious onset of renal failure. In recessive NPH, the
668 VI. Tubular Disease

larly given the degree of chronic renal insufficiency. Due to

the lack of specificity of the initial symptoms with absence
of edema and hypertension, most children have already
developed chronic renal failure when they first come to clin-
ical attention. This presents a small but definite risk of sud-
den death from fluid and electrolyte imbalance. Disease
recurrence has never been reported after renal transplanta-
tion for NPH (79,80).
Extrarenal manifestations occur only in recessive NPH, but
are absent from MCKD. The most frequent extrarenal associa-
tion is retinitis pigmentosa seen in SLS (81,82). SLS occurs in
approximately 12% of all patients with the NPH-MCKD
complex (83), which would be equivalent to 18% of all auto-
somal recessive cases. An early-onset and a late-onset type of
SLS have been described. Intrafamilial dissociation of renal
and eye involvement has never been demonstrated convinc-
FIGURE 35.2. Progression chart representing the average course ingly in any published pedigree. In the early-onset type, chil-
of deterioration of renal function in 19 patients of eight families dren present with coarse nystagmus and/or blindness at birth
with juvenile nephronophthisis as proven by deletion detection.
Median (solid line) and quartile (dashed lines) curves were calcu- or within the first 2 years of life. Funduscopic alterations are
lated from 308 serial serum creatinine values. (From Hildebrandt present in all SLS patients by the age of 10 years. The late-
F, Strahm B, Nothwang HG, et al. Molecular genetic identification onset form is characterized by development of blindness dur-
of families with juvenile nephronophthisis type 1: rate of progres-
sion to renal failure. Kidney Int 1997;51:261–269, with permis- ing school age after a variable period of night blindness. Other
sion.) eye symptoms besides tapeto-retinal degeneration comprise
nystagmus, myopia, coloboma of the chorioidea, strabismus,
hyperopia, optical nerve atrophy, and amblyopia (5). Age of
symptoms of polyuria, polydipsia, decreased urinary con- onset, symptoms, and histology of renal disease is identical to
centrating ability, and secondary enuresis are the earliest what is known from NPH patients without ocular involve-
presenting symptoms in more than 80% of cases (5) and ment. In patients with recessive mutations in the NPHP1
occur at 4 to 6 years of age. The symptom of starting to reg- gene, no molecular difference in defects of the NPHP1 gene
ularly drink at night, once children are 6 to 10 years old, is have been identified that would distinguish patients with the
rather characteristic of NPH. Pallor, weakness, and general- purely renal form of NPH from patients with the association
ized pruritus are also common. Anemia and growth retarda- of NPH and retinitis pigmentosa. As in SLS, no specific
tion occur later and are usually pronounced. All affected molecular differences of the NPHP1 gene distinguish patients
individuals progress to end-stage renal disease and therefore with the purely renal form of NPH from patients with the
require renal replacement therapy. In a study conducted in association of NPH and Cogan syndrome (23). With ocular
19 patients of eight families with deletion-positive NPH1, a motor apraxia, infants with Cogan syndrome cannot move
serum creatinine of 5.5 mg/dL was reached at an average their eyes sideways, which leads to a pattern of compensatory
age of 13.1 years of age (range, 7 to 25 years) (76). From head thrusts. This symptom resolves within the first few years
this study, charts for prediction of progression toward renal of life. Additional forms of extrarenal involvement are congen-
failure were derived (Fig. 35.2). A difference in age of pro- ital hepatic fibrosis (84,85) and developmental defects of bone,
gression to renal failure was shown between patients with predominantly in the form of cone-shaped epiphyses (type 28
deletion-positive NPH1 and patients from a Venezuelan and 28A) (86). These can also occur in combination with reti-
kindred of adolescent NPH (NPH3), in which median age nal degeneration and cerebellar ataxia (87,88). In Joubert syn-
of onset of end-stage renal disease was 19 years (27). If renal drome type B, a developmental disorder with multiple organ
failure has not developed by the age of 25 years, the diagno- involvement, NPH occurs in association with coloboma of the
sis of recessive NPH should be questioned. A high concor- eye or retinal degeneration; aplasia of the cerebellar vermis
dance rate (77) of the development of renal failure has been with ataxia; and the facultative symptoms of psychomotor
noted in monozygotic twins (78). A characteristic early retardation, polydactyly, and neonatal tachypnea/dyspnea
finding in NPH is the decreased ability to concentrate the (89,90). An NPH-like phenotype has also been observed in
urine above 800 mOsm/kg of water after an 8-hour water Sensenbrenner syndrome (91).
deprivation test or vasopressin administration (1). Pub-
lished reports do not adequately answer the question of
Medullary Cystic Kidney Disease
whether renal sodium loss is a typical finding in the disease
complex. Typically, edema, hematuria, and urinary tract Goldman and associates were the first to report a large kin-
infections are absent in NPH. Hypertension is rare, particu- dred with autosomal dominant, adult-onset MCKD (92).
 35. Nephronophthisis-Medullary Cystic Kidney Disease 669

This was followed by the publication of two large pedigrees

from the United States by Gardner and Burke et al.
(93,94). The dominant form of the disease complex differs
from the recessive forms in that the onset of end-stage renal
disease is in the third decade of life or even later (95), with
an average at age 28.5 years (96). In MCKD, penetrance
appears to be very high by the age of 45 years. The second
feature distinguishing MCKD from NPH is the lack of
extrarenal involvement in dominant disease, with the
exception of hyperuricemia and gout. For MCKD1, an
association of the renal symptoms with hyperuricemia and
gout has been described (34,39), and MCKD2 has been
found to localize to the same chromosomal region as famil-
ial juvenile hyperuricemic nephropathy (45).
FIGURE 35.3. Renal ultrasound in nephronophthisis. Note loss
of cortico-medullary differentiation, cysts at the cortico-medul-
EPIDEMIOLOGY lary border of the kidney, and increased echogenicity, which ren-
ders the ultrasound pattern similar to the pattern of liver
NPH and dominant MCKD seem to be distributed evenly (arrow). In nephronophthisis, kidneys are typically normal in
size. (Courtesy of Dr. U. Vester, Essen, Germany.)
among males and females. NPH has been reported from
virtually all regions of the world (5). The incidence of the
disease has been estimated to be 9 per 8.3 million patients present, electroretinography and electro-oculography can
in the United States (97) or 1 in 50,000 live births in Can- be used to evaluate severity. Retinal degeneration is charac-
ada (98). The condition constitutes the most frequent terized by a constant and complete extinction of the elec-
genetic cause for end-stage renal disease in the first two troretinogram, preceding the development of visual and
decades of life and is a major cause of end-stage renal dis- funduscopic signs of retinitis pigmentosa (103). Ophthal-
ease in children, accounting for 10 to 25% of these patients moscopy should be performed in any patient to evaluate for
(5,99,100). In the North American pediatric end-stage signs of SLS, as it may occur in patients with NPH1,
renal disease population, pooled data indicate a prevalence NPH2, or NPH3. Liver function tests and hepatic ultra-
of less than 5% (101,102). MCKD appears to be somewhat sonography are important to facilitate detection of patients
less infrequent. with hepatic fibrosis.

DIAGNOSIS AND DIFFERENTIAL DIAGNOSIS Imaging

The most useful imaging technique in the NPH-MCKD
Laboratory Studies
complex is ultrasonography. Kidneys are of normal or mod-
Patients with NPH are usually diagnosed when an erately reduced size and show increased echogenicity
increased serum creatinine value is detected during evalua- (which renders the renal echo texture more echogenic than
tion for nonspecific complaints. History generally reveals that of the liver), loss of cortico-medullary differentiation,
prolonged nocturia since school age. Specific gravity of a and, in later stages, cyst formation at the cortico-medullary
morning urine specimen will be low. Renal ultrasound will border of the kidneys (104) (Fig. 35.3). Garel et al. have
then corroborate the diagnosis (see Imaging) (Fig. 35.3), seen medullary cysts in 13 of 15 children studied at the
which can be subsequently confirmed by molecular genetic time of renal failure (mean age, 9.7 years) (105). Roentgen-
diagnostics if NPH1, NPH2, or NPH4 is present (http:// ography contributes little to the diagnosis of the disease.
www.renalgenes.org). A diagnostic algorithm for NPH has Medullary cysts can sometimes also be demonstrated on
been suggested (76). Hematuria, proteinuria, and bacteri- magnetic resonance imaging or computed tomography
uria are uncommon in NPH. In rare cases when pro- (106,107). Histology is characteristic but not pathogno-
teinuria is present, it is usually mild and of the tubular type. monic in NPH-MCKD because cysts may be absent, and
Laboratory studies are needed to assess the severity of renal tubulointerstitial disease can be relatively unspecific. Renal
failure and generally demonstrate elevated serum creati- biopsy can be circumvented as an initial procedure due to
nine, blood urea nitrogen, phosphorus, metabolic acidosis, the availability of molecular genetic diagnostics in NPH1,
hypocalcemia, and anemia. In SLS, retinitis pigmentosa is NPH2, and NPH4. If molecular genetic diagnostics do not
diagnosed by its specific findings on ophthalmoscopy, detect a molecular defect, the diagnosis can be based on the
including increased pigment, attenuation of retinal vessels, combined results of typical history, with polyuria, polydip-
and pallor of the optic disk. If retinitis pigmentosa is sia, and anemia; the classical appearance of the kidney on
670 VI. Tubular Disease

ultrasound; and renal histology. A thorough pedigree analy- such as chronic pyelonephritis or drug injury. In oligomega-
sis should be documented for each of three successive gen- nephronic dysplasia, kidney size is reduced, and histology is
erations to rule out autosomal dominant MCKD. distinct from NPH. The paucity of urinary abnormalities,
the frequent lack of hypertension, normal kidney size, and
the localization of renal cysts (if present) readily differentiate
Molecular Genetic Diagnosis
variants of the NPH-MCKD complex from recessive or
Recently, molecular genetic diagnosis has become available dominant polycystic kidney disease. Finally, medullary
for NPH1, NPH2, and NPH4 through identification of sponge kidney (108) can easily be distinguished from the
the respective genes (see Laboratory Studies) (http:// complex, as it does not usually lead to chronic renal failure
www.renalgenes.org). A diagnostic algorithm should be fol- and shows calcifications and calculi on renal ultrasound.
lowed to avoid unnecessary renal biopsy (76). Molecular
genetic analysis is the only diagnostic procedure by which
the diagnosis of NPH1, NPH2, and NPH3 can be made PROGNOSIS AND THERAPY
with certainty. However, due to the presence of additional
loci for NPH, the lack of detection of mutations in the There is no specific therapy for NPH or MCKD. Therapy is
NPHP1 gene does not exclude the diagnosis of NPH. If symptomatic and is directed toward the treatment of hyper-
renal disease with features of the NPH-MCKD complex tension, if present, as well as the correction of disturbances of
occurs in a person older than 25 years, its presence should electrolyte, acid-base, and water balance. Hypokalemia may
be thoroughly sought in preceding generations. This may contribute to polyuria, and oral potassium supplementation
frequently result in detection of a pattern of autosomal may alleviate this symptom. Metabolic acidosis should be
dominant inheritance. In MCKD1, only indirect molecu- corrected and osteodystrophy and secondary hyperparathy-
lar genetic diagnosis by haplotype analysis is possible so far, roidism treated with adequate calcium supplementation,
as the respective genes have not yet been identified. For this phosphorus restriction, non-magnesium–containing phos-
purpose, a pedigree and blood samples from at least seven phate binders, and vitamin D therapy. Anemia should be
affected members and their first-degree relative will have to treated with iron supplementation and erythropoietin, and
be obtained. In MCKD2, mutational analysis of the growth retardation may require administration of growth
UMOD gene can be performed. hormone if the diagnosis is made early enough for an inter-
Molecular genetic testing should be performed only after vention. Adequate nutrition should be maintained with the
consent within the guidelines of the National and Interna- help of a dietitian. Psychological counseling of the patients is
tional Societies for Human Genetics. Before genetic counsel- an integral part of therapy because of the poor self-image
ing, a thorough pedigree analysis to distinguish recessive associated with growth retardation and to alleviate pressures
(early-onset) from dominant (late-onset) disease is manda- resulting from the need to comply with complicated medica-
tory, and extrarenal organ involvement should be sought. tions and dietary prescriptions. All patients require renal
Because nonsymptomatic potential carriers of recessive replacement therapy by dialysis and renal transplantation
defects should not be examined by molecular genetic diag- during childhood, adolescence, or, in dominant disease, in
nostics, unaffected siblings younger than 13 years of age early adult life.
should be re-evaluated yearly for maximal urinary concen-
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672 VI. Tubular Disease

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 36

POLYCYSTIC KIDNEY DISEASE

KATHERINE MacRAE DELL
RUTH A. McDONALD
SANDRA L. WATKINS
ELLIS D. AVNER

Polycystic kidney disease (PKD) is a heritable disorder with 12) (Fig. 36.1). Delineating the extent to which these fac-
diffuse cystic involvement of both kidneys without dyspla- tors, singly or in combination, contribute to cyst develop-
sia (1). All forms of PKD can have clinical manifestations ment and enlargement in ADPKD and ARPKD remains
in infants and children. The major clinical entities of auto- an area of active investigation.
somal recessive PKD (ARPKD) and autosomal dominant
PKD (ADPKD) have considerable overlap in clinical pre-
sentation and radiographic features. Glomerulocystic kid- TUBULAR HYPERPLASIA
ney disease (GCKD) can be a feature of several inherited,
sporadic, and syndromal conditions, as well as an expres- Tubular hyperplasia is a central morphologic feature of all
sion of ADPKD. described human renal cystic diseases (7,10). On the basis
of mathematic modeling of cyst growth, it has been shown
that tubular cell hyperplasia, with expansion of tubular wall
PATHOPHYSIOLOGY OF CYST FORMATION segments to accommodate an increased cellular mass, is an
IN POLYCYSTIC KIDNEY DISEASE essential factor in cyst formation and enlargement (12).
Furthermore, cyst-derived epithelial cells from ADPKD
In the 1990s, major advances were made in understanding and ARPKD demonstrate increased cell growth potential
the molecular genetics of PKD. Through a combination of compared with controls (9,13). Unlike normal renal epi-
positional cloning, direct sequencing, and use of the rapidly thelia, ADPKD cystic epithelia respond to increased intra-
expanding genome databases, the major causative genes for cellular cyclic adenosine monophosphate (cAMP) with
both ADPKD (PKD1 and PKD2) as well as ARPKD proliferation (14,15). Recent data demonstrate differences
(PKHD1) have been identified (2–5) (Table 36.1). Details in cAMP-dependent protein kinase and proliferation in
specific to the molecular genetics of ARPKD and ADPKD normal and polycystic kidney epithelia in a murine model
are addressed in the respective sections that follow. Despite of ARPKD (16). Additional in vivo data demonstrate
the advances in the identification of the PKD genes and increased renal tubular epithelial cell proliferation in both
proteins, the precise mechanisms by which these gene cystic and noncystic tubular epithelium from ADPKD and
defects result in cyst formation have not yet been fully elu- ARPKD kidneys (17).
cidated. Considerable insights into the understanding of A growing body of evidence implicates the epidermal
cyst formation pathogenesis have been provided by many growth factor receptor (EGFR) and its respective ligands,
studies of the cellular pathophysiology of PKD. These stud- including epidermal growth factor (EGF) and transforming
ies complement the advances made in the genetics of the growth factor-α (TGFα), in proliferation of cystic epithe-
diseases and may form the basis for future therapeutic lium. In both human ADPKD and ARPKD and in several
interventions. murine models of PKD, cystic kidneys display characteristic
On the basis of extensive in vivo and in vitro studies alterations in EGFR expression. Quantitative abnormalities,
using human tissue and animal models of PKD, at least including increased messenger RNA (mRNA) and protein,
three basic processes are operative in renal cyst formation and qualitative differences, in particular, the appearance of
and progressive enlargement: (a) tubular cell hyperplasia; “mislocalized” EGFR expressed on the apical surface of tubu-
(b) tubular fluid secretion; and (c) abnormalities in tubular lar epithelium, are seen (18–20). Apical EGFR is functional
extracellular matrix (ECM), structure, and/or function (6– and capable of transmitting mitogenic signals in vitro (21).
676 VI. Tubular Disease

TABLE 36.1. HUMAN POLYCYSTIC KIDNEY DISEASE GENES AND PROTEINS

Mode of Chromosome
Disease Gene inheritance location Protein Function/role

ADPKD PKD1 AD 16p13.3–p13.12 Polycystin 1 ?Receptor

ADPKD PKD2 AD 4q21–q23 Polycystin 2 Cation channel
ARPKD PKHD1 AR 6p21 Fibrocystin (polyductin) ?Receptor
GCKD (hypoplastic HNF-1β AD 17cen–q21.3 Hepatocyte nuclear fac- Transcription factor
variant) tor-1β

ADPKD, autosomal dominant polycystic kidney disease; ARPKD, autosomal recessive polycystic kidney disease; GCKD, glomerulocystic kidney disease.

Inhibition of EGFR function in vitro by treatment with either contains EGF or EGF-like peptides in mitogenic concentra-
an inhibitor of tyrosine kinase function or a blocking anti- tions, despite apparent reductions in EGF tissue expression
body inhibits formation of proximal tubule cysts and (9,29–31). Treatment with EGF transiently improves renal
significantly decreases explant growth and distal nephron dif- function in murine models (32) but has no effect on histo-
ferentiation (22,23). Additional support for a central role for pathologic abnormalities, and continued EGF treatment
EGFR in the pathogenesis of cyst formation is provided by in worsens disease and shortens survival (33). TGFα and EGF
vivo data. Inhibition or reduction of EGFR function, either are cystogenic in both murine embryonic organ cultures (34)
by treatment with a novel tyrosine kinase inhibitor (24) or and normal human kidney cells grown in a unique collagen
genetic manipulation (25), leads to a marked reduction in gel system in vitro (35). ADPKD kidneys and cells derived
cyst formation and enlargement in animal models. Of note, from ADPKD have increased mRNA or protein levels of
EGFR is part of a larger family of receptors (the c-ErbB/HER TGFα (18,36). Transgenic mice that overexpress TGFα
receptors), and there are limited data on the expression and develop cystic kidneys (37). Additional data suggest that
function of these related receptors in PKD. C-ErbB2 (HER2) inhibiting EGFR ligand function may also partially amelio-
overexpression is seen in some cysts of ADPKD kidneys but rate disease. Treatment with an inhibitor of TGFα processing
not in late-stage ARPKD kidneys (26). However, late-gesta- decreases cystic kidney disease in a murine model, although
tion/early postnatal human ARPKD samples showed less effectively than EGFR inhibition. (38). Recent data
increased ErbB-2 expression compared with normal human demonstrate that combining inhibition of EGFR ligand
fetal and postnatal kidneys (27). The functional significance function with EGFR inhibition may maximize therapeutic
of altered ErbB2 expression in PKD remains to be deter- effectiveness while minimizing toxicity (39). Additional
mined. Studies in a murine models of ARPKD demonstrate EGFR ligands, including amphiregulin and heparin-binding
that ErbB4 expression increases with progressive cystic dis- EGF, are abnormally expressed in PKD and may prove to
ease, whereas a decrease is seen in normal (noncystic) age- also have a role in proliferation of cystic epithelium (40).
matched controls (28). Epithelial cell hyperplasia may be mediated not only by
Abnormalities in EGFR ligand expression have also been factors that control cell proliferation (such as activation of
reported in both ARPKD and ADPKD. Renal cyst fluid the EGFR/EGFR ligand axis) but also by alterations of

FIGURE 36.1. Pathophysiology of renal cyst for-

mation. Studies in a variety of experimental mod-
els and human diseases implicate three major
factors in renal cyst formation and progressive
enlargement. Normal renal tubular absorptive
epithelium can become cystic if (a) hyperplasia,
localized to a distinct nephron segment, requires
accommodation of an increased cell mass; (b) secre-
tion, as opposed to absorption, leads to the net
accumulation of intratubular fluid with low (or
no) afferent tubular flow or efferent tubular
obstruction; or (c) extracellular matrix (ECM)
abnormalities alter the epithelial microenviron-
ment such that abnormal cell-matrix interactions
result in abnormal epithelial hyperplastic and
secretory activity. These processes are not mutu-
ally exclusive, may reflect some characteristics of
undifferentiated epithelium, and probably oper-
ate in concert during renal tubular cyst formation
and progressive enlargement.
 36. Polycystic Kidney Disease 677

normal apoptotic mechanisms. Emerging evidence suggests Studies in a variety of model systems have evaluated pos-
that dysregulation of apoptosis, or the balance between sible mechanisms involved in PKD tubular fluid secretion.
apoptosis and proliferation, may contribute to the progres- Work to date has identified a major role for cAMP-medi-
sion of ARPKD and ADPKD (41–43). Evidence in sup- ated chloride secretion during in vitro cyst formation
port of a role for apoptosis in the pathogenesis of PKD is (14,61–63). A putative lipid “secretagogue” isolated from
provided by studies of various components of the apoptosis cyst fluid of human ADPKD kidneys was found to stimu-
pathway, including several caspases and the bcl-2/bax fam- late intracellular cAMP and stimulate fluid secretion (64).
ily of proteins. Ali et al. (44) demonstrated a six- to seven- Additional in vivo and in vitro studies demonstrate a poten-
fold increase in caspase 3 and 4 activity in kidneys of cpk tial role for quantitative and qualitative alterations in Na+-K+-
mice, a murine model of ARPKD. Unlike previous studies adenosine triphosphatase (ATPase) activity in mediating tubu-
in which increased apoptosis was found in renal tubules, lar fluid secretion in cystogenesis (65–70). In proximal
this study localized apoptosis primarily to the interstitium tubules, it has been postulated that increases in Na+-K+-
with little evidence of cell death in either cystic or noncys- ATPase activity modulate tubular secretion and cyst formation
tic tubules. Increased rates of apoptosis in a murine model through activation of a secondary active transport process (e.g.,
of ARPKD were demonstrated in the interstitial cells sur- tubular organic anion secretion), which osmotically obligates
rounding cystic collecting tubules (CT) when compared to intratubular fluid accumulation and cystogenesis (65–67). In
interstitial cells surrounding normal CT (45). Ecder et al. collecting tubules, apical, as opposed to normal, basolateral cell
(46) have reported upregulation of caspase 3 in the Han/ surface Na+-K+-ATPase expression may mediate basal to apical
SPRD rat, a model of ADPKD. vectorial sodium transport and thus directly drive fluid secre-
Data regarding the antiapoptotic molecule, bcl-2, and tion in affected nephron segments in ADPKD and ARPKD
its related proteins are somewhat conflicting. Mice deficient (68–70). Apical Na+-K+-ATPase expression in murine
in bcl-2 develop severe multicystic hypoplasia characterized ARPKD may reflect an exaggeration of the normal develop-
by proximal and distal tubular cysts and hyperproliferation mental profile of collecting tubule sodium pump expression
of epithelium and interstitium (47). In contrast, increased (70). This, in association with the relatively undifferentiated
bcl-2 expression has been demonstrated in animal models ultrastructural and genetic profile of cystic tubular epithelium
of both ARPKD and ADPKD (42,44,46). Of note, in one (11), suggests that abnormalities in the differentiation pro-
study, decreased expression of another antiapoptotic mole- gram of cystic tubular cells may be basic to the process of cys-
cule, Bcl-X(L), suggests that the balance (rather than the togenesis. Of note, however, apical Na+-K+-ATPase expression
absolute expression levels) of different components of the has not been a consistent finding in all PKD models (71).
pro- and antiapoptotic pathways may be the critical factor It has been hypothesized that cAMP-stimulated chloride
in the development of cystic kidney disease (46). Abnor- and fluid secretion occurs through activity of the cystic fibrosis
malities in the apoptotic pathway may also directly or indi- transmembrane receptor (CFTR), the chloride channel
rectly impact on the normal cell-cell interactions (48–51). mutated in cystic fibrosis (72). CFTR Cl– channels exist in
Alterations in apoptosis may also be mediated, in part, by apical membranes of ADPKD and are major mediators of for-
abnormal expression of proto-oncogenes, in particular, c- skolin-stimulated chloride and fluid secretion by epithelial
myc. In both murine ARPKD and human ADPKD kid- cells of human polycystic kidneys in vitro (72,73). CFTR is
neys, c-myc is overexpressed in cystic tissue (42,52–54) and required for cAMP-dependent in vitro renal cyst formation
associated with a marked increase in both tubular cellular (74). In vivo support for a role of CFTR in the pathogenesis of
proliferation and apoptosis (55–57). Other genes, such as PKD was provided in a report of an ADPKD kindred in
pax2, may also have a role in apoptosis in PKD (58). which cystic fibrosis was also present. Patients with ADPKD
and CF (which results in a loss of functioning CFTR) were
found to have less severe disease than those with ADPKD who
FLUID SECRETION did not have CF (75). However, a subsequent report failed to
demonstrate such a protective effect (76). Because of the phe-
In addition to epithelial hyperplasia, some data demon- notypic variation within ADPKD families, it was difficult to
strate a significant role for tubular fluid secretion in cyst determine if CFTR was, in fact, functioning as a significant
formation and progressive enlargement (7,9,10). On a the- modifying disease gene, or if the phenotypic differences were
oretical basis, tubular fluid secretion, in addition to hyper- due to other modifying factors. This question was recently
plasia, fulfills the requirements for cyst growth predicted directly addressed in a murine model of ARPKD. Mice
by mathematic modeling (12). Cellular proliferation with- homozygous for the CFTR mutation (CFTR “knock-out”
out tubular secretion would produce solid tumor nests of mice) were bred with the bpk mouse, a murine model of
tubular cells rather than cysts. In ADPKD, more than ARPKD. No improvement in cystic disease in resultant dou-
70% of cysts have no afferent or efferent tubular connec- ble mutants was found (77). These data suggest that CFTR
tions, and thus must fill by transepithelial secretion of sol- activity does not play a central role in the pathogenesis of
ute and fluid (59,60). ARPKD, and that it is not necessary for cyst growth or
678 VI. Tubular Disease

enlargement. Chloride channels (other than CFTR) or abnor- cyst expansion in ADPKD as well. When cysts enlarge, their
malities in amiloride-sensitive sodium transport may have nutrient requirements may outstrip their blood supply in a
roles in fluid secretion in cystic ARPKD epithelium (77). The manner analogous to tumor progression in cancer. Recent stud-
role of CFTR in ADPKD remains to be determined. ies demonstrate increased vascularity around cysts and evidence
of ongoing angiogenesis (96). Whether angiogenesis has a role
in cyst expansion in ARPKD remains to be determined.
EXTRACELLULAR MATRIX Theories of renal cyst formation generated in experimental
models are not mutually exclusive and are largely complemen-
The third major mediator of tubular cyst formation and pro- tary. A mutant gene or environmental factors can directly lead
gressive enlargement is abnormalities involving the ECM (7,9– to alterations in tubular epithelial metabolism. Some environ-
11,68). Diffuse ultrastructural and biochemical abnormalities mental factors can modulate the expression of a mutant gene or
of tubular basement membranes have been demonstrated in directly lead to tubular cell death. An induced alteration of
human and animal models of PKD. Specific defects in the bio- tubular cell metabolism may subsequently lead directly to the
synthesis and transport of sulfated proteoglycans have also been abnormal sorting of transport proteins, growth factor receptors,
identified (9,11,78–80). Renal tubular cells from patients with or cell adhesion molecules, with resultant abnormal ECM pro-
ADPKD grown in vitro produce increased amounts of ECM duction or production of growth factors mediating tubular
when compared with normal tubular epithelia (81). hyperplasia. Induced changes in transtubular transport energet-
It does not appear that matrix abnormalities mediate sim- ics may lead to hyperplasia secondary to increased trans-
ple changes in the compliance or viscoelastic properties of membrane sodium flux, whereas programmed cell death may
tubular basement membranes, leading to distention under lead to further hyperplasia secondary to tubular regeneration.
normal intratubular pressures (82). Rather, it would appear Alterations in sodium- or chloride-mediated transtubular trans-
that altered matrix composition modulates cyst formation port could lead to net intratubular fluid accumulation. Subse-
through altered tubular epithelial cell matrix interactions. quent increases in tubular wall tension may further increase
These interactions regulate various aspects of cell growth, cell stimulation of epithelial proliferation, leading to tubular hyper-
surface protein expression, cytodifferentiation, and gene plasia. The presence of a particular pattern of tubular hyperpla-
expression (8,11). Conceivably, altered epithelial cell matrix sia, along with necrotic debris from cell death, may lead to
interaction could modulate or amplify the processes of partial tubular obstruction and further increases in tubular wall
hyperplasia and fluid secretion discussed above. tension. Finally, abnormal ECM production could alter the
Experimental evidence suggests that matrix metalloprotein- epithelial microenvironment, further increasing hyperplasia
ases (MMPs) and tissue inhibitors of metalloproteinases may and transtubular transport, thereby contributing to cyst forma-
play a role in progression of disease in PKD (83–85). Elevated tion and progressive cyst enlargement. Such an overall hypo-
serum levels of MMPs, including MMP-1, tissue inhibitor of thetical schema of renal cyst formation appropriately focuses
metalloproteinase-1, and MMP-9, have been demonstrated in future investigations on the molecular mechanisms by which
a cohort of ADPKD patients when compared to normal con- tubular epithelial hyperplasia is controlled and tubular metabo-
trols (86). Although it is difficult to determine whether abnor- lism is altered in both experimental and human cystic diseases.
mal MMP expression is a reflection of a primary abnormality
or a secondary effect, recent data suggest that inhibition of
MMPs may have an impact on the severity of disease in animal AUTOSOMAL RECESSIVE POLYCYSTIC
models of ADPKD and ARPKD (38,87). Overexpression of KIDNEY DISEASE
other basement membrane, ECM, and cell adhesion compo-
nents have also been demonstrated in PKD. Tenacin, an ECM ARPKD is an inherited disorder characterized by cystic
glycoprotein, is abnormally expressed in human ARPKD and dilations of renal collecting ducts and varying degrees of
ADPKD fetal kidneys and in a murine model of ARPKD hepatic abnormalities consisting of biliary dysgenesis and
(88,89). Irregular expression of α-integrin subunits has also periportal fibrosis (97). ARPKD has alternatively been
been demonstrated in fetal PKD kidneys (90). referred to as infantile PKD. This term, however, is gener-
Abnormal processes within the interstitium leading to inter- ally no longer used because of recognition that the disease
stitial inflammation and fibrosis contribute to progression in all can present any time from the prenatal period through ado-
cystic kidney diseases. For instance, monocyte chemoattractant lescence. Furthermore, other forms of PKD, including
protein-1, a chemoattractant and mediator of interstitial ADPKD, can present in the neonatal period (98).
inflammation, is upregulated in ADPKD rats (91). In addition,
oxidant stress is increased and protective effects of antioxidants
Epidemiology, Genetics, and
decreased in the kidneys of animal models of both ARPKD and
Prenatal Diagnosis
ADPKD models (92). Abnormalities in steroid and lipid
metabolism have also been demonstrated in murine ARPKD Based on published reports, ARPKD occurs at an incidence
(93–95). Angiogenesis may have a role in the pathogenesis of of 1 in 10,000 to 1 in 40,000 pregnancies (99,100). The fre-
 36. Polycystic Kidney Disease 679

quency of the gene in the population is estimated to be cally dependent on confirming the diagnosis of ARPKD in
approximately 1 in 70 (101). However, the exact incidence the affected sibling (104). Diagnostic criteria as modified
is unknown, because published reports vary in the popula- from Dell and Avner and Zerres et al. (97,105) include
tions studied (e.g., autopsied patients vs. survivors), and
1. Ultrasonographic features typical of ARPKD, includ-
affected children may die in the perinatal period without a
ing enlarged, echogenic kidneys with poor cortico-
definitive diagnosis. With improvements in neonatal man-
medullary differentiation
agement leading to improved survival rates, as well as formal
2. One or more of the following:
reporting mechanisms (such as a newly developed ARPKD
a. Absence of renal cysts in both parents when ages
registry), more accurate incidence rates may become estab-
older than 30 years
lished. Consistent with autosomal recessive disease, het-
b. Clinical, laboratory, or radiographic evidence of
erozygotes (carriers) are unaffected. The recurrence risk for
hepatic fibrosis
subsequent pregnancies is 25%, and unaffected siblings have
c. Hepatic pathology demonstrating characteristic duc-
a 66% risk of being a carrier for ARPKD (97). Males and
tal plate abnormality
females are affected equally, and ARPKD affects all racial
d.Previous affected sibling with pathologically con-
and ethnic groups.
firmed disease
In 1994, Zerres et al. (102) localized PKHD1 to a region
e. Parental consanguinity suggestive of autosomal reces-
on chromosome 6p21. To date, all kindreds with features
sive inheritance
typical of ARPKD have demonstrated linkage to the 6p21
locus (101). Thus, there is no evidence for genetic hetero- Although PKHD1 has been cloned and identified, direct
geneity in patients with the typical features of ARPKD. mutation analysis is currently not available for clinical pur-
However, a kindred with features of ARPKD as well as poses due to its large size and the presence of multiple exons.
additional extrarenal abnormalities, including skeletal and
facial anomalies, has recently been described, and linkage to
Pathogenesis
the 6q21 locus excluded (103).
In 2002, the gene for ARPKD was cloned by two indepen- Despite recent advances in the understanding of the molecu-
dent research groups (4,5). PKHD1 spans a region of more lar genetics of ARPKD, the pathogenesis remains undefined.
than 400 kilobases (kb) of genomic DNA and contains at least Analysis of PKHD1 mutations in 14 probands demonstrated
66 and possibly over 86 exons. The mRNA for the gene is pro- six truncating and 12 missense mutations throughout the
duced as multiple alternative transcripts. The primary trans- gene (4). Eight affected patients were actually compound
cript of approximately 14 to 16 kb in length encodes a novel heterozygotes. However, because the actual function of fibro-
protein termed fibrocystin (alternatively named polyductin). cystin has not been determined, the mechanism by which
Fibrocystin is a large protein with a predicted molecular weight abnormalities in this protein produce renal cysts and hepatic
of 447 kDa, similar in size to polycystin 1. The precise func- fibrosis is unknown. It is anticipated that with greater under-
tion of fibrocystin is unknown at present. However, protein standing of the function of fibrocystin, genotype-phenotype
modeling suggests that it is a membrane-bound protein with studies in the future may be informative.
immunoglobulin-like properties, including the presence of Insights provided by several murine models of ARPKD
several TIG/IPT domains (immunoglobulin-like folds shared (Table 36.2), especially those for which the causative gene
by plexins and transcription factors). These motifs suggest that has been identified, may shed light on the pathogenesis of
fibrocystin may function as a receptor. Several alternative this disease.
transcripts described lack the transmembrane domain, suggest- The pck rat developed spontaneously in a colony of
ing that (if translated) they may result in production of Sprague-Dawley rats and was initially reported to be a model
secreted forms of fibrocystin as well (5). Northern analyses and of ADPKD in 2001 (106). Affected animals develop progres-
reverse transcriptase-polymerase chain reaction demonstrate sive cystic enlargement of the kidneys after the first week of
that PKHD1 is expressed in both fetal and adult kidney, and to life. Renal cysts develop from several nephron segments,
a much lesser extent in liver, pancreas, and lung. Expression in including loop of Henle, distal tubules, and collecting ducts.
other organs was not seen (4,5). Liver cysts are evident at birth. In addition, pck rats develop a
Prenatal diagnosis may be made in a family with at least liver lesion consistent with Caroli’s disease (congenital hepatic
one known affected child through the technique of linkage fibrosis) (107). Segmental and saccular dilatation of the intra-
analysis (see Chapter 18). Linkage analysis uses analysis of hepatic bile ducts is first seen in utero and progresses with age.
polymorphic markers that flank the location of a known A ductal plate abnormality with overgrowth of portal connec-
disease gene to “track” the disease. This technique can also tive tissue is seen. Proliferative activity of biliary epithelium is
be used to identify whether the unaffected sibling is a car- increased compared to controls. Rates of apoptosis in biliary
rier of the disease. In informative families, the accuracy of epithelium are lower than controls in the first week of life, then
prenatal diagnosis using linkage analysis was greater than increase to greater than controls after 3 weeks (107). The sever-
95% (104). An accurate genetic diagnosis, however, is criti- ity of the kidney disease was found to be greater in males,
680 VI. Tubular Disease

TABLE 36.2. MURINE MODELS OF POLYCYSTIC KIDNEY DISEASE (PKD)a

Mode of Chromosome
Species Gene/mutant inheritance location Protein product Function/role/comments

Mouse
Bpk AR 10 Bicaudal RNA-binding protein
Cpk AR 12 Cystin Cilia-associated protein
Inv AR 4 Inversin Role in left-right axis development
Jck AR 11 Nek8 Function unknown
Kat AR 8 Nek1 Function unknown
Jcpk AR 10 ? Allelic with bpk
orpk (TgN737) AR 14 Polaris Role in left-right axis development; cilia-
associated protein
Pcy AR 9 ? Phenotype consistent with ADPKD
Rat
Han-SPRD(Cy) AD 5 ? Function unknown
Pck AR 9 Fibrocystin (poly- Some clinical features of ADPKD
ductin)
Wpk AR 5 ? Function unknown

AD, autosomal dominant; AR, autosomal recessive.

aKnock-out models for PKD are not included in this listing.

although the liver disease did not show a similar pattern. analysis demonstrates that cystin is a novel cilia-associated
Although phenotypically similar to both ADPKD and protein that localizes to the apical cilia of renal tubule epi-
ARPKD, the pck trait, which is inherited in an autosomal thelia (114). However, the mechanism by which the muta-
recessive fashion, was found to map to an area syntenic with tion results in polycystic kidneys remains unknown.
the region for human ARPKD. Subsequent genetic studies led The orpk model (Oak Ridge PKD) arose as the result of
to the cloning of human PKHD1 (see Epidemiology, Genetics, large-scale mutagenesis project (6). The mutated gene, Tg737,
and Prenatal Diagnosis) and confirmed that the mutation in has been localized to chromosome 13 (115). Mice homozy-
pck mice lies within the region of PKDH1, and, thus, pck is a gous for the orpk mutation develop polycystic kidneys and
genetic model for human ARPKD (4). The question of why hepatic fibrosis. Functional correction (“rescue”) of the renal
this model displays features of both ARPKD and ADPKD but not the liver disease was accomplished by expression of
remains unanswered at this time. cloned wild-type Tg737 in affected orpk animals (116,117).
Recent studies of two murine ARPKD models, cpk and The Tg737 protein product, polaris, appears to have a key role
orpk, suggest that ARPKD may result, in part, from disrup- in development, in particular left/right axis determination
tions in some function or functions of cilia present on the (118). Of note, however, polaris has also been found to localize
apical surface of the renal tubule cells. The cpk (congenital to the cilia of Madin-Darby canine kidney cells, and cells
PKD) mouse model arose from a spontaneous mutation in derived from collecting tubules of orpk mice demonstrate
C57BL/6J mice (108) and is the most well-studied ARPKD abnormal cilia development (119). Whether the emerging
murine model. Cpk mice appear normal at birth, but starting “cilia hypothesis” of PKD pathogenesis will have relevance to
at postnatal day 5, progressive proximal tubule and collecting human ARPKD (or to ADPKD) remains to be determined.
duct cystic enlargement occurs with prominent tubular cell The pathogenesis of congenital hepatic fibrosis in ARPKD
hyperplasia (109,110). Animals die in renal failure at 3 to 4 remains obscure. Increased expression of both the profibrotic
weeks of age with massively enlarged kidneys (109,111,112). molecules TGFβ and thrombospondin-1 have been demon-
Although the renal abnormalities are consistently present on strated in human ARPKD livers (120). The study of the
a number of genetic backgrounds, the liver phenotype and hepatic abnormality in the pck rat (discussed above) or the bpk
other extrarenal manifestations are highly influenced by mouse model of ARPKD may provide additional insights. The
genetic background effects (95,112,113). latter arose from a spontaneous mutation in an inbred colony
By positional cloning, the cpk gene was localized to of Balb/c mice and results, apparently, from a mutation in a
mouse chromosome 12, and the gene was recently cloned gene encoding the mRNA and targeting protein, bicaudal C
and identified (114). The cpk gene has five exons spanning (121,122). These animals develop massively enlarged kidneys
approximately 14.4 kb of genomic DNA. The complemen- and die of renal failure in the first month of postnatal life. In
tary DNA is 1,856 base pairs (bp) in length, and at least addition to cystic kidneys, bpk animals develop hepatic abnor-
two splice variants are recognized. The mutation in cpk/cpk malities consisting of proliferative intrahepatic biliary tract
mice is a 12-bp and 19-bp deletion, resulting in a frame ectasia and nonobstructive dilation of the common bile duct
shift. The gene encodes a novel protein, cystin. Expression (121). Biliary epithelial hyperplasia, like renal tubule hyperpla-
 36. Polycystic Kidney Disease 681

sia in this model, appears to be mediated by a mitogenic cycle by low columnar or cuboidal epithelium. The glomeruli and
driven by the TGFα/EGF/EGFR axis (123). In addition, both other tubular structures appear to be decreased in number
the renal and hepatic diseases of these mice are ameliorated by because of marked collecting duct ectasia and interstitial
treatment with EGFR tyrosine kinase inhibitor (24). edema. In fetal kidneys, proximal tubular cystic lesions have
also been identified (129), but are largely absent by birth.
The pelvicaliceal system and renal vessels appear normal.
Pathology
Unlike ADPKD, in which the cysts become discontinuous
In the infant and young child, the kidneys are reniform but with the tubule, the cystic tubules in ARPKD are fusiform in
grossly enlarged. Pinpoint opalescent dots are visible on the shape and remain in contact with the urinary stream. Micro-
capsular surface and correspond to cystic cortical collecting dissection studies and scanning electron microscopy demon-
ducts (124). Microscopically (Fig. 36.2A), the cysts are usu- strate that obstruction of urinary flow is not a component of
ally smaller than 2 mm in size (“microcysts”) and have been ARPKD (124,125). With increased patient survival, the
shown by microdissection (125), histochemical, and immu- development of larger renal cysts, interstitial fibrosis, and
nologic studies (126–128) to be dilated collecting ducts lined hyperplasia produces a pattern more like ADPKD (see later)
(130). Gang and Herrin (131) described increasing fibrosis
and inflammation in later specimens from patients who had
typical collecting duct microcysts during infancy. It is unclear
whether the fibrotic change is part of natural course of dis-
ease or caused by environmental factors such as toxins,
hypertension, end-stage renal failure, or hemodialysis.
Some degree of biliary dysgenesis and hepatic fibrosis is
always present in ARPKD. Even in the newborn, the liver
demonstrates microscopic abnormalities. The classic liver
lesion shows a typical ductal plate abnormality consisting of
portal fibrosis surrounding increased numbers of hyperplas-
tic, ectatic biliary ducts with normal hepatocellular histology
(130,132). With time, hepatomegaly and portal hyperten-
sion become evident in many patients. Intrahepatic biliary
ectasia may result in macrocysts and dilation of extrahepatic
bile ducts sometimes resulting in an enlarged gallbladder
(133) or choledochal cysts (134). Although the combination
of collecting tubule and biliary ectasia with periportal fibrosis
is unique to ARPKD, portal fibrosis and bile duct prolifera-
tion may be associated with other types of renal diseases,
including ADPKD (135,136) and cystic dysplasia.

Clinical and Radiographic Features

Historically, ARPKD was originally separated into four dis-
tinct clinical entities based on age at presentation and relative
degrees of renal and hepatic involvement (137). Although
such distinctions present a useful clinicopathologic classifica-
tion, there is often much overlap between the degrees of renal
and hepatic involvement present. ARPKD and congenital
FIGURE 36.2. Kidney histology and ultrasonography of autoso- hepatic fibrosis with renal tubular ectasia are likely different
mal recessive polycystic kidney disease (ARPKD). A: Microscopic phenotypic manifestations of the same genetic abnormality
appearance of a kidney biopsy from a 7-month-old with ARPKD (138). In contrast to ADPKD (see later), intrafamilial variabil-
demonstrating multiple radially oriented collecting tubule cysts
extending from the medulla to the peripheral cortex. A small ity in ARPKD disease phenotype is reportedly small (139).
amount of residual parenchyma contains glomeruli and noncystic The majority of patients with ARPKD present in infancy
tubules situated between the cysts. No glomerular cysts or signs (105,140). In a series of 33 patients with ARPKD who sur-
of renal dysplasia are present. (Hematoxylin and eosin stain; orig-
inal magnification ×1.) (Specimen kindly provided by Dr. Steven vived the neonatal period, 11 were diagnosed in the first days
Emancipator, Case Western Reserve University.) B: Renal ultra- of life, 18 between 1 and 18 months, and four between 6 and
sound (right kidney) of a newborn with ARPKD demonstrates the 11 years of age (141). Rare presentations late in adolescence
typical appearance of echogenic, enlarged kidneys (length = 6.5
cm; normal for age = 4.48 ± 0.62 cm) with poor corticomedullary and even into adulthood have been described (142). With
differentiation. the widespread use of prenatal ultrasound, many patients
682 VI. Tubular Disease

with ARPKD are detected in utero. Prenatal ultrasound may an incidental finding during abdominal imaging. Liver syn-
demonstrate the findings of oligohydramnios, large renal thetic function is usually intact.
masses, or absence of fetal bladder filling (143). By ultrasonography, infants have characteristic large
At birth, patients usually present with large, palpable flank echogenic kidneys with poor corticomedullary differentia-
masses that may be large enough to complicate delivery. tion (Fig. 36.2B). Macrocysts are usually not present,
Urine output is usually normal; however, oliguric acute renal although they may be seen with worsening disease. Renal
failure may occur (99). In that subset of patients, increased macrocysts smaller than 2 cm and increased medullary
urine output and a corresponding improvement in renal echogenicity have been reported in older children (130).
function may be seen after improvement in respiratory status In a study of sonographic features of adult patients with
(144). Most patients (70 to 80%), even without overt renal ARPKD, Nicolau et al. (148) noted the presence of multi-
failure, have evidence of impaired renal function in the new- ple small cysts in a normal-sized kidney, increased cortical
born period (105,145). However, death from renal insuffi- echogenicity, and loss of corticomedullary differentiation
ciency is uncommon in that age group (137). Hyponatremia as common features.
related to a urine dilution defect is often present, but usually Ultrasonographic findings in the liver include hepatomeg-
resolves over time (105,140). Metabolic acidosis has also been aly, increased echogenicity, and poor visualization of the
reported (140,145). As might be predicted from a pathologic peripheral portal veins. Reversal of normal venous flow by
process that affects the collecting tubule, most patients have a Doppler study, suggestive of portal hypertension, may be
urinary concentrating defect and symptoms of polyuria and seen. Macroscopic liver cysts are uncommon (149), although
polydipsia (130,140,141,145). choledochal cysts have been reported (134). A smaller per-
Hypertension, which may be severe, is common in both centage of patients have overt evidence of biliary duct dilata-
infants and children and can be a presenting feature tion (Caroli’s disease).
(99,130). It can be present in patients with normal renal
function and eventually affects almost all children with the
Diagnosis
disease (145). Although the pathophysiology of hyperten-
sion in ARPKD is not clearly understood, peripheral vein Prenatal diagnosis may be suggested by antenatal ultrasound
renin levels are not usually elevated (140,141). If not aggres- findings of enlarged kidneys, oligohydramnios, and absence
sively managed, hypertension can result in cardiac hypertro- of urine in the bladder (150). Sonographic features of
phy and congestive heart failure (CHF), which may be a ARPKD may present in the second trimester but usually are
factor in progression of underlying renal insufficiency. not apparent until after 30 weeks’ gestation (151). Both false-
Pulmonary insufficiency, as manifest by respiratory dis- positive and false-negative results have been reported (152).
tress, is a major cause of morbidity and mortality in neo- However, with newer high-resolution obstetric ultrasonogra-
nates with ARPKD. Pulmonary hypoplasia, resulting from phy, it is probable that diagnostic sensitivity and detection
oligohydramnios, may be complicated by respiratory embar- rates will improve. Wisser et al. (153) reported a case of a fetus
rassment due to massively enlarged kidneys. Additional with pathologically confirmed ARPKD who demonstrated
causes of respiratory distress in these patients include pneu- echogenic, normal-sized kidneys at 15 ± 4 weeks’ gestation.
mothorax and atelectasis, or a variety of common neonatal Although less common, ADPKD in infants may have an
pulmonary disorders such as surfactant deficiency, bacterial antenatal sonographic appearance that is difficult to distin-
pneumonia, meconium aspiration, or persistent fetal circu- guish from ARPKD (98). Increased maternal alpha fetopro-
lation. Severely affected infants may demonstrate the full tein and amniotic fluid trehalase activity have been identified
components of the oligohydramnios sequence, including as potential markers for ARPKD, but neither has been con-
pulmonary hypoplasia, abnormal extremities, and character- firmed as specific or sensitive for disease detection in utero
istic Potter’s facies (146). Infants with true pulmonary hypo- (154,155).
plasia often die soon after birth secondary to pulmonary Although specific clinical diagnostic criteria for ARPKD
insufficiency. have not been established, the clinical constellation of bilat-
A subset of patients with ARPKD may present as older eral palpable flank masses, respiratory distress, and history
infants with abdominal enlargement secondary to enlarged of oligohydramnios and hypertension in an infant is sug-
kidneys or hepatosplenomegaly without the full spectrum gestive of ARPKD. Diagnostic criteria modified from
of clinical symptoms outlined above. Although hepatic Zerres et al. (105) are outlined in Epidemiology, Genetics,
involvement is invariably present microscopically, it may be and Prenatal Diagnosis. For the minority of children who
clinically absent in at least 50 to 60% of neonates (105). In present later in childhood, renal ultrasonographic features
contrast, the small percentage of patients who are diag- may be less reliable. Kidneys are echogenic and large, but
nosed with ARPKD as older children often present primar- massive enlargement is generally not seen. The echogenicity
ily with signs and symptoms of hepatic fibrosis and portal of the kidneys in older children may be similar to that of
hypertension, including hepatosplenomegaly (101,147). In ADPKD, and macrocysts more typical of ADPKD may be
fact, cystic renal disease in these patients may be detected as present (156). As noted, in the subset of patients who
 36. Polycystic Kidney Disease 683

present as older children and adolescents, hepatic abnor- has been reported as a common complication in at least one
malities are often the prominent presenting feature. uncontrolled series (145), but it is unclear whether children
Magnetic resonance imaging (MRI) shows enlarged kid- with ARPKD truly have an increased incidence of upper or
neys with hyperintense T2-weighted signals. A characteristic lower UTIs when compared with appropriately age-matched
hyperintense, linear radial pattern in the cortex and medulla controls. Thus, as in any child with an abnormal urinalysis,
representing microcystic dilatation has been described on clinical features and appropriately obtained urine cultures
RARE-MR urography (17). Radionuclide studies, such as must guide antibiotic therapy. If a UTI is documented, a
renal technetium-99m DMSA and liver HIDA imaging, voiding cystourethrogram and renal ultrasound should be
may also aid in establishing the diagnosis of ARPKD (158). performed to determine the possible presence of vesi-
As noted previously, genetic testing is available at present coureteral reflux and rule out obstruction or superimposed
only for families that already have an affected child. With upper tract structural abnormalities (131). Microscopic or
the recent cloning and ongoing characterization of PKHD1, gross hematuria and proteinuria may also be seen on occa-
however, it is possible that direct mutation analysis may sion (99,145). In infants and children who develop renal
become available in the future. insufficiency, the consequences of chronic renal failure (e.g.,
growth failure, anemia, and osteodystrophy) become appar-
ent as renal function decreases.
Treatment and Complications
Difficulties in feeding, even in patients without renal
Survival of neonates with ARPKD has improved in concert insufficiency, are often noted. This is presumably due to the
with overall advances in neonatal artificial ventilation and presence of enlarged kidneys or liver, or both, interfering
intensive care. It is currently impossible to predict which neo- with normal gastrointestinal function. Supplemental feed-
nates with ARPKD requiring immediate artificial ventilation ing via nasogastric or gastrostomy tubes may be required to
have critical degrees of pulmonary hypoplasia incompatible optimize weight gain and growth.
with survival (1,145). In some instances, severe pulmonary Dialysis or transplantation, or both, are indicated when
distress may be secondary to potentially reversible fluid children with ARPKD reach end-stage renal failure. Perito-
overload, neonatal lung disease, or restricted diaphragmatic neal dialysis in ARPKD is often successful even in the face
motion secondary to massively enlarged kidneys. In selected of large kidneys and hepatosplenomegaly. Kidney trans-
cases, some authors have advocated continuous venovenous plantation offers definitive renal replacement therapy in
hemofiltration, unilateral nephrectomy, or bilateral nephrec- children with ARPKD. Successful kidney transplantation
tomy coupled with peritoneal dialysis to allow optimal ventila- prolongs survival and often accelerates growth and develop-
tion and thereby assess the long-term pulmonary prognosis of ment in young uremic children. Nephrectomies may be
the patient (159–161). indicated before, or at the time of, transplantation to con-
Infants and young children without significant renal trol hypertension and/or to permit room for transplant
insufficiency must be followed closely. Because most chil- placement in patients with massively enlarged kidneys.
dren with ARPKD have a concentrating defect, significant With improved patient survival and advances in renal
dehydration is a particular risk during intercurrent illnesses replacement therapy, hepatic complications progressively
that may increase insensible water loss (e.g., through fever), dominate the clinical picture of many patients with ARPKD
limit free water intake (e.g., through nausea), or increase (105,147,162). The older child may have complications of
extrarenal water loss (e.g., through vomiting or diarrhea). hepatic fibrosis and portal hypertension (130,137). These
In patients with severe polyuria, thiazide diuretics may be include hepatosplenomegaly, bleeding esophageal varices,
of benefit to decrease distal nephron solute and water deliv- portal thrombosis, and hypersplenism causing thrombocy-
ery. Supplemental bicarbonate therapy is required for those topenia, anemia, and leukopenia.
with metabolic acidosis. A serious and potentially lethal complication in ARPKD
Hypertension can be difficult to manage and may require patients with significant hepatic involvement is bacterial
multiple medications (99). Despite the fact that peripheral cholangitis, which has been reported as early as a few weeks
vein renin values are not usually elevated in hypertensive of age (145). Fever or elevation of liver function tests at any
ARPKD patients, most patients respond well to angioten- time should lead to the suspicion of cholangitis and result
sin-converting enzyme (ACE) inhibitors, which are the in complete evaluation and appropriate antimicrobial ther-
treatment of choice. If additional medications are required, apy. However, patients may not present with the classic
calcium channel blockers, β-blockers (in those without clinical findings of cholangitis, and the diagnosis should be
chronic lung disease or signs of CHF), and diuretics are strongly considered in ARPKD patients with unexplained
appropriate choices. recurrent sepsis with gram-negative organisms (163).
Urinary abnormalities may be present or develop over the In infants and children with hepatic involvement, close
course of disease. Pyuria is a relatively common finding and monitoring for complications of portal hypertension is man-
can be seen in the absence of demonstrable bacteriuria or dated, particularly as typical liver function tests (e.g., serum
documented infection (130). Urinary tract infection (UTI) albumin and transaminases) are normal. Yearly ultrasonogra-
684 VI. Tubular Disease

phy to determine changes in liver or spleen size or identify or early adolescence. Life-table analysis of a cohort of
portal hypertension by reversal of venous flow is noninvasive patients surviving the first month of life revealed a renal
and may be of value. Endoscopy is necessary to evaluate sus- survival of 86% at 1 year and 67% at 15 years (147).
pected esophageal varices that can be treated by sclerother- As noted, with the success of renal transplantation and
apy or banding before life-threatening hemorrhage. Periodic improved survival of patients with ARPKD, morbidity and
monitoring should reveal the hematologic profile of hyper- mortality of complications related to congenital hepatic
splenism. Sudden worsening of anemia should raise the pos- fibrosis are more common and clinically relevant. Khan et
sibility of occult gastrointestinal blood loss secondary to al. recently reported the outcome of 14 patients with
splenic sequestration or variceal bleeding. Portosystemic ARPKD after renal transplantation (167). With a mean
shunting may be indicated in some cases (140,164). How- follow-up of 14 years, the study showed 1-and 5-year patient
ever, Tsimaratos et al. (165) recently reported recurrent survival rates of 93% and 86%, respectively. Overall, 36% of
hepatic encephalopathy leading to death in two children patients died, and in four of five of those patients, death was
with portocaval shunts who progressed to ESRD. It has been directly related to complications of hepatic disease. In those
hypothesized that loss of kidney function results in impaired who survived, 63% had portal hypertension. Thus, compli-
clearance of toxins that are shunted from the liver. This find- cations of CHF developed in almost 80% of patients after
ing has raised concerns about whether liver transplantation renal transplantation for ARPKD.
should be considered as an alternative therapy for ARPKD
patients with portal hypertension being evaluated for possi-
ble shunts. With the increased use and successful outcome of AUTOSOMAL DOMINANT POLYCYSTIC
living-related partial liver transplants, this option may be KIDNEY DISEASE
more viable than previously considered. In fact, sequential
liver and kidney living-related transplants have recently been ADPKD is a systemic inherited disease characterized by
reported (166). progressive cystic enlargement of the kidneys coupled with
In addition to the significant medical problems noted, variable extrarenal manifestations involving the gastrointes-
the psychosocial stresses of ARPKD on the patient and tinal tract, cardiovascular system, reproductive organs, and
family can be overwhelming. Social support measures and the brain (168). ADPKD has alternatively been called adult
periods of respite care are often necessary. A team approach PKD. However, this term is a misnomer because ADPKD
using the skills of pediatric nephrologists in concert with has been diagnosed in the fetus, newborn, and older child
other pediatric medical subspecialists, specialized nurses, and adolescent (99,141,169,170).
dietitians, social workers, psychiatrists, and other support
staff is required to provide optimal comprehensive care for
Epidemiology, Genetics, and
children with ARPKD.
Prenatal Diagnosis
ADPKD is the most common inherited human kidney dis-
Prognosis
ease and occurs at an incidence of approximately 1 in 1000.
Prognosis is difficult to assess, although it is now clear that It affects all races, and males and females are both affected;
survival of all but the most severely affected neonates who however, the phenotype may be more severe in males (171).
demonstrate pulmonary hypoplasia is possible (1,130). ADPKD is a rare cause of ESRD in the pediatric popula-
Published reports vary with respect to neonatal survival tion, but accounts for approximately 5 to 10% of ESRD in
rates, but suggest that approximately 70% of patients sur- adults. The two major disease-causing genes are PKD1 and
vive the newborn period with aggressive neonatal intensive PKD2. In the general population, PKD1 accounts for
care (140,145,147). Actuarial survival rates calculated from approximately 85% of ADPKD, with PKD2 accounting
birth for 55 patients with ARPKD referred to a pediatric for the remaining 15%. A third ADPKD locus (PKD3, or
tertiary care center revealed that 86% were alive at 3 non-PKD1 or PKD2) has been suggested by a few case
months, 79% at 1 year, 51% at 10 years, and 46% at 15 reports (172–174). Mutations in PKD1 and PKD2 produce
years (140). Calculations based on patients who survived to similar phenotypes; however, the age of onset of cystic dis-
1 year of age showed that 82% were alive at 10 years and ease, hypertension, and renal insufficiency is delayed in the
79% at 15 years (140). Patients who survive the neonatal latter (175–177).
period usually have a decreased glomerular filtration rate PKD1 has been mapped to chromosome 16p13.3 (178).
(GFR), but studies have demonstrated subsequent Its exact location was pinpointed because it was bisected by
improvement in renal function consistent with some degree a chromosome translocation; members with the balanced
of continued renal maturation (99). Indeed, GFR may be exchange had PKD (2). Like PKHD1, PKD1 is a very large
normal by 12 months of age, and those patients with neo- gene, spanning 53 kb of genomic DNA, with 46 exons
natal presentations who survive past 1 month of age gener- encoding a 14.5-kb transcript (179). A portion of the gene
ally do not develop renal insufficiency until late childhood is duplicated in the proximal portion of chromosome 16.
 36. Polycystic Kidney Disease 685

The gene encodes a large 4304–amino acid protein prod- and a calcium-dependent lectin domain. The presence of the
uct, polycystin-1, a novel 460-kDa protein that is predicted REJ domain is particularly intriguing because it may be a
to be involved in cell-cell or cell-matrix interactions (180). potential regulatory site for the molecule (192). These struc-
PKD2 has been linked to chromosome 4q13-q23 and was tural components, taken as a whole, suggest that the extracellu-
cloned and identified in 1996 (3,181). The PKD2 gene lar portion of polycystin-1 may be capable of binding an as yet
expresses a 5.4-kb mRNA, which encodes a 968–amino acid undefined ligand (191). Polycystin-1 is anchored to the cell
polypeptide, polycystin-2 (3). Polycystin-2 has been recently membrane by 7 to 11 putative transmembrane domains. The
shown to be a nonselective voltage-gated ion channel (182). carboxy-terminus (intracellular portion) of polycystin 1 con-
Prenatal genetic diagnosis is available for “informative” tains several phosphorylation sites and a putative coiled-coil
families with suitable pedigree structures using the technique domain, which has been demonstrated to interact in vitro with
of linkage analysis (141). In addition, techniques for accurate the carboxy-terminus of the protein product of the PKD2 gene
mutation screening by DHPLC for both PKD1 and 2 are (193,194). These numerous features suggest that polycystin-1
currently being refined, but the mutation detection rate has is a large, multifunctional molecule that is involved in carbohy-
not been fully determined (168,183). As with any prenatal drate motif recognition, ligand binding, and Ca2+ regulation. It
genetic testing, the question arises as to whether termination may engage in cell-cell and/or cell-matrix interactions, which
of an affected pregnancy is an acceptable option for couples, regulate signal transduction pathways mediated by cell-surface
especially given the wide clinical spectrum and the treatable protein-protein interactions.
nature of the disease (i.e., treatment of chronic renal failure Defining the expression pattern of polycystin-1 has been
and dialysis or transplantation) (184). In the majority of kin- hampered, in part, by discrepancies in the reported tissue and
dreds, fetuses harboring an ADPKD mutation will not show cellular localization of the protein. This is likely due to issues
any obvious renal or other abnormalities, and patients may related to antibody specificities (191). However, it appears that
be asymptomatic for two to three decades. Potential benefits polycystin-1 is expressed in multiple tissues, including kidney,
of prenatal diagnosis, either by genetic testing or ultrasonog- liver, pancreas, intestine, and cerebral blood vessels (all sites of
raphy, include early genetic counseling, careful preparation cystic changes in ADPKD), as well as in the lung, testis, and
for management of an affected fetus at and after delivery (in other tissues (195–198). The protein is overexpressed in the
the case of a fetus with clinical evidence of disease), and close cystic epithelium of ADPKD kidneys (195), and these cells
follow-up of an affected child. However, diagnosing remain phenotypically immature. Immunohistochemistry
ADPKD years before the onset of symptoms has potential studies located the protein in renal tubular epithelial cells of
major adverse outcomes that must be considered, including human fetuses, diminishing in abundance with age but with
psychological stress on the child and family and potential dis- persistent expression into adulthood (195,199), suggesting a
crimination in employment, financial, and insurance matters role in renal development. Further evidence of the importance
(185,186). Surveys of ADPKD families indicate that only of polycystin-1 in development was provided by knock-out
4% would consider pregnancy termination if the fetus were mouse models in which a variety of mutations have been
affected (187). Genetic linkage analysis for PKD1 or PKD2 introduced that result in the loss of functional polycystin. Ani-
is not routinely performed for prenatal diagnosis in the mals lacking polycystin-1 die in utero or soon after birth and
United States or Europe (188). demonstrate abnormalities in multiple organs, including the
heart, blood vessels, kidneys, and pancreas (200–202). The
subcellular localization of polycystin-1 in renal tubular epithe-
Pathogenesis
lium appears to shift during development from a predomi-
Despite the extraordinary advances in the 1990s in defining nantly basal staining in fetuses to apical/lateral staining in
the genetics of ADPKD, the precise mechanisms by which adult kidneys (203).
mutations in the PKD1 and PKD2 genes result in the for- Although the precise function of polycystin-1 is unknown,
mation of renal and extrarenal cysts is unknown (189). emerging data regarding its binding partners may shed light on
its role in normal and cystic kidney epithelium. As noted,
polycystin-1 has recently been shown to bind polycystin-2 at
PKD1
the coiled-coil domain. Polycystin-2 is a cation channel, and it
Polycystin-1 possesses a large extracellular domain, transmem- has been suggested that polycystin-1 plays a role in regulation
brane spanning regions, and an intracellular carboxy-terminus of that channel. Results of immunohistochemistry and func-
(190,191). The extracellular domain is dominated by the pres- tional studies suggest that polycystin co-localizes with and
ence of 16 copies of an 80–amino acid immunoglobulin-like forms large complexes with a wide variety of proteins, includ-
repeat, the PKD domain. Although the function of this ing those involved with cell-matrix interactions (such as α2β1
domain is unknown, PKD domains in several other proteins integrins and focal adhesion complexes) as well as cell-cell
are located on their extracellular surface and are highly glycos- interactions (including E-cadherin-β-catenin complexes)
ylated. The amino-terminal domain of polycystin 1 also con- (191,203,204). Additional data demonstrate that polycystin-1
tains a leucine-rich repeat, an LDL-A domain, an REJ domain, induces resistance to apoptosis and spontaneous tubulogenesis
686 VI. Tubular Disease

in Madin-Darby canine kidney cells (205). Nickel et al. (206) loop of Henle and the distal convoluted tubule, where it
reported that the C terminal of polycystin-1 triggers branching localized to the basolateral plasma membrane of kidney
morphogenesis and migration of inner medullary collecting tubular epithelium (213). Polycystin-2, like polycystin-1, is
duct cells and supports in vitro tubule formation. These studies expressed in the vasculature, including porcine aorta and
suggest a potential connection between altered polycystin-1 normal human elastic and intracranial arteries (214).
function and cystogenesis (204). Recently, Koulen et al. (182) confirmed by single channel
ADPKD is characterized by considerable intrafamilial studies that polycystin-2 (a member of a subfamily of the
phenotypic variation. By analyzing two closely linked poly- transient receptor potential channel superfamily) functions
morphic markers within the PKD1 gene, Qian et al. revealed as a calcium-activated intracellular ion release channel in
that the renal epithelia from single cysts are monoclonal, vivo and hypothesized that PKD results from the loss of reg-
containing only the mutant haplotype (207). A subsequent ulation of an intracellular calcium release signaling pathway.
study by Brasier et al. (208) confirmed these findings. These Multiple mutations in PKD2 have been identified in
two studies suggest that patients harboring a germline muta- affected families, and as with PKD1, most families have
tion in the one allele of a PKD gene undergo a somatic “sec- unique mutations (215). These mutations truncate polycys-
ond hit,” which results in the loss of the remaining normal tin-2 and appear to be loss-of-function mutations. Similar
allele and genetic homozygosity in the affected cell or cells. to PKD1, mice homozygous for targeted mutations in
These studies provide a possible molecular explanation for PKD2 die in utero or soon after birth and demonstrate car-
both the focal nature of cysts (only 1 to 2% of tubules may diac defects in septum formation as well as kidney and pan-
be cystic in ADPKD) as well as the phenotypic variability creatic cysts (216). Although there is significant genetic and
within families harboring the same germline mutation. phenotypic variability in patients with PKD2 mutations, it
To date, multiple different mutations throughout the has recently been suggested that certain clusters or groups
PKD1 gene have been identified in patients with ADPKD, of mutations may be associated with more severe clinical
with no particular “hot spots” for mutations identified disease (217).
(209). The majority of mutations are predicted to result in
truncation of the polycystin-1 protein. Of note, several kin-
Pathology
dreds were identified that had tuberous sclerosis coexistent
with severe childhood-onset ADPKD. These kindreds were In ADPKD (Fig. 36.3A), kidney cysts form in glomeruli and
subsequently found to have large deletions in an area con- all tubular segments, although the proximal tubule is a com-
taining both PKD1 and adjacent tuberous sclerosis 2 mon site of involvement. Glomerular cysts may be seen as a
(TSC2) genes, resulting in a contiguous gene syndrome component of ADPKD or as a separate disease entity. Unlike
(210). Another kindred has been identified exhibiting ARPKD, in which the cystic dilatations are fusiform in nature
cosegregation of an overlapping connective tissue disorder and remain in connection with the tubular lumen, in ADPKD
and ADPKD 1 (211). Although consistent genotype/phe- the enlarging cysts eventually “pinch off” and become discon-
notype correlations have not been demonstrated to date, nected from the tubular lumen and urinary space.
Rosetti et al. (212) recently found that even in light of sig-
nificant inter- and intrafamilial phenotypic heterogeneity,
Clinical and Radiographic Features
patients with mutations in the 5' region of PKD1 had sig-
nificantly more severe disease than those with mutations in Patients with ADPKD are usually diagnosed and become
the 3' portion of the gene. The location, rather than the symptomatic in adulthood. However, children affected with
type of mutation, was found to be the factor that correlated ADPKD may become symptomatic or be diagnosed as an
with the onset of ESRD. incidental finding. The clinical spectrum of pediatric
ADPKD ranges from severe neonatal manifestations indis-
tinguishable from ARPKD to renal cysts noted on ultra-
PKD2
sound in asymptomatic children (1,98,99,141,169). The
Polycystin-2 contains six transmembrane regions and has diagnosis of ADPKD has been made in utero by ultrasound,
intracellular domains at both its amino- and carboxy- and affected newborns can present with Potter’s phenotype
termini. The transmembrane regions share significant and die from pulmonary hypoplasia. Affected infants can be
homology with voltage-activated Ca2+/Na+ channels, which born with large hyperechoic kidneys with or without macro-
suggests that polycystin-2 could be a channel protein. The cysts and variable degrees of renal insufficiency. As with
carboxy-terminus contains an EF-hand domain that binds ARPKD, hypertension can present during the newborn or
Ca2+. The carboxy-terminus also contains several potential infant periods and is common even in pediatric patients
phosphorylation sites. By immunostaining of human and with normal renal function (141,169,218). Hypertension
murine renal tissues, polycystin-2 was found to be widely appears to be mediated by reduced renal blood flow, activa-
expressed and was present in the kidney. The highest levels tion of the renin-angiotensin system, and increased sodium
of expression within the kidney were the thick ascending retention (219,220).
 36. Polycystic Kidney Disease 687

than ADPKD (145,222). Renal infections are common in

adult patients with ADPKD and can be a presenting feature
in the affected infant and child (141). Potential complica-
tions of pediatric renal infection include pain, perinephric
abscess, hemorrhage, chronic pyelonephritis, sepsis, and
death. It has been reported that the risk of pyuria and bacte-
riuria in ADPKD increases progressively from 2% in the sec-
ond decade to 32% in the seventh decade.
In families with known ADPKD, asymptomatic children
may be identified by ultrasonographic examination (Fig.
36.3B) (169). In children at risk, the presence of even single
cysts in normal-sized kidneys is highly predictive of future
development of symptomatic ADPKD (169). In children with
ADPKD, renal involvement is commonly asymmetric (includ-
ing asymmetric kidney enlargement) and is unilateral in a
small minority (223). The extrarenal features of ADPKD seen
commonly in adults (224,225) are only rarely seen in pediatric
patients. When present, they can sometimes help to clinically
differentiate ADPKD from ARPKD. Although hepatic, pan-
creatic, or ovarian cysts are rarely detected before puberty, they
have been reported in affected children in the first year of life
(226,227). Liver cysts in children are not generally associated
with pain, infection, and hepatomegaly as noted in adult
patients. Congenital hepatic fibrosis with severe portal hyper-
tension in children and adults with ADPKD has been reported
(135,136). The presence of pancreatic cysts has been found
exclusively in PKD1 patients, and they do not appear to con-
tribute to morbidity or mortality (228). Similarly, although
rarely detected before the age of 20 years, there are reports of
clinically significant cerebral vessel aneurysms in pediatric
ADPKD patients (229). The increased incidence of cardiac
valvular abnormalities, such as mitral valve prolapse, com-
monly seen in the adult ADPKD population (224,230), has
also been reported in children with ADPKD (231). There
have also been several reports of endocardial fibroelastosis in
FIGURE 36.3. Kidney histology and ultrasonography of autoso- children with ADPKD (232,233). Increased left ventricular
mal dominant polycystic kidney disease (ADPKD). A: Microscopic mass and Doppler abnormalities consistent with early diastolic
appearance of a kidney biopsy from an adult with ADPKD demon-
strating multiple thin-walled cysts of varying sizes involving differ- dysfunction has been reported to occur in normotensive chil-
ent nephron segments. Focal hemorrhage is noted within some dren and young adults with ADPKD (234). An increased risk
cysts. (Hematoxylin and eosin stain; original magnification ×1.) of coronary aneurysms has been reported in adults, but no
(Specimen kindly provided by Dr. Steven Emancipator, Case West-
ern Reserve University.) B: Renal ultrasound (right kidney) of a 13- pediatric cases have been reported to date (235).
year-old with ADPKD demonstrates several cysts (the largest mea-
suring 1.5 cm × 1.6 cm). The left kidney also had several cysts not
present on an ultrasound 2 years before this study. Kidneys are 11.4 Diagnosis
cm (right) and 11.7 cm (left) (normal for age = 9.79 cm ± 1.5 cm).
There are no specific clinical diagnostic criteria for children
with suspected ADPKD. Prenatal diagnosis is suggested by
In older children, presenting symptoms include abdomi- antenatal ultrasound findings of enlarged kidneys with or
nal pain, palpable abdominal masses, gross or microscopic without cysts and absent urine in the bladder. However,
hematuria, UTIs, abdominal or inguinal hernias, and hyper- these findings may not be evident until the third trimester.
tension. The occurrence of gross hematuria after seemingly (236,237). In families not known to have ADPKD, diag-
minor trauma to the flank region should raise the possibility nosis of ADPKD in the fetus or young child can lead to the
of ADPKD (or obstructive uropathy). Renal insufficiency is diagnosis of ADPKD in asymptomatic adults after parental
rare but can occur in childhood (169,221). A concentrating radiographic studies. Because simple cysts are extremely
defect may be present, leading to polyuria and polydipsia, rare in childhood (238), the finding of even one renal cyst
but this is more consistently a clinical feature of ARPKD should alert the clinician to the possibility of ADPKD. In
688 VI. Tubular Disease

pediatric patients with a 50% risk of ADPKD, the finding features of UTIs or renal cyst infections in pediatric ADPKD
of one cyst is considered diagnostic. Ultrasonographic patients, it is reasonable to assume that their clinical course is
screening of asymptomatic pediatric patients at risk is an similar to that described for adult ADPKD patients (254).
issue of some debate. Because cysts may not be evident Sterile pyuria is common, and appropriate cultures are needed
until adulthood, the finding of a negative ultrasound may to determine whether an infection is present. Most renal
be falsely reassuring (239). Conversely, there may be signif- infections are caused by gram-negative enteric organisms and
icant psychosocial and financial implications of a diagnosis can be complicated by cyst infection. Eradication of cyst
of ADPKD in an asymptomatic patient who may not infections is often difficult, despite in vitro sensitivity of
develop clinical signs of disease for several decades. If responsible organisms; thus, the use of antibiotics that pene-
ADPKD is clinically suspected, family members (including trate cyst walls is mandated (255). Antibiotics that generally
the grandparents if the parents are younger than 30 years) penetrate cyst walls include ciprofloxacin (256) and sulfon-
should be considered for radiographic evaluation (240). As amides. Penicillins and aminoglycosides (standard treatments
noted previously, genetic testing in the form of linkage for UTI) are generally ineffective in treating cyst infection
analysis is available. Direct mutation screening is available, (255,257). Aggressive antibiotic treatment is critical because
but accuracy has not been fully determined, particularly for recurrent or ineffectively treated UTIs appear to be a definite
PKD1 mutations. An up-to-date listing of laboratories cur- risk factor in progression of renal disease (258). Occasionally,
rently performing genetic testing for PKD for clinical or cyst drainage may be required to control infection, and MRI
research purposes is available at www.geneclinics.org. may be a useful modality for identifying which cyst is infected
(259). In extreme cases, nephrectomy may be indicated (255).
Prophylactic antibiotics should be considered before the
Treatment and Complications
introduction of any urinary tract instrumentation in children
At present, there are no disease-specific therapies for ADPKD. with ADPKD.
However, a number of interventions have been shown to slow Episodes of flank pain are unusual in pediatric patients
progression of disease in animal models, including dietary flax- with few cysts. With progressive disease, however, particu-
seed, soy protein or protein restriction, sodium citrate, caffeine larly in certain adolescents, flank pain may become a more
restriction, and EGFR tyrosine kinase inhibition (241–247). prominent feature. In the majority of instances, the painful
To date, none has proven to significantly alter the clinical episodes resolve within a few days. Pain relief is accomplished
course of disease in humans (248). with acetaminophen or brief courses of oral narcotics. Non-
Asymptomatic children at risk for ADPKD should be steroidal antiinflammatory agents should be avoided. Long-
followed annually for the development of hematuria, term narcotic use is discouraged due to abuse potential.
hypertension, or palpable abdominal masses. Any of these Nonpharmacologic interventions and referral to a chronic
findings is an indication for ultrasound examination and pain management center should be considered (260). In
close clinical follow-up. Renal insufficiency and ESRD in cases of severe pain, laparoscopic cyst decortications may be
the child with ADPKD is treated with standard medical performed (261).
management of chronic renal failure and renal replacement As noted, hepatic cysts are rare in the pediatric popula-
therapy as indicated (see Chapters 70 through 74). As with tion. Patients with hepatic cysts may develop cyst infections.
other forms of chronic kidney disease, hypertension man- Typically, these infections present as right upper quadrant
agement is essential in slowing progression to ESRD in pain, fever, leukocytosis, and a rise in liver enzymes (262).
PKD. It has been suggested that ACE inhibitors, angioten- Antibiotics alone may be ineffective, and the addition of sur-
sin II receptor antagonists, and chymase inhibitors may gical drainage is generally recommended (263). Intestinal
offer benefits in addition to antihypertensive effects (249– diverticular disease (224) has not been reported in pediatric
251); however, this has not been confirmed. A longitudinal ADPKD patients.
study of children with ADPKD treated with ACE inhibi- Routine cerebral arteriography is not recommended for
tors is currently under way, and a larger scale, multicenter screening of possible intracranial aneurysms in pediatric
National Institutes of Health trial is being planned to ADPKD patients. The optimal age for screening and the nat-
address the question in adults. Of note, reversible acute ural history of aneurysm development are unknown in pedi-
renal failure may be precipitated by ACE inhibitors in atric patients. Although aneurysms are found in patients
ADPKD patients with diminished kidney function and with negative family histories, intrafamilial clustering of
massive cystic involvement (252). aneurysms has been reported in ADPKD populations
A number of renal and extrarenal complications may occur (264,265). Thus, it may be reasonable to consider investiga-
in patients with ADPKD. In pediatric patients, gross hema- tion of children with positive family histories of cerebral
turia and UTI, in particular, cyst infection, are the more com- aneurysms or those with symptoms such as headache. Use of
mon. Flank pain may also be seen. Renal calculi, a common magnetic resonance angiography may permit effective, non-
finding in adult patients with ADPKD (253), are rare in invasive detection of significant aneurysms (266). Even in
childhood. Although no data are available regarding specific adults, however, routine screening by magnetic resonance
 36. Polycystic Kidney Disease 689

angiography is generally reserved for those patients with or a sporadic occurrence. Reports exist of infants with
symptoms or a positive family history. GCKD who have family members affected with ADPKD,
which raises the question of whether these two entities are
different expressions of the same genetic defect. Sporadic
Prognosis
GCKD and GCKD occurring in the context of familial
The prognosis of ADPKD presenting in the fetus or neonate is ADPKD are clinically, sonographically, and histopathologi-
uncertain, given the small number of cases and short follow-up cally indistinguishable. The sporadic cases are conceivably
periods reported. Although neonatal ADPKD was once new mutations of the same disease.
thought to have an ominous prognosis, small series suggest Reports of kindreds with GCKD suggest autosomal domi-
that it may be compatible with favorable long-term patient nant inheritance in some cases (273). Carson et al. described a
and renal survival (141,145,169,236). Prognosis in the older family of 18 first-degree relatives with six affected individuals
child generally better. Progression to ESRD in childhood is (274). Their review of the literature for reported cases revealed
rare in this latter group, and the majority maintain normal several other such families, although sporadic cases constitute
renal function through adolescence (267). However, progres- the bulk of patients with this diagnosis. An apparently distinct
sion of disease does occur during childhood, especially in chil- entity is hypoplastic GCKD, a dominantly inherited disease
dren with evidence of severe renal enlargement at a young age reported in only a few families (275,276). These kidneys, apart
(267). Proteinuria has been identified as a potential early from being glomerulocystic, are small, and imaging studies
marker of severe cystic disease in children (268). Approxi- show abnormal pyelocaliceal anatomy. Recently, mutations in
mately 50% of adult patients with ADPKD progress to the hepatocyte nuclear factor-1β gene were identified in four
ESRD. On average, patients with PKD1 typically progress at kindreds with this hypoplastic GCKD variant (277).
an earlier age, with a mean age at ESRD of 53.0 years, whereas GCKD can be associated with such syndromes as orofa-
those with PKD2 progress to ESRD at a mean age of 69.1 ciodigital syndrome type I (278), brachymesomelia-renal
years (176). In light of the significant inter- and intrafamilial syndrome (279), trisomy 13 (280), Majewski-type short rib
phenotypic heterogeneity, it is difficult to predict at what age a polydactyly syndrome (280), and Jeune syndrome (281) and
given patient with ADPKD will develop renal failure. can be seen as a component of the renal abnormalities in
Once renal insufficiency occurs, GFR typically decreases nephronophthisis (282). Although tubular sclerosis generally
at a rate of approximately 2 to 3 mL/min/1.73 m2 per year, includes tubular cysts, glomerular cysts can be present (282).
with GFR declining at a faster rate in men than women Glomerular cysts also occur as a minor component in several
(171). Decline in GFR correlates with an increase in renal other syndromes, including Zellweger cerebrohepatorenal
cyst volume and a decrease in parenchymal volume as syndrome (280,281) in which the cysts are typically present
determined by CT (269,270). Moreover, increased renal but rarely serious enough to affect renal function.
volume growth rate positively correlates with decline in Other syndromes that may be associated with glomerular
renal function in patients with early disease and preserved cysts as a component of renal dysplasia include Meckel syn-
GFR (171). Thus, renal volumes and rate of renal growth drome, glutaric aciduria type II, and renal-hepatic-pancreatic
may be markers for disease progression in ADPKD. dysplasia (282). The glomerular cysts are minor in compar-
ison with the dysplastic components of the renal disease,
although they may be present in sufficient numbers to cre-
GLOMERULOCYSTIC KIDNEY DISEASE ate confusion with other glomerulocystic conditions.

The term glomerulocystic kidney disease (GCKD), coined by

Pathogenesis and Pathology
Taxy and Filmer in 1976, is used to describe the morpho-
logic appearance of glomerular cysts, which occur in a vari- The pathogenesis of GCKD remains unknown. Clinically,
ety of conditions (271). GCKD was first described GCKD can be difficult to distinguish from other cystic kid-
clinically by Ross in 1941 (272). GCKD can be categorized ney diseases. The diagnosis can only be established by his-
into three major groups: (a) nonsyndromal heritable and tologic examination of renal tissue. Sporadic GCKD in
sporadic forms of GCKD, (b) GCKD as the major compo- young infants is histopathologically indistinguishable from
nent of heritable malformation syndromes, and (c) glomer- ADPKD-related GCKD. The kidneys in both the familial
ular cysts as a minor component of abnormal or dysplastic and sporadic forms are variably enlarged, with the degree of
kidney disease, some of which are syndromal. renal enlargement related to the degree of cyst formation
(282). The cysts in both groups may be diffuse but can also
be clustered, which may be responsible for asymmetric and
Epidemiology, Genetics, and
asynchronous clinical presentations. Diffuse involvement is
Prenatal Diagnosis
associated with interstitial edema, whereas patchy involve-
Primary GCKD with isolated renal involvement can be an ment is associated with better preservation of overall renal
autosomal dominant disease, a familial hypoplastic disease, structure and function.
690 VI. Tubular Disease

Characteristically, the cysts are dilated Bowman’s spaces, echogenicity of the cortex and medulla, loss of corticomed-
comprising a sphere lined with cuboidal or columnar cells ullary junction differentiation, and small cortical cysts
and containing abortive or primitive-appearing glomeruli (283,291). Radiographically, a feature that can help distin-
(271), which occur as small scattered cysts separated by guish GCKD from ARPKD is abnormal medullary pyra-
normal parenchyma. The cysts are located in the cortex, mids in the latter. In the future, CT and nuclear MRI may
with preservation of the medulla. This lack of tubular be of some value in differentiating between these two dis-
involvement differentiates GCKD from other cystic dis- eases (292). Reduced intensity of cortex on T1-weighted
eases in which cysts generally arise from tubular dilation. In images and abnormalities of corticomedullary differentia-
rare cases, they are more diffuse, surrounded by atrophic tion may help confirm the diagnosis.
and fibrotic parenchyma. They may be found in association In summary, GCKD represents a heterogeneous collec-
with tubular cysts and dysplasia (282). tion of heritable and nonheritable clinical entities. The
The kidneys in sporadic GCKD and the GCKD form of clinical course and prognosis are quite variable and often
ADPKD often contain abnormally differentiated pyramids, dependent on the presence of associated disorders.
a type of medullary dysplasia. Both forms of GCKD are
associated with biliary dysgenesis in approximately 10% of
cases (282). POLYCYSTIC KIDNEY DISEASE ASSOCIATED
WITH CONGENITAL SYNDROMES
Clinical and Radiographic Features
Many diseases can present with enlarged kidneys or cysts in
Most GCKD patients described in the literature have some the infant and young child and can initially be confused with
degree of renal failure, and many have hypertension at pre- PKD (Table 36.3). Abdominal ultrasound examination and
sentation. The typical presentation is that of an infant with nuclear isotope scanning help rule out multicystic renal dys-
abdominal masses, renal insufficiency, and enlarged cystic plasia and obstructive uropathy. Multicystic dysplastic kidney
kidneys on sonography. GCKD may manifest in adulthood is generally unilateral with macrocysts of varying sizes, differ-
with hypertension, flank pain, and hematuria. Variable
degrees of renal dysfunction are seen. Later detection may
be consistent with a milder course (169,274). Clinically, TABLE 36.3. DIFFERENTIAL DIAGNOSIS OF
hepatic cysts have also been described (282). POLYCYSTIC KIDNEY DISEASE IN THE
PEDIATRIC PATIENT
Patients with the familial hypoplastic GCKD variant have
small kidneys with abnormal collecting systems and abnor- Cystic diseases
mal or absent papillae (275,276). Family studies show a pat- Autosomal recessive polycystic kidney disease
tern compatible with autosomal dominant inheritance. Most Autosomal dominant polycystic kidney disease
Glomerulocystic kidney disease
patients appear to have chronic renal failure early in life but Congenital and hereditary diseases
subsequently have stable courses without progression to Tuberous sclerosis
ESRD. Multicystic dysplastic kidney
Several reports of GCKD describe patients with no clear Nephronophthisis complex
familial or syndromic association (280,283,284). Histologi- Glycogen storage disease
Congenital nephrosis
cally and clinically, these patients resemble familial cases with Syndromes
large, hyperechoic kidneys. It remains unclear whether these Meckel-Gruber syndrome
sporadic cases are a distinct entity or are associated with unrec- Jeune syndrome and other chondrodysplasia syndromes
ognized syndromal or familial cases. Reports on an infant with Ivemark syndrome
GCKD and multiple cardiac rhabdomyomas and an infant Bardet-Biedl syndrome
Zellweger cerebrohepatorenal syndrome
with severe GCKD who later developed skin findings consis- Beckwith-Wiedemann syndrome
tent with tuberous sclerosis strongly suggest an association of Trisomy 9 and 13
GCKD with tuberous sclerosis (285,286). This, together with Neoplasia
the new information regarding the molecular basis of ADPKD Nephroblastomatosis
and tuberous sclerosis and the reported familial association of Bilateral Wilms’ tumor
Leukemia or lymphoma
GCKD and ADPKD, raises the possibility that autosomal Miscellaneous
dominant GCKD, ADPKD, and tuberous sclerosis are geneti- Pyelonephritis
cally linked in some kindreds. Single case studies have also Glomerulonephritis
reported GCKD in association with Henoch-Schönlein pur- Radiocontrast nephropathy
pura (287), hepatoblastoma (288), and as a sequela of Bilateral renal vein thrombosis
Transient nephromegaly
hemolytic-uremic syndrome (289,290).
Ultrasonography demonstrates bilateral renal enlarge- Adapted from McDonald RA, Avner ED. Inherited polycystic kidney
ment without distortion of the renal contour, increased disease in children. Semin Nephrol 1991;11:632–642.
 36. Polycystic Kidney Disease 691

entiating it from ARPKD (see Chapter 5). On radionuclide TABLE 36.4. DIFFERENTIAL CLINICAL FEATURES OF
scanning, it demonstrates no function, permitting differenti- CHILDHOOD POLYCYSTIC KIDNEY DISEASE
ation from ADPKD. Most syndromic and other inherited Major clinical features of both ARPKD and ADPKD
disorders can usually be differentiated from ARPKD and Enlarged kidneys
ADPKD by associated clinical features, with the exception of Hypertension
GCKD, occasionally tuberous sclerosis, and von Hippel- Concentrating defect
Sterile pyuria
Lindau disease (1,97,293). GCKD can be a feature of several
Clinical features suggesting ARPKD rather than ADPKD
inherited, sporadic, or syndromic conditions as discussed Neonatal presentation
above. In addition, GCKD may be an early histopathologic Progression to end-stage renal disease as a child
expression of the ADPKD gene in young patients. Tuberous Hepatosplenomegaly
sclerosis is an autosomal dominant neurocutaneous disorder, Portal hypertension and esophageal varices
Bacterial cholangitis
in which hyperplastic cystic lesions may affect any portion of
Negative family history
the nephron (285). Genetic linkage of the chromosome 16 Clinical features suggesting ADPKD rather than ARPKD
loci for tuberous sclerosis and ADPKD1 has been demon- Positive family history
strated (294). The TSC2 gene has been identified and Extrarenal cysts
encodes a novel protein, tuberin. Uncommonly, patients Cerebral aneurysms
Asymptomatic presentation
show polycystic renal involvement without clinical neurocu-
Unilateral renal presentation
taneous involvement or positive family history. Several kin- Hematuria
dreds have been identified with tuberous sclerosis and severe Urinary tract infection
childhood-onset ADPKD; they have large deletions in the
area containing PKD1 and adjacent TSC2 gene (210). Analy- Adapted from Avner ED. Polycystic kidney disease. In: Drukker A,
Grushkin A, eds. Pediatric nephrology. In: Branski D, series ed. Pedi-
sis of the deletions indicates that they inactivate PKD1, in atric and adolescent medicine. Basel: AG Karger, 1993.
contrast to mutations reported in ADPKD patients in which
abnormal transcripts have been detected. von Hippel-Lindau
disease is a dominantly inherited cancer syndrome character- tions (168). Radiographic studies may clearly distinguish
ized by renal cell carcinoma, pheochromocytoma, and ARPKD and ADPKD in some cases (Table 36.4). However,
hemangioblastomas of the eye, spine, and cerebellum. Cystic in clinical practice, 20 to 30% of all cases show certain fea-
kidneys and pancreas may be seen and, rarely, patients may tures of both diseases on radiographic studies, making defin-
present with “typical” features of ADPKD (295). To differen- itive diagnosis difficult if not impossible. If the constellation
tiate GCKD, tuberous sclerosis, and von Hippel-Lindau of physical and radiologic findings is equivocal, a tissue
from ARPKD and ADPKD, detailed family history, physical diagnosis may be required (Figs. 36.2 and 36.3). Renal
examination, and close clinical follow-up are necessary. biopsies, particularly when stained with segment-specific
lectins (127,296), clearly differentiate the isolated fusiform
cortical collecting tubular cysts of ARPKD (Fig. 36.2) from
DIFFERENTIAL DIAGNOSIS OF POLYCYSTIC the heterogeneous cystic nephron involvement of ADPKD
KIDNEYS IN CHILDHOOD (Fig. 36.3). In certain instances, liver biopsy may provide
additional information and reveal the characteristic biliary
In most clinical settings, the major problem in differential dysgenesis of ARPKD. However, hepatic portal fibrosis and
diagnosis of PKD in the pediatric patient is clearly delineat- bile duct ectasia have been associated with other types of
ing ARPKD from ADPKD. In fact, ADPKD presenting in renal cystic diseases, including ADPKD. In the future, fur-
the neonatal period may be indistinguishable clinically from ther molecular analysis of ARPKD and ADPKD mutations
ARPKD (98,145). In such instances, a staged evaluation, will permit definitive genetic diagnosis and guide genetic
including careful history, physical examination, imaging, counseling in childhood PKD.
and histologic examination, is recommended. As noted in
Table 36.4, certain clinical features can help differentiate
between ARPKD and ADPKD, although no single finding ACKNOWLEDGMENTS
is diagnostic. A complete family history is often the most
important element in difficult cases. Parents should have Dr. Avner is the director and Dr. Dell a member of the
renal and liver ultrasonography. If the parents of a child with National Institutes of Health–supported Rainbow Center
undiagnosed PKD are younger than 30 years, the grandpar- for Childhood PKD (#P50-DK27306), Rainbow Babies
ents should also be evaluated because 4 to 5% of patients and Children’s Hospital, and Case Western Reserve Univer-
with ADPKD may not have visible renal cysts before age 30. sity. Dr. Dell is supported by a Mentored Clinical Scientist
Absence of any cystic disease in family members makes the Award (#K08 DK-59488). Dr. Avner is also supported in
diagnosis of ARPKD more likely because less than 5 to 8% part by grants from the Polycystic Kidney Research Foun-
of all ADPKD cases appear to result from new gene muta- dation and Wyeth-Ayerst Research.
692 VI. Tubular Disease

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270. Sise C, Kusaka M, Wetzel LH, et al. Volumetric determina- of GCKD. Clin Nephrol 2002;57:386–391.
tion of progression in autosomal dominant polycystic kidney 288. Greer ML, Danin J, Lamont AC. Glomerulocystic disease
disease by computed tomography. Kidney Int 2000;58:2492– with hepatoblastoma in a neonate: a case report. Pediatr
2501. Radiol 1998;28:703–705.
271. Taxy JB, Filmer RB. Glomerulocystic kidney. Report of a 289. Amir G, Rosenmann E, Drukker A. Acquired glomerulo-
case. Arch Pathol Lab Med 1976;100:186–188. cystic kidney disease following haemolytic-uraemic syn-
272. Ross A. Polycystic kidney. Report of a case studied by recon- drome. Pediatr Nephrol 1995;9:614–616.
struction. Am J Dis Child 1941;61:116–127. 290. Emma F, Muda AO, Rinaldi S, et al. Acquired glomerulo-
273. Sharp CK, Bergman SM, Stockwin JM, et al. Dominantly cystic kidney disease following hemolytic uremic syndrome.
transmitted glomerulocystic kidney disease: a distinct Pediatr Nephrol 2001;16:557–560.
genetic entity. J Am Soc Nephrol 1997;8:77–84. 291. Cachero S, Montgomery P, Seidel FG, et al. Glomerulocys-
274. Carson RW, Bedi D, Cavallo T, et al. Familial adult glomer- tic kidney disease: case report. Pediatr Radiol 1990;20:491–
ulocystic kidney disease. Am J Kidney Dis 1987;9:154–165. 493; discussion 494.
275. Rizzoni G, Loirat C, Levy M, et al. Familial hypoplastic glom- 292. Egashira K, Nakata H, Hashimoto O, et al. MR imaging of
erulocystic kidney. A new entity? Clin Nephrol 1982;18:263– adult glomerulocystic kidney disease. A case report. Acta
268. Radiol 1991;32:251–253.
276. Kaplan BS, Gordon I, Pincott J, et al. Familial hypoplastic 293. Chatha RK, Johnson AM, Rothberg PG, et al. Von Hippel-
glomerulocystic kidney disease: a definite entity with domi- Lindau disease masquerading as autosomal dominant poly-
nant inheritance. Am J Med Genet 1989;34:569–573. cystic kidney disease. Am J Kidney Dis 2001;37:852–858.
277. Bingham C, Bulman MP, Ellard S, et al. Mutations in the 294. Kandt RS, Haines JL, Smith M, et al. Linkage of an important
hepatocyte nuclear factor-1beta gene are associated with gene locus for tuberous sclerosis to a chromosome 16 marker
familial hypoplastic glomerulocystic kidney disease. Am J for polycystic kidney disease. Nat Genet 1992;2:37–41.
Hum Genet 2001;68:219–224. 295. Browne G, Jefferson JA, Wright GD, et al. von Hippel-Lindau
278. Stapleton FB, Bernstein J, Koh G, et al. Cystic kidneys in a disease: an important differential diagnosis of polycystic kidney
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Dis 1982;1:288–293. 296. Holthofer H, Kumpulainer T, Rapola J. Polycystic disease of the
279. Langer LO Jr, Nishino R, Yamaguchi A, et al. Brachyme- kidney: evaluation and classification based on nephron segment
somelia-renal syndrome. Am J Med Genet 1983;15:57–65. and cell-type specific markers. Lab Invest 1990;62:363–369.
 37

AMINOACIDURIA AND GLYCOSURIA

ISRAEL ZELIKOVIC

Only negligible amounts of amino acids and glucose are nor- intracellular catabolism of peptides, and from de novo syn-
mally present in the final urine, reflecting very efficient reab- thesis within cells. More than 99% of the load of free
sorption mechanisms for these organic solutes in the proximal amino acid filtered by the kidneys of humans and other
tubule. Renal tubular transport defects or specific metabolic mammals is reabsorbed in the renal tubule and returned to
abnormalities result in excretion of significant quantities of plasma (1,2). Amino acid reabsorption occurs predomi-
amino acids or glucose in the urine. Although hereditary nantly in the pars convoluta of the proximal tubule and, to
defects in renal tubular transport of most of these substances a small extent, in the pars recta (1,2). Amino acids are reab-
are uncommon, they are of major biologic importance. First, sorbed primarily from tubular lumen by an active uphill
some of these membrane transport disorders [e.g., cystinuria, transport across the luminal membrane (1). In studies
lysinuric protein intolerance (LPI), Hartnup disease] are asso- using renal brush-border membrane vesicles (BBMV) from
ciated with significant morbidity. Second, the study of these various animals to explore amino acid transport across this
disorders has provided much insight into the physiology of membrane, the rate of accumulation by vesicles and the
renal tubular reclamation of amino acids and glucose and into magnitude of the overshoot, which indicates active concen-
the specific metabolic pathways that control their reabsorption trative transport, were greatly augmented by an external
and has been crucial in understanding the genetics of tubular Na+ gradient across the vesicle membrane (1). Hence, it is
transport systems. widely accepted that uptake of most amino acids at the
This chapter summarizes the general characteristics of brush-border surface occurs by Na+-amino acid cotransport
renal tubular transport of amino acids and glucose, reviews driven by the electrochemical Na+ gradient from tubular
recent studies on the molecular biology of the transporters, lumen to cell (1,2) (Fig. 37.1). The energy maintaining the
describes the ontogeny of these transport processes, and dis- Na+ gradient is established by the Na+-K+–adenosine tri-
cusses the specific hereditary membrane transport disorders phosphatase (ATPase), which is located at the basolateral
that result in abnormal aminoaciduria and glycosuria. Special membrane and translocates Na+ out and K+ into the cell.
emphasis is given to classic cystinuria, including molecular Active amino acid transport across the brush-border
genetic aspects of this disease. Not discussed in this chapter membrane is followed by efflux, mainly via carrier-medi-
are overflow aminoaciduria and glucosuria, which occur ated, Na+-independent, facilitated diffusion or exchange
when the filtered load of these solutes exceeds the transport from the cell into the peritubular space across the basolat-
capacity of the renal tubule. This tubular overload is charac- eral membrane (2–4) (Fig. 37.1). Thus, under normal con-
teristic of various inborn errors of amino acid metabolism ditions, net transepithelial movement of amino acids occurs
and diabetes mellitus, which result in elevated plasma levels from the tubular lumen to the peritubular space. However,
of amino acids and glucose, respectively. Fanconi syndrome, a net transepithelial flux of amino acids is composed of
proximal tubular disorder characterized by generalized ami- amino acid transport in both directions, namely lumen →
noaciduria and urinary hyperexcretion of glucose, bicarbon- interstitium and interstitium → lumen (3,4). Indeed, the
ate, phosphate, and other solutes, is discussed in Chapter 41. basolateral membrane harbors active Na+-dependent and
Na+-independent transport and exchange systems mediat-
ing amino acid uptake in the tubular cell (Fig. 37.1), and
AMINOACIDURIA
diffusional backflux of amino acids from cell into the tubu-
lar lumen is a well-documented phenomenon (3). Intersti-
General Characteristics of Tubular
tium → lumen–oriented backflux through paracellular
Amino Acid Transport
pathways also occurs. The sum of these vectorial fluxes
Circulating free amino acids are derived from dietary pro- determines the direction and the rate of transepithelial
tein that is hydrolyzed and absorbed in the intestine, from amino acid transport. This notion may be of major impor-
702 VI. Tubular Disease

be achieved in the proximal convoluted tubule (3,6). This

axial heterogeneity of Na+-linked amino acid uptake systems
with respect to kinetic characteristics has been demonstrated
for several amino acids (1) in BBMV derived from pars con-
voluta and pars recta of the proximal tubule. The recognition
of several transport systems for the same amino acid as well
as their axial heterogeneity is of major importance in under-
standing the pathophysiology of hereditary aminoaciduria.
One or more Na+ ions are transported for each amino
acid molecule translocated, and with most amino acids this
process is electrogenic-positive favored by a negative cell
interior (1,7). Na+-amino acid stoichiometry determines
the electrogenicity and efficiency of the transport system
(3). Additional ions besides Na+ are involved in the translo-
cation of the amino acid carrier complex across the brush-
border membrane (1). Taurine (8), glycine (9), and proline
(10) transports, for example, operate by means of 2 or 3
FIGURE 37.1. Summary of amino acid transport mechanisms in Na+:1 Cl–:1 amino acid carrier complex.
the proximal tubule. Filled circles indicate Na +-dependent,
active, carrier-mediated cotransport or antiport. Empty circles
indicate Na+-independent antiport or uniport (facilitated diffu- Specificity of Transport
sion). Half-filled, half-empty circles indicate various combina-
tions of the previously described circles. AA 0, neutral amino At least seven distinct group-specific Na+-dependent transport
acids; AA+, basic amino acids; AA–, acidic amino acids; CssC, cys-
tine; Cys, cysteine. See text for details. systems exist in the tubular luminal membrane (1,2). Evidence
for these systems is derived from a variety of microperfusion
experiments and vesicle studies and, in humans, from the exis-
tance in understanding renal tubular amino acid transport, tence of inborn errors of renal tubular transport that can be
particularly in disease states and during maturation (4,5). explained only by defects in specific transport pathways (1,11).
Na+-amino acid symport across the luminal membrane These include systems for dibasic amino acids, acidic amino
is a carrier-mediated saturable process obeying Michaelis- acids, neutral α-amino acids, imino acids, glycine, β- and γ-
Menten kinetics (1,2). The effectiveness of the active reab- amino acids, and cystine/cysteine.
sorption process for a specific amino acid depends on the There is good evidence to suggest that in addition to sepa-
ratio Vmax:Km (1). A low Vmax (decreased transport capacity) rate systems for L-cystine and the dibasic amino acids (L-
or a high Km (diminished transporter-substrate affinity) for lysine, L-arginine, and L-ornithine), these amino acids share a
a given amino acid results in decreased reabsorption rate of common transport pathway (12–14), as also suggested by
this amino acid. Changes in efficiency or capacity of amino the urinary hyperexcretion of all four amino acids in classic
acid transport also play an important role in both neonatal cystinuria (see Classic Cystinuria). Transport of L-cystine but
aminoaciduria and hereditary aminoacidurias. not L-cysteine can also proceed via Na+-dependent transport
In analyzing the data obtained from microperfusion and pathways for neutral amino acids (1,2). L-Cysteine is proba-
micropuncture experiments and studies using BBMV for bly reabsorbed by an additional, separate, and specific trans-
various amino acids (1), two or more Na+-linked transport port system (2).
systems with different kinetic characteristics have been The broad-specificity transport pathway for neutral α-
described. The demonstration of multiple transport systems amino acids, which is a low-affinity, high-capacity system, is
for the same amino acid becomes meaningful if the reduced located in the proximal convoluted tubule (11). However,
concentrations of filtered amino acid presented to the proxi- there are several alternative specific renal transport systems
mal straight tubule are considered. Thus, in the case of gly- for neutral amino acids, including high-affinity systems
cine, for example, two Na+-dependent, active transport located in the proximal straight tubule. The presence of
systems have been demonstrated along the luminal mem- such pathways is also suggested by the finding of isolated
brane of the isolated perfused proximal tubule (6): a low- transport defects for neutral amino acids (see Neutral Ami-
affinity, high-capacity system in the convoluted segment and noaciduria). The imino acids proline and hydroxyproline
a high-affinity, low-capacity system in the straight segment. are reabsorbed by at least two systems, a low-affinity/high-
The latter system, which also operates in parallel to lower capacity system in the proximal convoluted tubule shared
apical membrane backflux permeability in the proximal with glycine and an imino acid–specific, high-affinity/low-
straight tubule (3), absorbs less glycine against a greater con- capacity system in the proximal straight tubule (6,15).
centration gradient and probably permits the reduction of There also is a separate high-affinity/low-capacity system
the luminal glycine concentration to lower levels than could for glycine in the late proximal tubule (6).
 37. Aminoaciduria and Glycosuria 703

TABLE 37.1. AMINO ACID TRANSPORT SYSTEMS OPERATING IN THE RENAL TUBULE
Amino acid
transport system Cloned cDNA Amino acids transported Mechanism of action Localization

Neutral
A — Short chain neutral Na+-AA cotransport BLM
ASC ASCT 1–2 Short chain neutral Na+-dependent AA antiport BLM
B0 ATB0 Most neutral Na+-AA cotransport BBM
Gly GLYT 1–2 Glycine, sarcosine 2–3 Na +/1Cl–/1AA cotransport BBM
IMINO — Proline, hydroxyproline 2–3 Na +/1Cl–/1AA cotransport BBM
BETA TAUT, GAT 1–3, BGT-1 Taurine, β-alanine, GABA, betaine 2–3 Na+/1Cl–/1AA cotransport BBM
L 4F2hc/LAT-1, 4F2hc/LAT-2 Large, branched chain neutral AA antiport BLM
Cationic
B0,+ ATB0,+ (?) Cationic and neutral Na+/Cl–/AA cotransport BBM
b0,+ rBAT/b0,+AT Cationic and neutral (including cys- AA antiport BBM
tine)
y+ CAT 1–4 Cationic and neutral (excluding cys- AA uniporter (facilitative trans- BLM
tine) porter)
y+L 4F2hc/y+LAT-1 Cationic and neutral (excluding cys- Na+-dependent AA antiport BLM
4F2hc/y+LAT-2 tine)
Anionic
X–AG EAAT 1-5 Anionic 3Na+/AA cotransport, 1K+-anti- BBM
port

AA, amino acid; BBM, brush-border membrane; BLM, basolateral membrane; GABA, γ-aminobutyric acid.

The investigation of amino acid transport pathways in the factors are known to modulate transmembrane amino acid
plasma membrane of mammalian cells has delineated several transport (1,22). These include ionic and voltage condi-
transport systems (16–20) (Table 37.1; Fig. 37.1). Most of tions (discussed earlier), as well as availability of amino acid
these transport systems also have been identified in the kidney substrate, osmotic changes, and protein phosphorylation.
(16,17,19,21). These systems include the Na+-dependent con- Reabsorption of amino acids in the proximal tubule
centrative A and ASC systems (for small neutral amino acids), increases during periods of reduced amino acid intake and
B0 (for most neutral amino acids), B0,+ system (for dibasic and decreases with dietary excess (1). This renal adaptive
some neutral amino acids), X–AG system (for acidic amino response to diet is expressed at the tubular luminal mem-
acids), Gly system (for glycine and sarcosine), IMINO system brane surface. It has been suggested (22,23) that both new
(for proline and hydroxyproline), and β system (for taurine, synthesis of transporter protein and shuttling of preformed
β-alanine, and γ-aminobutyric acid), as well as the Na+- transporters are required for expression of the adaptive
independent nonconcentrative L system (for bulky neural, response. A recent expression study in Xenopus oocytes (24)
branched- chain amino acids and cysteine), y+ system (for the has demonstrated that the rat renal taurine transporter is
dibasic amino acids lysine, arginine, and ornithine), y+L sys- regulated by dietary taurine at the level of both messenger
tem (for dibasic and neutral amino acids excluding cystine), RNA (mRNA) accumulation and protein synthesis.
and b0,+ system (for dibasic and neutral amino acids including Amino acids are known to serve as regulatory osmolytes
cystine). Systems A, ASC, L, y+, and y+L operate in the basolat- in mammalian cells, including kidney cells (25–27). The
eral membrane (18,19,21) (Fig. 37.1). All these basolateral main amino acids involved in this function are taurine,
transporters (except for system A), as well as the luminal mem- proline, and glutamic acid (25,26,28). Studies using
brane-bound b0,+ system, function as antiport (exchange) sys- MDCK cells, a cell line of distal tubular origin, demon-
tems (19,20) (Fig. 37.1). Recent progress in molecular cloning strate changes in taurine transport in response to changes in
of amino acid transporters has helped to characterize and osmolarity of the medium (29). It has been shown that
define the nature and the role of most of these tubular amino osmotic regulation of taurine transport depends on changes
acid transport mechanisms (see Molecular Structure of Amino in taurine transporter gene expression (30).
Acid Transporters). Serine/threonine protein kinases play a central role in sig-
nal transduction by phosphorylating and thereby activating
effector proteins (31,32). Recent studies suggest that the
Adaptation and Regulation
three main groups of serine/threonine protein kinases,
of Amino Acid Transport
namely cyclic adenosine monophosphate–dependent protein
Although the exact mechanisms regulating renal tubular kinase [protein kinase A (PKA)], Ca2+- and phospholipid-
amino acid reclamation have not been established, several dependent protein kinase [protein kinase C (PKC)], and
704 VI. Tubular Disease

multifunctional Ca2+/calmodulin-dependent protein kinase and Na+-proline (44) cotransporters of the brain, which serve
II (CaMK II) alter amino acid transport across the tubular as neurotransmitters, as well as the Na+-Cl–-taurine (45)
brush-border membrane (33–35). However, the exact role of cotransporter of the kidney. In addition to the taurine trans-
protein kinase–induced phosphorylation in renal tubular porter of the kidney, Northern hybridization and polymerase
amino acid transport remains to be established. chain reaction analysis have identified two GABA transporter
isoforms, called GAT 2 and GAT 3 (46,47), as well as glycine
transporter isoforms called GLYT 1 and GLYT 2 (48–50) in
Molecular Structure of Amino Acid Transporters
the kidney. The GAT family also includes the renal Na+-Cl–-
Extensive efforts to isolate and identify the amino acid trans- betaine transporter. All these transporters show high homol-
port proteins in various tissues, including the kidney, have ogy in sequence and structure, and most of them have an
been largely unsuccessful. The isolation and molecular char- absolute requirement for Cl–.
acterization of amino acid carrier proteins has been hindered The proposed model of the GABA transporter (GAT 1) is
by their very low abundance in the membrane, their poor sta- shown in Figure 37.2. It is a 655-residue protein with a relative
bility in vitro, and the lack of specific tight-binding labels or molecular mass of 73,925 d. The predicted secondary struc-
inhibitors. Indirect approaches such as solubilization of pro- ture shows 12 hydrophobic, membrane-spanning domains
teins by organic solvents and incorporation into proteolipo- with a large extracellular hydrophilic loop between spans 3 and
somes, lectin-affinity chromatography, immunoprecipitation 4. The molecular structure contains putative N-glycosylation
using monoclonal antibodies, and radiation inactivation anal- and phosphorylation sites. The similarities between the family
ysis (36,37) have yielded little structural data. Since 1990, members may point toward domains important in Na+, Cl–,
however, by using molecular biology techniques, much and substrate coupling (51). The Na+-Cl–-betaine and Na+-
progress has been made in elucidating the molecular structure Cl–-taurine transporters are thought to play a major role in cell
of various membrane-bound transport proteins, including volume regulation in the renal medulla (26) (see Adaptation
amino acid transporters. This new area in the study of brush- and Regulation of Amino Acid Transport).
border membrane transporters was pioneered by Hediger et
al. (38), who cloned the small intestinal Na+-glucose cotrans- EAAT/ASCT Family
porter using the powerful method of expression cloning in The EAAT/ASCT family of transporters is made up of five
Xenopus oocytes. This strategy involves isolation of mRNA Na+- and K+-dependent, Cl–-independent anionic amino
from the tissue of interest, microinjection into Xenopus acid transporters, and three neutral amino acid transport-
oocytes, and analysis of expressed transport activity by mea- ers (19,20). The anionic amino acid transporters include
suring uptake of radiolabeled substrate. After size fraction- the GLAST (or EAAT1) (52), GLT (or EAAT2) (53), and
ation of mRNA, complementary (cDNA) synthesis, and EAAC1 (or EAAT3) (54), as well as EAAT4 (55) and
DNA library screening by functional expression in oocytes, a EAAT5 (56), which serve as neurotransmitters and show
single clone encoding the transporter activity is isolated. A marked similarity in sequence and structure. EAAC1, a
similar approach has been used to clone, functionally express, neuronal and epithelial high-affinity glutamate transporter
and sequence various amino acid transporters. first cloned from rabbit small intestine (54), was also iden-
The structural information and functional characteristics tified in the kidney by Northern hybridization analysis
of amino acid transporters have led to their classification into (54). In situ hybridization and immunofluorescence stud-
four different gene families (19,20,39,40): Na+- and Cl–- ies revealed that EAAC1 is expressed predominantly in the
dependent transporters (the GAT family), the anionic/neutral S2 and S3 segments of the proximal tubule (57).
amino acid transporters (the EAAT/ASCT family), cationic The proposed model of EAAC1 (Fig. 37.2) shows a
amino acid transporters (the CAT family), and cationic/neu- protein of 524 amino acids with a predicted molecular
tral amino acid transporters [the rBAT/4F2 heavy chain mass of 57,000 d and ten hydrophobic, membrane-span-
(4F2hc) family]. Investigation of the primary structure of ning domains. The kinetics and specificity of this protein
amino acid transporters has elucidated two main types of when expressed in Xenopus oocytes were similar to those of
membrane proteins involved in amino acid transport (19): the X–AG transport system (see Specificity of Transport;
(a) members of the GAT, EAAT/ASCT, and CAT families, Fig. 37.1). The gene for human EAAC1 (termed SLC1A1)
which present multiple transmembrane domains (Fig. 37.2) has been localized to chromosome 9p24 (58). A defect in
and are therefore considered putative carriers; and (b) mem- the EAAC1 gene is a likely cause of dicarboxylic aminoaci-
bers of the rBAT/4F2hc family, which do not fit this model duria (see Dicarboxylic Aminoaciduria).
(Figs. 37.3 and 37.4) and are known to function as compo- The neutral amino acid transports include the ASCT1 (or
nents or subunits of heteromultimeric carriers. SATT) (59), ASCT2 (or AAAT) (60), and ATB0 (61).
ASCT1 and ASCT2, which were cloned from human brain
GAT Family (59) and mouse testis (60), respectively, have structural simi-
The GAT family includes Na+-Cl––γ-aminobutyric acid larity to the anionic amino acid transporter gene family and
(GABA) (41), Na+-Cl–-taurine (42), Na+-Cl–-glycine (43), appear to encode Na+-dependent neutral amino acid trans-
 37. Aminoaciduria and Glycosuria 705

FIGURE 37.2. The putative transmembrane orientations of the γ-aminobutyric acid (GABA)
transporter (GAT-1), the glutamate transporter (EAAC1), and the cationic amino acid transporter
(CAT1) are shown as representative of three families of amino acid transporters. (Adapted from
McGivan JD, Pastor-Anglada M. Regulatory and molecular aspects of mammalian amino acid
transport. Biochem J 1994;299:321–334.)

porters with specificity characteristics of system ASC (see CAT Family

Specificity of Transport; Fig. 37.1). Northern blot analysis The CAT genes were the first mammalian amino acid
(59,60) revealed ubiquitous expression of these genes in sev- transporters cloned (40). Expression studies in Xenopus
eral tissues, including expression in the kidney, consistent oocytes identified the ecotropic murine leukemia virus as
with the general metabolic role ascribed to system ASC. the ubiquitous y+ system (now called CAT1), an Na+-inde-
ATB0 was cloned from human choriocarcinoma cells (61), pendent transport system that accepts dibasic amino acids
and its transcripts were detected in the kidney among other and excludes cystine (65,66). In addition, it catalyzes trans-
tissues. Expression studies in the HeLa cells and oocytes port of neutral amino acids only in the presence of Na+ (19)
showed an Na+-dependent uptake of neutral amino acids (Fig. 37.1). Northern hybridization analysis revealed the
with characteristics of the brush-border membrane system B0 CAT1 gene in various mouse tissues, including the kidney
(see Specificity of Transport; Fig. 37.1). It is noteworthy that (65). Since then, three other homologous murine cDNAs
the transport systems EAAT1-5 (19,62), ASCT1 (63), and have been found to express a similar amino acid transport
possibly ATB0 (64) have a Cl– channel mode of action in activity, CAT2, CAT3, and CAT4, all of which (except
addition to their amino acid transport mode of activity. CAT3) are expressed in the kidney (19,67,68). The CAT
706 VI. Tubular Disease

by studies demonstrating that CAT2 and nitric oxide syn-

thase 2 transcripts are co-induced in concert with system y+
activity (70). The CAT1 cDNA predicts a 629–amino acid
protein with 14 membrane-spanning domains and a
molecular mass of 68,000 d (Fig. 37.2).

rBAT/4F2 Heavy Chain Family

To gain insight into the transport defect in cystinuria, recent
research has focused on the molecular structure of the family
of carrier proteins responsible for transport of cystine and
other dibasic amino acids. Several groups have demonstrated
the expression of cystine (71) and dibasic and neutral amino
acids (72–74) in Xenopus oocytes injected with mRNA of
small intestine and kidney. In 1992 (75,76), kidney cortex
FIGURE 37.3. Transport pathways for cystine and dibasic amino
acids at the luminal and basolateral membranes of a proximal cDNAs from rabbit and rat [named rBAT (75) and D2 (76),
tubular cell. Large circles represent the heavy subunits and small respectively] have been cloned. On in vitro transcription to
circles the light subunits of the heteromeric amino acid trans- chromosomal RNA and injection into oocytes, they induce
porters b0,+ and y+L. Depicted are hereditary aminoacidurias
caused by defects in these transporters. (Modified from Zelikovic system b0,+, the Na+-independent transporter for neutral
I. Molecular pathophysiology of tubular transport disorders. amino acids, dibasic amino acids, and cystine (see Specificity
Pediatr Nephrol 2001;16:919–935.) of Transport; Figs. 37.1 and 37.3). The predicted proteins for
rBAT and D2 demonstrate significant homology with a fam-
ily of the carbohydrate-metabolizing enzymes α-glucosidases.
transporters allow accumulation of cationic amino acids Similarly, chromosomal RNA from the human 4F2hc surface
within the cell for general metabolic purposes. Also, it has antigen, a glycoprotein highly regulated at the onset of cell
been postulated that CAT-mediated arginine flux into cells proliferation, stimulated system y+L amino acid transport (see
could play a role in modulating nitric oxide synthesis in Specificity of Transport; Figs. 37.1 and 37.3) in Xenopus
various cell types (67,69). This notion has been supported oocytes (77).
The rBAT cDNA contains an open reading frame of 2049
nucleotides coding for a protein of 685 amino acids with a
molecular mass of approximately 90,000 d (40). The pre-
dicted proteins for rBAT and D2, as well as the 4F2hc antigen
(all of which show high structural similarity), were found to
contain only one (19) or four (78) putative membrane-span-
ning domains, depending on different typologic models. This
structure, which is atypical of the known membrane transport
proteins, raised the possibility that these proteins function as
activators of transport systems y+L and b0,+ or as regulatory
subunits of these transporters. The functional promoter of the
rat rBAT gene has been identified (79), and the gene for the
human rBAT, named D2H, was localized to chromosome
2p21 (79,80). The human rBAT (D2H) gene has been called
SLC3A1 (see Classic Cystinuria).
In situ hybridization and immunolocalization studies have
localized rBAT mRNA expression in the brush-border mem-
brane of the proximal straight tubule (S3 segment) and the
small intestinal mucosa (81,82). In contrast to rBAT, 4F2hc
mRNA is almost ubiquitous with marked expression in kid-
FIGURE 37.4. Schematic representation of the heteromeric
amino acid transporters (HAT). The heavy subunit of HAT ney, where it localizes to the basolateral membrane of proxi-
(HSHAT; dark gray) is linked by a disulfide bridge to the corre- mal tubular cells (19). Studies from several laboratories have
sponding light subunit of HAT (LSHAT; light gray). The cysteine demonstrated that both rBAT and 4F2hc are type II mem-
residues involved in this bond (S-S) are located extracellularly,
just after the transmembrane (TM) domain of HSHAT and in the brane glycoproteins, which constitute heavy subunits of het-
proposed extracellular loop 2 (EL2) of LSHAT. Loops and TM eromeric amino acid transporters (83–86). These studies
domains are not drawn to scale. See text for details. (Adapted showed that rBAT (90 kDa) and 4F2hc (85 kDa) associated
from Chillaron J, Roca R, Valencia A, et al. Heteromeric amino
acid transporters: biochemistry, genetics, and physiology. Am J by disulfide bridges with a light subunit (40 kDa) forming a
Physiol 2001;281:F995–F1018.) heterodimeric complex of 125 kDa (85,86) (Fig. 37.4).
 37. Aminoaciduria and Glycosuria 707

The light subunit of rBAT, termed b0,+AT, and the light Future investigations into the molecular and biochemi-
subunit of 4F2hc, termed y+LAT-1, which were identified cal characteristics of this expanding group of cloned amino
in 1998–1999 (87–89), are members of the family of light acid transporters may yield important insight into the
subunits of the heteromeric amino acid transporters inherited human diseases that result from a defective trans-
(LSHAT) (85,86). As of 2000, seven members of the port of amino acids (see Hereditary Aminoacidurias).
LSHAT family have been identified (85,86), to which the
light subunits LAT-1 and LAT-2 that combine with 4F2hc
Maturation of Tubular Amino Acid Transport
to form the L system (see Specificity of Transport; Table
37.1; Fig. 37.1) also belong. Recent research on the charac- Urinary fractional excretion of almost all amino acids in
teristics of LSHATs (83,85) has revealed that they are ung- humans (92) (Fig. 37.5) and animals (4) is higher in the
lycosylated proteins that contain 12 putative transmembrane newborn than later in life. A very high rate of urinary
domains (Fig. 37.4), they need coexpression with the cor- amino acid excretion is found in immature, very-low-birth-
responding heavy subunit to reach the plasma membrane, weight infants (93). Some amino acids, including glycine,
they confer the specific amino acid transport activity to the alanine, proline, dibasic amino acids, and taurine (4,93),
heteromeric complex, and, finally, all the amino acid trans- have been shown to contribute more to neonatal aminoaci-
port activities associated with the LSHATs behave as amino duria than other amino acids. Theoretically, a structural,
acid exchangers (85,86). In the kidney, system b0,+ quantitative, or regulatory change in any one of the mem-
(induced by rBAT and b0,+AT; Figs. 37.1 and 37.3) acts as brane-related events depicted in Figure 37.1 may underlie
tertiary active exchange mechanism of tubular reabsorption the diminished reabsorptive capacity of the renal tubule
of dibasic amino acids and cystine. This tertiary transport during early life (4,5).
mechanism is linked to a high intracellular concentration As indicated earlier, proline and glycine reabsorption in
of neutral amino acids (Fig. 37.1). System b0,+-mediated the proximal tubule occurs predominantly by a shared
efflux of neutral amino acids from renal epithelial cells is low-affinity/high-capacity transport system in the proximal
the driving force for cystine and cationic amino acid reab- convoluted tubule as well as by two specific, high-affinity/
sorption from lumen to cell (84,90,91) (Fig. 37.1). Reab- low-capacity systems located in the proximal straight tubule—
sorption of cystine and cationic amino acids is also favored one for proline and one for glycine (1,6). Lasley and Scriver
by the intracellular-negative membrane potential and by studied infants affected with familial renal iminoglycinuria to
the reduction of cystine to cysteine (Fig. 37.1) (84,91). explore the ontogeny of proline and glycine reabsorption in
System y+L (induced by 4F2hc and y+LAT-1; Figs. 37.1 the renal tubule (94). In iminoglycinuria, the shared trans-
and 37.3) mediates an electroneutral exchange mechanism port system for proline and glycine is affected by mutation
in which efflux of cationic amino acids (against the intra- (see Iminoglycinuria). Using the occurrence of ontogeny and
cellular-negative voltage) is enhanced by the influx of neu- maturation together, Scriver’s group provided evidence for
tral amino acids in the presence of Na+ (Fig. 37.1) (84,91). the appearance of the two specific high-affinity transport sys-
The tissue distribution of rBAT/b0,+AT and 4F2hc/y+LAT- tems in succession—the proline transporter by 3 months of
1 and their role in renal uptake of cystine and dibasic age and the glycine transporter by 6 months of age (94).
amino acids have made them candidates for the defective Similarly, earlier studies on glycine and proline transport in
genes in cystinuria and LPI, respectively (see Hereditary rat renal cortical slices (4) showed that the specific high-affin-
Aminoacidurias). ity transporters were not present at birth, yet appeared when

FIGURE 37.5. Tubular reabsorption of free amino

acids (% TAA) compared between infants (open cir-
cles) and children (black dots). Ala, alanine; Arg, argi-
nine; Cys, cysteine; Gly, glycine; His, histidine; Ile,
isoleucine; Leu, leucine; Lys, lysine; Met, methionine;
Orn, ornithine; Phe, phenylalanine; Pro, proline; Ser,
serine; Thr, threonine; Tyr, tyrosine; Val, valine.
(Adapted from Brodehl J, Gellissen K. Endogenous
renal transport of free amino acids in infancy and
childhood. Pediatrics 1968;42:395–404.)
708 VI. Tubular Disease

amino acid reabsorption reached adult levels. In contrast, related events play a role in the maturation of amino acid
subsequent animal studies demonstrated the existence of transport. Most studies indicate that transport maturation
both low-affinity/high-capacity and high-affinity/low-capac- does not represent the acquisition of new transport systems
ity systems for several amino acids (4) in neonatal and adult but rather a change in affinity or capacity of transporters.
kidneys; no new systems were acquired with maturation. The role of protein kinase–induced phosphorylation in the
Studies using cortical slices and isolated tubules from maturation of renal tubular amino acid transport remains to
several species have provided evidence for an impaired be established. The exact cellular mechanisms and the bio-
basolateral membrane exit step of amino acids from imma- logic signals responsible for the observed developmental
ture tubular cells (4). Decreased Na+-dependent taurine changes await future studies. Studies into the molecular
transport has been demonstrated in basolateral membrane structure of amino acid transporters will undoubtedly shed
vesicles from hypertaurinuric mice (95). It remains to be light on the mechanisms underlying the development of
established whether a similar alteration in basolateral mem- tubular amino acid reclamation.
brane amino acid transport during early life contributes to
neonatal hyperaminoaciduria.
Hereditary Aminoacidurias
Several studies explored the maturation of the first step
of amino acid reabsorption, namely transport across the Aminoacidurias are a group of disorders in which a single
brush-border membrane. A gradual age-related increase in amino acid or a group of amino acids are excreted in excess
Na+-coupled uptake of taurine (96) and proline (97) by rat amounts in the urine. The defective tubular reabsorption is
renal BBMV and of cystine by isolated dog renal cortical assumed to result from a genetic defect in a specific trans-
tubules (98) has been demonstrated. Whereas the matura- port system that directs the reabsorption of these amino
tion of proline transport involved an increase in affinity acids under normal conditions. Some of these disorders also
(decrease in Km) of transport (97), the maturation of cys- involve a similar transport abnormality in the intestine. As
tine transport was associated with an increase in capacity opposed to inborn errors of amino acid metabolism, in which
(increased Vmax) of transport (98). plasma levels of amino acids are elevated, resulting in over-
Alterations in phospholipid composition have been docu- flow aminoaciduria, plasma levels of amino acids in heredi-
mented during rat tubular brush-border membrane matura- tary aminoacidurias are largely normal.
tion (4), suggesting that changes in membrane fluidity may The aminoacidurias are generally categorized into five
account for the observed maturational changes in Na+-linked major groups according to the group-specific transport
tubular amino acid transport. In addition, an increased per- pathway presumed to be affected (Table 37.2). The groups
meability to Na+ (97) and an enhanced amiloride-sensitive are further subdivided into several disorders based on the
Na+-H+ exchange activity (99) have been demonstrated in profile of the affected amino acids within that group.
neonatal rat renal BBMV. This alteration in ionic permeabil-
ity and the increased luminal membrane Na+-H+ antiport
Cationic Aminoaciduria
[coupled with a diminished Na+-K+-ATPase activity known
to exist in the basolateral membrane of the neonatal proximal Five distinct inborn errors of cationic amino acid transport
tubular epithelium (100)] may result in a rapid dissipation of have been identified: (a) classic cystinuria, (b) isolated cysti-
the electrochemical Na+ gradient necessary for Na+-amino nuria, (c) hyperdibasic aminoaciduria, (d) LPI, and (e) iso-
acid cotransport, thereby contributing to the aminoaciduria lated lysinuria. These diseases differ in defined or putative
of early life (97,99). transport systems affected, pathophysiology, organs involved,
Protein kinases modulate renal tubular amino acid trans- and clinical features. Classic cystinuria is the prototype for
port (see Adaptation and Regulation of Amino Acid Trans- this group of hereditary aminoacidurias.
port). Recent studies (32,101) demonstrate higher activity
of PKC and CaMK II in the cytosol and the brush-border Classic Cystinuria
membrane derived from immature kidneys than in adult Cystinuria is a disorder of amino acid transport characterized
kidneys. Furthermore, the studies provide evidence for dif- by excessive urinary excretion of cystine and the dibasic
ferential regulation of PKC (101) and CaMK II (32) isoen- amino acids lysine, arginine, and ornithine. The pathogenic
zymes during kidney development. Age-related changes in mechanism of cystinuria is defective transepithelial transport
the activity and expression of protein kinases may underlie of these amino acids in the proximal tubule and the small
the developmental changes in tubular reclamation of amino intestine (102). The very low solubility of cystine in the urine
acids and other solutes. results in cystine stone formation in homozygous patients.
In summary, hyperaminoaciduria is a characteristic of the Lysine, arginine, and ornithine do not form urinary stones.
immature mammalian tubule. Although the mechanisms Urinary cystine calculi may produce considerable morbidity,
governing the developmental changes in tubular amino acid including urinary obstruction, colic, infection, and in severe
transport have not been fully established, evidence has accu- cases, loss of kidney function. Cystinuria accounts for 1 to
mulated that both luminal and antiluminal membrane- 2% of all urolithiasis and 6 to 8% of urolithiasis in children
TABLE 37.2. HEREDITARY AMINOACIDURIAS

Amino acid
transport
Defective system Individual amino acids Localization Mode of Clinical Organs OMIM
Disorder gene Locus defective affected of defect inheritance Prevalence manifestations involved no.a

Cationic aminoaciduria
Classic cystinuria
Type I SLC3A1 2p16.3-21 rBAT (heavy Cystine, lysine, argin- BBM Autosomal 1:7000–1:15,000 Urolithiasis Kidney, intes- 220100
subunit of ine, ornithine recessive tine
b0,+)
Type II and III SLC7A9 19q13.1 b0,+ AT (light Cystine, lysine, argin- BBM Incompletely 1:7000–1:15,000 Urolithiasis Kidney, intes- 600918
subunit of ine, ornithine autosomal tine
b0,+) recessive
Isolated cystinuria — — Cystine Cystine BBM Autosomal Two siblings Benign Kidney 238200
recessive reported
Hyperdibasic ami- — — Dibasic amino Lysine, arginine, orni- BBM Autosomal Two families Mental retardation Kidney, intes- 222690
noaciduria type I acids thine dominant reported tine
Lysinuric protein SLC7A7 14q11-13 y+LAT-1 (light Lysine, arginine, orni- BLM Autosomal 1:60,000 (Fin- Protein malnutri- Kidney, intes- 222700
intolerance subunit of thine recessive land) tion, failure to tine, liver
(hyperdibasic y+L) thrive, hyperam-
aminoaciduria monemia, sei-
type II) zures, coma
Isolated lysinuria — — Lysine (?) Lysine BBM (?) Autosomal One patient Failure to thrive, Kidney, intes- —
recessive reported seizures, mental tine
retardation
Neutral aminoaciduria
Hartnup disease ATB0 (?) 19q13.3 B0 (neutral, Alanine, serine, threo- BBM Autosomal 1:20,000 Skin rash, cerebel- Kidney, intes- 234500
monoamino nine, valine, leu- recessive lar ataxia, psy- tine
monocar- cine, isoleucine, chiatric illnesses
boxylic α- phenylalanine,
amino acids) tyrosine, tryp-
tophan, histidine,
glutamine, aspara-
gine
Methioninuria — — Methionine Methionine BBM Autosomal Two cases Edema, seizures, Kidney, intes- 250900
recessive reported mental retarda- tine
tion
Histidinuria — — Histidine Histidine BBM Autosomal Four cases Mental retardation Kidney, intes- 235830
recessive reported tine
Imminoaciduria and glycinuria
Imminoglycinuria — — Immino acids Proline, hydroxy- BBM Autosomal 1:15,000 Benign Kidney, intes- 242600
and glycine proline, glycine recessive tine

(continued)
TABLE 37.2. CONTINUED.

Amino acid
transport
Defective system Individual amino acids Localization Mode of Clinical Organs OMIM
Disorder gene Locus defective affected of defect inheritance Prevalence manifestations involved no.a

Isolated glycinuria — — Glycine Glycine BBM Autosomal Two families Benign Kidney 138500
recessive reported
Dicarboxylic ami- EAAC1 9p24 X–AG (acidic Glutamate, aspartate BBM Autosomal 1:29,000 Benign Kidney, intes- 222730
noaciduria (SLC1A1 (5p15 amino acids) recessive (French- tine
?) ?) Canadian)
β-amino aciduria — — β-amino acids Taurine BLM (?) Autosomal — Benign Kidney —
(mouse) recessive

BBM, brush-border membrane; BLM, basolateral membrane.

aOnline Mendelian Inheritance in Man (database at http://www.ncbi.nlm.nih.gov/omim).
 37. Aminoaciduria and Glycosuria 711

(103,104). The defective gastrointestinal transport of cystine ment of the proximal tubule and identified as system b0,+
and dibasic amino acids in cystinuria does not result in intes- (see Specificity of Transport; Figs. 37.1, and 37.3) that is
tinal disease. defective in classic cystinuria. The low-affinity, high-Km,
The disease was first recognized in 1810 by Wollaston unshared cystine system and the low-affinity, unshared diba-
(105) and later by Berzelius (106), who called the stones sic amino acid system, both located in the S1-S2 segments of
“cystic oxide” and “cystine,” respectively, assuming that the the proximal tubule, may be abnormal in isolated cystinuria
stones they analyzed originated in the bladder. In 1908, and dibasic aminoaciduria, respectively. The antiluminal
Garrod (107) postulated that cystinuria was an inborn error membrane harbors two specific systems, one for dibasic
of cystine metabolism. In the 1950s, Dent and Rose (108) amino acids identified as system y+L (see Specificity of Trans-
first recognized the true nature of the disease, suggesting port; Figs. 37.1 and 37.3) and one for cystine, but no shared
that cystine and the dibasic amino acids lysine, arginine, system. These transport mechanisms mediate uptake and
and ornithine that have structural similarity (two amino efflux of these amino acids across the basolateral membrane.
groups separated by four to six chemical bonds) share a car- The brush-border membrane of the intestinal cell has a sin-
rier protein in the brush-border membrane of the renal gle high-affinity, low-Km, shared b0,+ transport system for
tubule and the small intestine. They postulated that this cystine and dibasic amino acids that is defective in classic
transport mechanism was defective in cystinuria. cystinuria. Like kidney cells, intestinal cells have two basolat-
eral membrane-bound, unshared specific transport systems;
Transport Defect. Normally, 1% of the filtered cystine and one (system y+L) for dibasic amino acids; and one for cystine
dibasic amino acids is excreted. In classic cystinuria, cystine (5). The basolateral membrane-bound y+L transporter for
clearance may be near or equal to the glomerular filtration dibasic amino acids in the kidney and the intestine is defec-
rate (GFR), and in some patients even twice as high as the tive in LPI (see Lysinuric Protein Intolerance). Amino acid
GFR, suggesting active cystine secretion (109). Lysine and transport in parenchymal cells and leukocytes from cystinu-
ornithine clearance is 30 to 80% of the GFR, and arginine ric patients is not impaired (5) because the defect is not
excretion is less abnormal. expressed in the plasma membrane of these cells.
Dent’s postulate about a defective shared transport sys-
tem for cystine and dibasic acids in cystinuria has been sup- Genetics. Classic cystinuria is inherited in an autosomal
ported by the in vivo experiments of Kato (110) and recessive fashion. It is a common disorder with an overall
Robson and Rose (111), demonstrating that in healthy sub- prevalence of 1:7000 to 1:15,000 and estimated gene fre-
jects and cystinuric patients, increasing the filtered load of quency of 0.01 (102). A very high prevalence, 1:2500, is
one of these amino acids decreased reabsorption of the oth- observed in Israeli Jews of Libyan origin (118).
ers. However, this hypothesis was challenged by studies Although it is a recessive disease, phenotypic heterogene-
showing that cystine uptake was not impaired in kidney ity in homozygotes and heterozygotes is evident. The excre-
slices from cystinuric patients (112) and that cystine and tion patterns of cystine and dibasic amino acids in
dibasic amino acids did not share a common transport sys- heterozygotes and analysis of amino acid transport in jejunal
tem in kidney slices of healthy and cystinuric patients mucosa have delineated three cystinuric subtypes (119). In
(112). The reports of isolated cystinuria (113), isolated type I, the most common phenotype, heterozygotes have
dibasic aminoaciduria (114), and isolated lysinuria (115) normal urinary amino acid excretion, and homozygotes
cast further doubts on Dent’s hypothesis and suggested the have impaired intestinal transport of cystine and dibasic
existence of specific transport systems for these amino amino acids. In type II, heterozygotes have high excretion of
acids. Furthermore, the occurrence of several transport sys- cystine and dibasic amino acids, and homozygotes show
tems for cystine and dibasic amino acids was supported by impaired intestinal transport of dibasic amino acids but not
the observations of Brodehl and Gellissen (92) and Scriver of cystine. Type III heterozygotes have excretion rates inter-
et al. (116) that tubular reabsorption capacity matures at mediate between the other two, and homozygotes have nor-
different rates for different amino acids. mal intestinal transport of the four amino acids involved.
Subsequent studies using isolated cortical tubules and The three subtypes were considered to be allelic, namely
BBMV (12,13,117) have clarified the picture of tubular mild; moderate; and severe mutations at a single cystinuria
amino acid transport. These studies, coupled with the recog- gene locus (11). However, Goodyer et al. (120) provided
nition that kidney slices preferentially expose the basolateral evidence that type I and type III cystinuria mutations might
membrane, provided evidence for three brush-border mem- involve two distinct genetic loci. This was demonstrated by
brane-bound and two basolateral membrane-bound carrier the finding that type I/III compounds excreted less cystine
systems for cationic amino acids in the kidney. Transport at than type I/I probands, although type III/N heterozygotes
the brush-border membrane occurs by a system shared by excrete higher levels of cystine than their type I/N counter-
cystine and the dibasic amino acids, a system specific for cys- parts. These findings could be explained by genetic comple-
tine, and a system specific for dibasic amino acids. It is the mentation between nonallelic cystinuria genes. Subsequent
shared high-affinity, low-Km system located in the S3 seg- genetic studies have supported this hypothesis (see Molecu-
712 VI. Tubular Disease

lar Genetics). Whereas type I cystinuria is inherited as a fully

recessive trait, type II and III (collectively termed non-type I)
subtypes are inherited as incompletely recessive traits.
Renal ontogeny has important implications for genetic
counseling in cystinuria. This was demonstrated by Scriver
and colleagues’ finding that heterozygous infants under 6
months of age who have immature tubular function can
excrete cystine and dibasic amino acids at levels equivalent to
those found in homozygous adults (116). Urinary excretion
of these amino acids decreased steadily with age to reach the
variant parental value in heterozygous infants but not in
homozygotes. Hence, final classification of a cystinuric phe-
notype should not be done before the age of 6 months.

Molecular Genetics. The identification and characterization

FIGURE 37.6. Representation of the cystinuria-specific missense
of the rBAT/D2H gene have led to the speculation that a or single amino acid point mutations identified in the b o,+AT
defect in the human form of rBAT (SLC3A1) causes cysti- amino-acid transporter. Twenty-three missense or single amino
nuria. Pras et al. (121), using linkage analysis in 17 cystinuric acid point mutations in SLC7A9 (bo,+AT) are depicted. Amino
acid residues conserved in all the human members of the light
families, demonstrated linkage between cystinuria and three subunit of heteromeric amino acid transporter family are indi-
genetic markers on chromosome 2p, providing strong evi- cated in black. Mutations that are associated mainly with a
dence that SLC3A1 was indeed the gene causing the disease. severe urinary phenotype in heterozygotes (urine cystine levels
similar to or below 200 μmol/g of creatinine and the sum of
Calonge et al. (122) identified six specific mutations in the urine levels of cystine and the three dibasic amino acids similar
SLC3A1 gene that segregated with a cystinuria phenotype, to or below 1000 μmol/g of creatinine) are boxed. Mutations
thereby establishing the rBAT gene as the cystinuria gene (Fig. that are associated mainly with a mild urinary phenotype in het-
erozygotes (urinary levels of amino acids below the above indi-
37.3). Subsequent studies have revealed additional mutations cated limits) are indicated in italics. The rest of mutations (with
in SLC3A1 (123–125). As of 2000, more than 60 different undefined or ambiguous phenotype) are indicated in smaller
rBAT mutations have been reported in patients with type I font. See text for details. (Adapted from Palacin M, Borsani G,
Sebastio G. The molecular bases of cystinuria and lysinuric pro-
cystinuria (126). These mutations include nonsense, mis- tein intolerance. Curr Opin Gen Dev 2001;11:328–335.)
sense, splice site, frameshift mutations, and large deletions
(102,126). Defective transport of cystine and dibasic amino
acids has been demonstrated for many of these mutations uptake activity when cotransfected with rBAT in COS cells.
when expressed in Xenopus oocytes (84). The most commonly As of 2000, more than 35 different B0,+AT mutations have
occurring mutation is methionine 467 to threonine (M467T), been identified in non–type I cystinuria patients (126,134)
which causes a defect in trafficking to the plasma membrane (Fig. 37.6). These mutations explain 79% of the alleles in 61
(127,128). This trafficking defect, which has also been dem- non–type I cystinuria patients included in the recent study
onstrated for other rBAT mutations (126,129), is consistent by the International Cystinuria Consortium (134). The
with the proposed role of rBAT as a “helper” of the corre- unexplained alleles might be due to mutations outside the
sponding light subunit in the heteromeric amino acid trans- open reading frame of the SLC3A1 gene, although muta-
porter. No SLC3A1 mutations have been found in type II or tions in other genes might cause non–type I cystinuria
III patients (125,130). (85,126). One patient with I/III phenotype had a dual muta-
In 1997, Wartenfeld et al. (131) and Bisceglia et al. (132) tion in both SLC3A1 and SLC7A9, suggesting the existence
demonstrated that cystinuria type III (and possibly cystinuria of a digenic form of the disease (133).
type II) is linked to a locus on chromosome 19q13.1. In Interestingly, although heterozygous for SLC7A9 muta-
1999, the International Cystinuria Consortium (133) identi- tion show type II or III trait, a minority have type I pheno-
fied the gene SLC7A9, which encodes the 487–amino acid type (84,135). These data have prompted the International
protein B0,+AT that belongs to the family of light subunits of Cystinuria Consortium to consider reclassification of cysti-
amino acid transporters (see Molecular Structure of Amino nuria subtypes based on genotype rather than phenotype.
Acid Transporters; Fig. 37.3). The gene localized to the non– This new classification includes the following:
type I cystinuria 19q locus. Cotransfection of B0,+AT and
rBAT in COS cells resulted in trafficking of rBAT to the Type A—due to two mutations on SLC3A1 on chromo-
plasma membrane and induced L-arginine uptake by cells. some 2
SLC7A9 mutations were found in Spanish, Italian, North Type B—due to two mutations on SLC7A9 on chromo-
American, and Libyan-Jewish cystinuria patients (133). The some 19
mutations in Jews of Libyan origin is valine 170 to methio- Possible AB type—one mutation on each SLC3A1 and
nine, which leads to complete loss of B0,+AT amino acid SLC7A9 (135)
 37. Aminoaciduria and Glycosuria 713

Diagnosis and Clinical Features. The simplest diagnostic ing and voiding, with particular attention to nighttime
test is the microscopic examination of the urinary sediment hours, when urine may become supersaturated with cys-
of a freshly voided morning urine (102). The presence of tine. Water should be taken at bedtime and whenever the
typical flat hexagonal cystine crystals is diagnostic. Acidifi- patient awakens at night. Rigid adherence to fluid ther-
cation of the urine precipitates cystine crystals and may apy is effective in approximately 70% of patients (102).
improve the yield of the test. The best screening procedure ■ Oral alkali in addition to high fluid intake to further
is the cyanide-nitroprusside test (102). A positive reaction increase cystine solubility in the urine (104). A urine pH
occurs with as little as 75 to 125 mg cystine per gram of of 7.5 to 8.0 can be maintained by the provision of 1 to 2
creatinine, which is well below that of homozygotes, which mEq/kg/day of bicarbonate or citrate in divided doses.
excrete at least 250 mg/g creatinine. Some heterozygotes Because high sodium intake increases cystine excretion
may also be detected by this procedure (105). The test is (see Dietary Therapy), potassium citrate is preferred
not specific and may detect acetone or homocystine as well. (104). Because urine alkalinization may result in forma-
The definite test is a measurement of urine cystine and tion of mixed calcium-containing stones, adherence to
dibasic amino acid concentration by ion exchange chroma- high fluid intake is crucial.
tography. The upper limits of normal are 18, 130, 16, and ■ Dietary therapy to reduce cystine production and excre-
22 mg/g creatinine for cystine, lysine, arginine, and orni- tion. Studies examining the effect of dietary restriction of
thine, respectively (104). In establishing the diagnosis of methionine (a metabolic precursor to cystine) on urine
classic cystinuria, it is important to exclude other condi- cystine excretion have yielded variable results (104). Also,
tions associated with increased urinary cystine excretion, diets low in methionine are very difficult to follow and
including isolated cystinuria, tubular immaturity in young may be harmful to growing children. Therefore, dietary
infants, generalized aminoaciduria (Fanconi syndrome), methionine restriction is not recommended (104). Urinary
and organic acidemias (102,104). excretion of cystine and dibasic amino acids in cystinuric
Cystine stones are radiopaque because of the density of patients has been shown to correlate with urinary sodium
the sulfur molecule, and on a roentgenogram, they appear excretion (140,141). Hence, dietary sodium restriction is
smooth. Occasionally, they form staghorn calculi. Cystine recommended by some authors as a safe approach to the
also may act as a nidus for calcium oxalate so that mixed treatment of cystinuria (102). L-glutamine administered
stones may be found (136). Factors contributing to mixed orally or intravenously in conjunction with low salt intake
stone formation in cystinuria include alkalinization of urine reduces cystine excretion (140,142), but this effect was not
and urinary tract infections. observed in patients receiving a normal salt diet (143). The
The disease usually presents with renal colic. Occasion- mechanism of the anticystinuric effect of glutamine is
ally, infection, hypertension, or renal failure may be the unclear.
first manifestation (102). Cystinuria occurs with equal fre- ■ Pharmacologic therapy to increase cystine solubility and
quency in males and females, but males are more severely decrease cystine excretion. The sulfhydryl-binding com-
affected because of a greater likelihood of urethral obstruc- pound D-penicillamine (β-dimethylcysteine) leads to the
tion in the male. Clinical manifestations usually occur in formation of the mixed disulfide penicillamine-cysteine after
the second and third decades of life, with a 62% probability a disulfide exchange reaction. This mixed disulfide is far
of stones by age 25 years (137). Most patients have recur- more water-soluble than cystine. Hence, penicillamine acts
rent stone formation. Cystinuric patients who receive a kid- by reducing cystine excretion as well as by permitting the
ney transplant have normal urinary cystine and dibasic excretion of a more soluble compound. Penicillamine, given
amino acid excretion after transplantation (138,139). at a dosage of 1 to 2 g/24 hours (30 mg/kg in children), is
highly effective and reduces urinary cystine excretion to
Treatment. Cystine crystalluria occurs when the cystine under 200 mg/g creatinine (144). Unfortunately, penicil-
content of the urine exceeds 300 mg/L at pH 4.5 to 7.0. lamine produces serious side effects in 50% of patients
Cystine solubility increases sharply at a urine pH above 7 (145). These reactions include rashes (including pemphi-
(104). The major therapeutic approaches to cystinuria are gus), fever, arthralgia, nephrotoxicity (including nephrotic
designed to increase the solubility of cystine, reduce excre- syndrome in up to 30% of patients and rapidly progressive
tion of cystine, and convert cystine to more soluble com- glomerulonephritis), pancytopenia, and loss of taste. Penicil-
pounds (102). Therapies used in the management of lamine also increases copper and zinc excretion in the urine.
cystinuria include the following: The loss of taste may be reversed by copper administration
(104). Pyridoxine metabolism may be impaired, and pyri-
■ Increased oral fluid intake to increase urine volume and doxine supplementation should be provided for patients
cystine solubility. Because cystinuric patients excrete 0.5 receiving D-penicillamine. Most of these side effects revert to
to 1.0 g cystine/day, intake of 3 to 4 L could be required normal on discontinuation of the drug. Because of the seri-
to keep the urinary cystine concentration below 300 mg/ ous side effects, D-penicillamine therapy should be reserved
L. Patients should develop a 24-hour schedule for drink- for patients unresponsive to conservative management, and
714 VI. Tubular Disease

stepwise dosing is recommended (102). D-acetyl penicil- for patients in whom conservative therapy fails. Urologic
lamine, another anticystinuric sulfhydryl agent, has fewer intervention may be indicated in selected patients.
side effects than D-penicillamine. Mercaptopropionyl gly-
cine (MPG), another agent undergoing a disulfide exchange Isolated Cystinuria
reaction, is as effective as D-penicillamine in the treatment of Brodehl et al. (113) report two siblings who showed high
cystinuria (146). MPG has the same toxicity as D-penicil- urinary excretion rates of cystine but normal dibasic amino
lamine, but serious renal and hematologic reactions requir- acid excretion. The children did not develop renal stones.
ing cessation of therapy are much less common with MPG This report, along with the detection of a similar abnormal-
(146). Because of the lower incidence of side effects and ity in dogs (154), provides evidence of a separate cystine
because this compound can be used in patients who develop transporter not shared with dibasic amino acids in the
allergic reactions to D-penicillamine, MPG is the pharmaco- tubular brush-border membrane, which appears to be
logic agent of choice in the therapy of cystinuria (146,147). defective in isolated cystinuria. The molecular nature of
this proposed transporter is unknown.
Several studies have examined the effect of captopril, an
angiotensin-converting enzyme inhibitor, on urinary cystine Lysinuric Protein Intolerance (Hyperdibasic
excretion in cystinuric patients (148–150). This nontoxic sulf- Aminoaciduria Type II)
hydryl compound builds highly soluble captopril-cysteine LPI is a rare autosomal recessive disorder characterized by
disulfides. Although a reduction in cystine excretion has been excessive urinary excretion of dibasic amino acids (especially
demonstrated in some studies (148,149), others (150) have lysine), normal cystine excretion, and poor intestinal absorp-
failed to show an effect. Further studies are needed to evaluate tion of dibasic amino acids (155–157). Plasma values of
the efficacy of captopril therapy in cystinuria. It has been pro- dibasic amino acids are subnormal. The disease is relatively
posed that meso-1,3 dimercaptosuccinic acid (DMSA), an common in Finland, where the prevalence of the disease is
additional compound forming disulfide linkage with cysteine, 1:60,000 (157). Approximately 100 patients, one-half of
might be a useful therapeutic agent in cystinuria (151). The them Finns, have been described (157). Homozygous
efficacy of this agent in cystinuria remains to be established. patients show massive dibasic aminoaciduria as well as hyper-
Ascorbic acid, which acts as a reducing agent to convert ammonemia after a protein overload; heterozygotes have
cystine to the more soluble cysteine, has been suggested as a normal urinary amino acid excretion but impaired renal and
therapeutic modality in patients with cystinuria (147). To intestinal transport of dibasic amino acids at increased loads.
date, the results of the use of this compound on a limited The clinical manifestations in homozygotes for LPI are those
number of patients have been variable (104,147). Also, con- of protein malnutrition and postprandial hyperammonemia.
cerns have been raised that ascorbic acid therapy in cysti- They include failure to thrive, marked protein intolerance,
nuria is potentially lithogenic because of the hyperoxaluric anorexia, vomiting, diarrhea, hepatosplenomegaly, muscle
and hypocitraturic effect of this agent. hypotonia, interstitial lung disease, osteoporosis, seizures,
Several urologic procedures have been used to treat cys- and coma (157).
tine stones: The pathogenic mechanism of LPI appears to be a
defective transport of dibasic amino acids in the basolateral
■ Chemolysis of stones by irrigation through a percutaneous membrane of renal and intestinal epithelial cells, resulting
nephrostomy. Successful dissolution of stones has been in impaired efflux from cell to interstitium (158,159). This
achieved using N-acetylcysteine, D-penicillamine, α- has been confirmed by a measurement of fluxes in jejunal
MPG, and a very alkaline agent, tromethamine (103,104). biopsy specimens from LPI patients (158), as well as by the
■ Extracorporal shock wave lithotripsy. This therapeutic observation that infusion of citrulline to these patients
modality has been only partially successful because of the results in massive argininuria and ornithinuria (159). Cit-
organic nature and the uniform crystal structure of cys- rulline is reabsorbed from the tubular lumen by a neutral
tine stones (103,152). Percutaneous ultrasonic lithotripsy amino acid transport mechanism and is converted to argi-
has been somewhat more effective (153). nine and ornithine in the renal cell. Impaired exit at the
■ Lithotomy. Surgical removal of stones is necessary only in antiluminal membrane results in backflux of accumulated
rare patients with obstructing or infected stones unre- arginine and ornithine at the brush-border membrane sur-
sponsive to a more conservative approach. face. It is the high-affinity, specific dibasic amino acid trans-
porter y+L that is affected in this disease (84,128). Because
In summary, the mainstays of therapy in cystinuria basolateral membrane transporters of epithelial cells and
include hydration, alkalinization of the urine, and dietary plasma membrane transporters of parenchymal cells are
sodium restriction. Full compliance with this regimen homologous carriers, it is not surprising that hepatocytes
results in significantly reduced urinary cystine excretion (160), granulocytes (160), and cultured skin fibroblasts
and good long-term prognosis in most patients. Pharmaco- (161) from patients with LPI show impaired transport of
logic treatment with sulfhydryl agents should be reserved dibasic amino acids. Erythrocytes from LPI patients, which
 37. Aminoaciduria and Glycosuria 715

do not have the y+L system, show normal cationic amino ornithine, and, most important, citrulline (170). Adminis-
acid transport (162). tration of the latter amino acid, which corrects the hepatic
It is presumed that the defective hepatic transport of diba- deficiency in ornithine and arginine, results in clinical
sic amino acids deprives hepatic cells of ornithine and argi- improvement and catch-up growth.
nine, which are necessary for urea production (157). This
results in protein intolerance, hyperammonemia, and low Hyperdibasic Aminoaciduria Type I
urea formation. This notion is supported by the observation An autosomal dominant cationic aminoaciduria has been
that L-citrulline supplements improve protein tolerance in described in two families containing several heterozygotes
LPI patients (159). This amino acid, a metabolic precursor of and a single homozygote (114,171). This disease, called
ornithine and arginine, is absorbed in the intestine, enters hyperdibasic aminoaciduria type I, is characterized by
the hepatic cell via neutral amino acid transport mechanisms, impaired renal reabsorption and intestinal absorption of the
is metabolized in the liver to ornithine and arginine, and dibasic amino acids lysine, arginine, and ornithine but not
restores the pathway for ammonia disposal (157). of cystine. Plasma values of dibasic amino acids are normal,
In 1992, the rBAT homologous protein, the human cell and there is no protein intolerance or hyperammonemia.
surface glycoprotein 4F2hc (encoded by the gene CD98), The reported homozygous patient had mental retardation
was shown to induce y+L cationic amino acid transport in (171). Hyperdibasic aminoaciduria type I heterozygotes
Xenopus oocytes (77). Like rBAT, 4F2hc represents the have modest cationic aminoaciduria, whereas LPI heterozy-
heavy subunit of a disulfide-linked heteromeric amino acid gotes have no hyperaminoaciduria. It has been speculated
transporter (163) (see Molecular Structure of Amino Acid that the brush-border membrane–bound, high-capacity
Transporters; Figs. 37.3 and 37.4). Molecular analysis transporter for dibasic amino acids, which excludes cystine,
excluded CD98, which has been localized to chromosome is defective in this disease. The molecular nature of this
11, as a candidate gene for LPI (164). Linkage studies in transporter is unknown.
Finnish and non-Finnish LPI families placed the gene of
the disease to 14q11-13 (165,166). Isolated Lysinuria
In 1998, Torrents et al. (87) identified a human cDNA, Omura et al. (115) reported a single child with increased
SLC7A7, as encoding y+LAT-1—a member of the family of urinary excretion of lysine; impaired intestinal absorption
light subunits that combine with 4F2hc to form heteromeric of this amino acid; low plasma lysine levels; and normal
amino acid transporters (see Molecular Structure of Amino renal and intestinal transport of ornithine, arginine, and
Acid Transporters; Fig. 37.3). The 4F2hc/y+LAT-1 trans- cystine. The patient did not have hyperammonemia but
porter has been shown to have the activity of amino acid had failure to thrive and mental retardation, probably sec-
transport system y+L that is responsible for the efflux of basic ondary to the deficiency of the essential amino acid, lysine.
amino acids at the basolateral plasma membrane of epithelial This case implies a defect in a selective transport system for
cells (128) (see Specificity of Transport; Figs. 37.1 and 37.3). lysine in the kidney and the intestine. Such a system has
The SLC7A7 gene localized to the LP1 locus (87). In 1999, not been identified in physiologic or molecular studies.
the two groups (164,167) demonstrated that mutations in
SLC7A7 cause LPI in Finnish, Italian, and Spanish patients.
Neutral Aminoaciduria
As of 2000, more than 25 SLC7A7 mutations, spread along
the entire SLC7A7 gene, have been found in LPI patients Three distinct disorders of neutral amino acid transport
from different ethnic groups (126). Expression studies in have been identified: Hartnup disorder, methioninuria, and
Xenopus oocytes showed that various LPI mutations result in histidinuria.
proteins that fail to co-induce amino acid transport activity
when expressed with 4F2hc (168). Two of the mutants Hartnup Disease
reached the oocyte plasma membrane when coexpressed with Hartnup disease, which may have afflicted Julius Caesar
4F2hc, demonstrating that they are transport-inactivating and his family (172), was first recognized in two siblings in
mutations (168). England in 1956 (173). This disease is characterized by
LPI is characterized by poor genotype/phenotype corre- intestinal malabsorption and massive aminoaciduria of the
lation. Even Finnish patients, who share the same founder neutral monoamino monocarboxylic amino acids alanine,
mutation, show variation in the clinical picture (168). Sim- serine, threonine, valine, leucine, isoleucine, phenylalanine,
ilarly, both interfamilial and intrafamilial phenotypic vari- tyrosine, tryptophan, histidine, glutamine, and asparagine
ability were observed in Italian LPI patient heterozygotes (174). Most patients also have increased excretion of
for the same mutation (169). This suggests that, in addition indolic compounds that originate in the gut from bacterial
to SLC7A7 mutations, hitherto unknown factors play a degradation of tryptophan (175). Transport of other neu-
role in the pathogenesis of LPI (126,128). tral amino acids, including cystine, imino acids, glycine,
Therapy of LPI consists of protein restriction to prevent and β-amino acids, is unaffected. The disease is inherited as
hyperammonemia, as well as oral supplements of arginine, an autosomal recessive trait and has an estimated incidence
716 VI. Tubular Disease

of 1:20,000 live births. Heterozygotes have normal urinary soluble form of tryptophan, has been shown to increase
acid excretion under physiologic conditions. Clinical fea- serum tryptophan and reverse clinical symptoms in patients
tures in homozygotes may include photosensitive rash, cer- with Hartnup disease (181).
ebellar ataxia, and a variety of psychiatric manifestations, as
in the features of pellagra (171). The pellagra-like manifes- Methioninuria
tations are primarily caused by intestinal malabsorption There are two case reports of patients with isolated increased
and urinary loss of tryptophan, an amino acid that is urinary excretion of methionine and its metabolic break-
required for niacin synthesis. The diagnosis should be sus- down products (182,183). The patients had malodorous
pected in any patient with pellagra who has no history of urine, edema, episodic hyperventilation, seizures, and mental
niacin or nicotinamide deficiency and should be made by retardation. The underlying defect appeared to be abnormal
chromatographic analysis of the urine (174). transport of methionine in the kidney and in the intestine.
A defect in the broad-specificity neutral α-amino acid α-Hydroxybutyric acid, a bacterial degradation product of
transport mechanism in the renal and intestinal brush- unabsorbed intestinal methionine, appeared in the urine of
border membrane is presumed to be the pathogenic mecha- these patients. This organic acid may have been responsible
nism underlying this disorder (11,176). Two lines of evi- for the neurologic manifestations in these patients. A low-
dence support the hypothesis that the lesion is localized in methionine diet resulted in significant clinical improvement.
the brush-border membrane (11). First, plasma amino acid These cases of isolated methioninuria, as well as the observa-
response in patients with Hartnup disease was attenuated tion that methionine, which shares the broad-spectrum
after oral feeding of free α-amino acids but not when amino acid transport system with other neutral amino acids,
appropriate dipeptides were given orally (177). The dipep- are not hyperexcreted in Hartnup disease, provided evidence
tides are presumed to be reabsorbed by a dipeptide-specific for the existence of a specific transport pathway for methio-
brush-border membrane transporter and hydrolyzed in the nine in renal and intestinal epithelium.
enterocyte; the free amino acids exit the cell via a nondefec-
tive basolateral membrane-bound carrier. Second, tryp- Histidinuria
tophan transport is normal in various parenchymal cells Two siblings (184) and two other patients (185,186) have
from patients with Hartnup disease, including leukocytes, been reported with isolated histidinuria. All showed significant
placenta, and cultured skin fibroblasts (174,178). As indi- mental retardation. Investigation revealed impaired transport
cated earlier, carriers in plasma membranes of parenchymal of histidine in the kidney and intestine. Parents of the two
cells are homologous to basolateral membrane carriers in affected siblings showed normal urinary histidine excretion
epithelial cells, which are supposed to be normal in Hart- under normal conditions but hyperhistidinuria after an oral
nup disease. histidine load. The mode of inheritance of this disease is
The transport characteristics and the epithelial distribu- uncertain, but autosomal recessive inheritance has been sug-
tion of the Na+-dependent neutral amino acid transport Bo gested (11). Although the broad-specificity transporter of neu-
(see Specificity of Transport; Fig. 37.1), which is encoded tral amino acids is the major carrier for histidine, as suggested
by the recently identified ATBo gene (61) (see Molecular by in vitro studies as well as by fractional excretion rates above
Structure of Amino Acid Transporters), raise the possibility 50% for this amino acid in Hartnup disease (11), the case
that this transporter is defective in Hartnup disorder. The reports of histidinuria suggest that an isolated histidine carrier
role of this gene, which localizes to chromosome 19q13.3 is operating in the renal tubule and the intestine.
(61), in the pathogenesis of Hartnup disorder has not been
investigated.
Iminoaciduria and Glycinuria
The presence of alternative transport pathways for neu-
tral amino acids in the renal tubule explains why the defect Two distinct disorders belong to this group of aminoaci-
in the broad-specificity neutral amino acid transport in durias: iminoglycinuria and isolated glycinuria.
Hartnup disease results in only partial loss of some amino
acids (e.g., phenylalanine) (11). These alternative systems, Iminoglycinuria
as well as the oligopeptide-preferring carriers in the kidney Iminoglycinuria is an autosomal recessive membrane trans-
and intestine, are responsible for normal plasma amino acid port defect characterized by excretion of excessive amounts of
values and lack of symptoms in most patients (179). As proline, hydroxyproline, and glycine in the urine (187). Imi-
suggested by Scriver et al. (179), Hartnup disease is multi- noglycinuria is a benign condition with an estimated inci-
factorial in its pathogenesis, and only patients genetically dence of 1:15,000 live births (187,188). It is the shared,
predisposed to low plasma amino acid levels and impaired brush-border membrane–bound, group-specific transport
tryptophan metabolism develop symptoms. pathway for imino acids and glycine (see Specificity of Trans-
Patients with Hartnup disease respond well to oral ther- port) that is defective in this condition (94,187). The selective
apy with nicotinamide, 40 to 100 mg/day (180). More glycine-specific and imino acid–specific transport systems are
recently, oral administration of tryptophan ethylester, a lipid- not affected. The activity of these selective transporters
 37. Aminoaciduria and Glycosuria 717

accounts for the normal plasma levels of glycine and imino Dicarboxylic Aminoaciduria
acids observed in iminoglycinuria. As discussed earlier, the late
Selective urinary hyperexcretion of the acidic amino acids
maturation of these selective transporters in normal infants is
glutamate and aspartate was first reported in two children
responsible for neonatal physiologic iminoglycinuria (92,94).
(193,194). One of the children also had impaired intestinal
The absence of the selective transporters during early life also
absorption of these amino acids. The condition is inherited
explains why infants with iminoglycinuria who lack the shared
as an autosomal recessive trait and appears to be benign;
transporter have fractional excretion values for glycine and
screening in a French-Canadian population revealed a large
proline approaching 100%, which decline after the first
number of healthy probands with hyperdicarboxylic ami-
months of life (11). Defective intestinal transport of proline
noaciduria and an incidence of 1:29,000 live births (195).
has been found in some patients with iminoglycinuria (187).
The pathogenic mechanism appears to be a defect in the
As expected, proline transport is not defective in leukocytes or
dicarboxylic amino acid transport system in the brush-
skin fibroblasts because these cells do not possess carriers cor-
border membrane. The excretion of dicarboxylic amino
responding to a brush-border membrane carrier (11).
acids in homozygous patients may greatly exceed the GFR,
Iminoglycinuria is a genetically heterogenous condition
suggesting tubular secretion or backflux of dicarboxylic amino
with several mutant alleles (11,187). This view is supported
acids from cell to lumen (11). Treatment with glutamate and
by several observations. First, obligate heterozygotes may
aspartate corrected the hypoglycemia observed in one
have hyperglycinuria (but not iminoaciduria) or may have
patient (193). This hypoglycemia probably resulted from
normal urinary amino acid excretion (189). Second, some
the absence of these gluconeogenic amino acids.
homozygous patients have an intestinal transport defect.
The neuronal and peripheral anionic amino acid trans-
Third, a variant exists with normal transport maximum
porter EAAC1 (EAAT3) (see Molecular Structure of Amino
(Tm) for proline and a defect affecting glycine transport
Acid Transporters) is an obvious candidate for the transporters
more than proline transport, indicating a Km defect (190).
defective in dicarboxylic aminoaciduria. The transporter is
The genetic defect responsible for iminoglycinuria is
highly expressed in the kidney and intestine (54). Peghini et al.
unknown. The transport characteristics of the amino acid
(196) have shown that EAAC1-deficient mice develop dicar-
transport system expected to be defective in this disorder fit
boxylic aminoaciduria, a finding that strongly suggests that the
those of the IMINO and/or the Na+- and Cl–-dependent
EAAC1 gene is a candidate gene for dicarboxylic aminoaci-
transporters of the GAT transporter family (see Specificity
duria. In 2001, Nozaki et al. (197), by using homozygosity
of Transport). The latter group includes GLYT1, GLYT2,
mapping, assigned the Hartnup disease locus to chromosome
and PROT, which transport glycine and/or proline. The
5p15. Nevertheless, there are no functionally characterized
GLYT transporters are expressed in the kidney and may be
genes mapped to this locus that seem to be obvious candidates
good candidates for the immunoglycinuria phenotype.
for the Hartnup disorder.
Differential diagnosis of iminoglycinuria includes neo-
An interesting feature of dicarboxylic aminoaciduria is the
natal iminoglycinuria; Fanconi syndrome, in which gener-
decreased uptake of anionic amino acids by cultured skin
alized aminoaciduria occurs; and hyperprolinemia, an
fibroblasts from patients with this condition (198). It has been
inborn error of proline metabolism that exhibits overflow
shown that anionic amino acid–preferring carriers in epithelial
prolinuria as well as hydroxyprolinuria and glycinuria sec-
brush-border and parenchymal cells have similar characteris-
ondary to a proline-induced inhibition or hydroxyproline
tics (11,198). As pointed out by Scriver and Tenenhouse (11),
and glycine transport. This inhibition occurs at the shared
the apparent expression of the mutant gene in both the brush-
luminal carrier for these amino acids.
border membrane of the epithelium and the plasma mem-
brane of parenchymal cells is unique and contrary to the
Isolated Glycinuria
expected pattern of analogy between basolateral membrane-
Isolated glycinuria without iminoaciduria has been reported
bound and plasma membrane-bound carriers (see Classic
in two families (190,191). It has been suggested that this
Cystinuria, Hartnup Disease, and Lysinuric Protein Intoler-
condition is a manifestation of a mutation affecting the spe-
ance). The possibility that the EAAC1 transporter, likely
cific high-affinity carrier for glycine, the molecular identity
defective in dicarboxylic aminoaciduria, is expressed at both
of which remains to be established (11). It is possible, how-
the luminal and basolateral membranes of the proximal tubule
ever, that hyperglycinuria represents a defect in the shared,
awaits future investigation.
imino acid-glycine transport system affecting affinity rather
than capacity of the system (Km variant). Because the condi-
tion appears to be inherited as an autosomal dominant trait,
β-Aminoaciduria
it also is possible that these cases represent hyperglycinuric
heterozygotes for the iminoglycinuria allele. Glycinuria has No inborn error of the β/γ-amino acid transport system
also been reported in association with glycosuria, called gly- that carries taurine, β-alanine, β-aminobutyric acid, and
coglycinuria (192). The defect underlying this autosomal GABA has been reported in humans. An impaired taurine
dominant condition is unknown. transport, however, has been found in an inbred mouse
718 VI. Tubular Disease

strain (C57BL/6J) (199). Studies using kidney slices (199) concentration is progressively elevated, the amount of glu-
and isolated basolateral membrane vesicles (95) have local- cose reabsorbed increases linearly until a maximum value is
ized the transport defect to the antiluminal membrane of reached (Fig. 37.7). Beyond this maximum rate of glucose
the proximal tubular epithelium. In analogy with the reabsorption, further increases of filtered D-glucose are
defect in LPI, an impaired taurine exit across the basolat- excreted in the urine. As shown in Figure 37.7, the glucose
eral membrane and backflux from cell to lumen may titration curve provides parameters of glucose transport
underlie the hypertaurinuria observed in the C57BL/6J such as minimum threshold (FminG), defined as the filtered
mouse (95,199). glucose load at which 1 mg of glucose per minute appears
in the urine, and the tubular maximum for glucose reab-
sorption (TmG). Reported TmG values for glucose in human
GLYCOSURIA
adults and children have ranged between 260 and 350 mg/
min/1.73 m2 (202–204), with lower values in infants
General Characteristics of
(204,205). When corrected for the GFR (Tm/GFR), Tm
Renal Glucose Transport
value for glucose in infants, children, and adults is approxi-
Under normal conditions, the reabsorption of filtered glu- mately 2.5 mg/mL (204).
cose by the renal tubule is almost complete. Less than The glucose titration curve (Fig. 37.7) is characterized
0.05% of the renal glucose load is excreted in the human by a splay—a rounding of the curve during the transition
urine (200); 90% of the filtered glucose is reabsorbed in the from virtually complete reabsorption of filtered D-glucose
proximal convoluted tubule; and the rest is reclaimed in the to complete excretion of excess glucose load. This deviation
proximal straight tubule, the loop of Henle, and, to some from the theoretical curve is explained by the nephron
extent, the collecting duct (201). functional heterogeneity (the gradual progression from the
Reabsorption of glucose across the proximal tubular first to the last nephron to saturate), as well as by the varia-
brush-border membrane occurs by an active, carrier-medi- tion in affinity for glucose or Km values between carriers
ated, concentrative, Na+-dependent transport process (the magnitude of the splay is inversely proportional to the
(200,201). The Na+ electrochemical gradient driving glu- affinity of the transporter for the D-glucose) (200).
cose transport across the brush-border membrane is main- Studies in isolated perfused tubules (206) and later exper-
tained by the activity of the basolateral membrane-bound iments using isolated BBMV from pars convoluta (S1 and S2
Na+-K+-ATPase that pumps Na+ out and K+ into the cell. segments) and pars recta (S3 segment) of the proximal tubule
Na+-glucose cotransport across the luminal membrane is (207–209) demonstrate that the kinetic properties and stoi-
electrogenic positive and phlorizin inhibitable. Glucose exit chiometric relationships of D-glucose reabsorption change
from the cell occurs by an Na+-independent–facilitated dif- along the length of the nephron. These studies provide
fusion down the glucose concentration gradient (201). This evidence for two Na+-dependent transport mechanisms, a
diffusional exit of glucose is mediated by a carrier that is low-affinity/high-capacity cotransport system in the early
distinct from that found at the luminal membrane surface proximal tubule and a high-affinity/low-capacity system in
(see Facilitative Glucose Transporters). the late proximal tubule. The Na+-glucose coupling ratio was
The mammalian kidney is characterized by a limited found to be 1:1 in the convoluted proximal tubule and 2:1 in
capacity to reabsorb D-glucose (200). As plasma glucose the straight proximal tubule (208,209).

FIGURE 37.7. Renal glucose titration curves. Theoreti-

cal and observed normal curves are compared to
abnormal curves observed in type A and type B renal
glycosuria. TmG, maximum rate for glucose reabsorp-
tion; FminG, minimum threshold. (From Elsas LJ, Longo
N. Glucose transporters. Annu Rev Med 1992;43:377–
393, with permission.)
 37. Aminoaciduria and Glycosuria 719

FIGURE 37.8. Schematic structure of the Na+/glucose

cotransporter (SGLT) and the facilitative glucose
transporter (GLUT). The two mutations shown in SGLT
were identified in one of the original families with
glucose-galactose malabsorption. (Adapted from
Longo N. Human glucose transporters. Adv Pediatr
1998;45:293–313.)

The efficiency of a coupled carrier system increases as the of sodium cotransport proteins, which includes the renal Na+-
power of the stoichiometry increases (201,209). Whereas nucleoside, renal Na+-myoinositol, and the Escherichia coli
the early proximal tubule glucose transporter is responsible Na+-proline cotransporters (51,211).
for the reabsorption of the bulk of filtered D-glucose from The SGLT1 transporter (Fig. 37.8) consists of 662 to 664
the tubular lumen, the late proximal tubule glucose trans- amino acids and has 14 membrane-spanning sequences that
porter is responsible for the removal of the last traces of glu- are presumed to be α-helical, with the NH2 and COOH ter-
cose from the urine (209). The arrangement of transporters mini located on the cytoplasmic side of the membrane. A
in series along the proximal tubule enables the kidney to simple glycosylation site is found in the hydrophilic domain
reabsorb glucose from the urine in a more energy-efficient between transmembrane segments 5 and 6 (214). SGLT1
mechanism than can be achieved by either of the cotrans- from rabbit (38), rat (216), pig (216), and human (212)
porters acting alone. show high homology in sequence and structure. The gene
encoding the human intestinal SGLT1 has been recently
localized to the q11.2lqter region of chromosome 22 (217).
Molecular Biology of
Recent studies using Xenopus oocytes (218) demonstrate that
Na+-Glucose Cotransporters
protein kinases (PKA and PKC) regulate SGLT1 activity by
Various approaches have been used to identify and isolate the controlling the distribution of transporters between intracell-
Na+-glucose cotransporters. Earlier methods, including solu- ular compartments and the plasma membrane and that this
bilization and reconstitution techniques, semiselective and occurs by exocytosis and endocytosis.
photoaffinity labeling, phlorizin affinity chromatography, Studies by Pajor et al. (219) and Lee et al. (215) using
radiation inactivation, and immunoaffinity absorption (201), expression studies in Xenopus oocytes, Western and Northern
have been inconclusive and yielded very limited biochemical analysis, and in situ hybridization and immunocytochemistry
data. However, considerable progress has been made in the have provided evidence that the renal Na+-glucose cotrans-
past decade in elucidating the molecular structure of mem- porter SGLT1 is the high-affinity/low-capacity transporter
brane proteins that catalyze sugar transport processes, includ- found predominantly in the straight proximal tubule (S3 seg-
ing the Na+-glucose cotransporter (51,210,211). Hediger et ment). Evaluation of the stoichiometry of SGLT1-mediated
al., using expression cloning in Xenopus oocytes (see Molecu- transport in Xenopus oocytes revealed an Na+ to glucose cou-
lar Structure of Amino Acid Transporters), cloned and pling ratio of 2:1 (215).
sequenced the Na+-dependent D-glucose transporter from A second low-affinity Na+-glucose cotransporter named
rabbit (38) and human (212) intestine. Subsequently, molec- SGLT2 was isolated from rat (220) and human (221,222)
ular analysis revealed that rabbit intestinal and renal Na+- kidney. The amino acid sequence of SGLT2 is 59% identical
glucose cotransporters are essentially identical (213). The to that of SGLT1 (221). SGLT2 has an Na+ glucose coupling
Na+-glucose cotransport proteins belong to the SGLT1 family ratio of 1:1, does not recognize galactose (which is a substrate
720 VI. Tubular Disease

for SGLT1), and is strongly expressed in proximal tubule S1

segments (222). Human SGLT2 gene was mapped to chro-
mosome 16p11.2 close to the centromere (223).
A third Na+-dependent glucose transporter, termed SGLT3,
was isolated from an LLC-PK1 pig renal cell line (224). It is a
low-affinity, Na+-glucose (not galactose) cotransporter (225).
Human SGLT3 gene is located on chromosome 22 (211).

Facilitative Glucose Transporters

Glucose concentrated inside tubular epithelial cells flows to
the interstitium down its concentration gradient through
facilitative glucose transporters located in the basolateral
membrane (200,226). The basolateral Na+-independent
and the luminal Na+-dependent glucose transport systems FIGURE 37.9. Schematic model for the distribution (luminal or
also differ with respect to inhibition and specificity (201). basolateral) of cloned glucose transporters in renal tubular epi-
thelium. Orientation permits net reabsorption from lumen to
The basolateral glucose transporter is inhibited by the mold interstitium. (Adapted from Silverman M. Structure and function
metabolite cytochalasin-B but not by phlorizin. It also of hexose transporters. Annu Rev Biochem 1991;60:757–794.)
accepts D-glucose.
The basolateral membrane glucose transporters belong to
a family of Na+-independent facilitative glucose transporters, luminal Na+-dependent, high-affinity/low-capacity SGLT1
of which five different isoforms called GLUT1, GLUT2, coupled with the basolateral Na+-independent, high-affinity
GLUT3, GLUT4, and GLUT5 have been identified (226– GLUT1 reabsorb the remaining low concentration of glu-
228). These subspecies have extensive structural homologies cose in the late part of the proximal tubule.
but differ in their tissue distribution, specific function, insu-
lin sensitivity, sugar specificity, and kinetic characteristics.
Maturation of Glucose Transport
The human erythrocyte glucose transporter GLUT1 was the
first glucose transporter to be cloned and sequenced (229). It The immature renal tubule in animals (234,235) and humans
is the most ubiquitously distributed of the transporter iso- (204,236,237) is characterized by decreased ability to reabsorb
forms (227). Isoforms GLUT2 to GLUT5 have been cloned glucose. Tubular reabsorption of glucose relates directly to
by screening cDNA libraries from various tissues and species postnatal age, and glycosuria occurs commonly in infants less
with a GLUT1 DNA probe (226). The facilitative glucose than 30 weeks’ gestational age (236–238). There are few data
transporters (Fig. 37.8) have 12 membrane-spanning on the development of renal glucose transport mechanisms.
domains, with both the NH2 and the COOH termini of the The fetal kidneys of various animal species (234,239,240)
protein facing the cytoplasm. A single glycosylation site is have been shown to reabsorb glucose. In the fetal rat kidney
located between transmembrane domains 1 and 2. (239), glucose reabsorption appeared to be Na+ dependent and
GLUT2, a low-affinity transporter, is the predominant phlorizin inhibitable. A decreased initial rate uptake of α-
facilitative glucose transporter in hepatocytes and in the methyl-D-glucoside was found in isolated renal tubules from
basolateral membrane of intestinal and renal tubular cells neonatal rat (241) and dog (235). BBMV studies (242) dem-
(230,231). In the kidney, GLUT2 is present only in the onstrated a concentrative, Na+-dependent, electrogenic and
basolateral membrane of cells in the proximal convoluted phlorizin-sensitive glucose transport in the fetal rabbit kidney.
tubule (S1 and S2 segments) (230,231). Studies using anti- The fetal glucose transport mechanism, however, had a signifi-
peptide antibodies specific for GLUT1 have detected this cantly lower capacity than the adult glucose transport system.
high-affinity transporter in the kidney (231,232). It was Recent experiments exploring the expression of SGLT1 and
found in the basolateral membrane of cells forming the SGLT2 mRNAs in embryonic rat kidneys (220) revealed that
proximal straight tubule (S3 segment). GLUT5 has also the two Na+-glucose cotransporters appeared early in gestation
been identified in the kidney (233). and that they were developmentally regulated. Recent evidence
Thus, it appears that transepithelial glucose transport in suggests that the increase in SGLT1 mRNA-accompanying
the proximal tubule occurs by two different pairs of apical cell differentiation in the pig kidney cell line LLC-PK1 is regu-
Na+-dependent and basolateral Na+-independent glucose lated by PKA (243) and PKC (244).
transporters (200,226) (Fig. 37.9). A luminal Na+-depen- Little is known about the maturation of the facilitative
dent, low-affinity/high-capacity glucose transporter (desig- glucose transporters in the kidney. A study (245) examining
nated SGLT2) and a basolateral Na+-independent, low- the developmental pattern of these transporters in the rat
affinity GLUT2 are responsible for the bulk of glucose kidney showed that renal GLUT1 and GLUT5 gene expres-
reabsorption in the early part of the proximal tubule. A sion was unchanged throughout development, whereas
 37. Aminoaciduria and Glycosuria 721

GLUT2 was most abundant before weaning. The latter strated a defect in intestinal Na+-dependent glucose trans-
finding may be related to the fact that the kidney alone port (252). In 1991, molecular genetic studies in two sisters
seems to be responsible for gluconeogenesis before expres- afflicted with glucose-galactose malabsorption revealed a
sion of gluconeogenic enzymes by the liver. missense mutation that causes a change in residue 28 from
Further studies are needed to explore the activity, expres- aspartate to asparagine in the intestinal brush-border SGLT1
sion, and distribution of various glucose transporters dur- Na+-glucose cotransporter (253). This made glucose-galac-
ing kidney development and to elucidate the molecular tose malabsorption the first reported disease that is caused by
mechanisms underlying the maturation of renal tubular a mutation in a membrane transport protein. As of 2000,
glucose transport. more than 40 mutations have been identified in patients
with glucose-galactose malabsorption (246). The mutant
proteins, when expressed in Xenopus oocytes, cause a marked
Glycosuria
reduction in Na+-glucose transport activity (246).
Hereditary renal glycosuria is an abnormality in which variable Patients with glucose-galactose malabsorption who have
amounts of glucose are excreted in the urine at normal concen- been studied show a mild defect in renal tubular reabsorp-
trations of blood glucose (246). The renal defect is specific for tion of glucose, with normal Tm for glucose but decreased
glucose, and there is no increase in the urinary excretion of minimal threshold (FminG) (246,252). In contrast, patients
other sugars. Renal glycosuria is a benign condition without with renal glycosuria show no defect in intestinal D-glucose
symptoms or physical consequences except during pregnancy absorption. This indicates that the SGLT1 Na+-glucose
or prolonged starvation—when dehydration and ketosis may cotransporter affected in glucose-galactose malabsorption is
develop (246). The metabolism, storage, and use of carbohy- shared between the intestine and the kidney, as also sug-
drates as well as insulin secretion are normal. The condition gested by the molecular studies of Pajor et al. (219) and Lee
exists from infancy throughout adult life, and diagnosis usually et al. (215) (see Molecular Biology of Na+-Glucose Cotrans-
is done on routine urine analysis. The distinction between porters). The glucose transporter impaired in glycosuria is
renal glycosuria and diabetes mellitus is made with a fasting not shared.
blood glucose level and a glucose tolerance test. The genetic The accumulating clinical, physiologic, and molecular
pattern in renal glycosuria is autosomal recessive, although gly- data on renal glucose transport have led to the following
cosuria in some heterozygotes has led some investigators to hypothesis of the pathogenesis of hereditary renal glyco-
postulate a dominant inheritance (246,247). suria (200,249). A defect in the low-affinity/high-capacity
The underlying pathogenic mechanism appears to be an 1 Na+:1 glucose cotransporter (SGLT2) of the early proxi-
isolated, selective defect in proximal tubular glucose trans- mal tubule, which reabsorbs most renal tubular glucose,
port. Renal glycosuria is not associated with impaired D- would produce type A classic renal glycosuria but has no
glucose transport in the intestinal epithelium (248). effect on glucose absorption in the intestine. By contrast,
Renal glycosuria is a heterogeneous condition resulting SGLT1, the high-affinity/low-capacity 2 Na+:1 glucose
from several mutations. Analysis of renal titration curves for cotransporter of the late proximal tubule (which also carries
glucose reabsorption reveals two types of renal glycosuria galactose), mediates residual glucose reabsorption in the
(246,249) (Fig. 37.7). In type A, or classic renal glycosuria, renal tubule, and when defective, as in glucose-galactose
FminG and TmG are reduced. In type B, FminG is reduced, malabsorption, causes only mild type B renal glycosuria.
whereas TmG is normal but has an increased splay. It has been It is noteworthy that mutations in the gene for GLUT2,
suggested that the type A mutation reflects reduction in the the facilitative glucose transporter, are also associated with
capacity of the glucose transport system, which might arise glycosuria in the Fanconi-Bickel syndrome (254,255). This
from a uniform defect in all nephrons, and type B reflects a autosomal recessive disorder is characterized by hepatorenal
decrease in the affinity of the transport system, which might glycogen accumulation, Fanconi syndrome, and impaired
also be a consequence of nephron heterogeneity (248,250). A use of glucose and galactose. The renal loss of glucose is due
third type of glycosuria, termed type O, was described by to the transport defect for monosaccharides across the renal
Oemar et al. (251). In this rare condition, tubular reabsorption basolateral membrane. A recent study (256) has demon-
of glucose is virtually absent, and all glucose filtered is excreted strated that knock-out mice lacking the GLUT2 gene
in the urine. exhibit extreme glycosuria.
It is important to consider the relationships between renal
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 38

TUBULAR DISORDERS OF
ELECTROLYTE REGULATION
JUAN RODRÍGUEZ-SORIANO

HYPOKALEMIC STATES Neonatal Bartter’s Syndrome

Hypokalemia may be due to a great number of causes, Genetics

including inadequate dietary intake; increased losses by The known appearance in the same sibship, with apparent
the gastrointestinal tract, skin, or kidney; and abnormal normality of the parents and equal incidence in both sexes,
distribution of K in the body (see Chapter 8). In this points to an autosomal recessive inheritance. Recent findings
section we discuss only the renal tubular disorders in have established the genetic heterogeneity of neonatal Bart-
which hypokalemia constitutes a prominent biochemical ter’s syndrome (Table 38.1). In so-called type 1, frame shift,
feature, as occurs in Bartter’s and Gitelman’s syndromes nonsense, and missense mutations in the gene locus
and in states of pseudohyperaldosteronism. For descrip- SLC12A1 have been found (7–9). Probably these mutations
tion of other hypokalemic tubulopathies, such as renal are quite rare in the population because almost all patients
tubular acidosis or Fanconi’s syndrome, the reader is studied to date are homozygous for an identical disease
referred to Chapters 39 and 42, respectively. mutation in both chromosomes, as a consequence of parental
consanguinity (7). The gene, located on 15q15-q21 and con-
taining 26 exons, encodes the renal bumetanide-sensitive
Bartter’s-Like Syndromes
Na-K-2Cl cotransporter (also termed NKCC2) of the thick
In 1962, Bartter and coworkers (1) reported two ascending limb of the loop of Henle.
patients presenting with a new syndrome characterized Frame shift, nonsense, and missense mutations in the
by hypokalemia, metabolic alkalosis, hyperaldosteronism ROMK gene (locus symbol KCNJ1) have been demon-
with normal blood pressure, decreased pressor respon- strated in patients affected with type 2 of the neonatal form
siveness to infused angiotensin II, and hyperplasia of (10–14). This gene, located on chromosome 11q24, encodes
the juxtaglomerular complex. Since then, many reports, an adenosine triphosphate (ATP)-sensitive, inwardly rectify-
both in children and in adults, have appeared in the lit- ing K channel (ROMK) that recycles reabsorbed K back to
erature under the heading of Bartter’s syndrome. It is the tubular lumen. There are 5 exons that are used in varying
now evident that this term encompasses a variety of combinations to produce three isoforms of ROMK proteins
inherited disorders of renal tubular transport that are that share a core of 372 amino acids encoded by exon 5.
characterized by a biochemical picture of hypokalemia, Most of the mutations causing neonatal Bartter’s syndrome
hypochloremia, metabolic alkalosis, hyperreninemia, are located in this common exon 5 and disrupt an arginine-
normal blood pressure, and increased urinary excretion lysine-arginine triad necessary for the normal pH gating of
of Na, Cl, and K. Thus, the term Bartter’s-like syndromes the Kir channels (15). However, there are reports showing
better defines this ensemble of closely related tubular that homozygous deletions removing exons 1 and 2 also
disorders. result in neonatal Bartter’s syndrome (13,16).
At the present time, patients with Bartter’s-like syn- In some families, mutations in genes coding for NKCC2
dromes can be divided into three different genetic and or ROMK have not been found, reflecting further genetic
clinical entities (2–6): (a) neonatal Bartter’s syndrome, heterogeneity. In fact, mutations in a third gene have been
(b) classic Bartter’s syndrome, and (c) Gitelman’s syn- recently identified as a cause of a peculiar type of neonatal
drome. Obviously, this classification may require modifi- Bartter’s syndrome that is associated with neurosensorial
cation in the future as new mutations are described and deafness and early chronic renal insufficiency (17). This
genotypic-phenotypic correlations are better known. gene, called BSND, is localized on 1p32.3 and is formed by
730 VI. Tubular Disease

TABLE 38.1. GENETICS OF INHERITED DISORDERS OF ELECTROLYTE TRANSPORT

Type of
Inherited disorder inheritance Gene localization Gene symbol Gene product

Neonatal Bartter’s syndrome (type 1) AR 15q15-21 SLC12A1 NKCC2

Neonatal Bartter’s syndrome (type 2) AR 11q24 KCNJ1 ROMK
Neonatal Bartter’s syndrome with neurosenso- AR 1p32.3 BSND Barttin
rial deafness (type 4)
Classic Bartter’s syndrome (type 3) AR 1p36 CLCNKB ClC-Kb
Gitelman’s syndrome AR 16q13 SLC12A3 NCCT
Glucocorticoid-remediable aldosteronism AD 8q22 CYP11B1/CYP11B2 11β-hydroxylase/aldo syn-
thase
Liddle’s syndrome AD 16p12 SNCC1B, SCNN1G β, γ ENaC
Apparent mineralocorticoid excess AR 16q12 HSD11B2 11β-HSD2
Pseudohypoaldosteronism type 1
AD renal form AD 4q31.1 MLR Mineralocorticoid recep-
tor
AR multiple-organ form AR 16p12 SNCC1B, SCNN1G β, γ ENaC
AR 12p13 SNCC1A α ENaC
Pseudohypoaldosteronism type 2 AD 1q31-42, 12p13.3, WKN1, WKN4 WNK1, WNK4 kinases
17p11-21
Hypomagnesemia with secondary hypocalcemia AR 9q22 TRPM6 TRPM6
Isolated familial hypomagnesemia
AD form AD 11q23 FXYD2, Na+,K+-adenosine tri-
phosphatase γ subunit
AR form AR ? ? ?
Familial hypomagnesemia and hypercalciuria AR 3q PCN1 Paracellin-1 (claudin-16)

AD, autosomal dominant; AR, autosomal recessive.

four exons and encodes a novel protein that has been nent forehead, large eyes, protruding ears, and drooping
termed barttin (18). This protein has two transmembrane mouth. Strabismus and sensorineural deafness have been
α helices and is preferentially expressed in the ascending reported (22,23). Failure to thrive and growth retardation
loop of Henle and inner ear. It acts as an essential β subunit are the rule, but appropriate therapy is followed by satisfac-
for ClC-Ka and ClC-Kb chloride channels. It seems that tory growth (24). The presence of systemic manifestations
heteromers formed by ClC-K channel and barttin are cru- such as osteopenia, fever, secretory diarrhea, convulsions,
cial for renal NaCl reabsorption and K recycling in the and increased susceptibility to infection has been noted (4).
inner ear (19). Prenatal diagnosis can be made by the high Cl content
The correspondence between genotype and phenotype of the amniotic fluid. Of interest, concentrations of amni-
may not be as consistent as presented here, because there are otic fluid Na, K, Ca, and prostaglandin E2 are normal (25).
patients with the hypercalciuric variant and abnormalities in The biochemical examination of the mother’s urine may
the NKCC2 gene who have clinical onset beyond the neo- also be useful for prenatal diagnosis because it may show
natal period (9), and there are also occasional patients with very low concentrations of Na, Cl, and Ca (26). The defin-
classic Bartter’s syndrome type 3, due to mutations or dele- itive way to establish a prenatal diagnosis is through the
tions in the CLCNKB gene, who may present with polyhy- mutational analysis of genomic DNA extracted from cul-
dramnios and symptoms during the neonatal period (20). tured amniocytes obtained by amniocentesis (27).
Patients with type 2 often present at birth with mild
Clinical and Biochemical Features NaCl wasting and a biochemical picture that mimics pri-
This subject has been reviewed by Proesmans (21). Clinical mary pseudohypoaldosteronism type 1 (i.e., hyponatremia,
signs may be observed antenatally or immediately after hyperkalemia, and metabolic acidosis). Renal potassium
birth and include marked polyhydramnios (appearing wasting leading to hypokalemia and metabolic alkalosis may
between 24 and 30 weeks’ gestation and caused by intra- not be apparent until 3 to 6 weeks postnatally outside of the
uterine polyuria), premature delivery, massive polyuria, life- neonatal period, the biochemical features (hypokalemia,
threatening episodes of dehydration, hypercalciuria, and metabolic alkalosis, impaired concentrating ability, hyper-
early-onset nephrocalcinosis (Table 38.2). Urine output reninism, and hyperaldosteronism) are identical to those
may be as great as 12 to 50 mL/kg/hr and continue to be later described with more detail in the section Classic Bart-
greatly increased for at least 4 to 6 weeks after birth. Some ter’s Syndrome.
patients have a distinctive appearance: they are thin, with Urinary Ca excretion always remains very elevated in
small muscles and a triangular face characterized by promi- spite of therapy. The presence of marked hypercalciuria and
 38. Tubular Disorders of Electrolyte Regulation 731

TABLE 38.2. CLINICAL AND BIOCHEMICAL FEATURES OF BARTTER’S-LIKE SYNDROMES

Features Neonatal Bartter’s syndrome Classic Bartter’s syndrome Gitelman’s syndrome

Clinical onset Neonatal Infancy or childhood Childhood or adulthood

Polyhydramnios/prematurity Present Often present Absent
Polydipsia/polyuria Present Present Absent
Salt craving Present Present Absent
Growth retardation Present Present Absent or occasional
Dehydration Present Often present Absent
Muscle weakness/tetany Absent Occasionally present Present
Nephrocalcinosis Present Absent Absent
Sensorineural deafness Occasionally present Absent Absent
Metabolic alkalosis Present (it may be delayed) Present Present
Hypokalemia Present (it may be delayed) Present Present
Hypomagnesemia Absent or exceptional Occasionally present? Present
Urinary NaCl excretion Very high High Normal or high
Urinary Ca excretion Very high Normal or high Low
Urinary concentrating ability Impaired Impaired Normal
Hyperrenin/hyperaldosteronism Present Present Present
Hyperprostaglandinism Present (it may be delayed) Present Absent
Hypertrophy of juxtaglomerular apparatus Present Present Occasionally present

nephrocalcinosis in an infant or child with apparent Bart- The absorptive Na-K-2Cl cotransporter or NKCC2 is a
ter’s syndrome is consistent with the neonatal form beyond member of a family of four proteins also including secre-
the neonatal period. Increased urinary excretion of prosta- tory Na-K-2Cl, NaCl, and KCl cotransporters. All are
glandin E2 has been considered a characteristic feature of structurally similar and are formed by 12 membrane-span-
neonatal Bartter’s syndrome, but this is secondary to the ning helixes and long cytosolic amino- and carboxy-termi-
basic defect of renal NaCl reabsorption and, therefore, may nal segments (31). Structural abnormalities of this protein
not be present in all patients, especially during the early due to mutations of the encoding gene obviously lead to
neonatal period (28). Renal function is generally well pre- altered NaCl transport. The importance of NKCC2 in
served, but an occasional child may develop end-stage renal NaCl transport is underlined by findings in a knock-
chronic renal disease. out mouse model of Bartter’s syndrome. These mice with
an abated SLC12A1 gene manifest severe salt-wasting,
Pathology which results in rapid neonatal death unless they are res-
Placental pathology in two cases showed large placentas cued by indomethacin treatment (32).
with extensive subtrophoblastic basal membrane mineral- ROMK is a protein with two intramembranous domains
ization (29). The cardinal renal biopsy finding is hyperpla- containing the channel pore and two cytosolic amino- and
sia of the juxtaglomerular apparatus. Occasional absence of carboxy-terminal segments. It is found along the distal neph-
Tamm-Horsfall protein in the kidney probably represents a ron and is believed to be responsible for K secretion in the
secondary phenomenon (30). cortical collecting duct and K recycling in the thick ascend-
ing limb of the loop of Henle (33). Loss of ROMK function
Pathophysiology impairs the ability to recycle K from cells back into lumen,
Seyberth et al. (4) suggested that the neonatal cases repre- resulting in luminal K concentrations too low to permit con-
sented a different pathogenic entity, related to systemic tinued Na-K-2Cl activity (34).
overproduction of prostaglandins, so the term hyperprosta- Normal function of the Na-K-2Cl cotransporter is
glandin E2 syndrome was proposed. The increased renal and responsible for the reabsorption of approximately 30% of
systemic prostaglandin E activity was indicated by symp- filtered sodium. Therefore, it is not surprising that altered
toms such as fever, diarrhea, vomiting, osteopenia, and gen- function of this cotransporter results in renal sodium wast-
eralized convulsions, and by elevation of the urinary ing, volume contraction, hyperreninism, hyperaldoster-
excretion of prostaglandin E-M metabolite (the major uri- onism, hyperkaluria, hypokalemia, metabolic alkalosis, and
nary metabolite of the E prostaglandins). However, molec- hyperprostaglandinism, thus closing the pathogenic circle
ular biology findings demonstrate abnormalities in the (Fig. 38.2). As expected, patients with neonatal Bartter’s
genes coding for either NKCC2 (genetic type 1) or ROMK syndrome exhibit a blunted natriuretic and hormonal
(genetic type 2), and the prostaglandin abnormalities response to furosemide when compared to controls (35).
appear to be secondary phenomena. Elevated prostaglandin E2 secretion aggravates the picture
The channels and transporters implied in NaCl reab- further by independent stimulation of the renin-aldosterone
sorption in the distal nephron are depicted in Figure 38.1. axis and inhibition of both ROMK channel activity and
732 VI. Tubular Disease

FIGURE 38.2. Schematic representation of pathophysiologic

events leading to hypokalemia and metabolic alkalosis. Each of
the biochemical and hormonal abnormalities observed in Bart-
ter’s syndrome could represent a proximate defect turning on
the vicious circle. However, only extrarenal Cl loss (causing
pseudo-Bartter’s syndrome) and renal transport defects (causing
Bartter’s-like syndromes) appear as clinically relevant. ECF, extra-
cellular fluid.

The presence of a primary NaCl transport defect in the

loop of Henle was denied in the past by Seyberth et al. (4)
on the basis that no abnormality in distal NaCl reabsorp-
tion could be demonstrated when children with this syn-
drome were studied beyond the neonatal period by oral
hypotonic fluid administration. However, in our experi-
ence, when patients are studied after hypotonic saline infu-
FIGURE 38.1. Schematic models of NaCl reabsorption in thick sion, a profound defect in distal NaCl reabsorption can be
ascending limb of Henle (TALH) and convoluted distal tubule. In
TALH, NaCl enters the cell from the lumen via an Na-2Cl-K identified. The percent of distal NaCl reabsorption is dra-
cotransporter (NKCC2). K is recycled into the lumen via the K matically low during infancy due to the increased distal
channel ROMK to permit the continuous functioning of this NaCl delivery characteristic during this age.
cotransporter. Cl leaves the cell across the basolateral mem-
brane via a Cl channel (ClC-Kb) or in cotransport with K, while The characteristic presence of hypercalciuria is not sur-
Na exits the cell through the Na,K-ATPase system. Barttin acts as prising because approximately 25% of filtered Ca is reab-
an essential subunit of ClC-Kb. In the distal convoluted tubule, sorbed in the thick ascending limb of Henle coupled to
NaCl reabsorption in the luminal membrane is mediated by the
NaCl cotransporter (NCCT) and leaves the cell through Cl chan- Na-K-2Cl activity. Impaired electrogenic Cl transport
nels and the Na,K-ATPase. A K-Cl cotransporter is also present at impairs voltage-driven, paracellular Ca and Mg reabsorp-
the luminal membrane. tion. Hypercalciuria has also been related to an excessive
synthesis of 1,25-dihydroxy-vitamin D (39) leading to aug-
mented bone resorption and renal leak (40,41). Recent
NaCl transport in the thick ascending limb of Henle (36). studies have shown that serum and urine of children with
However, although administration of prostaglandin syn- neonatal Bartter’s syndrome contain excessive amounts of a
thetase inhibitors results in the impressive clinical and bio- heparin-like calciotropic factor closely related to basic
chemical improvement, especially in type 2, it is not fibroblastic growth factor, providing additional evidence
followed by complete correction of the tubular defect. that hypercalciuria is not solely due to an intrinsic renal
Recent findings indicate that cyclooxygenase-2 is highly leak (42,43).
expressed in the macula densa of patients with salt-losing Reabsorption of approximately 60% of filtered Mg also
Bartter’s syndrome and hyperreninemia (37) and that pref- occurs in the loop of Henle and is dependent on the positive
erential inhibition of this enzyme by nimesulide in patients luminal voltage generated by the activity of the cotrans-
with neonatal Bartter’s syndrome results in impressive porter. Absence of hypermagnesiuria in neonatal Bartter’s
improvement of hyperprostaglandinuria, secondary hyper- syndrome may be explained by the compensatory Mg reab-
aldosteronism, and hypercalciuria (38). These data would sorption taking place in distal convoluted tubules.
indicate that cyclooxygenase-2–derived prostaglandin E2 is
an important mediator for stimulation of the renin-angio- Differential Diagnosis
tensin-aldosterone system in the kidney and that its prefer- The clinical picture of neonatal Bartter’s syndrome is so
ential inhibition may offer a potential therapeutic benefit. characteristic that diagnostic doubts rarely arise. A similar
 38. Tubular Disorders of Electrolyte Regulation 733

but apparently different syndrome has been described in a In general, therapy permits clinical stabilization and
female infant, aged 5 weeks, who, in addition to the usual catch-up growth. Bone age has been appropriate for chro-
clinical features of neonatal Bartter’s syndrome, had mas- nological age, and pubertal and intellectual development
sive urinary excretion of prostaglandins E2 and E-M metab- have been normal. An occasional patient may even have a
olite, normal calcium metabolism, hyperphosphaturia, and spontaneous resolution of all signs and symptoms at
severe hyperchloriduria and hyperkaluria with limited approximately 6 years of age (51). Although indomethacin
response to indomethacin. Molecular analysis determines if may reduce the degree of hypercalciuria, renal calcium
these findings really represent a new congenital renal tubu- excretion remains greatly elevated, thus creating a real con-
lar abnormality as proposed by the authors (44). cern for the progression of the nephrocalcinosis. The use of
Type 2 neonatal Bartter’s syndrome, due to mutations in preferential cyclooxygenase-2 inhibitors remains experi-
the ROMK gene, may exhibit hyponatremia and hyperkale- mental. All coadjuvant therapies (e.g., amiloride, hydro-
mia in the early neonatal period, thus mimicking the picture chlorothiazide, potassium phosphate) have been ineffective,
of primary pseudohypoaldosteronism type 1. The possible so one should expect a low decrease of glomerular filtration
existence of prematurity, polyhydramnios, hypercalciuria, rate (GFR) over the years as a result of chronic tubulointer-
and nephrocalcinosis in the latter syndrome may contribute stitial nephropathy.
to the clinical confusion (45–48). The syndrome of familial The neonatal form associated with neurosensorial deaf-
hypomagnesemia-hypercalciuria with nephrocalcinosis may ness due to mutations in the BSND gene is resistant to
also be confused with neonatal Bartter’s syndrome because therapy with indomethacin and always carries a bad long-
hypercalciuria and nephrocalcinosis are already present, but term prognosis due to the progressive development of
significant hypomagnesemia may be absent in the early neo- chronic renal insufficiency (52).
natal period.
Pseudo-neonatal Bartter’s syndrome is rare, but biologi- Classic Bartter’s Syndrome
cal abnormalities simulating Bartter’s syndrome were
observed in a preterm neonate with complex cyanotic heart Genetics
disease, in whom the ductus arteriosus was maintained The so-called classic form, or Bartter’s syndrome type 3, is
open by high doses of prostaglandin E1 (49). an autosomal recessive disease caused by large deletions or
nonsense, missense, and splice mutations in a renal chloride
Treatment and Prognosis channel gene (ClC-Kb) (20,53) (Table 38.1). In humans,
In the immediate neonatal period, all therapeutic efforts there are two closely related voltage-gated chloride channels
should be directed to correct dehydration and electrolytic (ClC-Ka and ClC-Kb), proteins of 687 amino acids specif-
imbalance. Continuous saline infusion may be needed to ically expressed in mammalian kidney and encoded by two
attain this goal. Administration of indomethacin in the genes (locus symbols CLCNKA and CLCNKB), closely
early postnatal period may not only be unnecessary but located on 1p36 where they are separated by only 11 kb
dangerous, given the risk of necrotizing enterocolitis. Pre- (54). The fact that a certain number of patients with classic
natal treatment with indomethacin has been advocated Bartter’s syndrome do not demonstrate abnormalities in the
when diagnosis is made prenatally (27). The apparent bene- CLCNKB gene indicates that other genes implicated in
ficial effects of indomethacin should be taken with extreme NaCl transport in the ascending loop of Henle may also be
caution, however, and should be weighed against the involved as a cause of this syndrome. A possible candidate
potential therapeutic risks (50). Administering indometha- gene could be the one encoding the synthesis of the basolat-
cin to a mother whose fetus is known to have mutations eral KCl cotransporter.
characteristic of neonatal Bartter’s syndrome may prevent
progression of polyhydramnios, but may also be dangerous Clinical Features
for the fetus. Frequently, there is a history of maternal hydramnios and
At 4 to 6 weeks of life, patients greatly benefit from the premature delivery. Symptoms start during the first 2
administration of indomethacin and, eventually, of potas- years of life and include polyuria, polydipsia, vomiting,
sium chloride supplements. Although indomethacin has no constipation, salt craving, tendency to dehydration, and
direct effect on the inherited renal tubular abnormality, it is failure to thrive (Table 38.2). Occasional cases may have a
beneficial because it neutralizes the amplifying effect of clinical onset during the neonatal period (20). Growth
prostaglandins on the features of Bartter’s syndrome. In my retardation occurs if early therapy is not instituted. How-
experience, patients with the type 2 genetic defect are espe- ever, normal adult height may be achieved with a delayed
cially sensitive to indomethacin, and doses below 1 mg/kg/ adolescent growth spurt. Associated growth hormone
day may be sufficient to maintain plasma K levels within deficiency has been found in occasional patients (55). The
the normal range. In fact, a high dosage may lead to hyper- clinical findings of fatigue, muscle weakness, cramps, and
kalemia, and K supplements may not be necessary with recurrent episodes of carpopedal spasm may be observed
appropriate therapy. late in childhood but are more characteristically observed
734 VI. Tubular Disease

in Gitelman’s syndrome. Some patients have a distinctive tained high levels of angiotensin II may theoretically
appearance: they are thin, with small muscles and a trian- cause progressive tubulointerstitial damage. However, if
gular face characterized by prominent forehead, large therapy is adequate, there is no long-term deterioration
eyes, and drooping mouth. Other possible signs are of renal function.
developmental delay, minimal brain dysfunction with Hyperreninemia is constantly present and is accompa-
nonspecific electroencephalographic abnormalities, neph- nied, in most cases, by an elevated plasma concentration
rocalcinosis, and polyuria-related ureterohydronephrosis. of aldosterone. In a few patients, however, aldosterone
If hypokalemia is profound and prolonged, it may give secretion is suppressed by K depletion. The renal K loss is
rise to the formation of medullary cysts (56). Nephrocal- not exclusively related to hyperaldosteronism, because it
cinosis is very rarely observed. Patients are always normo- persists after renin suppression or adrenalectomy.
tensive. On occasion, the diagnosis is suspected in an Paradoxically, blood pressure is normal despite increased
asymptomatic child because hypokalemia is found in a blood levels of renin, angiotensin II, norepinephrine, and
routine investigation. endothelin (61). In fact, stimulation of the renin-angioten-
sin-aldosterone axis might be necessary to maintain normo-
Biochemical Features tension, as administration of saralasin (a competitive
The outstanding laboratory finding is hypokalemia, with inhibitor of angiotensin II) or captopril (a blocker of angio-
K concentrations in blood ranging between 1.5 and 2.5 tensin-converting enzyme) induces a significant fall in
mEq/L. Hypokalemia is almost always accompanied by blood pressure. The blunted increase in blood pressure after
hypochloremia and metabolic alkalosis, although (excep- infusion of angiotensin II is not a constant diagnostic fea-
tionally) the patient may present with metabolic acidosis ture as initially reported by Bartter (1), because it improves
(57). Hyperuricemia, induced by contraction of extracellu- after expansion of extracellular fluid volume or administra-
lar fluid volume, may be observed in approximately 50% of tion of prostaglandin synthetase inhibitors. The beneficial
patients. Hypomagnesemia is rarely encountered, and its effect of indomethacin in this regard depends on its capac-
presence in a patient with features of Bartter’s syndrome ity not only to correct sodium depletion and thus suppress
necessitates exclusion of the diagnosis of Gitelman’s syn- angiotensin production but also to normalize the pressor
drome, in which hypomagnesemia is a cardinal finding. responsiveness to the latter hormone by inhibiting the
Other biochemical features rarely present are polycythemia, secretion of prostaglandins. Indomethacin also corrects the
hypercalcemia, and hypophosphatemia. hyperactivity of the adrenergic system, manifested by
The most characteristic urinary findings are the increased increased plasma levels of noradrenaline and vascular, but
fractional excretion of K, Na, and Cl. The demonstration of not metabolic, unresponsiveness to this hormone. Probably,
a urinary Cl concentration above 10 mEq/L in a patient the vascular hyporeactivity of Bartter’s syndrome is a com-
with hypochloremic metabolic alkalosis rules out extrarenal plex phenomenon dependent not only on vascular under-
causes of Cl depletion (e.g., dietary deficiency, vomiting, filling and increased prostaglandin synthesis but also on
cystic fibrosis) in which urinary Cl excretion is very low or nitric oxide–mediated vasodilation. Increased urinary
even absent (58). Urinary Na wasting is also present, and it excretions of nitrites NO2– and NO3– and cyclic guanosine
may be especially evident during dietary Na restriction. A monophosphate all point to increased nitric oxide synthesis
very important urinary finding is the presence of normal or in vascular endothelial cells (62). Patients with Bartter’s-like
high urinary Ca excretion (59). Hypocalciuria is a character- syndromes evidence a blunted Gq-mediated cell signaling
istic finding of Gitelman’s syndrome, but in exceptional that results in reduction in protein kinase C production
cases it may also be observed in the follow-up of patients and upregulation of the nitric oxide system (63).
with classic Bartter’s syndrome (60). A very important finding is the increased urinary excre-
Defects in concentrating and diluting abilities are tion of prostaglandins, principally prostaglandin E2 and
always present and are the result of both hypokalemia 6-keto-prostaglandin F1a, the main metabolite of prosta-
and impaired NaCl reabsorption in the ascending limb glandin I2 or prostacyclin (64). Urinary excretion of prosta-
of the loop of Henle. The role played by prostaglandin glandin F2a is more variable. Urinary kallikrein excretion is
oversecretion is probably secondary, because administra- also increased. Prostaglandin overproduction may cause
tion of prostaglandin synthetase inhibitors is not fol- defective platelet aggregation and inhibition of phytohe-
lowed by complete correction of hyposthenuria. Another magglutinin-induced lymphocyte proliferation.
relevant finding is the inability to appropriately decrease
urine pH during ammonium chloride loading. This is Pathology
due to high rates of ammonium excretion, caused by The cardinal renal biopsy finding is hyperplasia of the
both increased production and preferential diversion of juxtaglomerular apparatus, although it may be absent in
ammonia into the urine. GFR is normal in early stages occasional cases. Hyperplasia of interstitial renomedullary
of the disease but may become impaired in untreated cells is observed more rarely. Glomerular hyalinization, api-
patients as a result of chronic hypokalemia. The sus- cal vacuolization of proximal tubular cells, tubular atrophy,
 38. Tubular Disorders of Electrolyte Regulation 735

and interstitial fibrosis may be also observed as a long-term judged in relation to distal Cl delivery. In fact, distal Cl
consequence of chronic K deficiency. reabsorption is less affected or is even normal when renal
clearance studies are performed after an oral water load
Pathophysiology instead of during hypotonic saline infusion (69,70).
Different hypotheses (e.g., vascular resistance to angiotensin It has long been postulated that the membrane ion trans-
II, tubular defect in potassium secretion, primary hyper- port defect was not limited to tubular cells but represented a
prostaglandinism, increased secretion of atrial natriuretic generalized membrane transport defect, as intracellular Na
peptide) have been presented to explain the range of abnor- concentration in erythrocytes or muscle cells was found to be
malities seen in classic Bartter’s syndrome, but it is now well consistently increased. The reasons proposed were either an
established that most cases result from mutations in the increased Na permeability or a decreased passive Na efflux
renal Cl channel ClC-Kb or genetic defect type 3. Other related to the low number of ouabain-sensitive Na-pumping
patients who apparently have classic Bartter’s syndrome may sites (71). However, on the basis of recent data, it is more
indeed have neonatal Bartter’s syndrome with a delayed clin- probable that these transport abnormalities are simple conse-
ical onset, which may be caused by defects in NKCC2 or quences of the hypokalemic state. The response to therapy
ROMK genes. The presence of nephrocalcinosis in the latter has been variable; indomethacin corrects the intracellular
cases is an important clinical clue. action abnormalities but has little effect on the membrane
The ClC chloride channels are members of a single family transport defect, whereas complete correction of hypokale-
originally defined by the voltage-gated Cl channel ClC-0, mia apparently normalizes all parameters (72).
isolated from Torpedo electric organ and formed by at least
nine different proteins (65). The members of this family, Clinical Variants
ClC-K1 and ClC-K2, were shown in the rat to be kidney-
specific and expressed from the thin ascending limb of the Associated with Proximal Tubular Dysfunction. A few cases
loop of Henle onward (Fig. 38.1). The human homolog of Bartter’s syndrome have been associated with signs of the
genes are called CLCNKA and CLCNKB. These genes Fanconi’s syndrome (73–75). In a personal case and in the
encode the channels ClC-Ka and ClC-Kb, both formed of case reported by Fricker et al. (75), the renal biopsy showed
687 amino acids and containing 12 transmembrane domains hyperplasia of the juxtaglomerular apparatus but also
and cytosolic amino- and carboxy-terminal segments. Like marked interstitial fibrosis with hyalinized glomeruli and
other members of the family, these channels are gated by tubular atrophy. The interstitial fibrosis may not represent a
both voltage and chloride. differential finding and may only be the result of chronic
The Cl channel ClC-Kb is situated at the basolateral mem- hypokalemia (76). This entity should probably not be clas-
brane of distal nephron segments and is mainly responsible for sified as a different syndrome, because in our case a homo-
Cl exit from the cell into the bloodstream. It needs the β sub- zygous missense mutation in the CLCNKB gene could be
unit barttin for functional expression. Therefore, it is not sur- demonstrated. This clinical variant of Bartter’s syndrome
prising that structural defects of the genes encoding both the should be distinguished from the opposed situation (i.e.,
channel itself and barttin are followed by defective NaCl reab- cases of primary Fanconi’s syndrome) or cystinosis mimick-
sorption in the distal nephron. Alternative mechanisms of Cl ing Bartter’s syndrome due to the presence of hypokalemia
transit across the basolateral membranes, such as a KCl and metabolic alkalosis (77–79). Patients with mitochon-
cotransport, have also been proposed, so it must be assumed drial cytopathies often have signs of proximal tubular dys-
that overfunction of these other transporters cannot compen- function but, exceptionally, a 10-year-old child with the
sate completely for loss of function of the Cl channel. How- Kearns-Sayre syndrome presented with signs of Bartter’s
ever, some compensation probably takes place because renal syndrome (80).
salt loss is not as massive as that observed in neonatal cases, and
the phenotype of this genetic defect is quite variable. Associated with Distal Tubular Defects Rather Than Loop
The presence of a defective NaCl transport at the level of of Henle Defects. Some adult patients reported as cases of
the ascending loop of Henle in classic Bartter’s syndrome Bartter’s syndrome show a normal or near-normal capacity
has long been suspected on the basis of the study of frac- to form solute-free water after oral water loading and are
tional clearances of Cl during hypotonic saline diuresis. The therefore interpreted as having a distal tubular defect rather
index of distal Cl reabsorption (CH2O / CH2O + CCl × 100) than a loop of Henle defect (81–84). Most of these patients
is normally above 80% (66), whereas patients with this phe- exhibit hypocalciuria and should be considered to have
notype have values characteristically diminished, often Gitelman’s syndrome and not a clinical variant of Bartter’s
below 60% (67). This defect is not observed in patients syndrome (85).
with hypokalemia of different etiologies (68). To reveal the
tubular defect, the conditions of the study, especially NaCl Differential Diagnosis
intake, and route of administration must be rigorously stan- Familial hypokalemic alkalosis with proximal tubulopathy
dardized, and the diluting segment Cl reabsorption must be (Güllner’s syndrome) has been uniquely observed in three
736 VI. Tubular Disease

siblings of a sibship of four presenting with features very ketoprofen administration. The early effects of prosta-
similar to Bartter’s syndrome but with normal distal Cl glandin inhibition are spectacular, with improved well-
reabsorption and characteristic focal histologic lesions con- being, strength and activity, diminution of polyuria and
sisting of dark staining of proximal tubular cells. Hypouri- polydipsia, and reinstitution of normal, or even catchup,
cemia was the only biochemical abnormality that could be growth. There is an immediate increase in plasma K,
ascribed to impaired proximal tubular function (86,87). which, however, rarely exceeds a concentration of 3.5
Among the renal causes mimicking Bartter’s syndrome, mEq/L. Plasma renin activity and aldosterone concentra-
one should also consider the surreptitious use of loop diuret- tion decrease to normal, and the vascular response to
ics such as furosemide (88,89). Although this situation is angiotensin II or noradrenalin also normalizes. The distal
exceptional in children, the presence of diuretics in the urine tubular defect in Cl reabsorption remains unmodified.
may help to establish the diagnosis. There is a report of an Addition of Mg salts should always be considered when
11-year-old boy presenting with a chronic salt-losing hypomagnesemia is present because Mg deficiency may
nephropathy manifested by volume depletion, hypodipsia, aggravate K wasting.
absence of salt appetite, normokalemic metabolic alkalosis, The efficacy of long-term use of prostaglandin syn-
hyperreninemic hyperaldosteronism, hypertrophy of juxta- thetase inhibitors has been clearly established. When
glomerular apparatus, highly conserved capacities to con- patients reach adulthood, clinical improvement is generally
centrate and dilute the urine, and paradoxically increased maintained, but there is some recurrence of hypokalemia
levels in plasma of atrial natriuretic peptide. Although this and hyperreninemia, and there is a tendency to develop
patient clinically resembles Bartter’s syndrome, values of hypomagnesemia and hypocalciuria, thus mimicking the
fractional NaCl reabsorption during hypotonic saline diure- biochemical profile of Gitelman’s syndrome (60). Tetanic
sis were normal, and no clinical amelioration was observed episodes, which are always absent during childhood, may
while on indomethacin therapy (90). be observed in adults. Long-term prognosis remains
Bartter’s syndrome should also be distinguished from guarded, however, and lack of rigorous therapeutic control
states of Cl depletion from extrarenal causes: cyclical vomit- may lead to slow progression to chronic renal failure (91).
ing, laxative abuse, and cystic fibrosis. In these circum- If the symptoms reappear, the dose of indomethacin should
stances, the biochemical and histologic pictures may be be readjusted, and a combined therapy with indomethacin
identical to those present in Bartter’s syndrome, but diagno- and spironolactone should be initiated. In adults, the use of
sis is rapidly made if one measures urinary Cl excretion (58). angiotensin-converting enzyme inhibitors (e.g., captopril,
States of mineralocorticoid excess should be easily excluded enalapril) has been tried with conflicting results (92,93).
because all are associated with arterial hypertension. There are no data concerning their use in children with
Bartter’s syndrome, but one should be cautious given the
Treatment and Prognosis risk of developing symptomatic hypotension.
Treatment is designed primarily to correct the hypokale- Anesthetic procedures should be carefully managed in
mia. Potassium supplementation in the form of KCl (1 to 3 patients with Bartter’s syndrome and special attention
mEq/kg/day or more) is always necessary. The amount should be paid to maintain cardiovascular stability, con-
needed changes according to the patient and must balance trol plasma K, and prevent renal damage (94). Mainte-
the amount of K lost by the kidney. However, in most nance of indomethacin therapy is mandatory when
cases, K supplementation alone is almost completely inef- undergoing surgical procedures such as a percutaneous
fective, as administered K is quickly lost into the urine. renal biopsy to avoid the risk of bleeding due to defective
Addition of spironolactone (10 to 15 mg/kg/day) or triam- platelet aggregation.
terene (10 mg/kg/day) may be initially effective in the con-
trol of hypokalemia, but its effect is often transient. The
Gitelman’s Syndrome
administration of β-adrenergic inhibitors (e.g., propran-
olol) does not offer any additive advantage. The syndrome of familial hypokalemia-hypomagnesemia
Therapy of Bartter’s syndrome is today best accom- (Gitelman’s syndrome) was first described by Gitelman,
plished by the use of prostaglandin synthetase inhibitors: Graham, and Welt in 1966 (95). It is not as infrequent as
indomethacin (2 to 5 mg/kg/day), acetylsalicylic acid reflected in the literature, because in the past it has been
(100 mg/kg/day), ibuprofen (30 mg/kg/day), or ketopro- confused with Bartter’s syndrome (96). Adult patients with
fen (20 mg/kg/day). Indomethacin is the drug most fre- Gitelman’s syndrome are much more common than those
quently used and is remarkably well tolerated. However, with Bartter’s syndrome. During childhood the incidence is
attention should be paid to signs of intolerance or toxicity probably similar (97).
(e.g., nausea, vomiting, abdominal pain, peptic ulcer,
hematopoietic toxicity, or liver damage). The possibility Genetics
of developing pseudotumor cerebri should also be consid- The equal incidence in both sexes and the lack of signifi-
ered, as it has been reported after both indomethacin and cant abnormalities in the parents suggest an autosomal
 38. Tubular Disorders of Electrolyte Regulation 737

recessive inheritance (Table 38.1). Molecular biology find- differentiation from patients with Bartter’s syndrome,
ings have established that almost all families studied have who exhibit an enhanced calciuric response to furosemide
mutations in the gene locus SLC12A3 (98–102). This (112).
gene, located in 16q13 and containing 26 exons, encodes
the renal thiazide-sensitive NaCl cotransporter (also vari- Pathophysiology
ously abbreviated as TSC, NCC, NCCT, or ENCC1) that Gitelman’s syndrome results from diminished NaCl trans-
is mainly located in the distal convoluted tubule. port in the distal convoluted tubule caused by defective
function of the thiazide-sensitive NaCl cotransporter
Clinical and Biochemical Features NCCT (Fig. 38.1). This was the first member of the Na-
Patients with Gitelman’s syndrome are often asymptom- K-Cl cotransporter family to be identified and, as other
atic, with the exception of transient episodes of weakness members of this family, is formed by 12 membrane-span-
and tetany that are usually accompanied by abdominal ning helixes and long cytosolic amino- and carboxy-termi-
pain, vomiting, and fever (Table 38.2). The disease-free nal segments (31). Structural abnormalities of this
intervals may be prolonged, and in many cases, diagnosis transporter due to mutations of the encoding gene lead to
is only made during adult life. However, almost one-half defective processing with retention of the protein in the
of the patients complain of salt craving, musculoskeletal endoplasmic reticulum and lack of activity at the plasma
symptoms (e.g., cramps, muscle weakness, and aches), membrane (113). As in Bartter’s syndrome, the NaCl
and constitutional symptoms (e.g., fatigue, dizziness, cotransporter defect directly leads to NaCl wasting, mild
nocturia, and polydipsia) (103). In children, growth hypovolemia, metabolic alkalosis, and stimulation of the
retardation is usually absent, but occasional cases may renin-angiotensin axis.
present evidence of associated growth hormone defi- The exact mechanisms underlying hypocalciuria and
ciency that improves with recombinant human growth hypomagnesemia are not yet fully elucidated, but both
hormone therapy (104,105). Chondrocalcinosis has been abnormalities may be direct consequences of the primary
occasionally reported in adult patients and is due to defect. In fact, they are also observed after administration
deposition of calcium pyrophosphate dehydrate crystals of thiazide diuretics, which inhibit the distal luminal
(106,107). Ophthalmic examination may show choroidal NaCl cotransporter. Hypocalciuria may result from
calcifications (108). increased tubular reabsorption of filtered Ca via a basolat-
The outstanding biochemical features are hypomag- eral NaCa exchanger. No obvious abnormalities are found
nesemia, hypokalemia, and moderate metabolic alkalosis. A in plasma levels of parathyroid hormone or 1,25-dihy-
modest elevation in uric acid may be present, although, on droxy vitamin D3 levels (114,115). Hypermagnesuria also
the whole, renal function is normal. The presence of hyper- results from decreased tubular reabsorption of filtered Mg
reninism and hyperaldosteronism may contribute to the due to inhibition of an apical NaMg exchanger. Meta-
confusion with classic Bartter’s syndrome. However, a nor- bolic alkalosis may play an important role in the patho-
mal urinary excretion of prostaglandin E2 has been genesis of hypermagnesuria by making the distal tubule
reported (109). Generally, no abnormal findings are present cells resistant to Mg uptake (116). A study of renal tubu-
at renal biopsy, although hypertrophy of juxtaglomerular lar reabsorption of Mg, performed in three children with
apparatus may be occasionally observed. Gitelman’s syndrome (96), revealed normal rates of Mg
The most characteristic finding in the urine, besides reabsorption when the amount filtered exceeded 1.2 mg/
the presence of hypermagnesuria and hyperkaluria, is the dL glomerular filtrate. However, in contrast to normal
striking diminution of urinary Ca excretion, which rarely children, Mg excretion was also evident at levels of filtered
exceeds 0.5 mg/kg/day. Plasma levels of parathyroid hor- Mg below 1.2 mg/dL GF, indicating a low renal Mg
mone and urinary excretion of cyclic adenosine mono- threshold. These data are compatible with the known fact
phosphate are normal. The study of distal tubular that the bulk of filtered Mg is reabsorbed in the thick
function reveals normal or only slightly impaired concen- ascending loop of Henle and that the distal convoluted
trating and acidifying mechanisms, but modestly reduced tubule only reabsorbs approximately 5% of filtered Mg
distal fractional Cl reabsorption during hypotonic saline (117).
diuresis. Values obtained are intermediary between nor-
mal values and values present in children with classic Bar- Differential Diagnosis
tter’s syndrome (110). A striking finding is the increased Hypomagnesemia and hypocalciuria are universal in Gitel-
natriuretic and kaliuretic response to the intravenous man’s syndrome, thus allowing an easy differentiation from
administration of furosemide and a blunted natriuretic other hypokalemic syndromes (Table 38.2). Gitelman’s syn-
response to hydrochlorothiazide (111). This suggests that drome should be differentiated from a rare entity described
the defect in NaCl reabsorption is in the distal tubule in an adult man who presented with a clinical syndrome of
rather than in the ascending loop of Henle. The calciuric a probable proximal tubular origin, associating moderate
response to furosemide is blunted. This readily permits glomerular impairment, hypokalemia, metabolic alkalosis,
738 VI. Tubular Disease

hypomagnesemia, glycosuria, hypocalciuria, and relative Genetics

hypomagnesuria (118). Surreptitious diuretic ingestion
Glucocorticoid-remediable aldosteronism, an autosomal
may also mimic Gitelman’s syndrome. Besides the urinary
dominant disease, arises due to a chimeric gene formed at
determination of diuretics, the molecular genetic diagnosis
meiosis after genetic recombination between two homo-
may also help to establish the correct diagnosis (119).
logues, adjacent genes on chromosome 8q22, CYP11B1 and
CYP11B2, encoding the enzymes 11β-hydroxylase and
Treatment and Prognosis
aldosterone synthase involved, respectively, in the final path-
Patients with Gitelman’s syndrome are best treated with Mg
ways of cortisol and aldosterone synthesis within the adrenal
salts alone, with no need, in most cases, to add either K salts or
cortex (127) (Table 38.1). The final 11β-hydroxylation in
prostaglandin inhibitors. Many Mg salts have been used in the
the synthesis of cortisol in the zona fasciculata is regulated by
treatment of hypomagnesemic states (e.g., Mg oxide, Mg chlo-
11β-hydroxylase, which depends on adrenocorticotropic
ride, Mg pidolate, Mg lactate, Mg pyrrolidone carboxylate). In
hormone (ACTH) secretion. The final stages of aldosterone
Gitelman’s syndrome, we recommend the use of Mg chloride
synthesis are catalyzed by the P-450 enzyme aldosterone syn-
(MgCl2) that also compensates for ongoing urinary Cl losses.
thase, whose expression is limited to the zona glomerulosa
Magnesium chloride has a high Mg content, is very soluble,
and is regulated by angiotensin II and K.
and is not dependent on gastric acidity for absorption. Also, its
administration is less often followed by diarrhea, although this
complication may still develop with increasing dosage. We use Clinical and Biochemical Features
a 5% solution that contains approximately 0.5 mEq (0.6 mg)
Mg2+/mL. The total dose should be individualized for each Glucorticoid-remediable aldosteronism manifests clinically
patient and given at 6- to 8-hour intervals. In our experience, by the presence of familial low-renin hypertension. In
regular administration of MgCl2 not only partly corrects the affected family members, hypertension is already observed
hypomagnesemia and prevents the appearance of tetanic epi- in those younger than 21 years of age and often during
sodes, but also normalizes plasma K concentration, acid-base childhood (128). Hypertension is especially severe and may
equilibrium, the renin-aldosterone axis, and urinary Ca excre- lead to cardiovascular complications (e.g., hemorrhagic
tion. However, there is some disagreement whether the K stroke) in almost 20% of cases. Characteristic biochemical
wasting may or may not be exclusively attributed to hypo- findings are metabolic alkalosis and very low plasma renin
magnesemia (120,121). In fact, occasional cases may require activity. Hypokalemia may be present but is not an obliga-
the additional administration of potassium salts or antialdos- tory finding.
terone drugs (e.g., spironolactone or amiloride) to correct the
hypokalemia (122). High-dosage indomethacin may be espe- Pathophysiology
cially indicated in exceptional cases with growth retardation
and bad tolerance to Mg supplementation (123). Hypertension results from aldosterone excess, with aldos-
The long-term prognosis of Gitelman’s syndrome, in terone being under the control of ACTH rather than the
terms of preserving renal function and maintaining growth, normal secretagogue, angiotensin II. The chimeric gene
appears to be excellent, but sustained lifelong Mg supple- possesses the 5' 11β-hydroxylase and the 3' aldosterone
mentation remains necessary to reduce the risk of tetanic epi- synthase sequences, and although it promotes the synthesis
sodes (124). There is a single report of an adult patient who of aldosterone, it is under the regulatory control of ACTH
developed end-stage renal disease (125). and not of angiotensin II.

Hyperaldosteronism Diagnosis
Although the common adult syndromes of primary hyperal- The ratio between urinary concentrations of C-18 oxida-
dosteronism (e.g., renin-secreting tumor and Conn disease tion products (18-oxotetrahydrocortisol and 18-hydrocor-
due to adrenal adenoma, carcinoma, or micronodular hyper- tisol) and tetrahydroaldosterone is characteristically
plasia) (126) are outside the scope of this chapter, we will increased, but given the technical difficulty of these analyti-
refer to one rare disorder, which may be observed during cal procedures, the diagnosis is generally established by the
the pediatric age, the so-called glucocorticoid-remediable use of the dexamethasone suppression test, which is based
aldosteronism. on the decrease of aldosterone production after ACTH sup-
pression. A value of plasma aldosterone below 4 ng/dL
postdexamethasone correctly diagnoses glucocorticoid-
Glucocorticoid-Remediable Aldosteronism
remediable aldosteronism with high sensitivity and specific-
Glucocorticoid-remediable aldosteronism, also called famil- ity (129). However, definitive diagnosis is established by
ial hyperaldosteronism type 1, represents a rare cause of low- the use of molecular biology techniques (130). There are a
renin hypertension in children and adults. few patients who present with a positive dexamethasone
 38. Tubular Disorders of Electrolyte Regulation 739

suppression test without any evidence of abnormalities in medullary cysts have been noted in a few cases. Aldosterone
CYP11B1 and CYP11B2 genes. The pathogenesis of these secretion is not stimulated by salt restriction, and improve-
rare cases of glucocorticoid-remediable aldosteronism not ment is observed after administration of triamterene but
linked to a chimeric CYP11B1/CYP11B2 gene remains not after administration of spironolactone.
unknown (131).
Pathophysiology
Therapy
Liddle’s syndrome results from excessive renal tubular Na
Hypertension is refractory to therapy with the usual antihy- absorption through activation of the renal epithelial
pertensive drugs but quickly resolves after administration of sodium channel. As mentioned above, this channel is
K-retaining diuretics such as spironolactone or amiloride. formed by three subunit proteins (α, β, and γ) having short
Occasionally, patients may require a combined therapy cytosolic termini, two transmembrane domains, and a large
with nifedipine (132). Although administration of dexa- extracellular loop (140). The α subunit appears to be
methasone may also ameliorate the hypertension, its pro- required for the assembly or function of the whole com-
longed use is not recommended in childhood given its plex. The three ENaC subunits are homologous to each
deleterious effect on growth. other and have approximately 35% amino acid identity. A
possible membrane topology of the heterotetrameric com-
plex is formed by two α subunits, one β subunit, and one γ
Pseudohyperaldosteronism
subunit, because this complex maintains a high amiloride
Under the term pseudohyperaldosteronism, we include a few sensitivity and a very Na+-selective pore (Fig. 38.3). The
syndromes that present clinical and biochemical findings ENaC is not only present in the kidney but also in other
similar to those observed in primary hyperaldosteronism organs (e.g., lung, colon, exocrine glands, skin and hair fol-
but without evidence of increased secretion of mineralo- licles). The C terminal of each ENaC subunit contains a
corticoids. In fact, plasma aldosterone levels are often xPPxY motif, which is necessary for interaction with the
undetectable. WW domains of the ubiquitin-protein ligase, Nedd4
(141). Nedd4 regulates ENaC function by controlling the
number of channels at the cell surface. Liddle’s mutations,
Liddle’s Syndrome
leading to truncated C-terminus of the β or γ subunits,
Liddle’s syndrome is a very rare disorder of tubular trans-
port, simulating primary hyperaldosteronism but with neg-
ligible aldosterone secretion. It was described in 1963 by
Liddle, Bledsoe, and Coppage (133) in eight individuals of
the same family.

Genetics
A pattern of autosomal dominant inheritance has been
described, although some cases appear to be sporadic (134–
136) (Fig. 38.1). Recent studies have demonstrated that
this entity is caused by mutations of the genes encoding
two of the three constitutive subunits of the amiloride-
sensitive epithelial sodium channel (ENaC). Mutations of
the genes encoding β subunit (βENaC) (137,138) and γ
subunit (γENaC) (139) have been described. Both genes
SNCC1B and SNCC1G are located on 16p12. No abnor-
malities have been noted in the gene SNCC1A, located at
12p13, or in encoding the α subunit (αENaC).

Clinical and Biochemical Features

Characteristic clinical and laboratory findings start in
infancy and early childhood and include polyuria, polydip- FIGURE 38.3. Membrane topology of the epithelial Na + chan-
sia, failure to thrive, arterial hypertension, hypokalemic nel. The proposed model is a heterotetrameric structure formed
by two α subunits, one β subunit, and one γ subunit. Each sub-
metabolic alkalosis, and almost absent renin and aldoster- unit is formed by two membrane-spanning domains, a large
one secretion. Hypercalciuria, nephrocalcinosis, and renal extracellular loop, and cytoplasmic amino- and carboxytermini.
740 VI. Tubular Disease

alter or delete the xPPxY motif, interrupt the interaction HSD11B2, encoding 11β-HSD2 (renal isoenzyme), contains
with Nedd4 protein, and lead to an increased number of 5 exons and is located in 16q12 (152,153).
Na channels in the apical membrane, increased Na reab- AME is caused by autosomal recessive mutations in the
sorption, expanded plasma volume with resulting hyperten- HSD11B2 gene, which results in a deficiency of 11β-HSD2
sion, and inhibited renin-aldosterone axis with secondary (154–158). Most patients are homozygotes for one of the
potassium wasting (142–144). A transgenic mouse model different mutations, and few compound heterozygotes have
for Liddle’s syndrome reproduces, to a large extent, a been identified.
human form of salt-sensitive hypertension (145). It is pos-
sible that polymorphisms of the β and γ subunits of the
Clinical and Biochemical Features
ENaC are related to variations of blood pressure in humans
(146) and may contribute to the high incidence of salt- AME usually presents early in life and is characterized by
related hypertension in blacks (147). polyuria, polydipsia, failure to thrive, hypertension, hypokale-
mic alkalosis, suppressed plasma renin activity, and absence of
adrenal secretion of any known mineralocorticoid. Renal
Diagnosis
cysts, nephrocalcinosis, rickets, and hyperparathyroidism have
Diagnosis is mainly established with the syndrome of been sporadically reported (159). Damage of many organs
apparent mineralocorticoid excess. The presence of hyper- (e.g., kidneys, retina, heart, central nervous system) univer-
tensive individuals in successive generations is very sugges- sally follows long-standing hypertension. Intrauterine growth
tive of Liddle’s syndrome. The lack of hypotensive effect of retardation is a common finding and is probably secondary to
spironolactone or dexamethasone administration also sug- placental 11β-HSD2 deficiency. The human placenta con-
gests the diagnosis. Nowadays, genetic testing allows a sure tains abundant 11β-HSD2, which has an important regula-
and rapid diagnosis (148). tory role of fetal growth (160).
A mild form of AME has been described in a 12.5-year-
old girl presenting with mild hypertension and suppressed
Therapy and Prognosis
renin and aldosterone secretion but lacking hypokalemia
Therapy consists of the combined administration of K sup- and low birth weight. Despite these mild symptoms, it was
plementation (as KCl) and triamterene (10 mg/kg/day). proven that this girl was homozygous for a missense muta-
Improvement is manifest by normalization of Na and K tion in the HSD11B2 gene (150).
balances, resolution of hypertension, and reinstitution of
normal growth. However, catch-up growth rarely occurs,
Pathophysiology
and patients may remain significantly growth-retarded if
the syndrome has been manifest early in infancy. If the 11b-HSD (EC 1.1.1.146) plays a critical role in determin-
diagnosis is not firmly established, a therapeutic trial with ing the specificity of type I receptors. In the normal state,
spironolactone or dexamethasone should also be considered cortisol is converted by 11β-HSD to cortisone, thus allow-
given the clinical similarity with 11β-hydroxysteroid dehy- ing the preferential binding of aldosterone to type I recep-
drogenase (11β-HSD) deficiency. tors. In cases of 11β-HSD deficiency, there is a high
intrarenal cortisol concentration that facilitates binding to
the type I receptor, thus resulting in AME (161). Trans-
Apparent Mineralocorticoid Excess
genic mice lacking 11β-HSD2 present all major features of
This entity, identified by Ulick and New (149), gives rise to human AME (162).
a state of apparent mineralocorticoid excess (AME), which
is clinically and biochemically very similar to Liddle’s syn-
Diagnosis
drome. Approximately 40 affected patients have been
described worldwide (150). Biochemical diagnosis can be made by measuring the ratio
of cortisol to cortisone metabolites (tetrahydrocortisol plus
allotetrahydrocortisol or tetrahydrocortisone). The optimal
Genetics
diagnostic test is to measure the generation of titrated water
There are two isoenzymes of 11β-HSD: NADP(+)-dependent in plasma samples when 11-titrated cortisol is injected.
isoenzyme 1 (11β-HSD1), isolated from liver but widely Conversion of cortisol to cortisone is only 0 to 6% in AME
expressed in many tissues; and NADP(+)-dependent isoen- patients, whereas the normal conversion is 90 to 95% (150).
zyme 2 (11β-HSD2), highly expressed in kidney, placenta,
and adrenal gland (151) (Table 38.1). The genes encoding
Therapy
both isoenzymes have been recently cloned. The gene
HSD11B1, encoding 11β-HSD1 (liver isoenzyme), contains Therapy consists of the administration of the mineralocor-
6 exons and is located in chromosome 1, whereas the gene ticoid receptor blocker, spironolactone. After the adminis-
 38. Tubular Disorders of Electrolyte Regulation 741

tration of spironolactone, hypertension disappears, natriuresis to creams or nasal sprays containing 9α-fluorinated corti-
ensues, and plasma renin activity becomes detectable. The coids should also be considered.
daily dose of spironolactone ranges between 2 and 10 mg/
kg. As hypercalciuria and nephrocalcinosis are consistent
features of the disease, a thiazide diuretic may be added. HYPERKALEMIC STATES
Thiazide diuretics may not only contribute to improve
hypercalciuria but also may aid to lower blood pressure A situation of hyperkalemia may be caused by increased
and allow for the dose of spironolactone to be reduced intake or endogenous liberation of K, decreased renal out-
(157). Renal transplantation, performed in exceptional put, or abnormal distribution of K in the body (see Chap-
cases, completely normalizes the clinical and biochemical ter 8). Most children with persistent hyperkalemia, in the
picture (163). absence of oversupplementation, demonstrate decreased
renal excretion of K. In such cases, one must differentiate
extrinsic causes, such as mineralocorticoid deficiency due to
Hypertension Secondary to Activated
primary adrenal disorders, from intrinsic causes, such as
Mineralocorticoid Receptor
renal failure or tubular renal disorders limiting K transport.
Geller et al. (164) have reported on a family whose proposi- The term renal tubular hyperkalemia has been proposed to
tus, a 15-year-old boy, had severe hypertension and sup- describe a group of isolated renal tubular defects of K secre-
pressed plasma aldosterone levels. This patient and 11 of 21 tion in which hyperkalemia constitutes the outstanding
at-risk relatives, all hypertensive, were heterozygous for a biochemical feature (167).
missense mutation substituting leucine for highly conserved To examine whether sustained hyperkalemia is due to a
serine at codon 810 of the mineralocorticoid receptor. This tubular defect of K secretion, it is necessary to challenge the
mutation resulted in a gain of function of the receptor and mechanisms of tubular transport and to study the function-
alteration of its specificity because it was also activated by ing of the renin-aldosterone system. Although sulfate infu-
progesterone. Carriers not only presented with severe early- sion has been widely used to test the functioning of the
onset hypertension but also, although normokalemic, tended cortical distal nephron, identical results can be obtained by
to have lower plasma K values and manifested severe hyper- acute intravenous administration of furosemide (1 mg/kg
tension during episodes of pregnancy. body weight). This diuretic blocks NaCl reabsorption in
the ascending limb of the loop of Henle and thus increases
the delivery of fluid and NaCl to the cortical collecting
Secondary Pseudohyperaldosteronism
tubule. By enhancing Na reabsorption in excess of Cl reab-
An acquired syndrome of pseudohyperaldosteronism, in all sorption in this cortical segment, it creates a favorable elec-
ways identical to 11β-HSD2 deficiency, has been observed tric gradient (lumen-negative) and facilitates the exit of K
after chronic ingestion of licorice (165). Natural licorice, an and H. Furosemide, by causing volume depletion, also
extract of Glycyrrhiza glabra root, contains glycyrrhizinic enhances the secretion of renin and aldosterone. A sum-
acid, which is a potent inhibitor of 11β-HSD2. This com- mary of the results obtained by our group in a study of nor-
plication is exceptionally observed in children (166). Car- mal children is presented in Table 38.3 (167).
benoxolone, an antiulcer drug, also competitively inhibits When assessing urinary K excretion, one must take into
11β-HSD and causes sodium retention as a relevant side account that as GFR decreases, fractional K excretion increases
effect. The possibility that a syndrome of AME is secondary exponentially. Because many patients with renal tubular hyper-

TABLE 38.3. RENAL RESPONSE OF CHILDREN (N = 20) TO ACUTE FUROSEMIDE ADMINISTRATION

(MEAN ± STANDARD DEVIATION)
UTAV UNH4V PRA Paldo
UpH (μEq/dL GF) (μEq/dL GF) FENa (%) FECl (%) FEK (%) (ng/mL/h) (ng/dL)

Basal values before 5.93 13.1 19.6 0.7 1.1 11.5 3.1 18.3
furosemide ±0.57 ±6.1 ±6.7 ±0.7 ±1.1 ±4.6 ±3.5 ±12.2
Peak values after 4.96 22.7 30.9 8.4 12.6 35.4 8.6a 32.5a
furosemideb ±0.32 ±10.7 ±8.0 ±3.5 ±4.9 ±8.9 ±5.9 ±18.6

FECl, fractional excretions of chloride; FEK, fractional excretions of potassium; FENa, fractional excretions of sodium; GF, glomerular filtrate; Paldo,
plasma aldosterone concentration; PRA, plasma renin activity; UNH4V, urinary excretions of ammonium; UpH, urine pH; UTAV, urinary excretions of
titratable acid.
aValues obtained 180 minutes after administration of furosemide.
bMean of peak values obtained in each subject.

Adapted from Rodríguez-Soriano J, Vallo A. Renal tubular hyperkalaemia in childhood. Pediatr Nephrol 1988;2:498–509.
742 VI. Tubular Disease

kalemia may exhibit some degree of renal insufficiency, it is aldosterone. Patients become suddenly dehydrated, with
necessary to relate values of fractional K excretion to the corre- signs of extracellular fluid volume depletion. Hyponatremia
sponding values for GFR. To study the action of aldosterone at and hyperkalemia are present, and urinary NaCl excretion is
the level of distal and collecting tubules, it is useful to calculate inappropriately high. Urgent therapy is mandatory and
the so-called transtubular K concentration gradient (TTKG) includes NaCl supplementation and administration of
by the formula proposed by West et al. (168): hydrocortisone and 9α-fludrocortisone. Glucocorticoid and
mineralocorticoid therapy should be maintained for life.
K urine ⁄ (U/P) osmolality
TTKG = -------------------------------------------------------------
-
K blood
Isolated Defects of
This index provides a ratio of the estimated concentra- Aldosterone Biosynthesis
tion of K in the cortical collecting tubule (or urine K con-
Aldosterone biosynthetic defects other than 21-hydroxylase
centration adjusted for medullary water abstraction) to that
deficiency can also cause salt-wasting and hyperkalemia
of K in the peritubular fluid in the renal cortex. Values in
(171). The conversion of 11-desoxycorticosterone to aldos-
normal children follow a nongaussian distribution, with a
terone requires the successive steps of 11 β-hydroxylation,
median of 6.0 and a range of 4.1 to 10.5. Transtubular K
18-hydroxylation, and 18-oxidation, all catalyzed by a single
concentration gradient is significantly higher in infants,
enzyme, aldosterone synthase (P450c11AS) (172). There are
with a median value of 7.8 and a range of 4.9 to 15.5
two inborn errors caused by different mutations in the
(169). Any value below these limits should be interpreted
CYP11B2 gene encoding aldosterone synthase, the so-called
as indicating mineralocorticoid deficiency or mineralocorti-
corticosterone methyloxidase type I and type II defects (173).
coid unresponsiveness.
Infants with both defects, but especially with type I, present
with failure to thrive, recurrent episodes of dehydration and
salt-wasting, and hyperkalemia (174,175). Simultaneous
MINERALOCORTICOID DEFICIENCY
serum determination of aldosterone and 18-hydroxycorticos-
terone levels permits differentiation between type I and type
Hyperreninemic Hypoaldosteronism
II defects (176). As in salt-wasting congenital adrenal hyper-
Several adrenal disorders are associated with a primary plasia, mineralocorticoid replacement therapy should be
defect in aldosterone secretion that is accompanied by a maintained for life.
secondary increase in plasma renin activity. This situation is
almost always accompanied by salt-wasting and hyperkale-
Hyporeninemic Hypoaldosteronism
mia, and, therefore, it should be distinguished from tubular
resistance to aldosterone or pseudohypoaldosteronism. A Hyporeninemic hypoaldosteronism is a syndrome character-
detailed description of adrenal disorders is beyond the ized by inappropriately low aldosterone secretion without an
scope of this chapter. associated impairment in the synthesis of cortisol but with low
plasma renin activity. In fact, hypoaldosteronism apparently
results from insufficient stimulation of the adrenal gland by
Adrenal Insufficiency (Addison’s Disease)
the renin-angiotensin system. This condition was first reported
When adrenal insufficiency occurs in infancy, it is usually due by Hudson et al. (177), and since then, a great number of cases
to congenital adrenal hypoplasia, a condition that may be spo- have been documented in the literature, particularly in adults
radic or inherited in an autosomal recessive or a sex-linked pat- with moderate chronic renal insufficiency due to diabetic
tern. The X-linked adrenal hypoplasia is often associated with nephropathy or chronic tubulointerstitial disorders. Hyper-
gonadotrophin deficiency, progressive hearing loss, glyceroki- kalemia and metabolic acidosis constitute the cardinal bio-
nase deficiency, and muscular dystrophy. In older children and chemical features. Salt-wasting is not generally present. This
adults, other causes may be observed: autoimmune adrenalitis syndrome has been rarely reported in children, but its fre-
and, more rarely today, tuberculosis, amyloidosis, and acute quency is probably higher than is usually recognized in
adrenal hemorrhage or infarction. Adrenal leukodystrophy patients with moderate chronic renal insufficiencies of various
(Addison’s-Schilder’s disease) is a rare sex-linked inherited con- etiologies (178). This condition appears to be especially fre-
dition characterized by progressive neurologic damage associ- quent in children with lupus nephritis or with chronic neph-
ated with primary adrenal insufficiency. ropathy secondary to methylmalonic acidemia.
Hereditary enzymatic defects may also cause mineralo- The pathogenesis of hyporeninemic hypoaldosteronism in
corticoid deficiency. Salt-wasting is present in approximately patients with chronic renal insufficiency is not yet elucidated.
two-thirds of patients with the classic form of congenital The hyperkalemia may be ascribed solely to hypoaldoster-
adrenal hyperplasia resulting from 21-hydroxylase defi- onism, but pharmacologic doses of mineralocorticoids are
ciency (150,170). Salt-wasting occurs usually during the often needed to correct it. This indicates associated tubular
first weeks of life and results from inadequate synthesis of resistance to aldosterone’s action. Metabolic acidosis may
 38. Tubular Disorders of Electrolyte Regulation 743

TABLE 38.4. CHARACTERISTICS OF TWO MAJOR FORMS OF PRIMARY TYPE I PSEUDOHYPOALDOSTERONISM

Characteristics Renal Multiple

Affected organs Kidney Kidney, sweat and salivary glands, colon

Mode of inheritance Autosomal dominant Autosomal recessive
Genetic defect Mutations in the gene encoding the miner- Mutations in the genes encoding the α, β, or
alocorticoid receptor γ subunits of epithelial Na channel
Salt-wasting Variable Severe
Catchup growth on NaCl supplements Common Rare
PRA and aldosterone concentration Very high, PRA decreases with age Very high
Sweat and salivary electrolytes Normal Very high
High-salt diet 1–3 yr Lifelong?
Improvement with age Common Absent?

PRA, plasma renin activity.

Adapted from Hanukoglu A. Type I pseudohypoaldosteronism includes two clinically and genetically distinct entities with either renal or multiple
target organ defects. J Clin Endocrinol Metab 1991;73:936–944.

result from selective aldosterone deficiency and from the Primary Type 1 Pseudohypoaldosteronism
direct inhibitory action of hyperkalemia on renal ammonia
Type 1 pseudohypoaldosteronism (PHA1) is a hereditary
genesis and tubular transport of ammonia. Hypoaldoster-
condition characterized by salt-wasting, hyperkalemia, and
onism appears to be the consequence of hyporeninemia, but
metabolic acidosis in the presence of markedly elevated
the reason for this is unclear. Chronic extracellular fluid
plasma renin activity and aldosterone concentrations. Since
expansion, damage to the juxtaglomerular apparatus, reduced
its first description by Cheek and Perry in 1958 (184),
activity of the adrenergic system, impaired formation of active
many cases have been reported. In recent years, it has
renin, or decreased synthesis of prostacyclin have all been
become clear that PHA1 is a heterogeneous syndrome that
advocated as possible causes (179).
includes at least two clinically and genetically distinct enti-
Specific therapy is often not required if attention is paid
ties with either renal or multiple target-organ defects (185)
to avoidance of renin-suppressing agents (i.e., beta-blockers,
(Table 38.4). An exceptional case with apparent resistance
calcium channel blockers, and nonsteroidal antiinflamma-
to aldosterone limited to sweat and salivary glands without
tory agents), or K-retaining drugs (i.e., amiloride, spirono-
associated salt-wasting has also been reported (186).
lactone, heparin, captopril, trimethoprim, tacrolimus, or
cyclosporine) (180). However, when dangerous hyperkale-
mia or severe metabolic acidosis is present, therapeutic mea- Renal Type 1 Pseudohypoaldosteronism
sures become necessary. Good results have been reported
Genetics
with the sustained administration of furosemide, either
alone or combined with 9α-fludrocortisone. Renal PHA1 represents the most frequent form of PHA1
The syndrome of hyporeninemic hypoaldosteronism (Table 38.1). The mode of inheritance is autosomal domi-
may be occasionally observed as an isolated or familial nant, with variable expression. Many cases appear as spo-
entity, not necessarily in association with chronic renal radic, but familial studies reveal high plasma levels of
insufficiency (181–183). An interesting feature of all these aldosterone in one of the apparently asymptomatic parents.
cases is the presence of salt-wasting, a condition rarely The pathogenesis of renal PHA1 has been greatly clarified
observed in cases of hyporeninemic hypoaldosteronism with the identification of heterozygous mutations in the
associated with chronic renal insufficiency. mineralocorticoid receptor gene (187–189). The structure
of the gene encoding the human mineralocorticoid recep-
tor (MLR) has been elucidated. It is formed by 9 exons and
Apparent Mineralocorticoid
is located at 4q31.1. Two different isoforms have been iden-
Unresponsiveness
tified. Each isoform is expressed in approximately equiva-
The term pseudohypoaldosteronism has been coined to lent levels in kidney, colon, and sweat glands.
describe disorders of electrolyte homeostasis characterized
by an apparent state of renal tubular unresponsiveness to
Clinical and Biochemical Features
the action of aldosterone and manifested by salt-wasting,
hyperkalemia, and metabolic acidosis. Recent molecular Clinical features are extremely variable. In general, symp-
biology studies have shown that these syndromes do not toms appear in early infancy with failure to thrive, weight
always result from target-organ unresponsiveness to miner- loss, vomiting, and dehydration. Salt-wasting and polyuria
alocorticoids but more often are caused by inherited abnor- may be already present in fetal life and manifested by
malities of renal Na transport. maternal hydramnios. Occasional patients may present also
744 VI. Tubular Disease

with hypercalciuria and nephrocalcinosis. If therapy is restricted to specific cells of kidney distal and collecting
delayed, patients may become severely undernourished. tubules, colonic epithelium, and sweat and salivary gland
During the salt-wasting episodes, which may be triggered ducts. In all these epithelia, aldosterone stimulates Na reab-
by intercurrent infections, patients appear shocked and sorption. Mineralocorticoid receptor–deficient mice have
comatose. A partial form with mild course of the disease been generated by gene targeting technology. These knock-
and almost absent salt-wasting may be observed (190). out mice develop symptoms of pseudohypoaldosteronism
Sweat and salivary electrolytes are characteristically normal. soon after birth, which can be compensated by abundant
In exceptional cases, diagnosis can be made in asymptom- NaCl administration. These NaCl-rescued mice display a
atic newborn babies or even in cord blood on the basis of a strongly enhanced fractional renal excretion of Na, hyper-
positive family history. kalemia, a persistently activated renin-angiotensin-aldoster-
The outstanding laboratory findings are hyponatremia one system, and very reduced ENaC activity in the kidney
and hyperkalemia. Metabolic acidosis may be also present. and colon (194).
Hyponatremia may be masked by the hemoconcentration.
Hyperkalemia varies from moderately to greatly increased
Treatment and Prognosis
values for plasma K concentration. Urinary Na excretion is
inappropriately high, given the presence of hyponatremia. Renal PHA1 is treated with NaCl supplementation (3 to 6 g/
On the contrary, urinary K excretion is very low, with day), which is followed by marked clinical and biochemical
decreased values of fractional K excretion and transtubular improvement. The expansion of extracellular fluid volume
K concentration gradient. GFR is normal after sodium results in an increase of tubular flow and NaCl delivery to the
depletion is corrected. Renal biopsy findings are usually distal nephron, thus creating a favorable gradient for K secre-
negative, but in a few cases, hypertrophy of the juxtaglom- tion despite the lack of mineralocorticoid action. The amount
erular apparatus has been reported. Although plasma elec- of NaCl required is deduced from the normalization of plasma
trolyte findings are very similar to those seen in defects of K concentration and renin activity. Although the primary
aldosterone biosynthesis, both plasma renin activity and defect persists for life, improvement may occur beyond 1 or 2
aldosterone concentration are markedly elevated, urinary years of age, due to maturation of proximal tubular transport,
excretion of aldosterone and tetrahydroaldosterone are ele- development of salt appetite, and improvement in the renal
vated, and a urinary pattern of aldosterone metabolites tubular response to mineralocorticoids (195). Older children
conforms to a normal pattern (i.e., a normal 18-hydroxy- with renal PHA1 are generally asymptomatic when eating a
tetrahydrocompound A to tetrahydroaldosterone ratio) normal salt intake, but plasma aldosterone concentrations
(191). There is also a characteristic lack of improvement remain elevated. In contrast to aldosterone levels, plasma renin
despite administration of large doses of mineralocorticoids. activity decreases into the normal range with advancing age.
The observation that many adult gene carriers have ele- The discrepancy between renin and aldosterone secretion may
vated aldosterone levels but no history of clinical disease be explained by the development of autonomous, tertiary
raises the possibility that only a fraction of such carriers hyperaldosteronism, as demonstrated by the increased excre-
develop clinically evident disease (192). The reasons for the tion of 18-hydroxy-compound B (a characteristic marker of
phenotype differences are unknown, but they may be the functioning of the zona glomerulosa). Exceptionally, subtle
related to intercurrent volume-depleting events or to symptoms leading to growth failure or even overt salt-wasting
dietary habits of salt ingestion. Also, the coexistence of may persist to late childhood.
polymorphisms or mutations in the gene encoding ENaC
could play a potential contributory role. Polymorphisms
and mutations that lead to either loss of function or gain of Multiple Type 1 Pseudohypoaldosteronism
function of the epithelial Na+ channel may aggravate or
Genetics
attenuate, respectively, the consequences of deficient miner-
alocorticoid receptor function. A few kindreds have been reported with multiple end-organ
PHA1 since its original description (196,197) (Table 38.1).
mineralocorticoid resistance is evident in kidney, lung, colon,
Pathophysiology
and sweat and salivary glands. This variant is inherited as an
The pathogenesis of renal pseudohypoaldosteronism type 1 autosomal recessive disorder, with uniform expression. The
is related to the loss of function of the mineralocorticoid parents of affected children are asymptomatic and evidence
receptor. The mineralocorticoid receptor is a protein of 984 normal plasma aldosterone levels. It has been demonstrated
amino acids belonging to the superfamily of steroid recep- that this entity is caused by loss-of-function mutations of
tors. It contains an immunogenic domain, a zinc-finger genes encoding one of the three constitutive subunits (i.e., α,
DNA-binding domain, and a hormone-binding domain β, and γ) of the ENaC (198–201). Critical hot spots for loss-
(193). At variance with glucocorticoid receptor that is of-function mutations seem to be the cysteine-reach domains
almost ubiquitous, mineralocorticoid receptor expression is in the large extracellular loop of the proteins (202).
 38. Tubular Disorders of Electrolyte Regulation 745

Clinical and Biochemical Features Treatment and Prognosis

The multiple PHA1 presents with more severe salt-wast- There is a poor response to NaCl supplementation alone, and
ing and has a poorer outcome than the renal form. rectal administration of exchange resins and dietary manipu-
These patients manifest salt-wasting episodes early after lation reducing the intake of K are often necessary. Adminis-
birth, and death may ensue during the neonatal period. tration of indomethacin or hydrochlorothiazide or both may
Sweat and salivary electrolytes are markedly elevated, be useful in occasional patients (215). Improvement with age
and active Na transport in the rectal mucosa is impaired. is less apparent, and therapy must be maintained throughout
Neither abnormality is corrected by the administration childhood and probably throughout life (216).
of exogenous mineralocorticoids. The high incidence of
lower respiratory tract involvement may contribute to
the confusion with cystic fibrosis (203,204). As in the Early-Childhood Hyperkalemia
renal form, hypercalciuria may be present. McSherry (217) described 13 infants and young children
who presented with failure to thrive or growth retardation
and frequent vomiting. The only biochemical abnormali-
Pathophysiology ties were hyperkalemia and metabolic acidosis. Clinical
Multiple target-organ PHA1 is due to defective Na trans- salt-wasting or hypertension was absent. Functional evalua-
port in many organs containing the ENaC: kidney, lung, tion revealed a normal ability to acidify the urine, low NH4
colon, and exocrine glands. Multiple PHA1 is allelic to and K excretion, and a mild defect in HCO3 reabsorption
Liddle’s syndrome, but the mutations present result in loss (i.e., functional markers of type 4 renal tubular acidosis).
of the channel activity with ensuing renal and rectal Na Plasma renin activity and aldosterone excretion were con-
loss and increased sweat and saliva electrolyte values. The sistently normal or elevated. McSherry speculated that this
frequent lower respiratory involvement may be explained entity could be due to a primary or idiopathic unrespon-
by the dysfunction of the ENaC of the respiratory epithe- siveness of the distal nephron to aldosterone that mani-
lia (205). The impaired bacterial killing may be due to fested itself by defects in K and H excretion with integrity
increased NaCl concentration in the airway surface fluid of Na reabsorption. However, Appiani et al. (218) reported
(206). The state of secondary hyperreninism and hyperal- five infants with similar clinical features who demonstrated
dosteronism is not caused by peripheral resistance to min- a significantly increased fractional Na excretion. As in the
eralocorticoids but derives from sustained extracellular cases communicated by McSherry, no clinical evidence of
fluid volume depletion. salt-wasting was observed.
Insights into pathogenesis have been facilitated by the Early-childhood hyperkalemia represents a variant of the
development of transgenic mouse models in which renal form of PHA1. Therapy is generally required during
expression of the α, β, or γ subunits of ENaC has been the first years of life in the form of sodium bicarbonate alone
ablated. Of interest, only the αENaC-deficient mice or associated with caption-exchange resin. With treatment,
present marked failure to clear the fetal lung fluid that growth accelerates and normal height is achieved within 6
leads to early neonatal death (207). If the mice are rescued months. The disorder appears to be transient, and at approx-
from death by the engineered expression of the α subunit imately 5 years of age, therapy is no longer needed.
gene in the lung, they develop a full-blown picture of
PHA, with salt-wasting and hyperkalemia (208). The
Secondary Type 1
βENaC- and γENaC-deficient mice do not die of neona-
Pseudohypoaldosteronism
tal respiratory failure but develop a very severe picture of
neonatal salt-wasting and hyperkalemia from birth, simi- Secondary forms of PHA1 have been rarely reported. A
lar to human PHA1 (209–211). partial tubular insensitivity to aldosterone may account for
The neonatal lung findings present in αENaC- the hyperkalemia observed in some infants after unilateral
deficient mice are only exceptionally observed in humans renal vein thrombosis or for the salt-wasting and hyperkale-
(212), despite impressive truncations of the α subunit in mia that follow neonatal medullary necrosis. A syndrome of
some kindreds. However, some impairment in Na trans- apparent renal tubular resistance to aldosterone was first
port across airway epithelia may persist throughout life. documented in 1983 by Rodríguez-Soriano et al. in young
In fact, pulmonary symptoms in patients with autosomal infants with urinary tract infection and associated urinary
recessive PHA1 are not always related to infection and tract malformations (219). Since then, 62 cases have been
may be secondary (especially in young patients) to fail- reported (220). After medical or surgical therapy, all abnor-
ure to absorb liquid from airway surfaces (213). The malities generally disappear, although, in cases of severe
defect in Na transport can be demonstrated by measure- obstructive uropathy, renal sodium loss may become tran-
ment of transepithelial voltage across the nasal epithe- siently more severe during the early obstructive period
lium (214). (221,222). The picture of hyponatremia or hyperkalemia
746 VI. Tubular Disease

may be also observed in infants and children with acute Pathophysiology

pyelonephritis, even in the absence of associated uropathy
Schambelan et al. (237) proposed the name of chloride-shunt
(223–225). All these observations indicate that renal ultra-
syndrome (or type 2 pseudohypoaldosteronism), according
sonography and urine culture should be performed in any
to the hypothesis that the primary abnormality was a tubular
infant or child presenting with salt-wasting or hyperkale-
hyperreabsorption of NaCl such that the mineralocorticoid-
mia to exclude structural renal lesions or infection as the
induced, charge-dependent K and H secretion would be
cause of the electrolyte disturbances.
attenuated by a voltage-shunting defect. Increased NaCl
reabsorption would lead to expansion of plasma volume,
Type 2 Pseudohypoaldosteronism suppressed secretion of renin and aldosterone, and eventual
(Gordon’s Syndrome) development of hypertension. In favor of this hypothesis was
the complete reversal of the defect when distal NaCl delivery
A familial syndrome (type 2 pseudohypoaldosteronism) of
was increased by sodium sulfate or bicarbonate infusions or
arterial hypertension, hyperkalemia, metabolic acidosis,
by the acute administration of furosemide.
suppressed plasma renin activity, and normal glomerular
Molecular biology studies have confirmed that increased
function was first described by Paver and Pauline in 1964
distal tubular reabsorption of NaCl is the primary patho-
(226), although it was characterized as a new clinical entity
logic event in this syndrome. Both WNK1 and WNK4
by Gordon et al. in 1970 (227). Approximately 100 cases of
kinases localize to the distal nephron segments known to
so-called Gordon’s syndrome have been reported in the lit-
play a key role in the homeostasis of Na reabsorption and
erature (228). Hypertension represents a feature limited to
H and K secretion. WNK1 is cytoplasmic, whereas WNK4
adolescent or adult individuals. A similar condition has
localizes to tight junctions. The gain-of-function mutations
been reported in children with short stature, hyperkalemia,
in these kinases may serve to increase transcellular or para-
and metabolic acidosis but with normal blood pressure
cellular Cl conductance in the collecting duct, thereby
(Spitzer-Weinstein syndrome) (229–232). The finding in
increasing NaCl reabsorption and intravascular volume,
the same family of affected siblings with and without
while concomitantly dissipating the electrical gradient and
hypertension supports the conclusion that both syndromes
diminishing H and K secretion (236).
are indeed the same genetic entity.

Treatment and Prognosis

Genetics
Diuretic therapy results in complete reversal of both clinical
Type 2 pseudohypoaldosteronism is inherited as an autosomal
and biochemical abnormalities, with return of blood pres-
dominant entity. Linkage analysis has demonstrated genetic
sure to normal levels (when it is elevated), rise in plasma
heterogeneity of the trait with involvement of at least three dif-
renin activity and aldosterone concentration, and correc-
ferent loci in chromosomes 1q31-42, 12p13.3, and 17p11-
tion of hyperkalemia and metabolic acidosis. Furosemide
q21 (233–235) (Table 38.1). Recently, mutations in two dif-
administration is very effective, but it aggravates the hyper-
ferent genes belonging to the WNK family of serine-theonine
calciuria and thus increases the risk of urolithiasis. In our
kinases have been identified as a cause of type 2 pseudohypoal-
opinion, the best treatment is the administration of hydro-
dosteronism (236). The WNK1 gene, formed by 28 exons and
chlorothiazide (1.5 to 2.0 mg/kg/day) that is as effective as
located in chromosome 12p, encodes the WNK1 kinase. Dis-
furosemide in the reversal of biochemical abnormalities and
ease-causing mutations in WNK1 are large intronic deletions
also corrects the hypercalciuria. Long-term prognosis
that increase WNK1 expression. WNK4 gene, formed by 19
remains uncertain because follow-up data are still limited.
exons and located in chromosome 17p, encodes the WNK4
kinase. The mutations in WNK4 are gain-of-function mis-
sense and cluster in a short, highly conserved segment of the HYPOMAGNESEMIC STATES
encoded protein.
Chronic hypomagnesemia may be caused by inadequate
dietary intake, defective gastrointestinal absorption, or
Clinical and Biochemical Features
increased urinary excretion and may be associated with hypo-
Short stature and arterial hypertension are the cardinal signs in calcemia and hypokalemia. Mg depletion due to excessive
children and adults, respectively. Muscle weakness has also been urinary losses may occur as an acquired entity, in postob-
reported. The relevant laboratory findings are hyperkalemia, structive diuresis; the diuretic phase of renal transplantation
hyperchloremic metabolic acidosis, hyporeninemic hypoaldos- or acute tubular necrosis; states of hyperparathyroidism or
teronism, and normal GFR. Exceptionally, the metabolic man- hyperaldosteronism; or administration of diuretics (e.g., loop
ifestations may be already apparent in early infancy. Although diuretics, thiazides), antiinfectious agents (e.g., aminoglyco-
not mentioned by most authors, hypercalciuria may be present sides, capreomycin, amphotericin B, pentamidine), anti-
and give rise to the formation of calcium oxalate stones. neoplastic agents (e.g., cisplatinum), or immunosuppressive
 38. Tubular Disorders of Electrolyte Regulation 747

drugs (e.g., cyclosporine) (238). Cisplatinum may not only modes of inheritance have been described. Clinical features
cause an acute renal Mg loss but also a chronic disorder that are variable, from asymptomatic cases to occurrence of car-
persists after withdrawal of the drug and may be associated popedal spasm or generalized convulsions. The nosologic
with hypokalemia, metabolic alkalosis, and hypocalciuria classification of all these patients is difficult at present, but
(239,240). the absence of hypokalemia or nephrocalcinosis clearly sepa-
Hypomagnesemia may also have a hereditary renal origin. rates them from the other entities discussed.
This syndrome, known as familial or congenital magnesium-
losing kidney, was first described by Freeman and Pearson in
Autosomal Recessive Form
1966 (241) and includes several different conditions, includ-
ing familial hypokalemia or hypomagnesemia or Gitelman’s In the autosomal recessive form, the patients also have vari-
syndrome, as discussed above (242,243). To differentiate able symptoms, but the urinary Ca excretion is normal
between primary renal loss from intestinal loss of Mg, a 24- (251,252). It probably represents a distinct disease because
hour urinary Mg excretion or the fractional excretion of Mg no linkage to any known previously reported loci has been
in a random urine specimen should be obtained. Daily excre- found (243).
tion of more than 30 mg of Mg per 1.73 m2 or a fractional
excretion of Mg above 2% in an individual with normal
Autosomal Dominant Form
renal function points to renal Mg wasting (243).
The autosomal dominant form is also associated with few
symptoms other than chondrocalcinosis. Patients always have
Hypomagnesemia with
hypocalciuria and variable but usually mild hypomagnesemic
Secondary Hypocalcemia
symptoms (253,254). The disease locus, HOMG2, has been
Hypomagnesemia with secondary hypocalcemia is an autoso- mapped to chromosome 11q23 in two Dutch families (255),
mal recessive disorder that manifests in the newborn period by but not in an American family (256), pointing out the exis-
very low plasma Mg and Ca concentrations (244). Patients usu- tence of genetic heterogeneity. In the Dutch families, the dis-
ally present with restlessness, tremor, tetany, and overt seizures. ease was due to dominant-negative mutations in the gene
In older children, clouded sensorium, disturbed speech, and FXYD2, encoding the Na+,K+-ATPase γ subunit (257) (Table
choreoathetoid movements have been described. The hypocal- 38.1). This mutation leads to a routing defect of the protein
cemia is secondary to parathyroid failure and peripheral par- to the plasma membrane, thus decreasing the activity of
athyroid hormone resistance as a result of Mg deficiency. Na+,K+-ATPase. Depolarization, reduced intracellular K, or
Hypokalemia is occasionally present and is corrected after nor- increased intracellular Na could then determine a reduced Mg
malization of plasma Mg. The disease appears to be fatal unless influx and secondary Mg wasting (257).
treated with very high oral intakes of Mg or, preferably, with
continuous nocturnal nasogastric infusion of Mg (245).
Familial Hypomagnesemia with
Mg deficiency seems to depend on a double intestinal and
Hypercalciuria and Nephrocalcinosis
renal transport defect (246). Genetic linkage to chromosome
9q22 was found in inbred Beduin families (247), and, The syndrome of familial hypomagnesemia with hypercal-
recently, two groups of investigators have found indepen- ciuria and nephrocalcinosis should also be distinguished
dently that the disease is due to mutations in a new gene, from other hereditary renal conditions leading to chronic
TRPM6, belonging to the TRPM family (248,249) (Table hypomagnesemia. The first description was made by Mich-
38.1). The TRPM6 gene, formed by 39 exons, encodes a pro- elis et al. in 1972 (258), but it was further characterized by
tein with 2022 amino acids, which acts as a putative ion Castrillo et al. in 1983 (259) (Michelis-Castrillo syndrome)
channel that is highly similar to the transient receptor poten- and by Rodríguez-Soriano et al. in 1987 (96). Although
tial channel family. These channels are characterized by six only approximately 50 cases have been reported (260), the
transmembrane domains, a conserved pore-forming region, disease may be more common than usually appreciated.
and a Pro-Pro-Pro motif after the last transmembrane seg-
ment (250). TRPM6 is highly expressed in intestinal epithelia
Genetics
and kidney tubules and shows great similarity with TRPM7, a
bifunctional protein that combines Ca and Mg-permeable Hypomagnesemia with hypercalciuria and nephrocalcinosis
action channel properties with protein kinase activity. behaves as an autosomal recessive disorder (Table 38.1). In
favor of this type of inheritance is the fact that two sisters
were transplanted with their parents’ kidneys without reap-
Isolated Familial Hypomagnesemia
pearance of the disease (261). However, a study of 26 rela-
Isolated hypomagnesemia, caused by congenital impairment tives belonging to four families is more suggestive of
of renal tubular Mg reabsorption, is a rare inherited disorder autosomal dominant inheritance with variable clinical
for which both autosomal dominant and autosomal recessive expression in affected members (262). In the relatives, nei-
748 VI. Tubular Disease

ther hypomagnesemia nor nephrocalcinosis or ocular during hypotonic saline diuresis, is normal or very mildly
abnormalities were present, but 11 of the 26 relatives impaired.
(42%) exhibited isolated hypercalciuria. Praga et al. specu-
late that isolated hypercalciuria may represent a milder clin-
Pathophysiology
ical expression of the disease, whereas the syndrome of
hypomagnesemia with hypercalciuria and nephrocalcinosis The primary defect is related to impaired tubular reabsorp-
could be the complete expression of this familial disorder. tion of Ca and Mg in the thick ascending limb of Henle
Recent studies have demonstrated that this disease is (Fig. 38.4). The study of tubular Mg reabsorption during
caused by mutations in a novel gene, PCLN-1, located in continuous Mg sulfate infusion revealed a marked impair-
chromosome 3q and encoding a novel renal tight junction ment of Mg reabsorption at all levels of filtered Mg, indi-
protein involved in Mg and Ca transport, which was cating that both renal threshold and renal tubular maximal
named paracellin-1, that belongs to the claudin family reabsorption were decreased in this condition (96). These
(claudin-16) (263–266). data strongly suggested the existence of a defect in Mg reab-
sorption at the level of the ascending limb of the loop of
Henle, where most of filtered Mg is reabsorbed via the
Clinical and Biochemical Features
paracellular pathways and driven by the lumen-positive
The cardinal clinical finding, besides episodic tetany or transepithelial electrical gradient (268). Recent molecular
convulsions, is the development of nephrocalcinosis. This biology findings have shown that paracellin-1 plays a criti-
is constantly associated with polyuria and, more occasion- cal role in the control of Ca and Mg reabsorption in this
ally, with urinary tract infection or arterial hypertension. nephron segment.
Nephrolithiasis has also been documented in several
cases. Growth retardation is rarely present and, when
observed, is more dependent on the nephrocalcinosis-
related renal insufficiency than on the hypomagnesemia
itself. An outstanding feature is the presence of ocular
abnormalities such as myopia magna, horizontal nystag-
mus, chorioretinitis, and macular coloboma. Hearing
impairment has also been occasionally reported. In some
cases with profound hypomagnesemia, hypocalcemia, or
a picture of vitamin D–resistant rickets may be observed,
thus contributing to diagnostic confusion with distal
renal tubular acidosis.
In contrast to Gitelman’s syndrome, hypomagnesemia is
not accompanied by hypokalemia, and there is a normal
acid-base equilibrium or a tendency to metabolic acidosis
instead of metabolic alkalosis. Hyperuricemia is manifest
and may occasionally lead to gouty arthritis. The presence
of variable degrees of renal insufficiency is related to the
progression of the tubulointerstitial nephropathy induced
by the nephrocalcinosis.
Hypermagnesuria occurs to a greater degree than with
Gitelman’s syndrome, despite a similar degree of hypo-
magnesemia. Documentation of marked hypercalciuria is
the rule. Asymptomatic family members may exhibit iso-
lated hypercalciuria, without hypomagnesemia. Defects
in concentrating and acidifying mechanisms are probably
secondary to nephrocalcinosis and contribute to the con-
FIGURE 38.4. Schematic models of Ca and Mg reabsorption in
fusion with primary distal renal tubular acidosis (267). thick ascending limb of Henle (TALH) and convoluted distal
However, hypermagnesuria (but not hypomagnesemia) is tubule. In TALH, Ca and Mg are exclusively reabsorbed via para-
only transiently observed in acidotic patients with distal cellular pathway through the action of paracellin-1 (claudin-16).
In the distal convoluted tubule, Ca enters the cell from the
renal tubular acidosis and rapidly corrects with alkali lumen via an epithelial Ca channel (ECaC) and leaves the cell
therapy. Thus, the association of hypomagnesemia and through a Ca ATPase and in exchange with Na. Mg absorption in
defective distal urinary acidification should strongly sug- the luminal membrane is mediated by an Mg channel (TRPM6?)
and probably leaves the cell in exchange with Na. The functional
gest the diagnosis of the syndrome of hypomagnesemia- integrity of Na+,K+-ATPase subunit appears to be essential for
hypercalciuria. Distal fractional Cl reabsorption, studied Mg reabsorption in the distal tubule.
 38. Tubular Disorders of Electrolyte Regulation 749

Paracellin-1 belongs to the claudin family, and it has 8. Vargas-Poussou R, Feldmann D, Vollmer, et al. Novel
been renamed claudin-16. It shows sequence and structural molecular variants of the Na-K-2Cl cotransporter gene are
similarity to other members of the claudin family. More responsible for antenatal Bartter syndrome. Am J Hum
than a dozen members of this family have been identified; Genet 1998;62:1332–1340.
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 39

RENAL TUBULAR ACIDOSIS

JOHN T. HERRIN

Renal tubular acidosis (RTA) is a clinical syndrome in DRTA was the first recognized syndrome. Type 2, or
which either an inherited or acquired renal tubular defect proximal RTA (PRTA), was subsequently identified, then
leads to failure to maintain a normal plasma bicarbonate appreciated as the result of defective bicarbonate reab-
concentration in the presence of a normal rate of acid pro- sorption. The hybrid nature of the infantile variant of
duction from the diet and metabolism. Systemic metabolic DRTA, in which a distal tubular defect is coupled with an
acidosis (MA) results from a decrease in net acid excretion increased fractional excretion of bicarbonate, was initially
(NAE) occurring as a defect in handling bicarbonate reab- believed to represent a distinct disease entity and was des-
sorption or defect in hydrogen ion secretion. ignated type 3 RTA. This term was subsequently applied
RTA, clinically, is characterized by a normal anion gap, to the mixed defects of proximal and distal tubular acidifi-
MA with hyperchloremia, bicarbonaturia, reduced urinary cation, which result from a deficit in carbonic anhydrase
excretion of titratable acid (TA) and ammonia, and an ele- (CA) 2 in osteopetrosis. The term type 3 RTA was later
vated urinary pH. Disorders of bicarbonate reclamation by dropped from classification schemes, but only after aldos-
the proximal tubule are classified as proximal or type 2 RTA, terone-dependent RTA type 4 was described. The present
whereas disorders resulting from a primary defect in distal terminology in which type 1 = DRTA, type 2 = PRTA,
tubular net proton secretion or from reduced buffer trap- and type 4 = aldosterone-dependent RTA is thus histori-
ping in the tubular lumen (decreased generation of new cally, rather than pathophysiologically, based (Table 39.1)
bicarbonate for reabsorption) are called distal or type 1 RTA. (3,20,22).
Distal RTA (DRTA) results in low ammonium (NH4+) or Although DRTA is characterized by a low NH4+ excre-
in low TA excretion (1–8). Hyperkalemic RTA may occur tion, some clinical syndromes with proximal tubular defects
as a result of aldosterone deficiency or tubular insensitivity also demonstrate low NH4+ excretion. Bicarbonaturia most
to its effects (RTA type 4) (9–17). Low potassium excretion commonly results from proximal tubular loss but can also
in the absence of aldosterone abnormalities may also lead to result from distal H+ secretion. Because of such overlap syn-
a hyperkalemic DRTA in obstructive uropathy and sickle dromes, classification of RTA based on pathophysiology of
cell anemia (18,19). In children with type 1 RTA, nonspe- the disorders is preferred (20,23,24).
cific symptoms occur early. If the acidosis is not corrected, The classification of RTA into primary defects in bicarbon-
electrolyte abnormalities, growth retardation, rickets, and ate reabsorption, defects in net hydrogen ion excretion, or
nephrocalcinosis may follow. Growth retardation is a con- defects in aldosterone-sensitive acid secretion is shown in Table
sistent feature of RTA in infants. Experimentally, MA in 39.1. Combined defects in proximal HCO3– reabsorption and
rats leads to inhibition of growth hormone secretion, H+ ion secretion have been described and separate patients or
decreased caloric intake, and decreased levels of hepatic families have variations of standard patterns; a subgroup of
insulin-like growth factor I expression (20,21). Similar adolescents and adult patients with apparent DRTA can lower
abnormalities have been observed during systemic acidosis urinary pH in response to acidosis but cannot increase urinary
in childhood. Identification and correction of acidosis are partial pressure of carbon dioxide (PCO2) in response to alka-
important in preventing symptoms and guide approved linization of the urine by bicarbonate titration (25). Two pat-
genetic counseling and testing. terns of combined or hybrid RTA have been described:
(a) infants and young children with DRTA show high FEHCO3
of 5 to 15% that clears with treatment and growth—bicarbon-
CLASSIFICATION AND TERMINOLOGY ate loss is rare in older children but may occur with prolonged
periods of untreated RTA with concurrent hyperparathy-
The somewhat confusing nomenclature in RTA reflects roidism (4,26); and (b) adult patients with Sjögren’s syn-
the history of disease identification. Type 1, classic, or drome and renal amyloidosis (2,27,28) show significant
758 VI. Tubular Disease

TABLE 39.1. CLASSIFICATION AND NOMENCLATURE RENAL CONTROL OF ACID-BASE BALANCE

IN RENAL TUBULAR ACIDOSIS (RTA): CLINICAL AND
PATHOPHYSIOLOGIC CRITERIA
The following brief review of the kidney’s role in acid-base
Primary defect in bicarbonate reabsorption (proximal RTA, type 2) metabolism and renal acidification provides an outline to
Mild to moderate metabolic acidosis or acid loading lowers permit appropriate investigation, classification, and under-
the urine pH below 5.5. Low bicarbonate threshold FE HCO3 standing of the pathophysiologic basis of symptomatology
above 10% by titration.
and treatment in RTA.
Primary defect in distal net H+ secretion
Inability to lower urine pH below 5.5 if FE HCO3 exceeds 5%; The activity of almost all body enzyme systems is depen-
suspect an associated bicarbonate leak and test the ability dent on hydrogen ion concentration; hence, the balance of
to increase urinary PCO2 during bicarbonate loading hydrogen ion is tightly regulated. Normal body defenses
Inability to maximally acidify urine (DRTA, type 1) against inappropriate changes in hydrogen ion concentra-
Diminished Na+ to distal tubule; functional RTA
tion include (a) body buffering of generated or administered
(exchange defect)
Inability to secrete H+ (secretory defect) H+, (b) respiratory excretion of volatile acid as carbon diox-
Increased back diffusion H+ (gradient defect) ide, and (c) renal reabsorption of filtered bicarbonate and
Abolition of the electrical gradient for H + ion secretion generation of new bicarbonate by excretion of hydrogen ion
(short circuit or voltage defect) with concurrent from nonvolatile acid, produced as a result of protein
impairment to K+ secretion, furosemide decreases UpH
metabolism. Excretion of protons provides for reabsorption
to normal
Hyperkalemic DRTA: hyperkalemia with U pH above 5.5 of filtered bicarbonate and regeneration of bicarbonate
Combined defects in proximal HCO3 reabsorption and H+ when secreted hydrogen ion combines with a buffer other
ion secretion (hybrid RTA type 1 = type 3, incomplete than bicarbonate (33).
RTA) FEHCO3 above 5% Initial defense against change in pH occurs within seconds
RTA secondary to deficits in aldosterone activity or chloride
through the combination of H+ with chemical buffers. These
reabsorption: type 4 RTA
Hyperkalemia, UpH below 5.5, further differentiation by buffers do not remove the hydrogen ion from the body, but
assessment volume status, renin-aldosterone rather bind it until secondary excretion can occur as a result of
Subtypes respiration (within minutes) or renal excretion (within hours
Aldosterone deficiency or days). Bicarbonate is the major extracellular fluid buffer,
High renin hypoaldosteronism = primary adrenal
whereas phosphate acts as the major buffer in intracellular
abnormality with normal renal function
Low renin hypoaldosteronism = primary renal disease fluid and the urine, to increase H+ excretion as TA.
with secondary mineralocorticoid deficit In normal individuals, net acid production is balanced by
Attenuated renal response to aldosterone with relative NAE. In an average American diet (34,35), net endogenous
aldosterone deficiency noncarbonic acid production averages approximately 1
Chloride shunting (pseudohypoaldosteronism type 2);
mEq/kg/day in adults and 1 to 3 mEq/kg/day (1 to 3 mEq
abnormal Na+-K+-2Cl– exchange
Deficient renin per 100 calories) in healthy infants and children (36). Chil-
Attenuated renal response: pseudohypoaldosteronism dren generate as much as 2 to 3 mEq H+/Kg, particularly
Pseudohypoaldosteronism (infancy) during active growth, as a result of combination of OH– and
Primary transient early RTA4 phosphate to form hydroxyapatite during bone formation.
NAE results from excretion of ammonia and TA minus
DRTA, distal renal tubular acidosis; UpH, urine PH.
Note: Incomplete DRTA syndrome: low NH+4, high UpH, patient is not
excreted bicarbonate (NAE = UNH4V + UTAV). UNH4V may
acidotic unless stressed. be measured chemically or estimated clinically as UAG, or
urinary net charge. If UpH is low (6.1 to 6.5), bicarbonate
excretion may be omitted from calculations. UTAV is pro-
reduction in tubular reabsorption of bicarbonate at normal portional to urinary phosphorus (31,33,37).
plasma levels (i.e., a low threshold similar to type 2 RTA), Each day, the kidney prevents loss of a large amount of
combined with an inability to acidify maximally even with filtered bicarbonate (i.e., 180 L/day × 25 mEq HCO3–/L =
severe acidosis (25). A similar defect occurs in patients with 4500 mEq/day in an adult). This is accomplished by secre-
Leigh’s syndrome and osteopetrosis (29,30). tion of H+ into the tubular lumen by secondary active trans-
Incomplete RTA is a condition in which normal plasma port in tubular segments where filtered bicarbonate ions are
bicarbonate is maintained despite decreased ability to acid- reabsorbed by interaction with H+ in the tubule to form
ify the urine (5). It is possible that a high rate of NH4+ H2CO3 in the presence of CA. Renal acid excretion is regu-
excretion compensates for a decrease in TA excretion. Ini- lated by hormones that concurrently control sodium reab-
tially, the condition was described in patients with nephro- sorption and thus volume control (e.g., angiotensin II in the
calcinosis or recurrent nephrolithiasis (5), or in families in proximal tubule and aldosterone, antidiuretic hormone, and
which a propositus had complete RTA (31). It has been endothelin 1 in the distal tubule) (39). In addition, renal
suggested that this latter condition represents a develop- acid excretion is affected by factors that regulate bicarbonate
mental stage of RTA (32). reabsorption PCO2 and by potassium balance, because
 39. Renal Tubular Acidosis 759

hypokalemia increases H+ distally by decreasing potassium TABLE 39.2. SUMMARY OF ACIDIFICATION

exchange for NH4+ at the Na+-K+-2Cl– antiporter. K+ and MECHANISMS (39,40)
NH4+ compete for a common site in the triporter of the Glomerular filtration
thick ascending loop of Henle and for secretion in conjunc- Delivery of bicarbonate and anions from food metabolism
tion with basolateral Na+ pumps (39,40). Proximal tubule
Primary H+ secretion occurs actively in the intercalated Apical H+ secretion (leads to reabsorption bicarbonate)
H+-ATPase
cells of the late distal tubule and collecting ducts, whereas
Coupled Na+-H+ exchange (NHE3 antiporter)
excess H+ combines in the tubule with phosphate and Back leakage from Cl– (base exchange)
ammonia buffers (plus urate, creatinine, and citrate), gener- Na+ cotransporters (amino acid, glucose, phosphate, citrate)
ating new bicarbonate ions (37,38). In chronic acidosis, Luminal CA4 enhances buffering and CO 2 reabsorption
ammonia production and excretion increase (33,41–43). Intracellular
Production of NH3 and HCO3 (from metabolism of
This pH balance is linked to sodium and water balance,
glutamate and α ketoglutarate)
with aldosterone stimulation of intercalated cells leading to CA2 intracellular production H+ and HCO3–
excretion of hydrogen ion (44,45). Changes in plasma Basolateral
PCO2 are linked to renal H+ excretion, with increased PCO2 Electrogenic Na+–3 HCO3– cotransport (Na-HCO3–-CO3–)
leading to increased hydrogen ion secretion. Acid balance NBC1
Na+,K+-ATPase (produce sodium gradient allowing Na +
requires that dietary H+ (which results from the metabo-
reabsorption)
lism of methionine, cysteine, and the chloride salts of Na+-H+ antiporter
lysine, arginine, and histidine) be removed by combination Thick ascending loop of Henle
with bicarbonate to form an acid salt, water, and CO2 (46). Water reabsorption leads to bicarbonate reabsorption
As CO2 is exhaled (respiration), a deficit of bicarbonate is H+ secretion (NHE3 activity)
NH4+ substitution for K+ in the Na+-K+-2Cl– cotransporter
sustained. When reduced excretion of a metabolizable
Distal nephron
anion (e.g., ketones, hydroxy butyric acid) occurs in the Apical
presence of acid loading, there is an increased output of H+ secretion
NH4+ with Cl– (47,48). Retained anions are metabolized to H+-ATPase intercalated cells (ATP6B1)
neutral end products (48). H+,K+-ATPase
HCO3– secretion intercalated cells
NAE results in the generation of new bicarbonate,
H+ back leak (amphotericin B)
which exits the cell by electroneutral chloride exchange. Mineralocorticoids
The bicarbonate generated is equal to that lost in buffering Stimulate H+ secretion by the distal nephron in a sodium-
the endogenous load of noncarbonic (nonvolatile) acid dependent fashion
generated from dietary intake and metabolism. If the diet is Stimulate H+ secretion in the cortical collecting duct and outer
medullary collecting duct in a sodium-independent fashion
high in acid, increased acid excretion occurs, the urine is
Basolateral
free of bicarbonate, and urinary pH approaches 4.5. In the Cl–-HCO3– (AE1)
presence of an alkaline load, the tubule rejects excess fil- CA luminal (buffering)
tered bicarbonate, and urinary pH may approach 8 or 9. CA2 intracellular production H+ and HCO3–
Phosphate buffer activity, and hence TA, is limited by
dietary intake and reabsorption (22,38). Ammonium, on ATPase, adenosine triphosphatase; CA2, carbonic anhydrase 2.
the other hand, is generated in the presence of acidosis.
RTA is predominantly the result of either excess bicarbon-
ate excretion or low NH4+ excretion (22). proximal and distal tubules in osteoporosis, as well as ear
and kidney abnormalities in ATP6B1-β1 subunit muta-
tions in DRTA with deafness (49,52). Mutations in the
RENAL ACIDIFICATION MECHANISM gene encoding NBC 1 (Na-HCO3 transporter) lead to
autosomal recessive PRTA (53).
The schema for normal acidification and sites of potential
inherited or acquired defects are presented in Figure 39.1
Renal NH4+ Production and Transport
and Table 39.2. Genetic control of H+ excretion is medi-
ated through control of the expression of membrane trans- Ammonium is the source of H+ for reabsorption of bicar-
porters in α-intercalated cells of the distal nephron, where bonate in the proximal tubule. Ammonia generation and
luminal H+ secretion occurs by an adenosine triphosphatase excretion is the expected response to acidosis and the key to
(ATPase)-dependent proton pump in the apical membrane understanding RTA. At physiologic pH, NH3, and NH4+
that is linked to basolateral anion exchange (bicarbonate- production from glutamine is regulated through glutami-
chloride) AEI 3 (49). AEI mutations have been in DRTA nase and phosphoenol-pyruvate carboxykinase with the
(49,50). Enzyme defects in the CA2 system explain com- production of α-ketoglutarate as a byproduct, which leads
mon pathophysiology of bone and ear in addition to both to the generation of two bicarbonate molecules (37,54,55).
760 VI. Tubular Disease

FIGURE 39.1. The schema outlines the major mechanism of acidification and the nephron site
of activity for inherited and acquired defects in acidification. α KG, α-ketoglutarate; AE 1, anion
exchanger Cl– and HCO3–; Apical, apical surface (lumen) of cell; ATPase, adenosine triphos-
phatase; BSL, basolateral surface of the cell; CA 2, carbonic anhydrase 2; CA 4, carbonic anhy-
drase 4; Glom, glomerulus; Glut, glutamine; Na-Cotransport, Na cotransport at the apical surface
of the cell with H+ (includes Na– amino acid, Na– phosphate, Na– glucose, and Na– citrate); NBC 1,
Na, bicarbonate cotransporter; NHE 3, sodium hydrogen ion exchanger; PROX TUB, proximal
tubule; TALoop, thick ascending loop of Henle.
 39. Renal Tubular Acidosis 761

In chronic MA, an increase in formation of ammonia from centration that augments potassium secretion. Na+ absorp-
glutamine occurs with little change in phosphate excretion tion through the apical Na+-selective channel (affected by
(phosphate released from bone is used in blood buffering aldosterone) maintains the negative potential difference in
during acidosis). NH4+ is secreted into the first portion the tubular lumen. Aldosterone increases apical Na-selec-
of proximal tubule, substituting for H+ in the Na+-H+- tive channel activity, and with time, aldosterone can also
antiporter on the apical membrane (43,56,57). Increase in increase the basolateral Na+,K+-ATP to produce changes,
direct proximal convoluted tubule (PCT) ammonium which parallel systemic K balance. Measurement of the
secretion increases delivery distally. In the S3 segment of transtubular K+ gradient can thus be a clinical tool to assess
the proximal straight tubule, ammonium is increased by an K+ excretion and direct therapy.
acid disequilibrium pH. As fluid leaves the PCT to the loop
of Henle, ammonium and ammonia efflux leads to an
Factors Influencing Reabsorption
increased medullary gradient for ammonia. As fluid is
of Bicarbonate
removed from the thin loop, leading to alkalinization,
efflux of ammonia can occur as a result of nonionic diffu- Nearly all of the bicarbonate filtered at the glomerulus is
sion (58,59). reabsorbed. Urinary excretion of acid results in generation
Direct ammonium transport across the medullary thick of new bicarbonate equal in amount to that lost in buffer-
ascending limb apical membrane occurs by substitution of ing the endogenous load of noncarbonic acid generated
NH4+ for K+ in the Na+-K+-2Cl– cotransporter (46). Coun- from diet and metabolism.
tercurrent reentry of NH4+ in the proximal straight tubule In the proximal tubule, secretion of H+ results in absorp-
thus correlates with flow. Ammonium is secreted from the tion of 80 to 90% of filtered bicarbonate, by combining
interstitium into the medullary collecting ducts by a com- with H+ to form carbonic acid, which, in the presence of
bination of diffusion of ammonia and H+ secretion (active CA (isoenzyme 4 in the brush border), dissociates to form
transport by H+ ATPase and H+,K+-ATPase) (39,59,60). CO2 and H2O (62–64). Luminal CO2 diffuses into the
cells. In the presence of cytoplasmic CA (isoenzyme 2),
hydroxylation occurs to form bicarbonate (63,64). Bicar-
Relationship of K+ and NH4+
bonate exits the basolateral membrane of the cell by a
Hyperkalemia is a determinant of changes in acid-base bal- sodium-coupled mechanism (39). Hydrogen ion secretion
ance by both direct and indirect effects on distal acidifica- in the proximal tubule is mediated by electroneutral Na+
tion. Serum potassium is a determinant of aldosterone H+ exchange at the luminal membrane. This exchange is
secretion and thus of distal proton secretion (45). Chronic driven by the lumen to cytoplasm sodium concentration
potassium deficiency increases NH4+ secretion, whereas gradient maintained by the basolateral Na+,K+-ATPase
hyperkalemia suppresses ammonium production (59). activity. In the presence of acetazolamide, carbonic acid
Hyperkalemia has no effect on ammonium transport in the (H2CO3) accumulates in the lumen, with an increase in H+
PCT but impairs absorption from the thick ascending limb concentration, leading to a decrease in H+ secretion and
where NH4+ may substitute for K+ in the Na+-K+-2Cl– bicarbonate reabsorption. Because factors that regulate
exchanger (59,60). sodium reabsorption alter bicarbonate reabsorption as a
secondary effect, luminal bicarbonate concentration and
pH, luminal flow rate, peritubular PCO2, and angiotensin
Interactions of Sodium and
II are important determinants of bicarbonate reabsorption.
Potassium Handling
H+ secretion proximally varies inversely with peritubular
Sodium and potassium handling are homeostatically regu- bicarbonate, pH, and, to a lesser extent, potassium balance.
lated processes. Potassium secretion across the apical mem-
brane of principal cells in the cortical collecting duct is
Factors Influencing Hydrogen Ion Secretion
tightly regulated (61). Quantitative potassium secretion
depends on the apical membrane potassium conductance In the distal nephron, H+ secretion is responsible for reabsorp-
and the electrochemical driving force (EDF) across the api- tion of the remaining 10 to 15% of filtered bicarbonate (not
cal membrane. Potassium conductance is the sum of the absorbed proximally), decreasing the pH to less than 6.1. As
collective K channels in the apical membrane, so K secre- the luminal pH decreases further, secreted H+ is titrated to the
tion is regulated by urine flow and Na delivery and by fac- urinary buffers (phosphate and ammonium), completing a
tors that regulate EDF (e.g., K balance, transepithelial process initiated in the proximal tubule (33). In the adjacent
potential difference, pH and bicarbonate concentration of cortical and medullary collecting tubules (MCTs), secretion
tubular fluid, systemic acid balance, and aldosterone) (61). of H+ is mediated by a proton translocating ATPase (H+
The EDF for K+ exchange is maintained by the basolateral ATPase) located on the luminal surface of the α-intercalated
Na+,K+-ATPase. The sodium pump generates a negative cells (46). In the cortical collecting tubules (CCTs), principal
luminal potential and a high intracellular potassium con- cells mediate electrogenic transfer of sodium, thus creating a
762 VI. Tubular Disease

transepithelial electrical potential difference that is lumen Epidemiology

negative and favors H+ secretion. The principal cells are
PRTA occurs in a variety of conditions, most often associ-
responsible for the secretion of potassium. Sodium transfer
ated with multiple tubular dysfunction (e.g., Fanconi syn-
does not occur in the MCT. Secretion of H+ by the distal
drome) as part of cystinosis, galactosemia, tyrosinemia,
tubule is influenced by aldosterone, sodium delivery, buffer
Wilson’s disease, Lowe’s syndrome, and mitochondrial
delivery, changes in pH and PCO2, luminal pH, and potas-
myopathies. Isolated transient or persistent proximal tubu-
sium (65–67). Aldosterone directly stimulates H+ ATPase in
lar defects in bicarbonate reabsorption have been described,
the intercalated cells and sodium reabsorption by principal
predominantly in males (71,74).
cells. The resulting luminal electronegativity further stimu-
Adult-onset persistent type 2 RTA and isolated PRTA
lates H+ secretion. [Increased sodium delivery to the distal
defects have been associated with cyanotic congenital heart
tubule when the avidity for sodium is high (e.g., volume
disease (75) and renal vascular accidents (76). A familial
depletion; administration of mineralocorticoid hormone)
autosomal dominant form that presents in early childhood
leads to sodium reabsorption, or when sodium is excreted
(74) and a congenital form associated with growth retarda-
with poorly absorbed anions such as sulfate, phosphate, ferro-
tion, mental retardation, and ocular and dental abnormali-
cyanide, or penicillin, an increased luminal negativity is gen-
ties in two brothers has been described (77).
erated.] If sodium is accompanied by chloride, the stimulus
for H+ secretion is lower because the chloride is passively reab-
sorbed by the paracellular pathway, dissipating the electrical Genetics
gradient. An increase in distal delivery of buffer (e.g., phos-
phate loading or β-hydroxy butyrate during ketoacidosis) Genetically transmitted forms include persistent PRTA
leads to stimulation of H+ secretion and an increased forma- (both autosomal dominant and autosomal recessive forms),
tion of TA. H+ secretion is stimulated by acidosis and inhib- an isolated familial transmitted PRTA (74), or as a PRTA
ited by alkalosis. H+ secretion is inhibited when the luminal syndrome in association with mental retardation and ocular
pH reaches 4.5. Potassium depletion directly stimulates inter- and dental abnormalities (77). PRTA may also be associ-
calated cells to secrete H+ and stimulates ammonia produc- ated with other genetically transmitted disorders (Table
tion, distal delivery, and H+ ion excretion. 39.3) such as osteopetrosis with CA2 deficiency (30), pyru-
vate carboxylase deficiency, familial forms associated with
other hereditary dysfunction of proximal tubules (heredi-
CLINICAL SPECTRUM tary fructose intolerance) (2), mitochondrial myopathies,
and glycogen storage disease.
Patients with RTA may have nonspecific symptoms such as
anorexia, vomiting, constipation, polyuria, and polydipsia.
Pathophysiology and Complications
Untreated children are characteristically growth retarded.
Hyperchloremic MA (HCMA) with increased urinary pH, Mechanisms suggested for the defective bicarbonate absorp-
a lack of ammonia secretion (+UAG), and even life-threaten- tion include abnormality of the Na+-H+ exchanger, an
ing acidosis, may occur in infants and young children. impaired basolateral Na+ HCO3– exchanger, decreased
Nephrocalcinosis (68), renal calculi (69), and musculoskel- Na+,K+-ATPase activity attenuating the sodium gradient from
etal complaints (myalgia, arthralgia) (70) are the most com- lumen to cell, and inhibition of CA activity. Decrease in
mon presentations of adult type 1 RTA. Rickets may occur bicarbonate reabsorption in the proximal tubule results in
in type 1 (71) classic DRTA, although this is more com- increased delivery of bicarbonate to the distal tubule, over-
monly seen in association with PRTA resulting from Fan- whelming the distal tubular reabsorption capacity (normally
coni syndrome (72,73). A subgroup of children present approximately 15% of the filtered load) (26). Bicarbonate loss
with RTA in the course of adrenal insufficiency (aldoster- results in a decrease in NAE and secondary MA. As MA
one deficiency or tubular resistance) with signs of volume increases and plasma bicarbonate levels fall, the filtered load of
depletion. In adults, hyporeninemia and hypoaldoster- bicarbonate falls below the distal tubular capacity and bicar-
onism more commonly occur in patients with decreased bonate losses cease so that the urinary pH falls below 5.5 (2).
renal function in the presence of diabetes mellitus and Therefore, PRTA is characterized by MA, with a urinary pH
interstitial disease (59). above 5.5 under mild to moderate MA, and the ability to
lower urinary pH below 5.5 under maximal stress.
In the isolated proximal lesion, other proximal tubular
Proximal Renal Tubular Acidosis Type 2 functions are intact (i.e., glycosuria, phosphaturia, and ami-
noaciduria, which characterize Fanconi syndrome, are
Symptoms
absent); sodium bicarbonate losses stimulate aldosterone
Characteristic symptoms include growth retardation, fail- secretion, and loss of potassium by the principal cells in the
ure to thrive (FTT), and recurrent vomiting. CCT results in hypokalemia. Renin and aldosterone levels
 39. Renal Tubular Acidosis 763

TABLE 39.3. PRIMARY DEFECT IN BICARBONATE generally are elevated as a result of volume contraction (78).
REABSORPTION [PROXIMAL RENAL TUBULAR Hypokalemia, although common, is less prominent in iso-
ACIDOSIS (PRTA) TYPE 2]
lated PRTA. Polyuria is common and appears early in the
Primary disorder with hypokalemia and solute diuresis (bicarbonate
Sporadic and sodium loss) (79). Acidosis leads to vomiting, lower
Transient childhood (71) caloric intake, and a decrease in growth rate that is reversed
Persisting (adult onset) (73)
when acidosis is corrected (21). Bone age may be retarded.
Genetically determined
Primary PRTA (74) Serum calcium, phosphorus, and vitamin D levels usu-
Sporadic transient (71) ally are normal despite persistent acidosis and rickets or
Genetic osteomalacia (71,74). Abnormalities do not occur in pri-
Autosomal dominant mary isolated RTA unless vitamin D or calcium intake is
Autosomal recessive
marginal (73), but disturbances are common in secondary
Isolated PRTA with mental retardation and ocular and
dental abnormalities (75) forms of RTA with multiple tubular defects (Fanconi syn-
Pyruvate carboxylase deficiency (118,119) drome) and in patients with osteopetrosis and a mixed RTA
Mitochondrial myopathies (82,83) pattern (80). Nephrocalcinosis and nephrolithiasis are rare
Osteopetrosis (30,72,80) in PRTA as increased citrate excretion occurs together with
Carbonic anhydrase deficiency (120)
increased calcium reabsorption in the distal tubule.
Sporadic
Genetic
Drug-induced (acetazolamide, sulfanilamide, mafenide
acetate) Treatment and Prognosis
Secondary
Hereditary multiple proximal tubular dysfunction, Fanconi
Alkali supplement should be provided in amounts suffi-
syndrome cient to offset the losses, which are variable and may be as
Cystinosis high as 2 to 20 mmol/kg/day (59). Sodium restriction or
Galactosemia hydrochlorothiazide, 1.5 to 2 mg/kg/day, may decrease
Glycogen storage disease type 1 alkali requirements by enhancing proximal reabsorption of
Hereditary fructose intolerance (with fructose exposure)
Tyrosinemia
sodium and bicarbonate.
Wilson’s disease Patients with Fanconi syndrome may require potassium,
Heavy metals phosphate, and vitamin D supplementation in addition to
Drugs and toxins specific therapy aimed at the underlying cause, such as cys-
Carbonic anhydrase inhibitors teamine in cystinosis, chelating agents (i.e., penicillamine)
6-Mercaptopurine
Streptozotocin
in Wilson’s disease, or dietary manipulation in galactosemia
Ifosfamide and tyrosinemia.
Outdated tetracycline Prognosis in PRTA depends on the etiology. In transient
Sulfonamides DRTA, supplementation to preserve growth is all that is
Mafenide acetate required. Adequate chronic alkali therapy protects against
Valproic acid
Heavy metals (Cd, Pb, Hg)
growth abnormalities in those with persistent lesions. Out-
Associated with other metabolic disorders come in secondary lesions is determined by the underlying
Primary or secondary hyperparathyroidism process and its response to treatment. Early treatment of
Vitamin D deficiency and dependency galactosemia has an excellent prognosis, whereas patients
Leigh’s syndrome (11,29,119) with Lowe’s syndrome (81) or mitochondrial myopathies
Metachromatic leukodystrophy
Osteopetrosis (30,72)
(82,83) have less favorable outcomes.
Pyruvate carboxylase deficiency (118,119)
Lowe’s syndrome (81)
Miscellaneous Renal Tubular Acidosis Type 4
Amyloidosis (28)
Cyanotic congenital heart disease Symptoms
Fallot tetralogy (75)
RTA type 4 is the most common nonazotemic form of RTA
Hereditary nephritis
Hyperkalemia and may result from a number of disorders (Table 39.4).
Multiple myeloma Patients usually present with hyperkalemia and mild MA.
Nephrotic syndrome Ability to acidify the urine to a urinary pH below 5.5 is
Renal cystic disease intact and signs of aldosterone deficiency or resistance may
Renal transplant
be present. Infants may have FTT, hyperkalemic MA with
Renal vascular accident (76)
Sjögren’s syndrome (92,93) associated salt wasting (in aldosterone deficit), or resistance
(pseudohypoaldosteronism) (13,16). In pseudohypoaldoster-
Note: References are indicated in parentheses. onism, renal sodium loss and potassium retention usually
764 VI. Tubular Disease

TABLE 39.4. DISTAL RENAL TUBULAR ACIDOSIS (RTA): remit by 4 or 5 years of age, although renin and aldosterone
ALDOSTERONE-SENSITIVE TUBULAR DEFECTS (RTA 4) levels remain elevated. In adults, mild renal insufficiency and
Primary hyperkalemic acidosis out of proportion to the renal func-
Early childhood transient (56) tional abnormalities is common (59). In the rare syndrome
Secondary of chloride shunt-associated disorder (9,10,15,17), infants
Aldosterone deficiency have FTT (16), whereas adolescents have hypertension and
Aldosterone deficiency without intrinsic renal disease (pri-
increased blood volume associated with hyporeninemia and
mary adrenal insufficiency)
Addison’s disease lowered aldosterone levels (15,17).
Bilateral adrenalectomy In critically ill patients, a new syndrome of isolated
Congenital adrenal hyperplasia syndrome (21 hydroxylase hypoaldosteronism has been noted. This is believed to fol-
deficiency) (11) low hypoxia, cytokine release, and atrial natriuretic peptide
Isolated hypoaldosteronism (121–124)
Isolated hypoaldosteronism of critical illness?
inhibition of aldosterone synthase. It is characterized by ele-
Inherited corticosterone methyloxidase deficiency (123) vated adrenocorticotropic hormone and cortisol levels with
Hyporeninemia (hypoaldosteronism syndrome) (10,59,85) decreased aldosterone and hyperkalemic MA, which is
Diabetes mellitus potentiated by K+-sparing diuretics, K+ load from parenteral
Gout
nutrition, and heparin (54).
Pyelonephritis
Interstitial nephritis
Nephrosclerosis (17)
Genetics
Attenuated renal response to aldosterone
Pseudohypoaldosteronism Inherited defects resulting in type 4 RTA include absence
Pseudohypoaldosteronism (infancy) (13)
Chronic tubulointerstitial nephritis with salt wasting
of aldosterone and glucocorticoid hormones (e.g., congeni-
Obstructive uropathy in infancy tal adrenal hypoplasia with salt wasting) (11) and isolated
Drugs hypoaldosteronism (Table 39.4). Possible inheritance is
Spironolactone suggested by familial cases of a form of early childhood
Heparin hyperkalemic RTA (27).
Amiloride
Prostaglandin inhibitors
Triamterene
Pathophysiology
Captopril
Cyclosporine RTA 4 results from deficiency of aldosterone or tubular
Attenuated renal response to aldosterone with relative aldos-
terone deficiency
unresponsiveness to its effects. Aldosterone stimulates H+
Selective tubular dysfunction with hypertension and secretion by intercalated cells and sodium absorption in the
impaired renin principal cells. This process increases luminal electronegativ-
Synonyms (chloride shunt syndrome; pseudohypoaldoster- ity, further enhancing sodium and potassium secretion.
onism type 2) (17) Increased potassium secretion by principal cells leads to
With associated renin deficiency
Chronic tubulointerstitial nephritis with chronic renal
enhanced ammonium production and distal delivery. In
insufficiency associated with deficient renin secretion experimental aldosterone deficiency there is a decrease in
Obstructive uropathy NAE secondary to decreased ammonium formation; studies
Renal transplant in human subjects after adrenalectomy confirm decreased
Lupus erythematosus
ammonium secretion, which is corrected with mineralocor-
Acute glomerulonephritis
Renal amyloidosis ticoid supplementation (84). This suggests that renal acidifi-
Unclassified type 4 cation is under tonic stimulation by physiologic levels of
Distal tubular disease/interstitial disease mineralocorticoids (84).
Renal amyloidosis As a result of aldosterone deficiency, hyperkalemia results
Lupus nephritis with antitubular basement membrane
antibodies
from decreased potassium excretion (59,85). Bicarbonate
Renal venous thrombosis excretion is reduced if plasma bicarbonate is returned to nor-
Drugs (methicillin) mal by supplementation. Reduced bicarbonate excretion
Contributing or aggravating factors in RTA 4 allows a urinary pH of 5.5 to be attained under acidotic
KCl supplements stimulation. Exclusion of other proximal tubular dysfunction
Heparin
Potassium-sparing diuretics is necessary to differentiate between PRTA and RTA 4. How-
Prostaglandin inhibitors ever, a decrease in urinary ammonium secretion leads to
Captopril decreases in NAE and MA, with marked hyperkalemia after
Cyclosporine any increase in dietary potassium intake.
Citrate excretion usually is high in patients with RTA 4,
Note: References are indicated in parentheses.
and nephrocalcinosis and nephrolithiasis do not occur
despite the acidosis. In patients with the early childhood
 39. Renal Tubular Acidosis 765

form of RTA 4, calcium reabsorption is increased, further or autosomal recessive trait (26)—or secondary to nephro-
protecting against nephrocalcinosis (86). calcinosis (88,89–91), autoimmune disease (84–86), or
Treatment of hyperkalemic RTA includes exclusion of exposure to drugs such as amphotericin (91) (Table 39.5).
excess dietary potassium intake or removal of potential offend- The inherited forms may be present as an isolated defect
ing drugs and correction of volume contraction. If restriction (68) or associated with genetically transmitted systemic dis-
of dietary potassium intake does not produce correction, a trial ease, such as CA2 deficiency (76,80), erythrocyte CA B
of furosemide to ameliorate the acidosis and hyperkalemia deficiency, Ehlers-Danlos syndrome, hereditary elliptocyto-
should be undertaken (59,87). In an emergency, mineralocor- sis, hereditary fructose intolerance with nephrocalcinosis,
ticoid (fluorohydrocortisone) or sodium bicarbonate may be Marfan’s syndrome, sickle cell anemia, osteopetrosis (30),
necessary to correct the acidosis, promote intracellular translo- sensorineural deafness, carnitine palmitoyl transferase type
cation of potassium, and increase potassium excretion. How- 1 deficiency, and idiopathic hypercalciuria (89).
ever, long-term use of sodium bicarbonate in mild renal
insufficiency leads to volume overload and hypertension (6). In
Pathophysiology
adrenal insufficiency, mineralocorticoid supplementation leads
to correction of underlying pathophysiology. With pseudo- Disordered H+ secretion in the CCT and MCT leads to
hypoaldosteronism, sodium chloride supplementation alone decreased bicarbonate reabsorption in the distal tubule
most often leads to correction; however, some patients addi- with consequent decrease in TA and ammonium secre-
tionally require sodium bicarbonate (86). In the small sub- tion. The subsequent decrease in NAE leads to MA. Dis-
group with a chloride shunt, possibly as a result of a defective ordered H+ secretion results in the inability to reduce
Na+-K+-2Cl– transporter system, concurrent hypertension is urinary pH even with spontaneous or induced acidosis.
present. Treatment includes salt restriction and thiazide or Bicarbonate excretion in early childhood is greater than
furosemide administration to correct both the hypertension in adults, and FEHCO3 may range from 5 to 15% (4,78).
and hyperkalemia by increasing sodium delivery to the distal This large bicarbonate loss, in addition to the inability
tubule, resulting in increased H+, K+, and water excretion. to secrete H+, leads to potentially life-threatening acido-
sis and a much larger requirement for bicarbonate sup-
plementation in the younger age group. Functional
Prognosis
immaturity of the proximal tubule has been suggested as
Prognosis depends on the underlying cause. Pseudohypoal- the cause for this increased bicarbonate loss (4,26). Fur-
dosteronism appears to result from a relative decrease in ther increases in bicarbonate requirements may occur
receptor number, leading to end-organ resistance that remits during periods of subsequent growth when H+ is pro-
by 4 to 5 years of age (86). Primary adrenal lesions usually duced during bone formation. At approximately 4 to 5
require permanent supplementation. Drug-induced changes years of age, there is usually a decrease in bicarbonate
may remit with removal of the drug and further avoidance of supplementation to levels similar to those required for
such agents. Hyporeninemic hypoaldosteronism from tubu- correction in adults. Bicarbonate excretion does not vary
lointerstitial nephritis occurs in adult patients with diabetes with filtered load over a wide range of plasma bicarbon-
or hyperuricemia and is likely to be associated with progres- ate levels and results from the inability to reduce urinary
sive renal insufficiency. pH below 7, even with severe acidosis. This phenome-
non is consistent with impaired distal tubular bicarbon-
ate reabsorption (26).
Distal Renal Tubular Acidosis Type 1,
Most adults show a constant FEHCO3 below 5% over a
Classic Renal Tubular Acidosis
wide range of serum bicarbonate levels. However, some
Symptoms patients have been described with net base excretion result-
ing from FEHCO3 exceeding TA + NH4+ (26).
Symptoms differ in children and adults. Childhood presenta-
Concurrent sodium and potassium wasting occur.
tion includes vomiting, FTT, and life-threatening acidosis.
Sodium wasting is a result of decreased reabsorption in
Nephrocalcinosis, renal calculi, or bone disease, including rick-
both proximal and distal tubules with bicarbonate or
ets or osteomalacia, are less common as presenting symptoms
other anions (94,95). Volume depletion stimulates
in younger children but are regularly present in older untreated
renin-aldosterone secretion and leads to increased deliv-
patients (87). Adults have recurrent renal calculi and musculo-
ery of bicarbonate and poorly absorbed anions such as
skeletal complaints such as arthralgia, myalgia, and bone pain
sulfate to the distal tubule. This promotes sodium and
(70).
potassium wasting and consequent hypokalemia. Sup-
plementation with alkali increases urinary pH and
Genetics
decreases the EDF for H+ secretion, allowing correction
A reduced rate of H+ secretion may occur as a primary con- of acidosis. Urinary sodium and potassium excretion
dition—DRTA inherited as either an autosomal dominant subsequently fall, leading to improvement in electrolyte
766 VI. Tubular Disease

TABLE 39.5. DISTAL RENAL TUBULAR ACIDOSIS (RTA)

Inability to secrete H+ (secretory defect) Primary biliary cirrhosis

Primary distal RTA (persistent classic syndrome) Thyroiditis
In infancy, associated HCO3 wasting Fibrosing alveolitis
In adolescence, secondary hyperparathyroidism Systemic lupus erythematosus
Nerve deafness develops in adolescence Polyarteritis nodosa
Transient infantile form Rheumatoid arthritis
Genetics (26,68,125) Drug or toxin
Sporadic (8,26,27) Amphotericin B
Endemic (8) Toluene
Secondary Analgesic abuse
Disorders of calcium metabolism with nephrocalcinosis or Lithium
hypercalciuria Cyclamate
Primary hyperparathyroidism Mercury
Hyperthyroidism Associated with other renal disease
Vitamin D intoxication Obstructive uropathy
Genetics Pyelonephritis
Idiopathic hypercalciuria (89) Renal transplant rejection
Hyperthyroidism (with nephrocalcinosis) Sickle cell disease
Medullary cystic disease—familial juvenile nephronophthisis Leprosy
Hereditary fructose intolerance with fructose exposure Associated with endocrine disease
Associated with genetically transmitted disease Hypothyroidism
CA type 2 (30,80) Salt-losing congenital adrenal hyperplasia
Erythrocyte CA B deficiency (126) Functional RTA (exchange defect)
Ehlers-Danlos syndrome (127) Marked volume depletion
Hereditary elliptocytosis (128) Hyponatremic states (hepatic cirrhosis/nephrotic syndrome)
Marfan’s syndrome (129) Sodium depletion
Sickle cell anemia (130,131) Increased back-diffusion H+ (gradient defect)
Osteopetrosis (30) Amphotericin B
With associated deafness (125,132–136) Abolition of the electrical gradient for H + ion secretion
Carnitine palmitoyl transferase deficiency type 1 (137) Short-circuit or voltage defect
Autoimmune disorders Combined defects in proximal HCO3 reabsorption and H+ ion secretion
Hypergammaglobulinemia (91) Infantile form type 1 RTA
Sjögren’s syndrome (92,93) Incomplete RTA
Chronic active hepatitis

CA, carbonic anhydrase.

balance. Volume correction decreases aldosterone secre- (alkaline buffer salts) with development of metabolic bone
tion and leads to retention of potassium and correction disease (98). Correction of the acidosis leads to correction
of the hypokalemia (51). A small group of patients have of bone disease, whereas nephrocalcinosis and nephrolithia-
continued potassium wasting despite correction of the sis tend to persist (69,99).
acidosis, a condition that may be associated with persist- Some patients with DRTA appear to have impaired gas-
ing hyperaldosteronism. MA is associated with an trointestinal absorption of calcium (100), as well as
increased excretion of calcium and phosphorus (88) and increased renal excretion of calcium, leading to hypocalce-
a decrease in citrate excretion (5,31,96,97). In acidosis mia, hyperparathyroidism, hypophosphatemia, and rickets.
(spontaneous or induced by ammonium chloride), cit- This occurs in patients with untreated disease and is uncom-
rate enters the mitochondria through stimulation of a mon in patients receiving adequate alkali supplementation
citrate carrier in the inner mitochondrial membrane (68). MA itself may impair vitamin D metabolism and lead
(96,97). This leads to a decrease in cytosolic citrate and to intestinal malabsorption of calcium, with secondary bone
a gradient for reabsorption from lumen to cell. Medul- disease with RTA. In acidotic children and adults with RTA
lary nephrocalcinosis and recurrent calcium phosphate and radiographic evidence of rickets (101,102), serum levels
or oxalate nephrolithiasis occur in the alkaline urine as a of 1,25-dihydroxyvitamin D (1,25-OH vitamin D) have
result of hypercalciuria and the reduced intraluminal been reported to be normal (101,102). However, in phos-
citrate. phorus-restricted healthy men, in whom basal levels of 1,25-
Nephrocalcinosis, which was, historically, a universal OH vitamin D were increased twofold, a decrease occurred
finding in patients with DRTA, has become rare with rec- when acidosis was induced with ammonium chloride (103).
ognition and early institution of adequate alkali therapy. The decrease was mediated by decreased production and
Persisting MA leads to buffering of hydrogen ion by bone increased clearance of 1,25-OH vitamin D (101). Thus, aci-
 39. Renal Tubular Acidosis 767

dosis in humans can impair the renal production of 1,25- in previously untreated older children. After age 4 to 5 years,
OH vitamin D. there is reduced bicarbonate loss, and, therefore, lower dos-
Polyuria is always present in patients with DRTA and ages of alkali supplementation (1 to 2 mEq/kg/day or 2 to 4
has been attributed to hypokalemia, nephrocalcinosis, sol- mEq per 100 calories) maintain correction.
ute excretion in untreated patients (e.g., hypercalciuria,
phosphaturia, excess sodium, potassium, and bicarbonate
Prognosis
excretion), or a primary tubular defect (79).
Growth rates can be restored and bone disease prevented
by early and consistent therapy. Early treatment is also
Treatment
necessary to prevent nephrocalcinosis, which can lead to
Therapy is directed at correction of acidosis at a rate com- chronic interstitial nephritis with scarring and glomerulo-
mensurate with risk of electrolyte and acid-base abnormality. sclerosis (105).
Severe hypokalemia presents a risk to cardiac and respiratory
muscle function (104) and should be at least partially cor-
rected before correction of the acidosis (because the latter can DIAGNOSIS OF RTA
aggravate the degree of hypokalemia) (3). Sodium and potas-
sium supplementation may be supplied as citrate or bicar- Diagnosis of RTA is based on clinical findings, assessment of
bonate, with one-third to one-half of the supplement plasma acid-base status, assessment of urine acidification
provided as the potassium salt. Increased growth velocity is (urinary pH), urinary ammonia or urine anion gap, and
expected to follow appropriate therapy in 2 to 3 weeks in the urine PCO2. Tools for characterization are outlined in Table
infant, and increased bone formation is followed by an 39.6 and include acid loading (106), bicarbonate titration
increase in acid production (68). Increased supplementation (107), urine to blood carbon dioxide tension during bicar-
is required until stabilization occurs with normal growth rate bonate titration (18,108), neutral sodium phosphate infusion
(68). A longer period of therapy is required to restore growth (101), and furosemide challenge (18,59,108). Urine-blood

TABLE 39.6. FEATURES OF RENAL TUBULAR ACIDOSIS (RTA)

With
bicarbonate Distal classic HyperK distal RTA 4 with GFR
Proximal RTA wasting RTA RTA normal Low GFR

Diagnostic testing Bicarbonate Acid load Renin—Aldoster- Renin—Aldoster-

Response Bicarbonate one one
response
SHCO3 untreated Usually 15–20 10–15 mmol/L 10–15 mmol/L 15–20 mmol/L 15–20 mmol/L
mmol/L
Features with metabolic
acidosis
SK+ N/L N/L N/L I I I
Min UpH <5.5 >5.5 >5.5 >5.5 <5.5 <5.5
Unstressed UpH >7.0 6.0–7.5 5.8–7.0 >5.5 <5.5 <5.5
Urine excretion
NH4 N D D D D MD
K+ N/I I I D D D
Ca2+ N I I I N D
Citrate N/I D D ? N N/D
%FEHCO3 at normal SHCO3 >10–15% >5–15% <3–5% <5% >5–15% <3–15%
U—B PCO2 (alkaline N/I L L L N L
urine) >20 <15 <15 <15 >20 —
Nephrocalcinosis (renal –/+ ++ ++ + – –
stones)
Rickets + — –/+ — — —
Bicarbonate to correct SpH 4–10 10–15 1–2 adult 2–3 2–4 1–2
(mmol/kg/d) 4–15 child
Response to bicarbonate Refractory Refractory Good Good Variable Good
(2 mEq/kg)

+, often; ++, very common association; –, rare or absent; ?, unknown; D, decrease; GFR, glomerular filtration rate; I, increase; L, low; MD, marked
decrease; N, normal; N/D, normal or decreased; N/I, normal or increased; SpH, serum pH; UpH, urine pH.
768 VI. Tubular Disease

TABLE 39.7. PATHOPHYSIOLOGY OF DISTAL RENAL TUBULAR ACIDOSIS (RTA)

U–B PCO2
U–B PCO2 with
Plasma with HCO3 phosphate Na2SO4 Furosemide K
Physiologic disorder Clinical condition K+ load load loading UpH excretion

Secretorya defect Classic RTA N/L L L >5.5 N/I

Transplant rejection — — — — —
Voltage-dependent defectb
Severe distal Na transport defect Obstructive uropathy — — — — —
Congenital adrenal hyper- I L L >5.5 D
plasia
Mild distal Na transport defect Sickle hemoglobinopathy I L N <5.5 N
Impaired distal Na delivery Hepatic cirrhosis — — — — —
N ? ? <5.5 N/I
Gradient defectc Nephrotic syndrome N/L N/L ? D N/I
amphotericin B

?, unknown; D, decreased; I, increased; N, normal; N/I, normal or increased; N/L, normal or low; L, low; UpH, urine pH.
aFailure to secrete H+.
bFailure to generate luminal electronegativity, which occurs as a result of impaired distal Na transport or delivery.
cIncreased back-diffusion of secreted H+.

carbon dioxide tension may be used as a specialized labora- sulfate infusion, which increases the sodium delivery;
tory test to define impaired H+ secretion by the collecting because the sulfate ion is impermeable, the gradient is main-
tubule. Urine-blood PCO2 is the most sensitive test for distal tained. In normal subjects with MA acid, urinary pH is fur-
tubular H+ excretion. Impaired excretion is associated with a ther lowered by furosemide or sodium sulfate infusion and
urinary CO2 tension (PCO2) that does not increase to potassium excretion is increased.
expected higher-than-plasma values after loading with Patients with diffuse impairment of H+-ATPase from a
sodium bicarbonate. Under normal circumstances, urinary decrease in glomerular filtration rate (GFR) or nephron
pH and bicarbonate are increased by administration of number have a persistent alkaline urine with increased
sodium bicarbonate, and urinary PCO2 rises to values con- potassium excretion because principal cell function is usu-
siderably higher than those of blood (normally more than ally intact (Table 39.7). Patients with a pump defect limited
20 mm above blood levels) (109,110). The difference in to the MCT have a normal increase in both H+ and K+
PCO2 between urine and blood (U PCO2 to B PCO2 gradi- because cortical function is stimulated by intraluminal elec-
ent) varies directly with the urine bicarbonate concentra- tronegativity. Patients with impairment in cortical sodium
tion. Increase in PCO2 in alkaline urine reflects an increase reabsorption (voltage defect) have baseline hyperkalemia
in H+ secretion in the collecting tubule, augmented by the and after challenge there is no increase in H+ or K+ because
high, intraluminal bicarbonate concentration (18). The there is no increased intraluminal electronegative gradient.
secreted H+ and luminal bicarbonate combine to form car-
bonic acid, which dissociates to carbon dioxide and water at
Differential Diagnosis
an uncatalyzed rate (CA is not present in the lumen of the
distal nephron). Because carbon dioxide generation occurs 1. Other causes of metabolic acidosis
in the distal segments of the collecting duct, where medul- (a) Gastrointestinal bicarbonate loss (e.g., diarrhea, fis-
lary trapping of carbon dioxide can occur, and in the renal tula) or administration of drugs [e.g., CaCl2, MgSO4, cho-
pelvis and bladder, where conditions are unfavorable for lestyramine (U Cl greater than Na + K)] (Fig. 39.2).
reabsorption of carbon dioxide into blood, carbon dioxide In chronic MA, ammonia generation from glutamine is
appears in increased amounts in the urine. Similar findings stimulated with little change in phosphate excretion (phos-
follow the administration of neutral sodium phosphate, phate released from bone is used in blood buffering during
which enhances the distal delivery of buffer. This stimulates acidosis). Thus, it is helpful to focus on ammonia genera-
H+ secretion and increases the U PCO2 to B PCO2 gradient. tion and excretion as the expected response in acidosis, and
This can also be used as a test of distal tubular H+ secretion. review of ammonia excretion is the key to understanding
Furosemide challenge (Table 39.7) may be used to assist in RTA. Urinary pH values taken alone may be misleading in
differentiation of site and mechanism of the defect in acidi- chronic MA from gastrointestinal loss, particularly if
fication (18). Delivery of sodium and reabsorption in the hypokalemia is present. Here the increase in ammonia
CCT is followed by an increase in luminal electronegativity. excretion buffers most of the secreted H+, leading to a uri-
A similar increase in negativity can be produced by sodium nary pH of 5.5 to 6.0. Thus, if pH alone is considered
 39. Renal Tubular Acidosis 769

FIGURE 39.2. This algorithmic approach to the patient with metabolic acidosis illustrates the
differential features leading to a diagnosis of renal tubular acidosis (RTA). Useful confirmatory
tests and features of differing types of RTA are outlined at the steps in which they are used clini-
cally. ADRTA, adult distal renal tubular acidosis; AG, anion gap; Aldo, aldosterone; BP, blood
pressure; B Vol, blood volume; CRF, chronic renal failure; GI, gastrointestinal tract; HCMA, hyper-
chloremic metabolic acidosis; IDRTA, infantile distal renal tubular acidosis; PRTA, proximal renal
tubular acidosis; S, serum; UpH, urine pH.

without consideration of urine AG, DRTA is suggested (b) Acid loading that may occur with NH4Cl, acidic amino
(24,111). In aldosterone deficiency, ammonia secretion is acids in parenteral nutrition, or ingestion of acid ingestion
significantly impaired and H+ secretion as ammonium is (e.g., HCl, NH4Cl, lysine, arginine, sulfur, and phosphorus
reduced. Here, incomplete buffering of secreted H+ leads to from food). HCMA is associated with a negative UAG.
low urinary pH even in patients with DRTA (112,113). 2. Anion gap acidosis (Fig. 39.2)
770 VI. Tubular Disease

3. Pseudo-RTA: In malnourished patients, the catabolic state present, RTA should be suspected. Initial studies should
produces increased acid, ketones, a decrease in GFR, and a include repeat blood gas, SK, UAG, urinary pH (by pH elec-
decrease in phosphate output. This limits both TA (decreased trode measurement, not indicator strips, which are notori-
capacity to excrete H+ with an acid urine), a decreased genera- ously inaccurate), and urinalysis to identify proximal
tion of ammonia from a glutamine deficit that further limits tubular dysfunction (e.g., proteinuria glycosuria, aminoaci-
H+ excretion, and increased ketone excretion (or potential loss duria) (Figs. 39.2 and 39.3).
of bicarbonate), thereby producing a pseudo-RTA syndrome. Serum K+ is helpful in classification because hypokale-
4. Urinary diversion or augmentation of the bladder mia occurs in PRTA (type 2) and DRTA (type 1). Hyper-
using segments of ileum or colon leads to hypokalemia kalemia suggests aldosterone deficit or resistance (type 4),
with severe acidosis, particularly if there is poor drainage voltage defect, or obstructive uropathy. Normal S K+ occurs
leading to prolonged mucosal contact with urine. in RTA with renal insufficiency, uremia, and the McSherry
5. Uremic acidosis usually is associated with an increased syndrome (86).
anion gap (from phosphate and sulfate retention), whereas If preliminary studies suggest HCMA with RTA,
reduced nephron number is followed by severely decreased serum, and urine, chemistries should be evaluated with
ammonium excretion and reduced bicarbonate reabsorption blood gases to rule out respiratory alkalosis and other
(associated with a relative solute diuresis in remaining neph- potential causes of bicarbonate loss. Additional history
rons) (114). should be obtained to check for potential HCl adminis-
6. Urinary infection with urea-splitting organisms (e.g., Pro- tration (e.g., parenteral nutrition, drug administration),
teus), an alkaline urine, and, if there is parenchymal infection, renal or gastrointestinal losses of bicarbonate, or the pres-
MA. Urinalysis and culture are necessary to exclude such facti- ence of a bowel segment augmenting bladder capacity or
tious RTA patterns, particularly in patients with obstructive used as a urinary diversion. Exclude concurrent or under-
uropathy. lying renal disease with uremia or retention rather than
7. Hyperkalemic DRTA is a condition in which low K+ tubular acidosis. In the patient with HCMA, urinary pH
excretion is present together with a decreased ability to acidify and urinary ammonia excretion should be evaluated by
the urinary pH to less than 5.5 during spontaneous or induced direct measurement or UAG (calculate urinary net charge,
acidosis (18). Concurrent sodium wasting is common, so UAG + UNa + UK – UCl – UHCO3) to evaluate the site of
salt-losing congenital adrenal hyperplasia, pseudohypoaldos- renal tubular lesion. A positive UAG reflects low H+ secre-
teronism, must be considered in the differential diagnosis. tion and low ammonium excretion, whereas a negative
Furosemide or sodium sulfate testing demonstrates typical low UAG reflects urinary bicarbonate loss.
potassium excretion. Low NH4+ production (UAG +) suggests DRTA, PRTA
8. Abnormal anions, certain drugs, or toxic substances may with low NH4+ production, D lactic acidosis, or failure to
lead to decrease in value of U AG (e.g., ketonuria), and D lac- lower NH4+ in the lumen of the collecting duct because of
tic acidosis and toluene (glue sniffing) are associated with a (+) excess distal delivery of HCO3– from the PCT (22).
net charge, which may be misinterpreted. Formal measure- High NH4+ production with acidosis makes it necessary
ment of NH3 may be necessary to check anion gap if the his- to check for loss of metabolizable organic anions in the
tory suggests these are potential causes. Glue sniffing leads to a urine (these equal potential HCO3–). Here, the cause of
high NH3, U AG, and U osmolar gap (from conversion of tol- acidosis is overproduction of organic acids, and the renal
uene to hippurate). Check for D lactic acidosis if MA is associ- lesion does not cause the acidosis, which may be UAG+. Loss
ated with central nervous system confusion, particularly if of metabolizable anion is suspected when there is an exces-
there is history of bowel disease or resection. A trial of antibi- sive rate of urinary excretion of organic anions shown by
otics to decrease production is then warranted. Na+ + K+ greater than Cl– (22,48).
9. Functional RTA secondary to marked decrease in If there is a low NH4+ excretion, urinary pH should be
sodium delivery (UNa less than 10) limits distal H+ excretion. evaluated. If the urinary pH is above 6.0, bicarbonate
excretion should be evaluated by examining the urinary
Other considerations in differential diagnosis include
PCO2 in alkaline urine to examine for distal lesions and
tubular dysfunction (which may occur as a result of extrin-
fractional excretion of bicarbonate for proximal lesions.
sic renal factors such as hyperkalemia, extracellular fluid
Evaluation for other features of Fanconi syndrome should
expansion, and loss of potential bicarbonate during recov-
be performed.
ery from chronic hypocapnia), with volume expansion
Low ammonium secretion is rare. It may occur with a
(dilutional) acidosis, or secondary hyperparathyroidism.
urinary pH of 4 to 5 and a defect in NH3 availability in
the renal medullary interstitium with a decrease in buffer-
ing ability (22). This occurs with a low GFR (22), hyper-
Practical Approach to Diagnosis
kalemia (22), decreased levels of blood glutamine (e.g.,
Symptoms often are nonspecific and a high index of suspi- decreased substrate for ammoniagenesis) (115), or high
cion is necessary. If HCMA with a normal anion gap is levels of fat-derived fuels (e.g., total parenteral nutrition)
 39. Renal Tubular Acidosis 771

FIGURE 39.3. The figure shows the expected

correlation between urine pH (UpH) and arte-
rial pH. Captions highlight the most useful
testing at different points in the spectrum.
Alkali loading is most useful in the patient
with marked acidosis; here comparison of the
serum and urine pH allows differentiation
between normals, proximal renal tubular aci-
dosis (PRTA), and renal tubular acidosis (RTA)
type 4. Acid loading is most useful in patients
with normal serum pH (SpH) or mild acidosis.
Estimations of net acid excretion from urine
anion gap (Urine AG) are most helpful with
urinary pH of approximately 6; urine osmolar
gap (Osm G) or ammonium excretion mea-
surement is most useful at serum pH below
7.4. Urinary PCO2 measurement requires an
alkaline urine. Fractional excretion of bicar-
bonate is helpful in differentiating between
PRTA and the infantile form of distal RTA.
Serum K+ is helpful in differentiating RTA type
4 from other forms. ADRTA, adult distal renal
tubular acidosis; IDRTA, infantile distal renal
tubular acidosis.

competing with glutamine as a source for regeneration of nium chloride to prevent life-threatening complications of
ATP in the cells of the PCT with decrease in ammoniagen- hyperammonemia.
esis and in the hypothetical alkaline cell pH (22). The pH If the initial urinary pH is above 6.5 and serum bicar-
falls even with minimal H+ transfer (22). A high urinary bonate is normal or only mildly depressed, evaluation for
NH4+ or a negative net charge, negative UAG, is consistent PRTA is necessary. Proximal tubular function should be
with normal distal acidification occurring in the older evaluated because the defect (failure of bicarbonate reab-
child (116). sorption) in PRTA is, more commonly, part of a multiple
Advantage should always be taken of any episode of defect syndrome (Fanconi syndrome) than an isolated
spontaneous acidosis to screen for H+ excretion. Standard defect.
treatment in RTA is base replacement; careful selection of If MA is present and the serum potassium is high
testing during correction makes it possible to obtain (above 5 mEq/L) with a urinary pH below 5.5, one must
information, which otherwise requires bicarbonate titra- consider RTA 4 (Table 39.8). Evaluation includes review
tion and correction of sodium deficit. This often allows of serum AG and UAG (NAE), pH, assessment of volume
characterization of RTA without the need for formal pro- and circulatory status (e.g., blood pressure, pulse, weight),
vocative testing. Formal testing, if subsequently required, and plasma renin and aldosterone measurement under
should be delayed after correction of acidosis and a period stress (e.g., volume depletion or furosemide challenge).
of caloric adjustment to avoid the confounding effects of Persistently high urinary sodium loss suggests hypoaldos-
malnutrition, volume depletion, and other acid-base teronism or pseudohypoaldosteronism (117). Urine
abnormalities. osmolality should be greater than isotonic and UNa greater
The patient suspected of having RTA (Fig. 39.3) who is than 20 mmol/L for transtubular potassium gradient to
not overtly acidotic requires formal acid loading to induce be valid in assessing response to mineralocorticoid admin-
an acidosis. A dose of 0.1 g/kg ammonium chloride (110 istration and furosemide challenge.
mEq/m2) or 2.0 mEq/kg calcium chloride is administered If UNa is below 15 mmol/L, it is important to exclude an
orally or intravenously. Ammonia excretion and urinary pH exchange defect because reduced H+ may result from
are measured at 1, 2, and 8 hours. If secondary RTA from a reduced sodium delivery. This is sometimes called func-
metabolic disorder is possible, hyperammonemic syn- tional RTA because of its transient nature and relationship
dromes must be excluded before administration of ammo- to limited H+ ion secretion by sodium delivery and poten-
772 VI. Tubular Disease

TABLE 39.8. RENAL TUBULAR ACIDOSIS TYPE 4, HYPERKALEMIC ACIDURIA PHYSIOLOGIC SUBTYPING
Urine
aldosterone/ Finding blood
Plasma renin serum Clinical blood volume/plasma
Subtype activity aldosterone pressure volume Salt-wasting

Aldosterone deficiency ± renal disease

Primary mineralocorticoid deficiency (Addi- S/E L L/N L/N E
son’s congenital adrenal hyperplasia syn-
drome, isolated aldosterone deficiency)
Aldosterone deficit (hyporeninemia)
Primary hyporenin-hypoaldosterone diabetes L L E/N E/N L
mellitus, gout, pyelonephritis, interstitial
nephritis, nephrosclerosis
Adolescent hyperkalemia syndrome L/N L/N E E L
Reduced tubular response
Pseudohypoaldosteronism M/E M/E L L E
Early childhood type 4 E E N L/N L

E, elevated; E/N, elevated or normal; L, low; L/N, low or normal; M/E, markedly elevated; N, normal; S/E, significantly elevated.

tial aldosterone effect rather than a true tubular defect. If recting bicarbonate deficit. The bicarbonate deficit is
this defect is suspected, the patient should be carefully replaced over 1 to 2 days depending on presenting symp-
assessed for dehydration, severe cardiac failure, and sodium toms. Initial correction to serum pH 7.25 to 7.30 is made
chloride (deficit from vomiting, diuretic abuse, or overuse). using the Henderson equation (H+ = 24 PCO2/HCO3) to
If present, dehydration or sodium deficit should be cor- assess the desired bicarbonate supplementation. Remaining
rected with appropriate hydration or salt supplementation deficits should be calculated after equilibration with bicar-
before UpH and UAG are reassessed. If volume deficit is not bonate space of 0.6 body weight/kg (i.e., HCO3 required =
apparent, a trial dose of furosemide or response to sodium 0.6 – body weight/kg – [desired HCO3 – measured HCO3]),
sulfate infusion to increase sodium delivery to the distal to which maintenance dosing is added. The adequacy of
tubule (with the consequent enhanced transepithelial volt- replacement is checked and the dosage of administered
age) should be administered. Expected response to furo- bicarbonate is titrated.
semide administration in healthy subjects includes a
decrease in urinary pH and increase in ammonia and UK
Maintenance Therapy
excretion. In patients with hyporeninemic hypoaldoster-
onism, urinary pH is decreased. Once a deficit is corrected, it is necessary to provide suffi-
cient base supplementation to balance nonvolatile acid pro-
duction (DRTA) and to replace any continuing losses
Therapy
(PRTA). Base may be supplied as sodium bicarbonate (55
In the patient with significant spontaneous acidosis, initial mEq/teaspoon), sodium citrate, or mixed sodium and
correction based on an exact diagnosis is not necessary, nor is potassium citrate depending on the presence or absence of
it necessary to delay therapy until investigation is complete. continuing potassium losses. The aim is to maintain CO2
Bicarbonate administration and titration are effective in cor- greater than 20 to 22 mEq/L.
recting the acidosis with RTA regardless of type. However, in In DRTA, replacement covers the production of 2 to 4
DRTA type 1 with hypokalemia, and likely hypocalcemia, mEq of acid/kg from the diet equivalent to 2 mEq per 100
partial correction of potassium and calcium should be carried kcal (Table 39.6). In young children, after treatment is
out before total correction of serum bicarbonate to prevent commenced, an extra 2 to 3 mEq/kg acid may be generated
muscle weakness, respiratory failure, arrhythmia, or painful from bone formation, as hydroxyl and phosphate groups
tetany. Care should be taken to obtain appropriate diagnostic are incorporated into hydroxyapatite. In PRTA, dosages as
samples of blood and urine under spontaneous acidosis and high as 4 to 15 mEq/kg/day are sometimes required to
during base supplementation to allow diagnosis without the compensate for continuing losses secondary to the low
need for formal acid loading or bicarbonate. tubular bicarbonate threshold.
Patients with RTA 4 require treatment for hyperkalemia
(59). This may include correction of acidosis, sodium reple-
Acute Correction of Acidosis
tion, mineralocorticoid replacement, and, in some patients,
First, serum potassium and calcium should be assessed and glucocorticoid replacement (59,87). It is particularly
significant deficits should be partially replaced before cor- important to review drug and dietary intake in patients
 39. Renal Tubular Acidosis 773

with RTA type 4. Common contributing factors to hyper- 16. Spitzer A, Edelmann CM, Goldberg LD, et al. Short stat-
kalemia include low urine flow; decreased sodium delivery; ure, hyperkalemia and acidosis: a defect in renal transport of
rapid decline in GFR, particularly in acute or chronic renal potassium. Kidney Int 1973;3:251–257.
failure; hyperglycemia or hyperosmolality; and unsuspected 17. Schambelan M, Sebastian A, Rector FC Jr. Mineralocorti-
intake of potassium salt supplements or medications. Cal- coid-resistant renal hyperkalemia without salt wasting (type
2 pseudo-hypoaldosteronism). Role of increased renal chlo-
culation of transtubular potassium gradient and response to
ride reabsorption. Kidney Int 1981;19:716–727.
furosemide and fluorohydrocortisone allow clinical correc- 18. Battle DC. Segmental characterization of defects in collect-
tion of hyperkalemic syndromes while awaiting formal ing tubule acidification. Kidney Int 1986;30:546–554.
assessment of renin-aldosterone status. If there is a low 19. Battle DC. Hyperkalemic hyperchloremic metabolic aci-
transtubular potassium gradient (below 5) in a patient with dosis associated with selective aldosterone deficiency and
hyperkalemia and no response to mineralocorticoid supple- distal renal tubular acidosis. Semin Nephrol 1981;1:260–
mentation, a furosemide challenge and treatment with 274.
sodium or bicarbonate to increase excretion is appropriate. 20. Krieg RJ Jr, Santos F, Chan JCM. Growth hormone, insu-
lin-like growth factor and the kidney (Editorial review).
Kidney Int 1995;48:321–336.
21. Challa A, Krieg RJ Jr, Thabet MA, et al. Metabolic acido-
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 40

NEPHROGENIC DIABETES INSIPIDUS

NINE V. A. M. KNOERS
LEO A. H. MONNENS

HISTORY ing, and poor weight gain are usually the initial symptoms
(5). Patients are eager to suck but may vomit during or
The renal type of diabetes insipidus was appreciated as a shortly after the feeding. Dehydration is evidenced by dry-
separate entity more than 50 years ago, when it was ness of the skin, loss of normal skin turgor, recessed eye-
described independently by two investigators: Forssman (1) balls, increased periorbital folding, depression of the
in Sweden and Waring et al. (2) in the United States. Fami- anterior fontanelle, and a scaphoid abdomen. Intermittent
lies with this type of defect, however, had been described high fever is a common complication of the dehydrated
previously (3) without clear differentiation of nephrogenic state, particularly in very young children. Body tempera-
diabetes insipidus (NDI) from the central or neurohor- ture can be normalized by rehydration. Seizures can occur
monal form of the disorder. In 1947, Williams and Henry but are rare and most often seen during therapy, particu-
(4) noticed that injection of antidiuretic hormone (ADH) larly if rehydration proceeds too rapidly. Obstipation is a
in doses sufficient to induce systemic side effects could not common symptom in children with NDI. Nocturia and
correct the renal concentrating defect in some patients. enuresis are common complaints later in childhood.
They coined the term NDI. Subsequent studies revealed Untreated, most patients fail to grow normally. In a ret-
active hormone in the serum and urine of affected persons rospective study of 30 male NDI patients, most children
and lent further support to the theory of renal unrespon- grew below the 50th percentile, most of them having stan-
siveness to vasopressin. NDI is synonymous with the terms dard deviation (SD) scores lower than –1 and showing no
vasopressin- or ADH-resistant diabetes insipidus and diabetes clear catch up growth after the first year of life. Weight for
insipidus renalis. height SD scores were initially low, followed by global nor-
malization at school age (6). The exact reasons for the fail-
ure to thrive seen in NDI are unknown. Initial feeding
DEFINITION AND CLINICAL problems and the ingestion of large amounts of low-caloric
MANIFESTATIONS fluid resulting in a decreased appetite may play roles (7,8).
Furthermore, it is possible that repeated episodes of dehy-
Congenital NDI is a rare inherited disorder, characterized dration have some as yet undetermined negative effects on
by insensitivity of the distal nephron to the antidiuretic growth.
effects of the neurohypophyseal ADH, arginine vasopressin Mental retardation has long been considered an impor-
(AVP). As a consequence, the kidney loses its ability to con- tant complication of untreated NDI and assumed to be a
centrate urine, which may lead to severe dehydration and consequence of recurrent episodes of severe brain dehydra-
electrolyte imbalance (hypernatremia and hyperchloremia). tion and cerebral edema caused by overzealous attempts at
The defect in NDI is present from birth, and manifesta- rehydration (9,10). Additional evidence underscoring the
tions of the disorder generally emerge within the first weeks assumption that NDI has adverse effects on the cerebrum is
of life. With breast milk feedings, infants usually thrive and provided by several reports describing intracranial calcifica-
do not develop signs of dehydration. This is because human tions in NDI patients (11,12). Such lesions are generally
milk has a low salt and protein content, and therefore a low considered to be the result of hemorrhage or necrosis. Most
renal osmolar load. With cows’ milk formula feedings, the of the reported patients with cerebral calcifications were
osmolar load to the kidney increases, resulting in an mentally retarded.
increased demand for free water. If cow’s milk feedings are Currently, mental retardation is considered to be rare due
not supplemented by hypotonic fluids in infants with NDI, to earlier recognition and treatment of NDI. The frequency
hypernatremic dehydration appears. Irritability, poor feed- of mental retardation under modern treatment regimens is
778 VI. Tubular Disease

TABLE 40.1. COMMON CAUSES OF ACQUIRED OR

SECONDARY NEPHROGENIC DIABETES INSIPIDUS

Amyloidosis
Analgesic nephropathy
Chronic pyelonephritis
Chronic renal failure
Drug-induced
Lithium
Tetracyclines
Hypercalcemia/nephrocalcinosis
Hypokalemia
Juvenile nephronophthisis
Obstructive uropathy
Renal dysplasia
Sarcoidosis
Sickle cell anemia and trait

(10 mg for infants, 20 mg for children) is administered intra-

nasally. Urine is collected during the subsequent 5.5 hours.
The first collected portion of the urine should be discarded.
The maximal urine osmolality in any collected aliquot is cho-
FIGURE 40.1. Intravenous pyelogram of a patient with congen-
ital nephrogenic diabetes insipidus and severe hydronephrosis. sen as a measure of the concentrating capacity (15). After
DDAVP administration, NDI patients are unable to increase
urinary osmolality, which remains below 200 mOsm/kg H2O
unknown, but in the largest psychometric study ever (normal greater than or equal to 807 mOsm/kg H2O) and
reported, only 2 of the 17 male NDI patients (aged 3 to 30 cannot reduce urine volume or free-water clearance.
years) tested had a total intelligence quotient more than 2 Plasma vasopressin levels are normal or only slightly
SDs below the norm. Fourteen patients had an intelligence increased in affected children. Other laboratory findings
score within or above the normal range, and one patient had have been described, which mainly result from chronic
a general index score between –1 and –2 SD (13). dehydration. Serum sodium concentration is generally ele-
The psychological development of patients with NDI is vated and may be as high as 170 mmol/L. There is also an
influenced by a persistent desire for drinking and the need increase in serum chloride concentration and retention of
for frequent voiding, which compete with playing and urea and creatinine. All values are normalized by adequate
learning. Therefore, many NDI patients are characterized rehydration. In addition, reduced glomerular filtration rate
by hyperactivity, distractibility, short attention span, and and renal blood flow return to normal when a state of nor-
restlessness. In the psychometric study mentioned earlier, mal hydration has been achieved.
the criteria for attention deficit hyperactivity disorder were The primary congenital form of NDI must be differenti-
met in 8 of 17 tested NDI patients (13). ated from the secondary or acquired forms, which are much
Persistent polyuria can result in the development of more common. In our experience, the urinary osmolality
megacystis, hydroureter, and hydronephrosis (14) (Fig. obtained after DDAVP administration in these disorders is
40.1). We have recently found that this complication is rare always higher than in NDI. Several secondary causes, some
in patients with NDI. Dilation of the urinary tract of which are discussed later, are listed in Table 40.1.
occurred in only 2 of 30 patients studied (6).

CELLULAR PHYSIOLOGY OF ARGININE

DIAGNOSTIC PROCEDURES VASOPRESSIN’S ANTIDIURETIC ACTION
IN THE DISTAL NEPHRON
The observation of polyuria in a dehydrated infant, together
with the finding of a high serum sodium concentration, pro- The physiologic action of vasopressin on the renal collecting
vides presumptive evidence of a renal concentrating defect. To duct has been one of the most intensively studied processes in
confirm the concentrating defect and to distinguish the renal the kidney. AVP (ADH) is synthesized on the ribosomes of
from the central form of diabetes insipidus, a vasopressin the magnocellular neurons of the supraoptic and paraventric-
test is performed with 1-desamino-8-D-arginine vasopressin ular nuclei of the hypothalamus as a large, biologically inac-
(DDAVP), a synthetic analogue of natural AVP that produces tive bound form, and transported down the neuronal axons
a high and prolonged antidiuretic effect. In the test, DDAVP to the posterior pituitary where it is stored. After appropriate
 40. Nephrogenic Diabetes Insipidus 779

be essential for AQP2 delivery to the apical membrane

(16,17). In addition, it has been shown that anchoring of
PKA to PKA-anchoring proteins, which ensures targeting of
PKA to AQP2-bearing vesicles, is another prerequisite for
AVP-mediated AQP2 translocation (18). The identity of the
PKA-anchoring proteins involved has not yet been elucidated.
The increase in apical membrane permeability allows water to
flow from the tubule lumen, via AQP2 in the apical mem-
brane and via AQP3 and AQP4, constitutive water channels
in the basolateral membrane, to the hypertonic medullary
interstitium. This then leads to the formation of concentrated
urine. Withdrawal of vasopressin triggers the endocytosis of
AQP2-containing vesicles and restores the water-impermeable
state of the apical side of the cell.
The molecular machinery for the docking and fusion of
AQP2-containing vesicles with the apical membrane is most
likely similar to the process of synaptic vesicle fusion with the
presynaptic membrane and involves vesicle (v) SNAREs (solu-
ble NSF attachment protein receptors) and target membrane
(t) SNAREs (Fig. 40.2). Thus, in subcellular fractions from rat
kidney, enriched for AQP2-containing vesicles, the v-SNARE
synaptobrevin (VAMP-2) was found, as well as the fusion-
FIGURE 40.2. Schematic view of the antidiuretic arginine vaso- mediating protein SNAP23 (19–22). The potential t-SNARE
pressin (AVP) signaling pathway in collecting duct cells and the
proteins (microtubule, dynactin-dynein) and vesicle targeting syntaxin-4 was identified at the apical plasma membrane of rat
receptors (VAMP-2, syntaxin-4, NSF) that might participate in kidney collecting duct principal cells (23). Rab3A, a member
the specificity of aquaporin-2 (AQP2) water channel targeting to of the Rab family of guanosine triphosphatases, involved in
the luminal plasma membrane. AC, adenylate cyclase; AQP3,
aquaporin-3 water channel; AQP4, aquaporin-4 water channel; exo- and endocytotic membrane trafficking, is also believed to
G, stimulatory guanine nucleotide binding protein; NSF, N-ethyl- play a role in AQP2 translocation to the apical membrane
maleimide–sensitive fusion protein; PKA, protein kinase A; V 2, (21,24). In addition, it has been shown that both the micro-
vasopressin type-2 receptor. (See Color Plate 40.2.)
tubule-associated motor protein dynein and the associated
dynactin-complex, which catalyze transport of vesicles along
stimuli, AVP is secreted from the posterior pituitary into the microtubules, are important in vasopressin-regulated traffick-
circulation as biologically active hormone. AVP release is pri- ing of these vesicles (Fig. 40.2) (25). Recently, a critical role for
marily regulated by changes in plasma osmolality (by more calcium as an intracellular mediator of the vasopressin-induced
than 2%) but can also occur in response to nonosmotic stim- trafficking of AQP2 has been established (26). The transloca-
uli. These nonosmotic stimuli are generally related to changes tion of AQP2 from the intracellular vesicle to the plasma
in either total blood volume or the distribution of extracellu- membrane can also be stimulated by cAMP-independent
lar fluid. In addition, physical pain, emotional stress, and cer- pathways. Thus, Bouley et al. have shown that nitric oxide and
tain drugs (e.g., nicotine) influence the release of AVP. In its atrial natriuretic factor stimulate the insertion of AQP2 into
effector organ, the kidney, AVP binds to vasopressin type-2 renal epithelial cell membranes via a cyclic guanosine mono-
receptors (V2Rs) on the basolateral membrane of the principal phosphate–dependent pathway (27). The importance of this
inner medullary collecting duct cells and of the arcade cells pathway is as yet unclear.
(Fig. 40.2). The arcades are long, highly branched renal Long-term adaptation to circulating AVP levels, for
tubule segments that connect distal convoluted tubules of sev- instance, in a dehydrated state, is accomplished by increas-
eral deep and midcortical nephrons to the origin of cortical ing the expression of AQP2 messenger RNA (mRNA) and
collecting ducts. V2R occupancy results, via the intermediacy protein. A cAMP-responsive element in the AQP2 promo-
of a stimulatory G protein (Gs), in activation of adenylate tor has proven to be a key element in cAMP-dependent
cyclase and an increase in intracellular cyclic adenosine mono- transcriptional regulation of this gene, and as such for the
phosphate (cAMP) from adenosine triphosphate. The ele- long-term regulation of water permeability (28).
vated cAMP levels stimulate protein kinase A (PKA), which
in turn initiates a redistribution of aquaporin (AQP)-2 water
channels from intracellular vesicles to the apical plasma mem- GENETICS
brane, rendering this membrane water permeable. Phospho-
rylation of a PKA consensus site in AQP2 (serine at position Three different inheritance patterns of NDI have been rec-
256 in the cytoplasmic carboxy terminus) has been shown to ognized. In most cases (approximately 90%), NDI is trans-
780 VI. Tubular Disease

mitted as an X-linked recessive trait (MIM304800). In nected by extracellular and intracellular loops. The receptor
these families, female carriers who are clinically unaffected contains one unique consensus sequence site for N-linked
transmit the disease to sons, who display the complete clin- glycosylation in the extracellular amino-terminus (44) and
ical picture (1,4,29). In 1988, the major NDI locus was phosphorylation sites for G protein–coupled receptor
mapped to the distal region of the long arm of the X chro- kinases represented by a serine cluster in the carboxy-termi-
mosome (Xq28) (30), and in 1992, mutations in the V2R nus (45,46). The amino-terminal part of the protein
gene were shown to underlie X-linked NDI (31–33). In a including the first transmembrane domain and the posi-
minority of families (approximately 10%), the transmission tively charged first intracellular loop are important for
and phenotypic characteristics of NDI are not compatible proper insertion and orientation in the membrane (47). A
with an X-linked trait. In these families, females display the conserved glutamate or dileucine motif in the intracellular
complete clinical picture of NDI and are clinically indistin- carboxy terminal part of the receptor is essential for recep-
guishable from affected male family members (34–36). In tor transport from the endoplasmic reticulum (ER) to the
addition, linkage analysis in these families has excluded Golgi apparatus (48). Two conserved adjacent cysteines in
linkage between NDI and polymorphic DNA markers the C-terminus are palmitoylated, thereby anchoring the
from the Xq28 region. Family pedigrees suggested the exis- carboxy-tail to the plasma membrane and controlling the
tence of both an autosomal recessive (MIM 222000) and tertiary structure of this region of the receptor (49).
an autosomal dominant form (MIM 125800) of NDI. In To date, more than 180 distinct putative disease-causing
recent years, it has been demonstrated that both autosomal mutations in the V2R gene have been detected in families
forms of NDI are caused by mutations in the AVP-sensitive with X-linked NDI (see http://www.medicine.mcgill.ca/
AQP2 water channel (37,38). The prevalence of NDI is nephros). Remarkably, the mutations are not clustered in
not exactly known, but the disease is assumed to be rare. one domain of the V2R but are scattered throughout the
Even in large pediatric and nephrology clinics, it is protein, except for the part coding for the N- and C-termi-
observed infrequently. In the Dutch population of approxi- nal tails of the receptor. Approximately 50% of the muta-
mately 15 million, 35 different families are known. tions are missense mutations. Nucleotide deletions and
insertions causing frame shifts (27%), nonsense mutations
(12%), large deletions (5%), in-frame deletions or inser-
X-LINKED NEPHROGENIC DIABETES tions (4%), and splice-site mutations (2%) account for the
INSIPIDUS: MUTATIONS IN THE remainder of mutations (42). Several mutations are recur-
VASOPRESSIN TYPE-2 RECEPTOR GENE rent, as evidenced by the fact that these mutations were
found on different haplotypes in ancestrally independent
The V2R had long been considered a prime candidate for families. The most frequent of these recurrent mutations
the defective step in the AVP-mediated response in X- (D85N, V88M, R113W, R137H, S167L, R181C, and
linked NDI. The reason for this belief was the observation R202C) occur at potential mutational hotspots.
that in patients with X-linked NDI, not only the antidi- The impact of more than 80 mutations on the function
uretic but also the vasodilatory coagulation and fibrinolytic of the V2R has been studied in in vitro expression systems,
responses to the V2R-specific agonist DDAVP were lacking such as COS-7 cells (monkey kidney cells), allowing fur-
(39). This finding suggested a general V2R defect in these ther classification of the genotypes based on the cell-
patients. Independent support for the V2R being involved biologic outcome, similar to what has been done for other
in X-linked NDI was provided by the finding that a gene proteins (50–52). The first class of mutations (class I) yields
conferring V2-like binding activity colocalized with the defects in the synthesis of stable mRNA, which precludes
NDI locus in the subterminal region of the X chromosome the formation of sufficient amounts of protein. Mutations
long arm (Xq28) (40). Soon after the identification of the resulting in aberrant splicing, frame shifts, and premature
human V2R gene and complementary DNA in 1992 (41), termination of translation belong to this first category.
the role of the V2R in the pathogenesis of NDI was finally Most of the truncated receptor proteins found in NDI
proven by the demonstration of mutations in the encoding patients result in a complete lack of receptors present on
gene in affected individuals (31–33,42,43). The V2R gene the cell surface and, consequently, preclude specific func-
(AVPR2; Genbank association number L22206) is rela- tion of the receptor. The majority of mutations found in X-
tively small and consists of three exons separated by two linked NDI belong to the second class (class II), in which
short intervening sequences (introns). The mRNA has been the translation of the protein is completed, but the abnor-
found exclusively in the kidney, specifically in the cortical mal protein is misfolded or improperly assembled and
and medullary collecting ducts. The complementary DNA trapped in the ER.
encodes a receptor protein of 371 amino acids, has a pre- Subsequently, such mutant proteins are often targeted to
dicted molecular mass of approximately 41 kDa, and shares proteosomes for degradation. Therefore, class II mutations
the general structure of a G protein–coupled receptor con- also lead to severely reduced or absent receptor expression
sisting of seven hydrophobic transmembrane helices, con- on the cell surface. Most missense mutations and in-frame
 40. Nephrogenic Diabetes Insipidus 781

deletions and some nonsense mutations in the V2R fall in Genotype-Phenotype Correlations in
class II, and, except for some mutations in the extra- and X-Linked Nephrogenic Diabetes Insipidus?
intracellular loops (R104C, R113W, R143P, G201D,
Almost all mutations in the V2R gene result in a uniform
R337stop, E242stop), most of these mutations are located
clinical NDI phenotype with polyuric manifestations in the
in transmembrane domains (L44P, I46K, L59P, L62P, L62-
first weeks of life and poor growth. There are, however, a few
64del, L83P, L83Q, A84D, I130F, R137H, R143P,
exceptions to this rule. Three mutations (D85N, G201D,
W164S, S167T, S167L, S167A, I209F, V287del, Y280C,
and P322S) are associated with a milder form of NDI, char-
L292P, A294P) (43,53–60). Recently, Morello et al. dem-
acterized by a later clinical presentation, around the age of
onstrated a prolonged interaction time between the molec-
10, and without growth retardation. Functional studies of
ular chaperone calnexin and several class II V2R mutant
these mutations by in vitro expression systems have con-
proteins and suggested that calnexin could play a role in the
firmed the partial phenotype of the NDI. P322S is the most
intracellular retention of misfolded V2R proteins (61). This
remarkable of these three mutations, since another mutation
finding is consistent with the dual role proposed for cal-
substituting proline 322, namely P322H, is associated with a
nexin. On the one hand, this chaperone is believed to assist
severe phenotype. By in vitro expression of both P322H and
folding of neosynthesized wild-type proteins, whereas on
P322S in COS-7 cells, Ala et al. (58) have shown that the
the other hand it targets the incompletely synthesized or
P322H mutant had totally lost the ability to stimulate the
misfolded proteins toward the degradation pathway (62).
Gs/adenylate cyclase system, whereas the P322S mutant was
A few of the missense and frame-shift mutations, how-
able to stimulate adenylate-cyclase, albeit less than the
ever, have been shown not to significantly alter protein
wild-type receptor. Thus, the in vitro experiments closely
abundance at the cell surface, but either impair the cou-
correspond to the clinical phenotype. On the basis of three-
pling efficiency of the receptor to the stimulating G protein
dimensional modeling of the P322H and P322S mutant
(class III mutations) or reduce AVP-binding affinity (class
receptors, a plausible hypothesis to explain the molecular
IV mutations). As yet, only a few mutations have been
basis for the mild phenotype of the P322S has been pro-
identified that belong to class III. These mutations are
posed. Based on this modeling it is suggested that complete
found in the lower half of the transmembrane regions
loss of function of the P322H receptor could be due, in part,
(D85N, P322S/P) and within the second intracellular loop
to hydrogen bond formation between the His322 side chain
(R137H) (58,63). The study by Rosenthal et al. (63) dem-
and the carboxyl group of Asp85, which does not occur in
onstrating disturbed G protein activation of the in vitro–
the P322S receptor (58).
expressed R137H mutation, stresses the importance of the
Recently, a family was reported in which the R137H
highly conserved DRY/H motif in the second intracellular
mutation was associated with severe NDI in the proband
loop for G protein activation, which is also known for
but with very mild NDI in his affected brother (67).
other G protein–coupled receptors (64). In conflict with
Genetic or environmental modifying factors are likely to
the study by Rosenthal, others have reported that the
account for this intrafamilial phenotype variability.
R137H mutant is almost completely retained in the cell
interior (55).
Class IV mutations are mainly found in the first, sec- AUTOSOMAL RECESSIVE AND AUTOSOMAL
ond, sixth, and seventh transmembrane domains (L44F, DOMINANT FORMS OF NEPHROGENIC
A84D, V88M, Y128S, P286R, F287L, V277A, S315R) and DIABETES INSIPIDUS: MUTATIONS IN
in the first and second extracellular loops (W99R, R106C, THE AQUAPORIN-2 WATER CHANNEL
F105V, R181C, G185C, ΔR202, R202C, T204N, Y205C,
V206D, R113W) (43,53,56,59,61,65). It is well known Both the autosomal recessive and the autosomal dominant
that the conserved disulfide bridge between Cys112 in the types of NDI are caused by mutations in the AQP2 water
first and Cys192 in the second extracellular loop ensures channel gene (Genbank accession number z29491). The
correct folding of these loops and, as a consequence, the human AQP2 gene is a small gene consisting of 4 exons,
establishment of the ligand-binding domain. It is assumed comprising 5 kb genomic DNA. The 1.5-kb mRNA
that the introduction of additional cysteine residues as a encodes a protein of 271 amino acids, which has a pre-
result of mutations found in NDI (R106C, R181C, dicted molecular weight of 29 kDa (68). AQP2 belongs to
G185C, R202C, Y205C) might impair receptor function a family of membrane integral proteins, AQPs, which func-
by altering disulfide bonding of the conserved cysteine res- tion as selective water transporters throughout the plant
idues (58) or, more likely, by introducing an additional and animal kingdom. In mammals, ten different AQPs
disulfide bond (66). Based on the finding of class IV muta- have been identified to date, seven of which (AQPs 1 to 4
tions in transmembrane domains (I, II, VI), it can be con- and 6 to 8) are highly expressed in the kidney. Like other
cluded that several amino acids in these domains are AQPs, AQP2 is assembled in the membrane as a homotet-
involved in forming the conformation of the receptor for ramer in which each 29-kDa monomer, consisting of six
agonist binding. membrane-spanning α-helical domains and intracellular
782 VI. Tubular Disease

meability (71,73,75,76). This indicates that at high expression

levels, these AQP2 mutant proteins escape from the ER and
are routed to the plasma membrane, where they are functional.
Several families have been described with autosomal
dominant NDI, based on the transmission of the disease
from father to son. In one of these families, a point muta-
tion (G866A) in one allele of the AQP2 gene, resulting in
the substitution of a lysine for a glutamic acid at position
258 (E258K) in the C-terminal tail of AQP2, was identi-
fied. Expression studies in X. laevis oocytes have shown that
this E258K–AQP2 mutant is a properly folded functional
water channel but is retained in the Golgi region (38) (Fig.
40.3). In coexpression studies with wild-type AQP2, a dom-
inant-negative effect was observed, caused by impaired rout-
ing of wild-type AQP2 to the plasma membrane after
hetero-oligomerization with the E258K mutant (77). Subse-
quently, a similar impairment of wild-type AQP2 in its
FIGURE 40.3. Schematic representation of the postulated routing to the plasma membrane of oocytes was reported for
mechanisms in a collecting duct cell to explain recessive and three other dominant AQP2 mutants (721delG, 763-
dominant forms of nephrogenic diabetes insipidus (NDI) caused 772del, 812-818del) (78). Very recently, a novel AQP2
by aquaporin-2 (AQP2) water channel mutations. Normal AQP2
proteins are transported from the endoplasmic reticulum via the mutant (727delG) identified in a family with dominant
Golgi apparatus to the luminal side of the cell. In case of a reces- NDI was shown to interfere with the routing of wild-type
sive NDI mutation, AQP2 is retained in the endoplasmic reticu- AQP2 to the apical membrane by its mistargeting to the late
lum due to misfolding and is subsequently broken down by
proteosomes. In case of a dominant NDI mutation, there is a endosomes or lysosomes (79).
change in the transport signal of the AQP2 protein and conse- Remarkably, all mutations causing dominant NDI are
quent misrouting to the Golgi network or late endosomes/lyso- located in the C-terminal tail of AQP2. The cell-biologic
somes. (Adapted from Knoers NVAM, Deen PMT. Van Gen naar
Ziekte; van vasopressine-V2-receptor en aquaporine-2 naar basis of dominant NDI, as revealed by in vitro expression of
nefrogene diabetes insipidus. Dutch J Med 2000;144:2402– the mutations, underscores the importance of the C termi-
2404.) (See Color Plate 40.3.) nus of AQP2 in trafficking to the cell surface.
Kanno et al. (80) showed that AQP2 is detectable in the
N- and C-termini, is a functional water channel. The six urine of normal individuals and, after treatment with
transmembrane domains are connected by five loops (A DDAVP, in the urine of patients with central diabetes insipi-
through E). In the hourglass hypothesis, loops B and E are dus but not in that of patients with NDI. These initial find-
assumed to fold back into the membrane and to interact via ings, together with additional examinations of AQP2
their highly conserved motifs asparagine-proline-alanine excretion (81), raised the possibility of assessing AQP2 levels
(NPA boxes) to form the water pore (69). Cryo-EM analy- in the kidney by the measurement of urinary AQP2 levels.
sis of AQP1 crystals at atomic resolution revealed that this However, because the amount of AQP2 in the urine appears
was indeed the case (70). AQP2 is exclusively localized in to be determined largely by trafficking of AQP2 to the api-
the apical membrane and a subapical compartment of col- cal membrane of collecting duct cells in response to vaso-
lecting duct cells. It is upregulated by dehydration or AVP, pressin, rather than their content of AQP2 (82), AQP2
indicating that it is the AVP-regulated water channel. excretion in the urine must be interpreted with caution with
To date, 29 putative disease-causing mutations in AQP2 respect to predicting AQP2 expression levels.
have been identified in families with autosomal recessive NDI
(42,43,71,72). These include 23 missense mutations, two
Differential Diagnosis between the
nonsense mutations, two small deletions, and two splice-site
X-Linked and the Autosomal Forms
mutations. Approximately one-half of these mutations are
of Nephrogenic Diabetes Insipidus
found in the B and E loops of the protein, where it is likely
that they will destroy the pore function of the protein. Expres- Patients with X-linked NDI can be discriminated from
sion studies in Xenopus laevis oocytes have revealed that all 23 patients with autosomal NDI on the basis of their extrare-
AQP2 missense mutations that cause recessive NDI are class II nal reaction to administration of the synthetic V2-vaso-
mutations. Thus, these mutations lead to misfolding of the pressin analogue 1-DDAVP. Patients with autosomal NDI
mutant protein and retention in the ER (71,73,74) (Fig. show normal increases in von Willebrand factor, factor
40.3). At high expression levels in oocytes and Chinese ham- VIII, and tissue-type plasminogen activator levels, whereas
ster (CHO) cells, six of these AQP2 mutants (A147T, in patients with X-linked NDI, these extrarenal responses
T126M, G64R, L22V, A47V, and T125M) confer water per- are absent as a result of an extrarenal mutant V2R (83).
 40. Nephrogenic Diabetes Insipidus 783

NEPHROGENIC DIABETES reduced AQP3 expression also plays a crucial role in the
INSIPIDUS IN FEMALES development of lithium-induced polyuria. The mechanism of
AQP3 downregulation remains to be elucidated.
Several families have been described in which females show Other polyuric states that have been associated with
classic clinical and laboratory features of NDI. After the downregulation of AQP2 expression in rat renal medulla
identification of AQP2 mutations as a cause for autosomal include chronic hypokalemia, hypercalcemia, bilateral ure-
recessive NDI, and in some cases for autosomal dominant teral obstruction, low-protein diet, chronic renal failure,
NDI, a satisfying explanation for the complete manifesta- and puromycin aminonucleoside- or doxorubicin-induced
tion of the disease in some females had been found. How- nephrotic syndrome (91–95).
ever, several families have been reported in which In some families with autosomal primary nocturnal
symptomatic females do not have an AQP2 defect but are enuresis, linkage analysis has revealed cosegregation of this
heterozygous for a V2R defect (84–87). In some of these disorder with polymorphic microsatellite markers from
women, maximal urinary osmolality after DDAVP admin- 12q, in the region where the AQP2 gene maps (96). Deen
istration does not exceed 200 mOsmol/L. Of interest, in et al. sequenced the AQP2 coding region in affected pri-
some of the reported families, asymptomatic female family mary nocturnal enuresis individuals from these dominant
members shared the same V2R mutation with the manifest- families but did not identify any significant mutations.
ing females (84,88). The most likely explanation for the Thus, the AQP2 gene could be excluded as a candidate for
existence of different phenotypes in carriers of a V2R muta- autosomal primary nocturnal enuresis. (97).
tion, varying from no symptoms to complete manifestation
of the disorder, is skewed X-inactivation. This hypothesis
was underlined by the study of Nomura et al., investigating TREATMENT
the X-inactivation patterns of female carriers belonging to
one family via the detection of a methylated trinucleotide The treatment of NDI has been challenging since the origi-
repeat in the human androgen receptor gene (88). The V2R nal description of the disorder. Replacement of urinary
mutation carriers in this family displayed different degrees water losses by adequate supply of fluid is the most impor-
of manifestation of NDI. One woman, who had no symp- tant component of therapy. However, most infants with
toms at all, showed random X-inactivation, whereas her NDI cannot drink the required amounts of fluid. One
grandmother with complete NDI showed extremely approach to reduce urine output is provision of a low-
skewed methylation of one X chromosome. One should solute diet to reduce the renal osmolar load and decrease
keep in mind that this skewed X-inactivation pattern in obligatory water excretion. Initially, a diet low in sodium (1
blood cells does not necessarily reflect the situation in renal mmol/kg/day) as well as protein (2 g/kg/day) was recom-
collecting duct cells. mended. However, severe limitations of dietary protein
may introduce serious nutritional deficiencies. Therefore, it
is preferable to prescribe dietary restriction of sodium only.
ACQUIRED NEPHROGENIC Diuretics, such as hydrochlorothiazide (2 to 4 mg/kg per
DIABETES INSIPIDUS 24 hours), were the first class of drugs shown to be effective
in lowering the urine volume in NDI (98). When com-
Although the hereditary forms of NDI are relatively rare, a bined with a reduction of salt intake, hydrochlorothiazide
wide range of pathologic conditions and drug treatments can reduces urine volume by 20 to 50% of baseline values.
lead to acquired NDI (Table 40.1). In a variety of these However, thiazide-induced hypokalemia may cause further
acquired forms of NDI, AQP2 expression is downregulated. impairment of urine-concentrating ability in patients with
For example, it has been shown that prolonged treatment of NDI. Another possible risk associated with hypokalemia is
rats with therapeutic doses of lithium causes a 95% reduc- cardiac arrhythmia. Simultaneous administration of potas-
tion in AQP2 expression in the inner medullary collecting sium salt is therefore advised in most cases.
duct principal cells, concomitant with the development of There is ample evidence that the combined administra-
severe polyuria (89,90). Cessation of lithium infusion or tion of hydrochlorothiazide with either a prostaglandin-
DDAVP treatment for 1 week only partially reversed this synthesis inhibitor such as indomethacin (2 mg/kg/24 hr), or
downregulation, consistent with the clinical observation of the potassium-sparing diuretic amiloride, is much more
slow recovery from induced NDI in patients treated with effective in reducing urine volume than the thiazide diuretic
lithium. It is assumed that the inhibition of vasopressin- alone (99–103). Prolonged use of prostaglandin-synthesis
induced adenylate cyclase activity by lithium is the cause of inhibitors, however, is often complicated by gastrointestinal
the reduced expression of AQP2. and hematopoietic side effects. Gastrointestinal com-
It has recently been demonstrated that there is also marked plaints and complications include anorexia, nausea, vomit-
downregulation of AQP3, but not of AQP4, expression in ing, abdominal pain, ulceration, perforation, and hemorrhage.
kidneys of rats treated with lithium (90). It is assumed that Hematopoietic reactions include neutropenia, thrombocyto-
784 VI. Tubular Disease

penia, and, rarely, aplastic anemia. In addition, renal dys- mutants could be rescued by the same treatment is promis-
function has been described during indomethacin therapy, ing, given the diversity of mutations identified in NDI.
most often consisting of a slight reduction in glomerular fil- Similarly, it has been shown that treatment of cells
tration rate. expressing ER-retarded AQP2 mutants with chemical chap-
Amiloride counterbalances the potassium loss from pro- erones, such as glycerol, facilitated the translocation of these
longed use of thiazides and thus prevents hypokalemia. mutants to the plasma membrane (76). The feasibility of
Because amiloride appears to have only minor long-term side treatment with pharmacologic and chemical chaperones
effects, the combination of hydrochlorothiazide (2.0 to 4.0 awaits in vivo testing. It is important to appreciate that the
mg/kg/24 hr) with amiloride (0.3 mg/kg/24 hr) is the treat- exact ER-retention mechanisms of NDI causing V2R and
ment of choice for most patients. Our personal experience of AQP2 mutants remain to be elucidated. In vitro studies have
more than 12 years with the amiloride-hydrochlorothiazide suggested that, in the future, gene therapy for NDI may
combination, however, indicates that amiloride is less well become technically feasible (109). There are, however, ques-
tolerated in young children below the age of 4 to 6 years tions as to whether gene therapy would be the treatment of
because of persistent nausea. Therefore we recommend the choice for this disorder. Development of gene-transfer vec-
use of the combination of indomethacin-hydrochlorothiaz- tors and gene-delivery techniques as well as analysis of gene-
ide in these young children. therapy safety need to be pursued. Such studies determine
For a long time, the following mechanism for the para- the feasibility of gene therapy in renal diseases such as NDI.
doxic effect of thiazides in NDI has been proposed; thiazides In this respect, recently developed transgenic mice carrying
reduce sodium reabsorption in the distal tubule by inhibition a functionally inactive V2R receptor protein (110) and
of the NaCl cotransporter. This subsequently results in AQP2 knock-in mice (111) will be important reagents for
increased sodium excretion, extracellular volume contraction, preclinical testing.
decreased glomerular filtration rate, and increased proximal
sodium and water reabsorption. Consequently, less water and
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obstruction downregulates expression of the vasopressin- misfolded V2 vasopressin receptor mutants. J Clin Invest
sensitive aquaporin-2 water channel in rat kidney medulla. J 2000;105:887–895.
Am Soc Nephrol 1995;6:1012. 109. Schöneberg T, Sandig V, Wess J, et al. Reconstitution of
93. Teitelbaum I, Strasheim A, McGuinness S. Decreased aqua- mutant V2 vasopressin receptors by adenovirus-mediated
porin-2 content in chronic renal failure. J Am Soc Nephrol gene transfer of a receptor fragment: molecular basis and
1996;7:1273. clinical implication. J Clin Invest 1997;100:1547–1556.
94. Sands JM, Naruse M, Jacobs JD, et al. Changes in aqua- 110. Yun J, Schoneberg T, Liu J, et al. Generation and phenotype
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in rats fed a low protein diet. J Clin Invest 1996;97:2807– pressin receptor gene. J Clin Invest 2000;106:1361–1371.
2814. 111. Yang B, Gillespie A, Carlson EJ, et al. Neonatal mortality in
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medullary water channels expression in puromycin-amino- diabetes insipidus. J Biol Chem 2001;276:2775–2779.
 41

CYSTINOSIS AND FANCONI

SYNDROME
JOHN W. FOREMAN

In the 1930s, de Toni (1), Debré et al. (2), and Fanconi (3) 5. Dysfunction of the Na+,K+–adenosine triphosphatase
independently described several children with the combina- (ATPase) pump would increase the intracellular Na concen-
tion of renal rickets, glucosuria, and hypophosphatemia. The tration, diminishing the electrochemical gradient for Na
name of this clinical entity has been shortened to Fanconi syn- influx at the luminal membrane and its coupling to other
drome. Fanconi syndrome is a dysfunction of the proximal solute transport.
tubule leading to excessive urinary excretion of amino acids, 6. Finally, there could be a significant paracellular back-
glucose, phosphate, bicarbonate, and other solutes handled by flux of solute from increased permeability of the tight junc-
this nephron segment. These losses lead to the clinical prob- tions between proximal tubule cells.
lems observed in this syndrome (e.g., acidosis, dehydration,
electrolyte imbalance, rickets, and growth failure). Numerous In addition to membrane defects, abnormalities in cellu-
disorders, ranging from inborn errors of metabolism to exoge- lar organelle function could impair net solute reabsorption.
nous toxins, are associated with Fanconi syndrome. Examples include the apical endosome in Dent’s disease
and the lysosome in cystinosis. Numerous filtered proteins
are reabsorbed through binding to the endocytic receptors,
PATHOPHYSIOLOGY megalin and cubilin (Fig. 41.2). The protein-receptor com-
plex is then incorporated into an endosome. In the endo-
Currently, the sequence of events underlying Fanconi syn- some, the ligand and the receptor are disassociated and the
drome is incompletely defined. A number of possible receptor is recycled back to the luminal membrane and the
mechanisms exist that could lead to diminished net solute reabsorbed protein into lysosomes for further processing.
reabsorption in the proximal tubule (Fig. 41.1). At the This disassociation process is dependent on acidification of
membrane level, at least six possibilities exist: the endosome by an H+-ATPase and the ClC-5 chloride
channel. The importance of this pathway in Fanconi syn-
1. An intrinsic defect in carrier function could be the drome is underscored by the finding of low urinary levels of
cause. Because there are many different carriers, this is megalin in patients with Fanconi syndrome due to Dent’s
unlikely except for a defect in sodium binding common to disease and Lowe syndrome but not all causes of Fanconi
most or all of the carriers. syndrome (5). An abnormal endocytic pathway may affect
2. There may be a generalized defect in the insertion or the recycling of transport proteins, other than megalin,
recycling of these carriers into the brush-border membrane back to the luminal membrane, leading to decreased solute
or in a membrane lipid-protein carrier interaction necessary reabsorption. Furthermore, disturbances in energy genera-
for their complete function. tion could impair net transepithelial transport. Energy is
3. Solute reabsorption may be normal, but the resorbed necessary for operation of the Na+-K+-ATPase pump and
solute quickly leaks back into the lumen. This mechanism intracellular Na+ regulation and may be involved with sol-
has been implicated in the experimental Fanconi syndrome ute reabsorption in other undefined ways as well. A defect
induced by maleic acid (4). in energy generation has been raised as the underlying
4. There may be impairment in solute movement out of cause of Fanconi syndrome observed in a diverse group of
the cell at the basolateral membrane—this would lead to disorders, including hereditary fructose intolerance (HFI),
solute accumulation intracellularly, which would augment galactosemia, cytochrome-c-oxidase deficiency, and heavy
solute efflux across the luminal membrane, decreasing net metal poisoning. In experimental models of Fanconi syn-
transepithelial transport. drome (including those induced by maleic acid, succinylac-
790 VI. Tubular Disease

drome. Animals given cadmium or lead have abnormal ATP

levels and decreased Na+-K+-ATPase levels. Pathologically,
experimental heavy metal toxicity is associated with signifi-
cant abnormalities in renal tubule mitochondria. Outdated
tetracycline, with the formation of anhydro-4-epitetracy-
cline, causes reversible experimental Fanconi syndrome (15)
associated with decreased mitochondrial respiration (16).
An experimental model of cystinosis and Fanconi syn-
drome has been created by injecting rats and incubating renal
tubule cells with cystine dimethylester. Treated tubules had sig-
nificantly increased cystine levels, decreased tubular transport,
decreased metabolic fuel and O2 consumption, decreased ATP
levels, increased intracellular K+ concentrations, and decreased
mitochondrial respiration (17–19). ATP added to the incuba-
tion media partially restored the tubular transport function of
the cystine-loaded cells, suggesting that cystine storage in cysti-
FIGURE 41.1. Model of solute (S) transport by the human renal nosis impairs mitochondrial function and ATP generation,
proximal tubule cell. S uptake by the brush-border membrane
(BBM) from the lumen is coupled to Na+ influx. A favorable elec- leading to Fanconi syndrome. Succinylacetone has been pro-
trochemical driving force for luminal Na+ is maintained by the posed as the presumed toxin in tyrosinemia because of its
Na+-K+–adenosine triphosphatase pump. Transported S is then structural similarity to maleic acid. Normal rats injected with
either used by the cell or returned to the blood across the baso-
lateral membrane (BLM). Fanconi syndrome could arise because this compound develop proteinuria, glucosuria, and aminoaci-
of a defect of influx (1), leakage back into the lumen after influx duria (20). Renal tubules incubated with succinylacetone have
(2), decreased flux out of the cell across the BLM (3), a defect in reduced amino acid, sugar, and phosphate transport; ATP
energy generation or transduction to the plasma membrane (4),
an increase in backflux across the tight junctions between the levels; and O2 consumption. Succinylacetone inhibits renal
cells (5), and, finally, defective transporter recycling (6). ATP, brush-border membrane transport and alters membrane fluid-
adenosine triphosphate; TBM, tubular basement membrane; ity (21). Thus, succinylacetone may be the cause of Fanconi
MITO, mitochondrion.
syndrome in tyrosinemia.
Spontaneous Fanconi syndrome has been observed in
etone, and cystine dimethylester), decreased adenosine several dog breeds, especially the Basenji (22,23). These
triphosphate (ATP) production has been related to dogs developed polyuria, polydipsia, glucosuria, aminoaci-
impaired solute reabsorption. duria, and acidosis. The glomerular filtration rate (GFR)
was normal in most of the dogs, although some went on to
develop renal failure. Renal biopsies from affected dogs
Experimental Models of were normal except for large nuclei in a few tubular cells.
Fanconi Syndrome Solute uptake by renal cortical slices from the affected dogs
Maleic acid, the cis isomer of fumaric acid, has been used was also decreased. This model resembles the idiopathic
extensively to produce an experimental model of Fanconi human Fanconi syndrome.
syndrome (4,6–9). Injection of maleic acid in dogs and,
especially, rats leads to a reversible Fanconi syndrome with
aminoaciduria, glucosuria, phosphaturia, proteinuria, and SIGNS AND SYMPTOMS
bicarbonaturia. Maleic acid has been shown to inhibit
Na+-K+-ATPase (6,7) and decrease cellular ATP levels (6– Hyperaminoaciduria
8). The low ATP levels were associated with low kidney Aminoaciduria is one of the major characteristics of Fanconi
tissue coenzyme A levels (8), leading to the speculation syndrome (Table 41.1). Virtually every amino acid is found
that maleic acid irreversibly bound to coenzyme A and in excess in the urine, giving rise to the designation of gener-
impaired tricarboxylic acid cycle function. Maleic acid alized aminoaciduria. Reabsorption rates vary for each amino
also has been shown to impair the incorporation of phos- acid, with some approaching the GFR. The easiest method
phate into the cell membrane (7). Maleic acid also dis- for determining the renal handling of amino acids is to assess
rupts the apical endocytic pathway of the renal tubule the percentage of tubular reabsorption (Taa):
mediated by megalin, a receptor for a number of filtered
proteins (9) (Fig. 41.2). Whether any or all of these effects U aa × P aa
underlies Fanconi syndrome induced by maleic acid is T aa = 1– -------------------------- × ( 100% )
U cr ⁄ P cr
unclear.
Cadmium (10), lead (11,12), mercury (13), and uranium where Uaa and Paa are the urine and plasma amino acid con-
(14) have been used to induce experimental Fanconi syn- centrations and Ucr and Pcr are the urine and plasma creati-
 41. Cystinosis and Fanconi Syndrome 791

FIGURE 41.2. Megalin-endocytic pathway. Low-molecular-weight proteins in the luminal fluid

bind to megalin and are endocytosed. The recycling of megalin and further catabolism of these
proteins are dependent on acidification of the vesicle by a proton pump. The ClC-5 chloride
channel provides an electrical shunt for efficient functioning of the proton pump. This endocyto-
sis pathway may also play a role in membrane transporter recycling, and disruption of this path-
way could interfere with absorption of other luminal solutes.

nine concentrations. Normally, Taa for most amino acids is reflected by the excessive excretion of phosphate, except
more than 98%, except for glycine (95%) and histidine when Fanconi syndrome first develops. At a steady state,
(92%). The urinary amino acid losses in Fanconi syndrome urinary phosphate excretion simply reflects dietary intake
do not cause any clinical consequences because the losses similar to that observed in X-linked hypophosphatemic
are trivial in relation to dietary intake. However, plasma rickets, vitamin D deficiency, and normals. Excessive phos-
amino acid levels tend to be lower than normal. phate excretion is observed only when plasma phosphate is
above its steady-state level. Phosphate handling can be
assessed by measuring the percentage of tubular reabsorp-
Glucosuria
tion of phosphate, as described for amino acids. The tubu-
Glucosuria is one of the originally recognized features of lar reabsorption of phosphate ranges from 80 to 95% in
Fanconi syndrome. Glucosuria is the result of impaired normal children (24), with much lower values in patients
tubular reabsorption of glucose and is present despite nor- with Fanconi’s syndrome. A preferred way of estimating the
mal plasma glucose concentrations. It is often an early fea-
ture of Fanconi syndrome and indicates that the renal
threshold (i.e., the plasma level of a solute when it first
TABLE 41.1. FEATURES OF FANCONI SYNDROME
appears in the urine) is reduced.
Acidosis Hypokalemia
Dehydration Hypophosphatemia
Hypophosphatemia Glucosuria Hypouricemia
Growth retardation Polyuria
Hypophosphatemia secondary to impairment in phosphate
Hyperaminoaciduria Proteinuria
reabsorption is another important feature of Fanconi syn- Hypocarnitinemia Rickets
drome. Impaired phosphate reabsorption is usually not
792 VI. Tubular Disease

renal handling of phosphate is to derive the tubular maxi- drome. Despite dehydration, the urine is typically dilute.
mal rate of phosphate reabsorption (Tmp) related to GFR: Recurrent febrile episodes, as a consequence of the dehydra-
tion, are often the first sign of Fanconi syndrome in infants
Tmp/GFR = Sp – (Up × Scr/Ucr)
with cystinosis. Polyuria is secondary to the osmotic diuresis
where Sp is serum phosphate, Up is urine phosphate, Scr is from the excessive urinary solute losses. In some patients,
serum creatinine, and Ucr is urine creatinine, determined there also appears to be an associated defect in renal concen-
on simultaneous blood and urine specimens (25). trating ability, especially with prolonged hypokalemia.
Phosphate handling by the kidney is affected by a number
of factors, including parathyroid hormone (PTH) and vita-
min D levels. PTH levels have been found to be both normal Growth Retardation
and elevated in patients with Fanconi syndrome (26,27). Growth retardation is another common feature of Fanconi
Low levels of 1,25-dihydroxy vitamin D have been found in syndrome in children (34). The cause is multifactorial. Hypo-
some patients with Fanconi syndrome (28,29). Furthermore, phosphatemia, disordered vitamin D metabolism, and acido-
patients with the Fanconi syndrome appear to have impaired sis can impair growth. Chronic hypokalemia and extracellular
conversion of 25 vitamin D to 1,25-dihydroxy vitamin D volume contraction may be additional factors, but glucosuria
(30). Metabolic acidosis, another feature of Fanconi syn- and aminoaciduria probably are not. However, even with cor-
drome, also impairs the conversion of 25 vitamin D to 1,25- rection of all of these metabolic abnormalities, most patients
dihydroxy vitamin D. This may, in part, explain why alkali fail to grow, especially those with cystinosis.
therapy may heal rickets in some patients with Fanconi syn-
drome. Bone disease is common in patients with long-stand-
ing untreated Fanconi syndrome (31). Uricosuria
Impairment in renal handling of urate is often present in
Acidosis Fanconi syndrome, leading to hypouricemia, especially in
adults. Urolithiasis from the uricosuria has rarely been
Hyperchloremic acidosis, another feature of Fanconi syn- reported (35). This is probably because the urine flow and
drome, results from impaired bicarbonate reabsorption by pH are increased, inhibiting uric acid crystallization.
the proximal tubule [proximal renal tubular acidosis (RTA)].
In Fanconi syndrome, more than 30% of the normal filtered
bicarbonate load may be excreted when plasma levels are Proteinuria
normal, leading to low plasma bicarbonate levels (usually Proteinuria, another feature of Fanconi syndrome, is usu-
between 12 and 18 mEq/L), and a reduction in extracellular ally minimal, except when the Fanconi syndrome develops
fluid volume. Occasionally, there is an associated defect in in association with the nephrotic syndrome. The proteins
distal acidification, usually in association with long-standing lost in Fanconi syndrome (e.g., enzymes, immunoglobulin
hypokalemia or nephrocalcinosis. Ammoniagenesis usually is light chains, and hormones) are of low molecular weight
normal or increased because of the hypokalemia and acidosis, (1900 to 30,000 d) (36).
unless there is associated impairment of GFR.

Sodium and Potassium Losses ETIOLOGIES

Na+ and K+ losses may give rise to major clinical problems Patients with Fanconi syndrome can be classified into two
in patient’s with Fanconi syndrome. Far less than the nor- main categories: inherited and acquired (Table 41.2). In
mal 60 to 80% of the filtered Na+ load is reabsorbed in the adults, the most common cause of a persistent Fanconi syn-
proximal tubule in Fanconi syndrome. Part of this loss is drome is an exogenous toxin, such as a heavy metal,
related to impaired bicarbonate reabsorption, with the sub- whereas in children, the most common cause is an inborn
sequent urinary excretion of NaHCO3 and KHCO3. In error of metabolism, such as cystinosis.
some cases, Na+ and K+ losses are so great that metabolic
alkalosis and hyperaldosteronism result, simulating Bartter’s
syndrome despite the lowered bicarbonate threshold (32). Inherited Fanconi Syndrome
The clearance of K+ may be twice that of the GFR, and
Cystinosis
resultant hypokalemia has caused sudden death (33).
Cystinosis, or cystine storage disease, is an enigmatic disor-
der characterized biochemically by excessive storage of cys-
Dehydration
tine in the lysosomes of numerous organs, including the
Polyuria, polydipsia, and frequent bouts of severe dehydra- kidney (37). Three different types of cystinosis have been
tion are common symptoms in patients with Fanconi syn- distinguished on the basis of the clinical course and the
 41. Cystinosis and Fanconi Syndrome 793

TABLE 41.2. CAUSES OF FANCONI SYNDROME most common mutation is a 57-kb deletion, but numerous
Inherited Acquired
other mutations have also been described. Ocular cystinosis
is associated with one severe and one mild mutation leading
Cystinosis Amyloidosis to the production of some functional protein (49). Presum-
Galactosemia Azathioprine ably there is enough lysosomal cystine transport to prevent
Glycogenosis Chinese medicine
Hereditary fructose intolerance Cisplatin
the kidney and other organ dysfunction, but not enough to
Idiopathic Diachrome poisoning prevent corneal crystal formation. Patients with later onset
Lowe syndrome Gentamicin or slower disease progression have been shown to have
Mitochondrial cytopathies Glue sniffing mutations that result in some functional protein, which
Tyrosinemia Heavy metal poisoning results in less lysosomal cystine transport than occurs in
Wilson’s disease Ifosfamide
Dent’s disease Light-chain proteinuria
ocular cystinosis (50).
Mesenchymal tumors The mechanism(s) by which increased intracellular cys-
Multiple myeloma tine levels lead to renal injury and dysfunction remains unex-
Nephrotic syndrome plained in cystinosis. Lysosomal cystine concentration rises
Ranitidine to levels sufficient to form crystals, which presumably cause
Renal transplantation
Sjögren’s syndrome
cell injury and death. Foreman et al. (17) have shown that
Streptozotocin loading normal rat renal tubules with cystine impairs solute
Suramin transport. Furthermore, cystine-loaded tubules have reduced
metabolic fuel and O2 consumption, ATP levels, and mito-
chondrial oxidation (18,19,51). These studies suggest that
intracellular cystine content (38). Benign or ocular cystino- the intracellular storage of cystine may impair energy genera-
sis is associated with cystine crystals only in the cornea and tion, which leads to defects in solute reabsorption.
bone marrow and the smallest increase in intracellular cys- In nephropathic cystinosis, high intracellular cystine con-
tine levels (39). There is no renal disease. Infantile or neph- centrations can be found in virtually every cell and organ,
ropathic cystinosis is most common and is associated with including bone marrow, leukocytes, cornea, conjunctiva,
the highest intracellular levels of cystine (5 to 10 nmol/mg retina, lymph nodes, liver, spleen, intestine, thyroid, muscle,
protein) in leukocytes or cultured fibroblasts. Autopsy kid- and kidney. The brain was previously believed not to be
ney specimens contained 150 to 2000 nmol/mg protein of affected in cystinosis, but a few patients surviving into their
0.5 cystine (40). Children with nephropathic cystinosis 20s have developed neurologic disorders in association with
develop renal failure by late childhood. Patients with ado- increased cystine levels in brain tissue (52,53). The defini-
lescent cystinosis tend to have intracellular cystine levels tive method of making the diagnosis of cystinosis is the
between those of the infantile and the ocular forms. Renal demonstration of elevated intracellular levels of cystine, usu-
involvement occurs but does not appear until later and ally in polymorphonuclear leukocytes or fibroblasts. A pre-
progresses more slowly (41). natal diagnosis can be made by measuring the cystine
Cystinosis arises from a defect in the egress of cystine content of amniotic cells (54). Finding needle-shaped,
from lysosomes (42). Heterozygotes for cystinosis have rates tinsel-like refractile opacities in the cornea by slit-lamp
of lysosomal cystine egress that are one-half as fast as those examination is also usually diagnostic (Fig. 41.3). Such ocu-
from normal individuals, while homozygotes for nephro- lar findings are invariably present after 2 years of age.
pathic cystinosis have very low rates (42). This is due to a
mutation in the CTNS gene, encoding the protein cysti-
nosin, which is located on the short arm of chromosome 17
(43). Cystinosin is an integral lysosomal membrane protein
composed of 367 amino acids with seven transmembrane
domains and a C-terminal GYDQL sorting motif that
directs it to the lysosomal membrane (43). It mediates the
egress of cystine from the lysosome (44). Cystine transport
is stimulated by an outwardly directed pH gradient (44).
ATP also stimulates cystine egress through lysosomal acidifi-
cation (45). The origin of lysosomal cystine in cystinosis is
mainly from lysosomal protein degradation (46), but it is
also from plasma cystine (47). There is no defect in cystine
reduction or degradation in cystinosis (48).
Nephropathic cystinosis, with an estimated incidence of
1 in 200,000 births, is caused by a severe mutation on both FIGURE 41.3. Tinsel-like refractile opacities in the cornea of a
chromosomes that leads to no functional protein (43). The patient with cystinosis under slit lamp examination.
794 VI. Tubular Disease

The first clinical signs and symptoms of cystinosis are phy, interstitial fibrosis, and abundant crystal deposition
polyuria, polydipsia, anorexia, failure to thrive, dehydra- occur. In the glomerulus, there is giant-cell formation of
tion, and hyperchloremic metabolic acidosis, which usually the visceral epithelium, segmental sclerosis, and eventual
appear in the latter half of the first year of life (55). Subtle obsolescence. Hyperplasia and hypertrophy of the juxta-
abnormalities of tubular function can be demonstrated ear- glomerular apparatus have been noted and probably are
lier in families with index cases, including glucosuria, gen- related to abnormalities of the renin-angiotensin system.
eralized aminoaciduria, and mild proteinuria. Tubular Electron microscopy studies have demonstrated crystalline
reabsorption of phosphate is decreased with the develop- inclusions surrounded by a single limiting membrane,
ment of hypophosphatemia and rickets. The renal thresh- which electron diffraction has shown to be cystine. Peculiar
old for bicarbonate is usually between 12 and 15 mEq/L. dark cells, unique to the cystinotic kidney, have also been
Hypokalemia and hypouricemia are common. Rarely, observed. These cells have a uniform darkening of their
hyponatremia and metabolic alkalosis have been observed cytoplasm, probably reflecting a reaction between osmium
(32). Medullary calcinosis detected by renal ultrasound is tetroxide and cystine. These cells appear principally in the
relatively common (56), and a few patients have developed interstitium and visceral glomerular epithelium, but also in
renal calculi (35). Children with cystinosis usually have fair the loop of Henle, collecting duct, capillary endothelium,
complexion and blond hair, but this is not uniform. Cysti- mesangium, and arteriolar smooth muscle layer.
nosis occurs in blacks but is less common than in whites. Symptomatic treatment of nephropathic cystinosis involves
Before the use of cysteamine, the GFR always declined in early management of the tubulopathy and later management
patients with nephropathic cystinosis, and end-stage renal of renal failure. Acidosis and hypokalemia can be treated with
disease developed between 7 and 10 years of age. Chronic potassium citrate. Rickets responds to vitamin D and phos-
dialysis and transplantation are well tolerated by children phate supplementation. Low plasma and muscle carnitine
with cystinosis. Successful renal transplantation reverses the levels have improved with prolonged supplementation (61).
renal failure but does not improve the other symptoms of Careful attention to fluid and electrolyte replacement during
cystinosis (57). Cystine accumulates in the monocytes and gastroenteritis is particularly important.
interstitial cells of transplanted kidneys, but symptoms of The specific therapy for cystinosis is to lower tissue cystine
proximal tubule dysfunction typical of cystinosis have not levels. The best agent for this is cysteamine, which has been
been noted. Photophobia is common and usually appears shown to lower intracellular cystine levels (Fig. 41.4) and to
between 3 and 6 years of age. The severity of this symptom slow the decline in GFR, particularly in children with normal
increases with age. In addition to corneal and conjunctival renal function treated before 2 years of age (59,62) (Fig. 41.5).
crystal deposits, a peripheral retinopathy can occur. The Cysteamine therapy also improves linear growth (59,62) but
appearance of this is variable, with either hyperpigmented does not improve the Fanconi syndrome. The most common
areas or the juxtaposition of hypopigmented and hyperpig- problems associated with cysteamine are nausea, vomiting, and
mented areas in a salt-and-pepper pattern. Retinopathy a foul odor and taste. These problems preclude its use in some
affects the temporal more than the nasal side but occurs sym- patients. The best formulation is cysteamine bitartrate (Cysta-
metrically in both eyes. Retinopathy tends to appear later in gon). Absorption of this formulation is equal to cysteamine
the course of the disease and to be progressive, although it
has been observed in the first year of life. A few patients have
developed visual impairment and blindness (55,57).
Growth failure is another major feature of cystinosis and,
usually, is evident during the first year of life and before a sig-
nificant decline in GFR. It occurs despite careful attention to
electrolyte and mineral deficiencies. The appearance of renal
failure and hypothyroidism further aggravates growth failure.
Some patients have had improved growth after renal trans-
plantation; however, the final height of patients with cystino-
sis is reduced. Recombinant growth hormone has improved
linear growth (58), as has reducing intracellular cystine levels
with cysteamine (59).
The morphologic features of the kidney in cystinosis
vary with the stage of the disease (60). Early in the disease,
FIGURE 41.4. Effect of cysteamine (MEA) on lysosomal cystine
cystine crystals are present in tubular epithelial cells, inter- (CYS-CYS). In cystinosis, the transporter for cystine egress from
stitial cells, and, rarely, glomerular epithelial cells. A swan- the lysosome is defective. Cysteamine can easily enter the lyso-
neck deformity or thinning of the first part of the proximal some and combine with cystine, forming cysteine (CYS) and the
mixed disulfide cysteamine–cysteine (MEA-CYS). Both of these
tubule can be observed, but this is not unique to cystinosis. compounds can exit the lysosome via a transporter different
Later in the course of the disease, pronounced tubular atro- from the cystine carrier.
 41. Cystinosis and Fanconi Syndrome 795

late-onset complications highlight the need to continue cys-

teamine therapy throughout life.

Galactosemia
Galactosemia is an autosomal recessive inherited disorder of
galactose metabolism. The most common type is the result
of deficient activity of the enzyme galactose-1-phosphate
uridyl-transferase, known as transferase deficiency galac-
tosemia, with an incidence of 1 in 62,000 live births. The
gene for galactose-1-phosphate uridyl-transferase is found
on the short arm of chromosome 9 (69). This enzyme cata-
lyzes the reaction of galactose-1-phosphate (Gal-1-PO4)
plus uridine diphosphate glucose to uridine diphosphate
galactose plus glucose-1-phosphate. Uridine diphosphate
galactose can then be further metabolized to either glucose
or CO2 and H2O via glycolysis. Deficiency of this enzyme
leads to the intracellular accumulation of galactose-1-phos-
phate with damage to the liver, proximal renal tubule, ovary,
brain, and lens. A less common cause of galactosemia is a
FIGURE 41.5. Mean creatinine clearance as a function of age in deficiency of galactose kinase, which forms galactose-1-
normal subjects, 17 patients with cystinosis who had received ade- phosphate from galactose. Cataracts are the only manifesta-
quate therapy with cysteamine (started before 2 years of age and
leukocyte cystine level less than or equal to 2 nmol of 0.5 cystine/ tion of this form of galactosemia.
mg protein), 32 who had received partial treatment, and 67 who Affected infants ingesting milk containing lactose, the
had not received treatment. (From Markello TC, Bernardini IM, most common source of galactose in the diet, rapidly
Gahl WA. Improved renal function in children with cystinosis
treated with cysteamine. N Engl J Med 1993;328:1157–1162, with develop vomiting, diarrhea, and failure to thrive. Most also
permission.) manifest jaundice and unconjugated hyperbilirubinemia
and may have severe hemolysis. Continued intake of galac-
tose leads to hepatomegaly and cirrhosis. Cataracts appear
HCl, and leukocyte cystine levels were lower after 3 weeks of within days after birth, although they often are detectable
therapy compared with cysteamine HCl, possibly reflecting only by a slit-lamp examination. Mental retardation may
better patient compliance with this formulation (63). The cur- develop within a few months. Fulminant Escherichia coli
rent recommendation for treatment of cystinosis is to initiate sepsis has been described in a number of infants (70); it may
cysteamine soon after the diagnosis is made and to increase be a result of inhibition of leukocyte bactericidal activity.
dosage over 4 to 6 weeks to 1.3 g/m2/day in four divided In addition to these clinical findings, galactose intake in
doses. Slowly increasing the dosage minimizes the risk of a patients with galactosemia leads, within days, to hyperami-
serum sickness–like reaction. Leukocyte cystine levels should noaciduria and albuminuria. Melituria, which is a cardinal
be checked every 3 to 4 months to monitor effectiveness and feature of Fanconi syndrome, is principally a result of galac-
compliance. The goal of therapy is to maintain cystine levels tosuria and not glucosuria. There seems to be little or no
below 1.0 nmol 0.5 cystine/mg protein (63). impairment in glucose handling by the renal tubule. Galac-
With the use of dialysis and renal transplantation to tosemia should be suspected when there is a urinary reduc-
improve survival, a number of new complications of cystino- ing substance that does not react in a glucose oxidase test.
sis have been recognized (57). Common problems include However, finding galactose in the urine does not establish
hypothyroidism, splenomegaly, hepatomegaly, decreased the diagnosis because it can be seen in severe liver disease,
visual acuity, and corneal ulcerations. A few patients have in the first week of life in normal newborns (71) and even
developed insulin-dependent diabetes mellitus after renal for longer periods in premature infants (72), and in some
transplantation in association with cystine crystal deposition children with a high consumption of milk (73). Galacto-
in the pancreas (64). Of greater concern, several patients suria disappears in affected infants after the withdrawal of
have developed progressive neurologic disorders (52,53). galactose, which can obscure the diagnosis. Confirmation
Cortical atrophy has also been noted (65). A generalized of a suspected diagnosis should be made by demonstrating
myopathy and swallowing difficulties attributed to muscular deficient transferase activity in red blood cells, fibroblasts,
dysfunction have been observed in most patients older than leukocytes, or hepatocytes.
20 years (66). Patients also have a restrictive ventilatory The relationship of biochemical abnormalities to specific
defect attributable to this myopathy (67). Hypogonadism is symptoms of galactosemia is not clear. Accumulation of Gal-1-
common in adult male patients with cystinosis (68). These PO4 with the ingestion of galactose can inhibit a number of
796 VI. Tubular Disease

pathways for carbohydrate metabolism and correlates with RTA. The development of lactic acidosis adds significantly
some clinical symptoms. Defective galactosylation of pro- to the metabolic acidosis (82). Chronic fructose ingestion
teins has also been postulated. Formation of galactitol from may lead to nephrocalcinosis, impairing distal tubular
galactose by aldose reductase has been proposed as a pathoge- function as well. Although the onset of proximal tubule
netic mechanism and is at least responsible for cataract for- dysfunction is rapid after fructose loading, resolution of
mation. As to the renal tubular abnormalities, aminoaciduria these symptoms may take days or weeks (83).
has been induced in normal rats fed a high-galactose diet Acute fructose loading in the rat has been used as a model
(74) and in normal human subjects given galactose intrave- for HFI (84–86). Fructose loading leads to depletion of
nously (75). Experimentally, galactose inhibits amino acid intracellular phosphate as fructose-1-phosphate accumulates
uptake by slices of kidney cortex (76). and adenine deaminase is activated. Adenine deaminase con-
Galactosemia is treated by eliminating galactose from verts adenosine monophosphate to inosinic monophosphate
the diet. Acute symptoms and signs resolve within a few and ultimately to uric acid. A fall in adenosine monophos-
days. Cataracts also regress to some extent. However, even phate causes a drop in adenosine diphosphate and ATP,
with early elimination of galactose, developmental delay, because these levels are maintained at or near equilibrium by
speech impairment, ovarian dysfunction, and growth retar- adenylate kinase. Therefore, a drop in intracellular phosphate
dation are common outcomes in galactosemia. Profound can cause a depletion of ATP. Furthermore, phosphate deple-
intellectual deficits are rare even in suboptimally treated tion impairs ATP regeneration by mitochondria. Fructose
infants (77). infusion induces a fall in tissue ATP in normal humans as
well (87), and to an even greater extent in patients with HFI.
This fall in ATP may be the cause of Fanconi syndrome
Hereditary Fructose Intolerance
because, experimentally, ATP depletion impairs substrate
HFI is another disorder of carbohydrate metabolism associ- reabsorption in the proximal tubule.
ated with Fanconi syndrome (78,79). Patients with HFI Treatment of this disorder is strict avoidance of foods
experience nausea, vomiting, and symptoms of hypoglycemia containing fructose and sucrose. Most patients develop a
shortly after ingesting fructose, sucrose, or sorbitol. These strong aversion to such foods, making this interdiction easy.
symptoms may progress to convulsions, coma, and even The greatest risk occurs during infancy.
death, depending on the amount consumed. Concomitant
serum biochemical findings include decreases in serum in
Glycogenesis
glucose, phosphate, and bicarbonate and increases in serum
uric acid and lactic acid after fructose ingestion. Chronic A number of patients have been described with Fanconi syn-
exposure to fructose leads to failure to thrive, hepatomegaly, drome and glycogen storage in the liver and kidney (88–91).
jaundice, hepatic cirrhosis, and nephrocalcinosis. The syndrome is inherited as an autosomal recessive trait
HFI is inherited as an autosomal recessive trait with an and is characterized by heavy glucosuria along with other
incidence estimated to be 1 in 20,000. It is caused by a features of Fanconi syndrome. This has led to the name
deficiency of the enzyme fructose-1-phosphate aldolase B, renal glucose losing syndrome or Fanconi-Bickel syndrome
which cleaves fructose-1-phosphate into D-glyceraldehyde because the glucose losses can be massive. Fanconi syndrome
and dihydroxyacetone phosphate for further conversion may appear in the first few months of life and precede the
into glucose or CO2 and H2O. The gene for aldolase B is development of hepatomegaly from glycogen storage. The
found on the long arm of chromosome 9 (79). Aldolase B is degree of renal tubular dysfunction is often severe, and rick-
present in liver, small intestine, and proximal renal tubule ets, osteoporosis, and growth retardation are common. Glu-
cells. Symptoms of HFI appear at weaning when fruit, veg- cose tolerance tests are diabetic in pattern, but glucagon
etables, and sweetened cereals that contain fructose or stimulation leads only to a slight increase in blood sugar.
sucrose are introduced. Symptoms appear in the newborn Galactose metabolism also is impaired, but the activities of
period if the infant is fed a formula containing sucrose. the enzymes associated with galactose metabolism are nor-
Young infants with this disorder may have a catastrophic mal. Ketonemia, ketonuria, elevations in plasma lactate and
illness, with severe dehydration, shock, acute liver impair- pyruvate, and hyperlipidemia are also observed as in type I
ment, bleeding, or acute renal failure, with exposure to glycogen storage disease. A few patients with type I glycogen
fructose. The diagnosis should be suspected when charac- storage disease have been described with mild Fanconi syn-
teristic symptoms develop after the ingestion of fructose. drome (92).
Confirmation can be made either by performing a fructose Fanconi-Bickel syndrome is caused by a deficiency of the
tolerance test or by assaying the activity of fructose-1-phos- transporter, GLUT2, that facilitates the exit of glucose and
phate aldolase in a liver biopsy specimen. other hexoses from the basolateral side of the proximal
Abnormalities associated with Fanconi syndrome appear tubule and intestinal cell and the entry and exit of these
rapidly after the ingestion of fructose (80,81). Proximal sugars from the hepatocyte and pancreatic β-cell (93). The
bicarbonate reabsorption falls by 20 to 30%, leading to therapy of this disorder is directed at the renal solute losses;
 41. Cystinosis and Fanconi Syndrome 797

treatment of rickets, which can be severe; and frequent coni syndrome and may be the cause of the tubular
feeding to prevent ketosis. Uncooked cornstarch has been dysfunction of tyrosinemia. Experimentally, succinylacetone
shown to lessen hypoglycemia and improve growth (94). administration to rats leads to Fanconi syndrome (20,21).
Succinylacetone is also a potent inhibitor of D-aminolevulinic
acid dehydratase, leading to the accumulation of D-aminole-
Tyrosinemia
vulinic acid. This compound is neurotoxic and probably
Hereditary tyrosinemia type I, also known as hepatorenal causes the acute porphyria-like symptoms of tyrosinemia.
tyrosinemia, is an autosomally inherited defect of tyrosine Treatment with a low-phenylalanine and low-tyrosine diet
metabolism affecting the liver, kidneys, and peripheral dramatically improves the renal tubule dysfunction (99). In
nerves (95). The liver is the major organ affected in tyrosine- chronic cases, this diet leads to a rise in plasma phosphate,
mia, and liver dysfunction may be evident in the first month healing of rickets, a decrease in aminoaciduria, and the disap-
of life. Clotting abnormalities can be the initial sign of liver pearance of glucosuria and proteinuria. However, its efficacy
disease, with little elevation in transaminases. Infants may in halting the liver disease is uncertain. There is a risk of
also have obvious and severe signs of liver disease, and those inducing deficiencies of phenylalanine or tyrosine. There is
presenting at younger than 2 months of age have a high like- evidence for hepatic involvement prenatally, before any ele-
lihood of dying during the first year of life. Some children vations of tyrosine or methionine are evident, and this raises
are not diagnosed until after infancy; they have more indo- doubt that elevated levels of these amino acids are the cause
lent liver disease but are at risk for acute exacerbations of of liver injury (100). Liver transplantation has been success-
liver dysfunction. All children eventually develop macro- fully used to treat patients with severe liver failure and to pre-
nodular cirrhosis, and many develop hepatocellular carci- vent the development of hepatoma. Liver transplantation leads
noma. Acute, painful peripheral neuropathy may appear to rapid correction of Fanconi syndrome (101). Nitrotri-
and can lead to transient paralysis. Autonomic dysfunction fluorobenzoylcyclohexadione, which blocks p-hydroxyphenyl
with hypertension and tachycardia can be associated with pyruvate dioxygenase and the formation of maleylacetoace-
this acute neuropathy. Some renal tubular dysfunction is tate and fumarylacetoacetate, dramatically improves the renal
evident in all patients with tyrosinemia, especially those pre- and hepatic dysfunction (95).
senting after infancy. Rickets, secondary to the renal phos-
phate loss, can be severe. Generalized aminoaciduria, renal
Wilson’s Disease
tubular acidosis, and mild proteinuria are also often seen,
whereas glucosuria is less common because plasma glucose Wilson’s disease is a disorder of copper metabolism that
levels are usually low. Nephromegaly is common, and neph- affects numerous organ systems. Approximately 40% of
rocalcinosis may be seen. Glomerulosclerosis and impaired patients present with liver disease, 40% with extrapyramidal
GFR may be seen with time. Plasma tyrosine and methio- symptoms, and 20% with psychiatric or behavioral abnor-
nine levels usually are elevated in untreated patients. The malities (102). Although symptoms rarely occur before 6
hypermethioninemia imparts a cabbage-like odor to affected years of age, the most common presentation in children is
patients. In association with these amino acid abnormalities, with chronic active hepatitis or cirrhosis with little or no
p-hydroxyphenolic compounds, as a result of p-hydroxyphe- neurologic findings except those ascribable to hepatic failure.
nyl pyruvate dioxygenase inhibition, are elevated in the Greenish-brown rings at the limbus of the cornea, indepen-
urine. The presence of succinylacetone in blood or urine is dently described by Kayser (103) and Fleischer (104), are
diagnostic of hereditary tyrosinemia type I. usually present and are pathognomonic of Wilson’s disease.
A deficiency of fumarylacetoacetate hydrolase activity is The underlying problem in Wilson’s disease is a defect in a
the cause of hereditary tyrosinemia type I (96). The gene cod- P-type copper transporting ATPase in the liver. This leads to a
ing for this enzyme resides on the long arm of chromosome severe impairment in biliary copper excretion, the major excre-
15 (97,98). Patients with early onset of severe symptoms tend tory route for copper, and in the incorporation of copper into
to have no immunologically detectable fumarylacetoacetate ceruloplasmin (105). These abnormalities cause excessive
hydrolase peptide, whereas patients with a more chronic intracellular accumulation of copper in the liver, with subse-
course usually have detectable peptide. Decreased or absent quent overflow into other tissues such as brain, cornea, and
activity of fumarylacetoacetate hydrolase leads to accumula- renal proximal tubule. The gene for this enzyme has been
tion of maleylacetoacetate and fumarylacetoacetate in affected localized to chromosome 13q14.3, and multiple mutations
tissues. These compounds can react with free sulfhydryl have been described in patients with Wilson’s disease (105).
groups and reduce intracellular levels of glutathione. They Excessive storage of copper in the kidney leads to renal
also may be capable of acting as alkylating agents. Maleylace- tubular dysfunction in most patients and a full-blown Fan-
toacetate and fumarylacetoacetate are not detectable in coni syndrome in some (106). Hematuria also has been
plasma or urine but are converted to succinylacetoacetate. noted. Renal plasma flow and GFR decrease as the disease
Succinylacetone, a metabolite of succinylacetoacetate, is struc- progresses, but death from extrarenal causes occurs before the
turally similar to maleic acid, which is known to induce Fan- onset of renal failure. Tubular dysfunction overshadows the
798 VI. Tubular Disease

glomerular disease. Fanconi syndrome usually appears before retardation, hypotonia with diminished to absent reflexes,
the onset of hepatic failure and is characterized by intermit- and renal abnormalities (113,114). Initially, the renal
tent glucosuria; aminoaciduria; decreased phosphate reab- abnormalities are those of Fanconi syndrome but, later,
sorption leading to hypophosphatemia, osteomalacia, and GFR is impaired. The disorder is transmitted as an X-
rickets; and mild proteinuria. Hypouricemia secondary to a linked recessive trait mapped to Xp24-p26 (115). This gene
significant uricosuria is common and is a clue to the diagno- codes for a phosphatidylinositol bisphosphate phosphatase
sis (107,108). Hypercalciuria (109), with the development of (116) localized in the Golgi complex (117). A few females
renal stones and nephrocalcinosis (110), also has been have been reported (118,119). This may be explicable by
reported. Besides proximal tubular dysfunction, abnormali- the Lyon hypothesis, or there are several modes of inheri-
ties in distal tubular function, decreased concentrating abil- tance. Cataracts and punctuate lens opacities have been
ity, and distal RTA have also been observed. noted in mothers of affected males, and these findings have
Histologically, the kidney in untreated Wilson’s disease been proposed as a method of carrier detection (120).
shows either no alterations on light microscopy or only flat- Generalized aminoaciduria with relative sparing of the
tened proximal tubule cells without recognizable brush bor- branched-chain amino acids is a constant feature of Lowe
ders (107). Electron microscopy shows loss of the brush syndrome after the first several months of life. Proteinuria is
border, disruption of the apical tubular network, electron- present from infancy and often exceeds 1 g/m2 body surface
dense bodies (probably representing metalloproteins) in the area per day. Glucosuria is minimal and only intermittently
subapical region of tubule cell cytoplasm, and cavitation of present. Phosphate reabsorption is variably impaired, but
the mitochondria with disruption of the normal cristae pat- this impairment appears to worsen with age. Potassium
tern. Rubeanic acid staining shows intracytoplasmic copper reabsorption is also impaired and some patients require
granules. The copper content of kidney tissue is significantly supplementation. Proximal RTA, polyuria, and impaired
elevated. concentrating ability are common. The GFR usually only
The diagnosis of Wilson’s disease should be suspected in slowly declines with the appearance of end-stage renal fail-
children and young adults with unexplained neurologic dis- ure in the fourth decade of life.
ease, chronic active hepatitis, acute hemolytic crisis, behavioral The histology of the kidney by light microscopy is nor-
or psychiatric disturbances, or the appearance of Fanconi syn- mal early in the disorder (121,122). Swelling of endothelial
drome. In such patients, the presence of Kayser-Fleischer rings cells with thickening and splitting of the glomerular base-
is an important clue in making the diagnosis, but slit lamp ment membrane and fusion of the epithelial foot processes
examination may be necessary. Serum ceruloplasmin levels are were observed by electron microscopy. In the proximal
decreased in 96% of patients with Wilson’s disease, making tubule cells, there was shortening of the brush border and
this a useful screening test. However, normal values may be enlargement of the mitochondria, with distortion and loss
seen in young children with severe liver disease. A significantly of the cristae. With progression of the disease, there was
increased urinary copper level is also useful in making the diag- further thickening of the basement membrane, increased
nosis, especially if it increases significantly with D-penicil- cellularity of the glomeruli, glomerular fibrosis, tubular
lamine. Liver copper levels are increased in untreated patients; atrophy, dilation, and interstitial fibrosis.
however, in advanced cirrhosis this may not be the case. Treatment of Lowe syndrome is only symptomatic. The
Treatment with D-penicillamine reverses the renal dys- eye abnormalities usually require therapy early in infancy.
function and may reverse the hepatic and neurologic disease, Electrolyte and vitamin D supplements are needed by
depending on the degree of damage before the onset of ther- many patients for treatment of Fanconi syndrome. Renal
apy (111). Recovery, however, is slow. Trientine can also che- insufficiency usually develops in adulthood.
late copper and is indicated in patients who cannot tolerate
penicillamine. Tetrathiomolybdate is a potent agent in
Dent’s Disease
removing copper from the body and may be the drug of
choice for patients with neurologic disease to prevent the Dent’s disease is an X-linked recessive disorder characterized
immediate worsening of symptoms that can occur with peni- by low-molecular-weight proteinuria, hypercalciuria, neph-
cillamine. Zinc salts, which induce intestinal metallothionein rolithiasis, nephrocalcinosis, and, in some cases, rickets (123).
and blockade of intestinal absorption of copper, are useful in Affected males also have aminoaciduria, phosphaturia, and
maintenance therapy. Liver transplantation has been success- glucosuria. They also excrete β2-microglobulin in amounts
ful in some patients but should be reserved for those with that are 100-fold greater than normal. Another characteristic
liver failure (112). of affected males is severe hypercalciuria that leads to recur-
rent stone formation. These stones are usually made up of
calcium phosphate or a mixture of calcium phosphate and
Lowe Syndrome
oxalate. In addition, most affected males and some females
Lowe syndrome (oculocerebrorenal syndrome) is character- have medullary nephrocalcinosis. Rickets is a frequent prob-
ized by congenital cataracts and glaucoma, severe mental lem in children and osteomalacia in adults. Hypokalemia is
 41. Cystinosis and Fanconi Syndrome 799

also common. Renal failure is common and may occur by TABLE 41.3. MITOCHONDRIAL CYTOPATHIES
late childhood. The renal pathology is not pathognomonic
MERRF Myoclonic epilepsy with ragged red
and mainly shows interstitial nephritis with scattered calcium fibers
deposits. Hemizygous females usually only have low-molecu- NARP Neuropathy, ataxia, and retinitis pig-
lar-weight proteinuria and mild hypercalciuria. mentosa
X-linked recessive hypophosphatemic rickets, X-linked MELAS Mitochondrial encephalopathy, lactic
acidosis, and stroke-like episodes
recessive nephrolithiasis, and Japanese idiopathic low-molecu-
LHON Leber hereditary optic neuropathy
lar-weight proteinuria have similar features, and all are part of Leigh syndrome Maternally inherited Leigh syndrome
the clinical spectrum of an absent or inactive renal ClC-5 (somnolence, blindness, deafness,
chloride channel from a mutation in the CLCN-5 gene peripheral neuropathy, degenera-
located at Xp11.22 (124). A number of mutations have been tion of brainstem)
Pearson syndrome Pancytopenia, exocrine pancreatic
described, and the same mutation may result in a different
deficiency, hepatic dysfunction
phenotype in different patients, indicating that there are other Kearns Sayre syndrome Ophthalmoplegia, pigmentary reti-
genetic or environmental modifiers. The ClC-5 chloride nopathy, heart block, ataxia
channel resides in the membrane of endocytic vesicles just Alpers syndrome Intractable epilepsy, liver disease,
below the luminal membrane of the proximal tubule. Entry neuronal degeneration
of Cl– via this channel is necessary for acidification of the ves-
icle by the H+-ATPase pump. Inactivation of this channel
interferes with protein reabsorption and cell surface receptor
recycling, which may explain the hypercalciuria, phospha- The mitochondrion has its own DNA, which encodes for 13
turia, glucosuria, and aminoaciduria. Targeted disruption of protein subunits of 4 biochemical complexes; 2 ribosomal
the ClC-5 channel in a mouse led to a phenotype that was RNAs; 22 transfer RNAs; and machinery for DNA replica-
quite similar to that of patients with Dent’s disease (125). tion, transcription, and translation. Mitochondrial DNA
Treatment of Dent’s disease is largely supportive. High mutates ten times more often than nuclear DNA; has no
fluid intake is helpful for the stone disease. Calcium restric- introns, so mutations are more likely to affect DNA coding
tion reduces calcium excretion but probably adds to the sequences; lacks protective histones; and has an ineffective
bone disease. A pharmacologic dose of vitamin D heals the repair system. Mitochondria with their DNA are inherited
rickets but may worsen the hypercalciuria. Thiazide diuret- maternally and multiple mitochondria are present in each
ics reduce the hypercalciuria, but these patients are quite egg. Therefore, normal and mutant mitochondria can coexist
sensitive to them and may develop dehydration and within a cell, a condition termed heteroplasmy, and this pro-
hypotension. portion can vary from cell to cell and tissue to tissue. This
allows otherwise lethal mutations to exist and gives rise to the
variation in the phenotype of a particular mutation. Some
Mitochondrial Cytopathies
mitochondrial DNA mutations, mainly missense mutations,
In 1977, Biervliet et al. (126) described a family in which can be homoplasmic (i.e., affecting every mitochondrion).
three infants died from a mitochondrial myopathy in asso- Mitochondrial cytopathies most often present with neu-
ciation with lactic acidemia and Fanconi syndrome. Subse- rologic disorders such as myopathy, myoclonus, ataxia, sei-
quent to this report, two more infants with similar clinical zures, external ophthalmoplegia, stroke-like episodes, and
presentations were reported (127,128). The infants were optic neuropathy. Other manifestations include pigmentary
noted to have weak cries and a poor suck, with generalized retinitis, diabetes mellitus, exocrine pancreatic insufficiency,
hypotonia in the neonatal period. The hypotonia and siderocrestic anemia, sensorineural hearing loss, pseudoob-
weakness progressed in all affected infants, leading to respi- struction of the colon, hepatic disease, cardiac conduction
ratory failure and death. Examination of muscle cells from disorders, and cardiomyopathy. These various manifestations
these babies showed lipid-filled vacuoles in type I fibers and tend to group together in specific syndromes and reflect spe-
swollen mitochondria with abnormal cristae. Cytochrome- cific mutations in mitochondrial DNA (Table 41.3).
c-oxidase activity, an important enzyme in the electron- The most common renal manifestation associated with
transport chain, was very low in both muscle and kidney. mitochondrial cytopathies is Fanconi syndrome, although a
This deficiency, with its consequent impairment of oxida- few patients have had focal segmental glomerulosclerosis
tive phosphorylation, appeared to underlie the myopathy with nephrotic syndrome. All of the patients with renal
and renal tubulopathy. abnormalities have had extrarenal disorders, mainly neuro-
After these reports, it has been recognized that there is a logic diseases. Most patients presented in the first months
diverse group of diseases related to dysfunction of mitochon- of life and died soon after.
dria in various tissues, termed mitochondrial cytopathies A clue to these disorders is an elevated serum or cere-
(129,130). Most are caused by abnormalities in mitochon- brospinal fluid lactate level, especially if associated with an
drial DNA that lead to impaired oxidative phosphorylation. altered lactate to pyruvate ratio, suggesting a defect in
800 VI. Tubular Disease

mitochondrial respiration. The presence of ragged red reported tubular atrophy with interstitial fibrosis interspersed
fibers, a manifestation of abnormal mitochondria, in a with areas of tubular dilation. Significantly dilated proximal
muscle biopsy is another clue, especially with large abnor- tubules with swollen epithelium and grossly enlarged mito-
mal mitochondria on electron microscopy of muscle tissue. chondria with displaced cristae have also been noted.
Specific abnormalities of respiratory chain enzyme activity Therapy for idiopathic Fanconi syndrome remains
are best made by spectrographic analysis of isolated mito- symptomatic. Careful follow-up of these patients is neces-
chondria, tissue homogenates, circulating lymphocytes, or sary to prevent serious dehydration and electrolyte imbal-
cultured fibroblasts. Analysis of the mitochondrial DNA ance as well as severe bone disease. Renal transplantation
for deletions, duplications, or point mutations adds further has been done in a few patients who have reached end-stage
insight into the nature of the abnormality. renal disease (140). Fanconi syndrome has recurred in the
There is little to offer these patients in terms of definitive allograft without evidence of rejection, suggesting that in
therapy (131). Low electron transport chain complex III some cases, there is an extrarenal cause of idiopathic Fan-
activity can be treated with menadione or ubidecarenone. coni syndrome.
Deficient complex I activity may be treated with riboflavin
and ubidecarenone. Ascorbic acid has been used to minimize
oxygen free-radical injury. A high-lipid, low-carbohydrate Acquired Causes
diet has been tried in cytochrome-c-oxidase deficiency.
Exogenous Intoxications
Numerous substances can injure the proximal renal tubule,
Idiopathic Fanconi Syndrome
and this injury can range from an incomplete Fanconi syn-
A number of patients develop complete Fanconi syndrome drome to acute tubular necrosis and renal failure. The
in the absence of any known cause. These cases have been extent of the tubular damage is variable and depends on the
called idiopathic or primary Fanconi syndrome (26– type of toxin, the amount, and the host. A detailed history,
28,34,132–139). Originally, only adults were believed to be therefore, is important in patients with tubular dysfunction
affected; hence, it was called adult Fanconi syndrome. How- to discover whether a toxin is the cause.
ever, it is now clear that idiopathic Fanconi syndrome A major cause of proximal tubular dysfunction is heavy
occurs in children as well. All of the features of Fanconi metal intoxication. Lead poisoning induces Fanconi syn-
syndrome may not be present when the patients are first drome, principally in children (141,142). However, the
studied. Idiopathic Fanconi syndrome can be inherited in tubular dysfunction usually is overshadowed by the involve-
an autosomal dominant (27,132), autosomal recessive (29), ment of other organs, especially the central nervous system.
and even an X-linked pattern (134). However, most cases Generalized aminoaciduria is regularly present in children
occur sporadically, without any evidence of genetic trans- with moderate lead poisoning. Glycosuria and hypophos-
mission. The possibility of an underlying enzymatic or phatemia with impaired phosphate reabsorption can also
other abnormality that has not yet been described as caus- occur, but not as often. There is a rough correlation between
ing Fanconi syndrome should be kept in mind, especially in the severity of the intoxication and the completeness of
those in whom the syndrome is inherited. A number of Fanconi syndrome. In acute lead poisoning, the alterations
patients, in whom the known causes of Fanconi syndrome in renal histology are mainly in the proximal tubule and
have been excluded, still appear to have an underlying met- include eosinophilic intranuclear inclusions with mito-
abolic abnormality (136,137). Deal et al. (138) described chondrial swelling (143).
six infants from consanguineous marriages with idiopathic Cadmium intoxication also leads to Fanconi syndrome
Fanconi syndrome, ichthyosis, dysmorphism, jaundice, and (142,144), usually after a long exposure to the toxin. This
diarrhea who failed to thrive and died in infancy. poisoning was dramatized by a large number of cases
Children with idiopathic Fanconi syndrome usually appearing in the Jintsu River Basin in Japan after World
exhibit growth failure, recurrent dehydration, and rickets, War II. It was characterized by severe bone pain, which
despite an adequate intake of vitamin D. They often have gave rise to the name Itai-Itai (ouch-ouch) disease. Ami-
the other features of Fanconi syndrome, including polyuria, noaciduria, glucosuria, hypophosphatemia with impaired
polydipsia, hypokalemia, hypophosphatemia, RTA, ami- phosphate reabsorption, hyposthenuria, and RTA were also
noaciduria, glucosuria, and proteinuria. The GFR, usually, observed. The cause was traced to industrial contamination
is normal in childhood. The prognosis is variable. These of the soil and water with cadmium.
patients require careful management to prevent death from Certain organic compounds cause Fanconi syndrome in
dehydration and electrolyte imbalance. However, some humans. Outdated tetracycline causes a reversible Fanconi
develop chronic renal failure 10 to 30 years after the onset syndrome even in therapeutic doses (15,145,146). Affected
of symptoms (26,132,135,139). patients experience dizziness, muscular weakness, acidosis,
Renal morphologic descriptions of such cases are scanty. and, occasionally, neurologic symptoms. They have a gener-
In some reports, no abnormalities were found, and others alized aminoaciduria, renal glucosuria, hypophosphatemia,
 41. Cystinosis and Fanconi Syndrome 801

RTA, hypokalemia, proteinuria, and hypouricemia. The or light-chain fragments (Bence Jones proteins) that crystal-
GFR is moderately reduced in some. Recovery is rapid lize within the tubule cells (164,168).
when the degraded drug is stopped. The compound Hypophosphatemia has been well described in patients
responsible for the tubular dysfunction is anhydro-4-tetra- with benign and malignant mesenchymal tumors of bone.
cycline formed from tetracycline by heat, moisture, and a A single patient was also reported who had Fanconi syn-
low pH. drome, as well as hypophosphatemia, in association with a
A number of other compounds also may give rise to Fan- nonossifying fibroma of bone (169). Removal of the tumor
coni syndrome, often in association with a reduced GFR, led to resolution of the tubular dysfunction, indicating a
but the renal injury usually is reversible. For example, a few humoral substance that impaired tubular transport.
patients ingesting methyl-3-chromone (diachrome) (147), The nephrotic syndrome has rarely been associated with
6-mercaptopurine (148), toluene (glue sniffing) (149), Chi- Fanconi syndrome (170). The renal pathology in most cases is
nese medicine (150), valproate (151), suramin (152), and focal segmental glomerulosclerosis, and Fanconi syndrome
Lysol (153) have developed Fanconi syndrome. Gentamicin heralds a poor prognosis. These patients have hypokalemia,
(154), streptozotocin (155), and ranitidine (156) have RTA, rickets, and growth retardation. Hypocalcemia and tet-
caused Fanconi syndrome and renal failure. Tubular dys- any can be significant problems in these children.
function during recovery from acute renal failure from any Fanconi syndrome has appeared rarely after renal trans-
cause can occur, diminishing the significance of some toxins plantation (171). The pathogenesis probably is multifacto-
as inducers of Fanconi syndrome. rial (e.g., sequels of acute tubular necrosis, rejection,
A number of cancer chemotherapy agents have been nephrotoxic drugs, ischemia from renal artery stenosis, and
associated with Fanconi syndrome and renal tubule dysfunc- residual hyperparathyroidism) (172).
tion, especially cisplatin and ifosfamide. The nephrotoxicity
of cisplatin is dose dependent and often irreversible (157).
Renal cisplatin toxicity is manifested by tubular proteinuria, THERAPY
aminoaciduria, hypercalciuria, hyperphosphaturia, and,
especially, hypermagnesuria. The hypermagnesuria can be Therapy, when possible, should be directed at the underly-
severe, persistent, and difficult to treat. Reductions in GFR ing cause, such as avoidance of the offending nutrient in
are common with prolonged use of cisplatin. Cisplatin prin- galactosemia, HFI, and tyrosinemia. Wilson’s disease can be
cipally affects the tubule, especially the proximal and distal treated with penicillamine and other copper chelators.
tubules at the corticomedullary junction. Strict attention to Heavy metal intoxication can be treated with chelation
hydration and avoidance of other nephrotoxins are helpful therapy. In these cases, resolution of Fanconi syndrome
in preventing cisplatin nephrotoxicity. usually is complete.
Ifosfamide is another useful chemotherapeutic agent In other instances, therapy is directed at the biochemical
against solid tumors. However, nephrotoxicity is a well- abnormalities secondary to the renal solute losses and the
documented side effect of this drug (158–162). Fanconi bone disease often present in these patients. Proximal RTA
syndrome may appear at any time but seems more likely to usually requires high dosages of alkali (2 to 10 mEq/kg/day
appear after doses greater than 60 g/m2 body surface area in given in divided doses throughout the day) for correction.
children under 2 years of age, in those with reduced renal Some patients require even higher dosages that may lead to
mass, and in those treated previously with cisplatin. Hypo- volume expansion and further bicarbonate wasting. In this
phosphatemic rickets as a consequence of Fanconi syn- situation, 1 to 3 mg/kg/day of hydrochlorothiazide to pre-
drome has occurred in a number of these children long vent volume expansion may lessen the dose of alkali needed.
after cessation of the drug, as has a reduced GFR. Experi- Hydrochlorothiazide aggravates the potassium losses, how-
mentally, the perfusion of isolated rat kidneys with chloro- ever. Potassium supplementation is also commonly needed,
acetaldehyde, a metabolite of ifosfamide, caused Fanconi especially if there is significant RTA. The use of potassium
syndrome, although this was not observed with ifosfamide citrate, lactate, or acetate corrects hypokalemia as well as
or acrolein, another metabolite of ifosfamide (163). acidosis. A few patients require sodium supplementation, as
well as potassium. Again, the use of a metabolizable anion
aids in the correction of the acidosis. Rare patients require
Other Acquired Disorders
NaCl supplementation. Usually these patients manifest
Dysproteinemia associated with a number of diseases is a alkalosis when untreated because of large urinary NaCl
cause of Fanconi syndrome. This cause is mainly observed losses leading to volume contraction, which overrides the
in adults because the diseases are rare in children. Dyspro- RTA. Magnesium supplementation is required in those
teinemia and Fanconi syndrome are associated with multi- patients who manifest hypomagnesemia. Ensuring adequate
ple myeloma (164), light-chain proteinuria (165), Sjögren’s fluid intake is essential, especially during gastrointestinal
syndrome (166), and amyloidosis (167). The appearance of diseases. This is of greater importance in the infant who
tubular dysfunction is correlated with specific light chains does not have free access to fluid. Correction of hypokale-
802 VI. Tubular Disease

mia and its effect on the concentrating ability of the distal 3. Fanconi G. Die nicht diabetischen glykosurien und hyper-
tubule may decrease polyuria. glykamien des alterm kindes. Jahrb Kinderheikld 193;133:
Bone disease is another important aspect of managing 257–300.
patients with Fanconi syndrome. Often, complaints referable 4. Rosenberg LE, Segal S. Maleic acid-induced inhibition of
to this problem (e.g., pain, fractures, rickets, or growth fail- amino acid transport in rat kidney. Biochem J 1964;92:345–
352.
ure) are the presenting feature. Factors implicated in bone
5. Norden AGW, Lapsley M, Igarashi T, et al. Urinary megalin
disease include hypophosphatemia, decreased synthesis of deficiency implicates abnormal tubular endocytic function
calcitriol, hypercalciuria, and chronic acidosis. Both low and in Fanconi syndrome. J Am Soc Nephrol 2002;13:125–133.
normal plasma levels of calcitriol have been found in patients 6. Kramer HJ, Gonick HC. Experimental Fanconi syndrome.
with Fanconi syndrome. Moreover, healing of rickets has I. Effect of maleic acid on renal cortical Na-K-ATPase activ-
occurred in patients receiving supplemental vitamin D with- ity and ATP levels. J Lab Clin Med 1970;76:799–808.
out phosphate and in some receiving phosphate without sup- 7. Eiam-Ong S, Spohn M, Kurtzman NA, Sabatini S. Insight
plemental vitamin D. In the presence of hypophosphatemia, into the biochemical mechanism of maleic acid-induced
oral phosphate should be started with the goal of normaliz- Fanconi syndrome. Kidney Int 1995;48:1542–1548.
ing serum phosphate levels. This often requires 1 to 3 g/day 8. Szczepanska M, Angielski S. Prevention of maleate-induced
of supplemental phosphate. This is best given in divided tubular dysfunction by acetoacetate. Am J Physiol 1980;239:
F50–F56.
doses, and the dosage should be slowly increased over several
9. Bergeron M, Mayers P, Brown D. Specific effect of maleate
weeks to reduce the gastrointestinal intolerance. With regard on an apical membrane glycoprotein (gp330) in the proxi-
to vitamin D supplementation, it is unclear whether stan- mal tubule of the rat. Am J Physiol 1996;271:F908–F916.
dard vitamin D (i.e., ergocalciferol) or a vitamin D metabo- 10. Gonick HC, Indraprasit S, Rosen VJ, et al. Experimental
lite is better. Currently, most clinicians would use a vitamin Fanconi syndrome: III. Effect of cadmium on renal tubular
D metabolite, such as calcitriol or dihydrotachysterol. This function, the ATP-Na-K-ATPase transport system and renal
obviates concern of inadequate hydroxylation by proximal tubular ultrastructure. Min Electro Metab 1980;3:21–35.
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hypercalcemia because of the shorter half-life of these metab- amino acids in lead poisoned rabbits. Acta Pharmacol Toxicol
olites. Vitamin D therapy improves the hypophosphatemia 1962;19:35–42.
and lessens the risk of hyperparathyroidism. Supplemental 12. Goyer RA. The renal tubule in lead poisoning: I. Mitochon-
drial swelling and aminoaciduria. Lab Invest 1968;19:712–
calcium is indicated in those with hypocalcemia after supple-
777.
mental vitamin D has been started. 13. Gyrd-Hansen N, Helleberg A. Methoxyethyl mercury toxic-
Hyperaminoaciduria, glucosuria, proteinuria, and hyper- ity in pigs: kidney functions and mercury distribution. Acta
uricosuria usually do not lead to clinical symptoms and do Pharmacol Toxicol 1976;38:229–240.
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136. Chesney RW, Kaplan BS, Teitel D, et al. Metabolic abnor- 156. Neelakantappa K, Gallo G, Lowenstein J. Ranitidine-associ-
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bohydrate metabolism in two patients. Pediatrics 1981;67: ney Dis 1993;22:333–336.
113–118. 157. Madias NE, Harrington JT. Platinum nephrotoxicity. Am J
137. Aperia A, Bergqvist G, Linne T, et al. Familial Fanconi syn- Med 1978;65:307–314.
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mal kinase and transferase activity. Acta Paediatr Scand ifosfamide. Arch Dis Child 1990;65:732–738.
1981;70:527–533. 159. Burk CD, Restaino I, Kaplan BS, et al. Ifosfamide-induced
138. Deal JE, Barratt TM, Dillon MJ. Fanconi syndrome, ich- renal tubular dysfunction and rickets in children with
thyosis, dysmorphism, jaundice and diarrhea: a new syn- Wilms tumor. J Pediatr 1990;117:331–335.
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139. Patrick A, Cameron JS, Ogg CS. A family with a dominant mide neurotoxicity, hematoxicity, and tubular nephrotoxicity
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ure in adult life. Clin Nephrol 1981;16:289–292. cer Res 1987;47:1457–1460.
140. Briggs WA, Kominami N, Wilson RE, et al. Kidney trans- 161. Suarrez A, McDowell H, Niaudet P, et al. Long-term follow-
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25. nant mesenchymal tumors: an International Society of Pedi-
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duria, hypophosphatemia and rickets in lead poisoning. Am 162. Raney B, Ensign LG, Foreman J, et al. Renal toxicity of
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142. Goyer RA, Tsuchuja K, Leonard DL, et al. Aminoaciduria sarcoma study for patients with gross residual tumor. Am J
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143. Galle P, Morel-Maroger L. Les lesions renales du saturnisme mide metabolite chloroacetaldehyde causes Fanconi syn-
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malfunction and pulmonary emphysema in cadmium pig- 164. Solomon A, Weiss DT, Kattine AA. Nephrotoxic potential
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146. Brodehl J, Gellisson K, Hagge W, et al. Reversibles renales proteinuria. N Engl J Med 1976;294:71–74.
Fanconi-Syndrome durch toxisches abbauprodukt des tetra- 166. Shearn MA, Tu WH. Nephrogenic diabetes insipidus and
zyklins. Helv Paediatr Acta 1968;23:373–383. other defects in renal tubular function in Sjögren syndrome.
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following intoxication with methyl-3-chromone. J Pediatr 167. Finkel PN, Kronenberg K, Pesce AJ, et al. Adult Fanconi
1968;73:422–425. syndrome, amyloidosis and marked light chain proteinuria.
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Ann Intern Med 1980;92:69–70. 169. Leehey DJ, Ing T, Daugirdas JT. Fanconi syndrome associ-
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1973;132:555–561. 1992;151:601–607.
 42

PRIMARY HYPEROXALURIA
PIERRE COCHAT
LAURE B. D. E. COLLARD

First report on oxalosis by Lepoutre, reprinted from Jour- verted into L-glycerate by lactic acid dehydrogenase, and into
nal d’Urologie Médicale et Chirurgicale 1927;20:424, with D-glycerate by hydroxypyruvate reductase (HPR), which also
permission. has a GR activity.
Calculs multiples chez un enfant; infiltration du parenchyme
rénal par des dépôts cristallins. — Chez un enfant de quatre ans
PRIMARY HYPEROXALURIA TYPE 1
et demi, opéré ou soigné pour calculs multiples de l’urètre, de
l’uretère et des deux reins, une biopsie fait découvrir un paren-
chyme rénal bourré de concrétions cristallines d’oxalate de PH1, the most common form of PH, is an autosomal reces-
chaux. Ce cas réalise en clinique l’expérience d’Ebstein et sive disorder caused by the functional defect of the hepatic,
Nicolaier, qui, faisant ingérer de l’oxamide à des chiens, peroxisomal, pyridoxal phosphate–dependent enzyme AGT,
voyaient de développer des calculs urinaires et de multiples the protein product of a gene that has been identified on
formations cristallines dans le parenchyme rénal. chromosome 2q37.3. The disease occurs because AGT
activity is undetectable or because AGT is mistargeted to
Primary hyperoxaluria (PH) results from endogenous over-
mitochondria, which may explain clinical and enzymatic
production of oxalic acid, as opposed to secondary hyperox-
heterogeneity.
aluria, which is attributable to increased intestinal absorption
or excessive intake of oxalate. Such derangement leads to
accumulation of oxalate within the body. The main target Metabolic Derangement
organ is the kidney because oxalate cannot be metabolized
PH1 is due to a deficiency of the liver-specific pyridoxal
and is excreted in the urine, leading to nephrocalcinosis,
phosphate–dependent enzyme AGT (EC 2.6.1.44, 392
recurrent urolithiasis, and subsequent renal impairment.
amino acids, 43 kDa) (Fig. 42.1) (1). The resulting
Hyperoxaluria, the hallmark of any kind of PH, is associated
decreased transamination of glyoxylate into glycine leads to
with increased urinary excretion of either glycolate in PH
subsequent increase in its oxidation to oxalate, a poorly sol-
type 1 (PH1) or L-glycerate in PH type 2 (PH2).
uble end-product. In patients with a presumptive diagnosis
of PH, 10 to 30% are identified as non-PH1 because AGT
OXALATE METABOLISM enzymatic activity and immunoreactivity are normal (2).
Among PH1 patients, 75% have undetectable enzyme activ-
Oxalate is a poorly soluble end-product of the metabolism of ity (enz–) and the majority of these also have no immunore-
a number of amino acids, particularly glycine, and of other active protein (cross-reacting material, crm–). In the rare
compounds such as sugars and ascorbic acid (Fig. 42.1). The enz–/crm+ PH1 patients, a catalytically inactive but immu-
immediate precursors of oxalate are glyoxylate and glycolate. noreactive AGT is found within the peroxisomes. The
The main site of synthesis of glyoxylate and oxalate is the remaining PH1 patients have AGT activity in the range of 5
liver peroxisome, which can also detoxify glyoxylate by to 50% of the mean normal activity (enz+), and the level of
reconversion into glycine, catalyzed by alanine:glyoxylate immunoreactive protein parallels the level of enzyme activ-
aminotransferase (AGT). In the cytosol, glyoxylate can be ity (crm+) (2). In enz+/crm+ patients, the disease is caused
converted into oxalate by lactic acid dehydrogenase. It can by a mistargeting of AGT: approximately 90% of the immu-
also be converted into glycolate by glyoxylate reductase (GR) noreactive AGT is localized in the mitochondria instead of
and into glycine by glutamate:glyoxylate aminotransferase. in the peroxisomes, where only 10% of the activity is found;
Glycolate can also be formed from hydroxypyruvate, a catab- almost all patients who are pyridoxine-responsive are in this
olite of glucose and fructose. Hydroxypyruvate can be con- group (3,4). Of interest, human hepatocyte AGT, which is
808 VI. Tubular Disease

FIGURE 42.1. Human hepatocyte. Major reactions

involved in oxalate, glyoxylate, and glycolate metabolism
in the human hepatocyte. AGT, alanine:glyoxylate ami-
notransferase; DAO, D-amino oxidase; GGT, glutamate:
glyoxylate aminotransferase; GO, glycolate oxidase; GR,
glyoxylate reductase; HPR, hydroxypyruvate reductase;
LDH, lactate dehydrogenase; PH1, primary hyperoxaluria
type 1; PH2, primary hyperoxaluria type 2; X, metabolic
block in PH1; O, metabolic block in PH2.

normally exclusively localized within the peroxisomes, is phism, which has a high allelic frequency (approximately
unable to function when diverted to the mitochondria, its 20%) plays an important role in determining the phenotypic
normal localization in several other species (e.g., cat, dog, manifestations of specific mutations (15).
frog) (3). In addition, patients with a primary peroxisomal DNA analysis among different ethnic groups has revealed
disorder (e.g., Zellweger syndrome) do not exhibit hyperox- the presence of specific mutations, founder effects, and phe-
aluria (5). notype-genotype correlations among North African, Japa-
Different AGT crystal forms have been recently obtained nese, Turkish, and Pakistani populations (2,18,19).
for some polymorphic variants [AGT, AGT (P11L), AGT Prenatal diagnosis using a combination of linked polymor-
(P11L, I340M)] (6). phism and detection of the two most common mutations has
an accuracy of more than 99% and can be performed from
chorionic villi or amniocytes according to gestational age (20).
Genetics
PH1 is the most common form of PH (1:60,000 to
Clinical Presentation
1:120,000 live births) (7,8). Due to autosomal recessive
inheritance, it is much more frequent when parental con- PH1 has four general clinical presentations: (a) a rare infantile
sanguinity is present (9–11). Indeed, it is responsible for form with early nephrocalcinosis and rapid kidney failure, (b) a
less than 0.5% of pediatric end-stage renal failure (ESRF) rare late-onset form with occasional stone passage in late adult-
in Europe versus 13% in Tunisia (7). hood, (c) the most common form with recurrent urolithiasis
Human liver AGT complementary DNA and genomic and progressive renal failure leading to a diagnosis of PH1 in
DNA have been cloned and sequenced; the normal AGT childhood or adolescence, and (d) a rare condition where PH1
gene (AGXT ) is a single copy gene, which maps to chromo- is diagnosed after the prompt recurrence of the disease after kid-
some 2q37.3 (11 exons spanning approximately 10 kb) (2). ney transplantation (7,21,22). In addition, some patients are
Polymorphic variations have been identified in AGXT (2); asymptomatic, and PH1 may be discovered by family history.
the two best-studied polymorphic variants are those
encoded by the major AGXT allele (80% frequency in the
Renal Involvement
European and North American populations) and the minor
AGXT allele, which has a frequency of 20%. PH1 presents with symptoms referable to the urinary tract in
More than 40 mutations in the AGXT gene have been more than 90% of cases [e.g., loin pain, hematuria, urinary
identified to date. However, some of them are more frequent tract infection, passage of stones, evidence of nephrocalcinosis
and may play a role either in enzyme trafficking or in expres- (Fig. 42.2), uremia, metabolic acidosis, growth delay, anemia].
sion of a specific clinical or biochemical phenotype (12–16). Calculi—multiple, bilateral, and radiopaque—are composed
The G630A mutation leading to a G170R substitution is of calcium oxalate. Nephrocalcinosis, best demonstrated by
found in one-third of European and North American ultrasound, is evident on plain abdomen x-ray at an advanced
patients and appears to act together with the P11L polymor- stage. The median age at presentation is 5 years, ranging from
phism leading to peroxisome-to-mitochondrion AGT mis- birth to the sixth decade; ESRF is reached by the age of 25
targeting. Double heterozygous patients seem to present at a years in one-half of patients (23). The infantile form of PH1
younger age and demonstrate a more aggressive disease. The often presents as a life-threatening condition because of rapid
T444C mutation is more frequent in the severe forms; the progression to ESRF due to both early oxalate load and imma-
G630A mutation is more frequent in less severe cases (17). In ture glomerular filtration rate (GFR); one-half of the patients
addition, G630A mutation homozygotes have higher AGT experience ESRF at the time of diagnosis, and 80% develop
residual activity (17). In any condition, the P11L polymor- ESRF by the age of 3 years (9).
 42. Primary Hyperoxaluria 809

FIGURE 42.2. Plain abdomen x-ray in a 20-month-old primary

hyperoxaluria type 1 boy: bilateral nephrocalcinosis, 13 months
after starting hemodialysis (5 × 3/wk).

Extrarenal Involvement
When GFR falls to below 40 to 50 mL/min per 1.73 m2, con-
tinued overproduction of oxalate by the liver along with
reduced oxalate excretion by the kidneys leads to a critical satu-
ration point for plasma oxalate (plasma oxalate concentration FIGURE 42.4. Spontaneous fracture of the left tibia in a 25-
month-old boy on hemodialysis.
greater than 30 to 50 μmol/L) so that oxalate deposition
occurs in many organs (24,25). Bone is the major compart-
ment of the insoluble oxalate pool (Fig. 42.3), and bone because of progressive vascular lesions—heart (e.g., cardiomy-
oxalate content is higher (15 to 910 μmol oxalate per gram opathy, arrhythmias, heart block), nerves (e.g., polyradiculo-
bony tissue) than that observed in ESRF patients without neuropathy, mononeuritis multiplex), joints (e.g., synovitis,
PH1 (2 to 9 μmol/g) (26). Calcium oxalate crystals accumu- chondrocalcinosis), skin (e.g., ulcerating calcium oxalate nod-
late first in the metaphyseal area and form characteristic, dense ules, calcinosis cutis, livedo reticularis), soft tissues (e.g.,
suprametaphyseal bands on x-ray (27). Later on, oxalate oste- peripheral gangrene), retina (e.g., flecked retinopathy, cho-
opathy leads to pain, erythropoietin-resistant anemia, and roidal neovascularization), and other visceral lesions (e.g.,
spontaneous fractures, sometimes in infancy (Fig. 42.4). Along intestinal infarction, hypothyroidism) (7,28–33).
with the skeleton, systemic involvement includes many organs Systemic involvement, named oxalosis, is responsible for
poor quality of life leading to both disability and severe
complications. Indeed, PH1 is one of the most life-threat-
ening hereditary renal diseases, mainly in developing coun-
tries where the mortality rate approaches 100% in the
absence of adequate treatment (34).

Diagnosis
The combination of both clinical and radiologic presenta-
tion is a strong argument for PH1 in a large number of
patients. In addition, family history may bring additional
information (e.g., other affected siblings, parental consan-
guinity, and unexplained death in siblings) (34). Physico-
chemical investigation is of major interest in diagnosis, and
infrared spectroscopy is helpful for the identification and
the quantitative analysis of stones, showing calcium oxalate
FIGURE 42.3. Bone histology from a 13-year-old girl with pri-
mary hyperoxaluria type 1 on hemodialysis for 2 years: oxalate monohydrate crystals (type Ic whewellite, CaC2O4.H2O)
crystals on polarized light microscopy. (35). Such crystals can also be identified in urine or biopsy
810 VI. Tubular Disease

TABLE 42.1. PLASMA AND URINE CONCENTRATIONS from the index case and parents is available (20). Such a pro-
OF OXALATE, GLYCOLATE, AND L-GLYCERATE: cedure identifies normal, affected, and carrier fetuses. In the
NORMAL VALUES
absence of identified mutations in an index case, the two most
Urine Oxalate per day Child <0.46 mmol/1.73 m 2 common mutations (G170R and I144T) can be checked (2).
Adult <0.40 mmol/1.73 m2 The detection of AGXT mutations using simplified reliable
Oxalate: creatinine <1 yr <0.15 mmol/mmol technology can be proposed to identify healthy carriers
1–4 yr <0.13 mmol/mmol
among family members, and sometimes for screening of poly-
5–12 yr <0.07 mmol/mmol
Adult <0.08 mmol/mmol morphisms in patients with nephrolithiasis (16).
Glycolate per day Child <0.55 mmol/1.73 m 2
Adult <0.26 mmol/1.73 m2
Glycolate: creatinine <1 yr <0.07 mmol/mmol Treatment and Prognosis
1–4 yr <0.09 mmol/mmol
5–12 yr <0.05 mmol/mmol Supportive Treatment
Adult <0.04 mmol/mmol
L-glycerate: creati- Child <0.03 mmol/mmol Conservative measures should be started as soon as the diag-
nine nosis of PH1 has been made or even suspected. The aims are
Plasma Oxalate Child <7.40 μmol/L to decrease oxalate production and to increase the urinary
Adult <5.40 μmol/L
solubility of calcium oxalate. The risk of stone formation is
Oxalate: creatinine Child <0.19 μmol/μmol
Adult <0.06 μmol/μmol increased when urine oxalate exceeds 0.4 to 0.6 mmol/L,
especially if urine calcium exceeds 4 mmol/L. Therefore,
Note: Oxalate (COOH-COOH): 1 mmol = 90 mg; glycolate (COOH-CH2OH): therapy should keep the concentrations of oxalate and cal-
1 mmol = 76 mg. cium below these limits (39). This should be accomplished
From Barratt TM, Danpure CJ. Hyperoxaluria. In: Barratt TM, Avner ED,
Harmon WE, eds. Pediatric nephrology, 4th ed. Baltimore: Williams & by maintaining a high fluid intake (greater than 2 L/m2/day),
Wilkins, 1999:609–619; Leumann E, Hoppe B. The primary hyperox- sometimes requiring a nasogastric tube or gastrostomy in
alurias. J Am Soc Nephrol 2001;12:1986–1993; and Gaulier JM, Cochat P,
Lardet G, et al. Serum oxalate microassay using chemiluminescence
infants, complemented by administration of calcium-oxalate
detection. Kidney Int 1997;52:1700–1703, with permission. crystallization inhibitors. Citrate (potassium or sodium), 100
to 150 mg/kg/day in three to four divided doses (8,40,41),
has been shown to be effective in PH1 patients. When it is
tissues (e.g., kidney, bone, marrow) by polarized light not available, crystallization inhibition may be accomplished
microscopy or infrared spectroscopy (36). Funduscopy may by sodium bicarbonate (aiming at keeping pH greater than
also show flecked retina as early as infancy. In patients with or equal to 7), magnesium or orthophosphate (20 to 60 mg/
normal or significant residual renal function, concomitant kg body weight per day) administration (40). Diuretics
hyperoxaluria and hyperglycoluria are indicative of PH1 require careful management: furosemide maintains a high
(Table 42.1), but 20 to 30% of PH1 patients do not urine output but increases the risk of calciuria, whereas the
present with hyperglycoluria (37). In dialysis patients, the diuretic effect of hydrochlorothiazide is less marked but is
biochemical assessment may include plasma oxalate or gly- associated with an appreciable decrease of calcium excretion.
colate to creatinine ratio, and oxalate or glycolate measure- The combination of both loop agents and thiazides is recom-
ment in dialysate (34,35,22). mended in most instances.
A definitive diagnosis requires assessments of AGT activity Restriction of dietary oxalate intake (e.g., beet roots,
and immunoreactivity in hepatic tissue (from a freshly frozen strawberries, rhubarb, spinach, coffee, tea, nuts) has very
liver biopsy specimen, minimum of 4 mg). Nevertheless, limited influence on the disease because dietary oxalate con-
there is controversial information about the relationship tributes very little to hyperoxaluria in PH (40). Calcium
between AGT activity and the severity of the disease (17). restriction is not recommended, because dietary calcium is
Liver biopsy is mandatory if liver transplantation is being con- required to bind oxalate and form insoluble calcium oxalate
sidered (see below). In selected populations, a direct molecu- complexes in the gut. Vitamin C supplementation is not
lar diagnosis can be performed, such as the search for the recommended because ascorbic acid is a precursor of
I244T mutation in patients from Maghreb (Basmaison, sub- oxalate. The effects of conservative measures can be assessed
mitted ). However, in most clinical settings, the search for fre- by serial determinations of crystalluria score and calcium
quent mutations (G630A, T853C, G170R) cannot currently oxalate supersaturation software (41,42).
replace enzymology in the diagnosis of PH1 (34,38). Prenatal Pyridoxine (cofactor of AGT, see Fig. 42.1) sensitivity is
diagnosis can be performed from DNA obtained from chori- found in 10 to 40% of patients, so that it must be tested
onic villi (10 to 12 weeks’ gestation) or amniocytes (13 to 17 early at a daily dose of 2 to 5 mg/kg with stepwise increase
weeks’ gestation). It is based on either mutational analysis up to 10 to 20 mg/kg (8). Response to pyridoxine may
using polymerase chain reaction amplification, or linkage delay the progression to ESRF. Pyridoxine sensitivity can be
analysis using the various intragenic and extragenic polymor- detected by monitoring of both oxalate and glycolate and
phisms; the latter is more generally applicable, provided DNA can be tested at any stage of the disease (2,3,41). However,
 42. Primary Hyperoxaluria 811

one should keep in mind that large doses of pyridoxine AGT activity (48), but graft survival is improved if trans-
might induce sensory neuropathy. Patients most likely to plantation is performed when there is substantial renal func-
respond are those with residual AGT activity (43), but pyri- tion (i.e., a GFR ranging from 20 to 30 mL/min/1.73 m2),
doxine responsiveness is still poorly understood at the and in the absence of major extrarenal involvement. In
molecular level. An attempt to inhibit hepatic synthesis of selected patients, good results have been reported after early
oxalate by using (L)-2-oxothiazolidine-4-carboxylate has renal transplantation and vigorous perioperative dialysis
been reported without clinically significant changes in uri- (49,50). However, at this point in time, living-related
nary oxalate excretion (44). donors should be avoided because the overall results are
The treatment of stones should avoid open and percuta- poor (47). Isolated kidney transplantation may be a tempo-
neous surgery because further renal parenchymal damage rary solution for PH in developing countries before manag-
alters the GFR (45). The use of extracorporeal shock wave ing the patient in a specialized center for further (combined)
lithotripsy may be an available option in selected patients, liver, if required, kidney transplantation. Even with previous
but the presence of nephrocalcinosis may be responsible for pyridoxine resistance, it is recommended to retest vitamin
parenchymal damage. Bilateral nephrectomy is recom- B6 responsiveness after isolated renal transplantation (49).
mended in most patients on renal replacement therapy to
limit the risk of infection, obstruction, and passage of stones.
Enzyme Replacement Therapy
Ideally, any kind of transplantation should precede accu-
Renal Replacement Therapy
mulation of large systemic oxalate loads. Assessment of the
Dialysis oxalate burden therefore needs to be regularly monitored
Conventional dialysis is unsuitable for PH1 patients who have by measurement of serial GFR, plasma oxalate (Table
reached ESRF because it cannot clear sufficient amounts of 42.1), calcium oxalate saturation, and systemic involve-
oxalate (3). In such patients, plasma oxalate levels range ment (assessment of bone mineral density by quantitative
between 80 and 160 μmol/L (normal is less than 7 μmol/L) measures, such as bone histology and computed tomogra-
(Table 42.1). Therefore, daily hemodialysis (6 to 8 hours per phy) (34,51–54).
session) using high-flux membranes would be required. Such a
strategy cannot be routinely used (46). A reasonable therapeu- Rationale for Liver Transplantation
tic goal is to keep predialysis plasma oxalate concentration Because the liver is the only organ responsible for glyoxy-
below 50 μmol/L to limit the progression of systemic oxalosis. late detoxification by AGT, the excessive production of
Daily hemodialysis in infants raises major technical and devel- oxalate continues as long as the native metabolically defec-
opmental problems and its association with nighttime perito- tive liver is left in place. Therefore, any form of enzyme
neal dialysis may be sometimes proposed. Conventional long- replacement succeeds only when the deficient host liver is
term hemodialysis is generally regarded as contraindicated concomitantly removed (55). Successful liver transplanta-
because it prolongs a deteriorating quality of life because of tion can supply the missing enzyme in the correct organ
unabated progression of extrarenal oxalate deposition. (e.g., liver), cell (e.g., hepatocyte), and intracellular com-
The benefit of pre- and posttransplantation hemodialy- partment (e.g., peroxisome) (2,55). The ultimate goal of
sis is still debated and should be limited to patients with organ replacement is to reduce endogenous oxalate synthe-
either oliguria or severe systemic oxalate loads. sis and provide good oxalate clearance via either the native
or the transplanted kidney.
Kidney Transplantation Of interest, the removed native liver from a PH1 patient
Successful kidney transplantation allows significant removal has been used for domino transplantation in a high-risk 69-
of soluble oxalate. However, because the biochemical defect year-old marginal recipient with initially a favorable post-
is in the liver, overproduction of oxalate and subsequent operative clinical outcome but evolution toward progressive
deposition in tissues continues unabated. High urinary renal insufficiency due to hyperoxaluria (56).
oxalate excretion originates from both ongoing oxalate pro-
duction from the native liver and oxalate deposits in tissues. Combined Liver-Kidney Transplantation
Due to oxalate accumulation in the transplanted kidney, In Europe, eight to ten combined liver-kidney transplanta-
overall graft survival is limited. Therefore, isolated kidney tions per year have been reported in the PH1 Transplant
transplantation is no longer recommended in most pyridox- Registry Report (47). The results are encouraging. Patient
ine-unresponsive patients (see later). The long-term out- survival approximates 80% at 5 years and 65 to 70% at 10
come of PH1 after renal transplantation remains uncertain, years. In addition, despite the potential risks for the grafted
with a 5- to 10-year patient survival rate ranging from 10 to kidney due to oxalate release from body stores, kidney sur-
50% (47). In addition, renal transplantation does not pre- vival is approximately 95% at 3 years posttransplantation,
vent the progression of skeletal and vascular complications. and the GFR ranges between 40 and 60 mL/min/1.73 m2
Outcome of renal transplantation is unrelated to residual after 5 to 10 years (47,54,55).
812 VI. Tubular Disease

Isolated Liver Transplantation tion and therefore should be limited to patients with anuria
Isolated liver transplantation might be the considered ini- or with evidence of significant systemic oxalate involvement
tial therapy in selected patients before advanced chronic (34,52,60).
renal failure has occurred (i.e., at a GFR between 60 and 40 Combined transplantation should be planned when the
mL/min/1.73 m2) (18,58). Such a strategy has a strong GFR ranges between 20 and 40 mL/min/1.73 m2 because, at
rationale but raises ethical controversies. Approximately 20 this level, oxalate retention increases rapidly (22,59). In
patients have received an isolated liver transplant to date. patients with ESRF, vigorous hemodialysis should be started
Overall outcome is difficult to evaluate because the course and urgent liver-kidney transplantation should be performed.
of the disease is unpredictable and a sustained improve- Even at these late stages, damaged organs other than the kid-
ment can follow a phase of rapid decrease in GFR (57–59). ney, such as the skeleton or the heart, do benefit from enzyme
replacement from the donor liver (57,60,61), which results in
Posttransplantation Reversal of Renal an appreciable improvement in quality of life.
and Extrarenal Involvement
Deposits of calcium oxalate in tissues can be remobilized Donors for Combined Liver-Kidney Transplantation
according to the accessibility of oxalate burden to the blood The type of donor (i.e., cadaver or living related) depends
stream (55). After combined liver-kidney transplantation, mainly on the physician and the country where the patient is
plasma oxalate returns to normal before urine oxalate does, treated (34,62,63) (Table 42.2). A living related donor must
and oxaluria can remain for several months posttransplanta- be considered because of the restricted number of potential
tion (3,47,49). Therefore, there is still a risk of recurrent biorgan donors. A living related donor can be proposed in a
nephrocalcinosis or renal calculi that might jeopardize renal preemptive procedure using either isolated liver or synchro-
graft function. Glycolate, which is more soluble than oxalate nous liver-kidney transplantation. In patients with end-stage
and does not accumulate, is excreted in normal amounts renal disease and systemic involvement, a metachronous
immediately after liver transplantation. Thus, independent transplantation procedure might be an option because a first-
of the transplantation strategy, the kidney must be protected step liver transplantation will then allow oxalate clearance by
from damage induced by the systemic oxalate load suddenly vigorous hemodialysis before considering further kidney
released from tissues. Forced fluid intake (3 to 5 L/1.73 m2/ transplantation from the same (living related) donor.
day) supported by diuretics and the use of crystallization
inhibitors is the most important strategy. Plasma oxalate con- Fate of Primary Hyperoxaluria Type 1 in Infants
centration, crystalluria, and calcium oxalate saturation are PH1 in early childhood raises specific problems due to
helpful tools in renal graft management after combined liver- (a) the severity of the disease in infants (approximately
kidney transplantation (42,53,60). The benefit of daily high- 50% death rate), (b) the access to available diagnostic pro-
efficiency pre- and posttransplant hemodialysis or filtration is cedures in this age group, and (c) the adequacy of manage-
still debated; it provides a rapid drop in plasma oxalate but ment, which directly reflects local economic particulars.
also an increased risk of urinary calcium-oxalate supersatura- Infantile PH1 presents two different issues worldwide. It is

TABLE 42.2. SUGGESTIONS FOR TRANSPLANTATION STRATEGIES IN PRIMARY HYPEROXALURIA

TYPE 1 PATIENTS ACCORDING TO RESIDUAL GLOMERULAR FILTRATION RATE (GFR),
SYSTEMIC INVOLVEMENT, AND LOCAL FACILITIESa
Estimated oxalate
GFR (mL/min/1.73 m2) burden Proposed transplantation strategy Comments

60–40 ± Preemptive CAD liver Tx? Hazardous, limited experience

Raises ethical problems
Do not reject LRD (in case of further kidney Tx)
30–10 ++ Preemptive synchronous CAD liver- No advantage with LRD
kidney Tx HD only if post-Tx acute tubular necrosis/delayed
graft function
End-stage renal disease +++ Metachronous LRD Tx procedure: Avoid synchronous CAD Tx:
1. Liver Tx Renal risk ++
2. Aggressive HD to clear oxalate Pre- and post-Tx HD required?
3. Ox store assessment Avoid metachronous CAD Tx:
4. Kidney Tx using the same donor Needs two different donors
See synchronous

±, mild; ++, moderate; +++, important; CAD, cadaver; HD, hemodialysis; LRD, living related donor; Tx, transplantation.
aAuthors’ personal opinion.
 42. Primary Hyperoxaluria 813

a very rare disease in developed countries where combined Clinical Presentation

liver-kidney transplantation is available in children older
PH2 has been documented in less than 40 published patients
than 6 months of age (or 5 to 6 kg body weight). Yet, it is a
(65,69). Median age at onset of first symptoms is 1 to 2
frequent cause of early end-stage renal disease in developing
years, and the classical presentation is urolithiasis, including
countries, often due to consanguinity, where therapeutic
hematuria and obstruction. However, stone-forming activity
withdrawal is widely applied (9,58). Management of PH1
is lower than in PH1 so that nephrocalcinosis and urinary
is, therefore, a major example of ethical, epidemiologic,
tract infection are less frequent (41). Glomerular filtration
technical, and financial dilemmas, which may be raised by
rate is usually maintained during childhood, and systemic
recessive, inherited diseases with early life-threatening onset
involvement is therefore exceptional (69).
presenting in different socioeconomic contexts.

Diagnosis
Future Trends
In the presence of hyperoxaluria without hyperglycoluria, a
Although gene therapy has been advocated, many years of
diagnosis of PH2 should be considered, especially when
research will be required before its potential may be actual-
AGT activity is normal. However, hyperoxaluria in PH2
ized (3). Amino acid changes found in AGT may affect its
tends to be less pronounced than in PH1. The biochemical
stability (6). A better understanding of such changes will
hallmark is the increased urinary excretion of L-glycerate
facilitate design pharmacologic agents that stabilize AGT,
(Table 42.1) (70), but the definitive diagnosis requires mea-
leading to appropriate targeting of functional proteins in
surement of GR activity in a liver biopsy (71) as some PH2
the potential of PH1 without the need for organ transplan-
patients have normal L-glycericaciduria (72).
tation (Danpure, personal communication, 1999).

Treatment and Prognosis

NON–TYPE 1 PRIMARY HYPEROXALURIA
The overall long-term prognosis in PH2 is better than for
PH1. ESRF occurs in 12% of patients between 23 and 50
In patients with overt hyperoxaluria, the pattern of urinary
years of age (69). As in PH1, supportive treatment includes
metabolites is indicative but not diagnostic of PH. In
high fluid intake, crystallization inhibitors, and prevention
patients with a clinical picture of PH1, 10 to 30% have
of complications; there is no rationale to use pyridoxine.
normal AGT activity that may lead to a diagnosis of PH2
Kidney transplantation has been performed in some end-
or of another disorder causing hyperoxaluria. Enzyme
stage renal disease patients, often leading to recurrence
activity measurement in a single-needle liver biopsy can
(nephrocalcinosis), including hyperoxaluria and L-glycerate
confirm or exclude PH1 and PH2.
excretion (69,73,74). The concept of liver transplantation
has therefore been suggested, but more data are needed
Primary Hyperoxaluria Type 2 concerning the tissue distribution of the deficient enzyme
and the biochemical impact of hepatic GR/HPR deficiency
PH2 is another rare inherited defect of oxalate metabolism
before such a strategy can be recommended.
causing raised urine oxalate and L-glycerate (Fig. 42.1).

Non–Type 1 Non–Type 2
Metabolic Derangement
Primary Hyperoxaluria
PH2 is characterized by the absence of an enzyme with GR,
Few reports have shown a possible association of PH (a) with-
HPR, and D-glycerate dehydrogenase activities (Fig. 42.1)
out AGT or GR/HPR deficiency, and (b) with hyper-
(64–66). Analysis of liver and lymphocyte samples from
glycoluria in the absence of AGT deficiency (75–77). It is
patients with PH2 showed that GR activity was either very
therefore likely that there is at least another form of PH yet
low or undetectable, whereas D-glycerate dehydrogenase
to be identified; hepatic glycolate oxidase is a candidate
activity was reduced in liver but within the normal range in
enzyme for a third form of inherited hyperoxaluria.
lymphocytes (67).

CONCLUSION
Genetics
There is evidence for autosomal recessive transmission, Children with nephrocalcinosis or urolithiasis should be
and the gene encoding the enzyme GR or HPR (GRHPR) screened for hyperoxaluria. Those patients with hyperox-
has been located on chromosome 9q11 (68). Several mis- aluria or recurrent calcium oxalate urolithiasis should be
sense, nonsense, and deletion mutations have been identi- referred for diagnosis and management to specialized clini-
fied (66). cal centers with interest and experience in the conditions
814 VI. Tubular Disease

and access to the appropriate biochemical and molecular 14. Nogueira PK, Vuong TS, Bouton O, et al. Partial deletion of
biologic facilities. Major advances in biochemistry, enzy- the AGXT gene (EX1_EX7del): a new genotype in hyperox-
mology, genetics, and management of PH have been aluria type 1. Hum Mutat 2000;15:384–385.
achieved during recent years. Further steps will assess geno- 15. Lumb MJ, Danpure CJ. Functional synergism between the
type-phenotype relationships and underlying metabolic most common polymorphism in human alanine:glyoxylate
aminotransferase and four of the most common disease-
defects of atypical PH. The ongoing analysis of data from
causing mutations. J Biol Chem 2000;275:36415–36422.
multicenter databases will improve transplantation/enzyme 16. Pirulli D, Giordano M, Lessi M, et al. Detection of AGXT
replacement strategies and subsequent patient survival and gene mutations by denaturing high-performance liquid
quality of life. chromatography for diagnosis of hyperoxaluria type 1. Clin
Foundations dealing with hyperoxaluria include http:// Exp Med 2001;1:99–104.
www.ohf.org and http://www.airg-france.org. 17. Amoroso A, Pirulli D, Florian F, et al. AGXT gene muta-
tions and their influence on clinical heterogeneity of type 1
primary hyperoxaluria. J Am Soc Nephrol 2001;12:2072–
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 43

TUBULOINTERSTITIAL NEPHRITIS
URI S. ALON

Involvement of the tubulointerstitial compartment in renal matrix is composed of a fibrillar net of basement membrane
diseases can be either primary or even more commonly secon- and interstitial collagens, glycoproteins, and proteoglycans,
dary to glomerular, vascular, or structural disease (Table 43.1). as well as interstitial fluid (12). The interstitium provides
However, even in the latter disorders the magnitude of structural support for the nephrons and capillaries and
involvement and damage to the tubulointerstitium can have a plays an important role in the transport of solutes. It is also
significant effect on the outcome of the primary disease (1,2). the site of production of cytokines and hormones such as
This chapter addresses primary tubulointerstitial nephritis prostaglandins and erythropoietin. The roles of the tubules
(TIN), which is a syndrome in which there is a spectrum rang- in maintaining homeostasis of fluid, electrolytes, minerals,
ing from acute to chronic diseases. TIN is characterized histo- and acid-base balance are described in previous chapters.
logically by inflammation and damage of tubulointerstitial Damage to different segments of the tubules result in meta-
structures, with relative sparing of glomerular and vascular ele- bolic abnormalities related to the affected segments.
ments (3,4). Acute TIN is typically associated with marked
tubulointerstitial inflammation, varying degrees of edema and
tubular epithelial cell damage, and mononuclear cell infiltra- PATHOPHYSIOLOGY
tion (5). In contrast, chronic lesions generally are characterized
by evidence of tubular epithelial cell damage and atrophy, with The similarity of tubulointerstitial lesions in all forms of
tubulointerstitial fibrosis (5). Although there are important TIN, which is characterized by predominantly T-cell lym-
clinical and pathophysiologic distinctions between acute and phocytic infiltrates, suggests immune-mediated mechanisms
chronic TIN, they are best viewed as a continuum of manifes- of renal injury (6,13). Such mechanisms are likely important
tations of renal injury (6,7). Acute TIN is often reversible; in either initiating tubular injury or amplifying damage
however, progression to chronic renal disease can occur (8,9). induced by both immune and nonimmune causes. Studies
from experimental models of tubulointerstitial disease indi-
cate that both cell-mediated and humoral immune mecha-
STRUCTURE AND FUNCTION OF THE nisms are relevant pathways that induce renal injury (14,15).
TUBULOINTERSTITIAL COMPARTMENT

The tubulointerstitial compartment comprises approxi- CELL-MEDIATED IMMUNITY

mately 80% of the renal parenchyma with the majority of
its volume contributed by the tubules (10). The intersti- Historically, abnormalities in cell-mediated immune response
tium is made of matrix and cells. Two main types of cells have been implicated as central to the pathophysiology of TIN.
are recognized: Analysis of infiltrating mononuclear cells in human TIN of
various etiologies demonstrated the majority of them to be T
1. Type I interstitial cells are fibroblast-like cells that are lymphocytes (16–18). The T-cell lymphocytic (CD4+ and
able to produce and degrade extracellular matrix, with CD8+) interstitial infiltrates generally occur in the absence of
a subgroup residing in the inner medulla producing antibody deposition (5). Immunohistochemical studies con-
prostaglandins. ducted on biopsies obtained in drug-induced TIN also indicate
2. Type II interstitial cells include dendritic antigen- the importance of cell-cell interactions in intrarenal inflamma-
presenting cells located mainly in the cortex. tion because there is a significant increase in interstitial expres-
sion of cellular adhesion molecules. In human forms of acute
In addition, a few monocyte-derived macrophages capable TIN, increased expression of lymphocyte function–associated
of phagocytosis are found in all renal zones (11,12). The antigen-1 and very late antigen-4 cell-surface receptors, as well
818 VI. Tubular Disease

TABLE 43.1. CLASSIFICATION OF cells promotes autoimmune injury by facilitating presentation

TUBULOINTERSTITIAL NEPHRITIS (TIN) of a self (tubular)-antigen by nonlymphoid cells (27). The
Primary TIN cause for antigen expression by the tubular cells is unknown.
Infection (bacterial pyelonephritis, hantavirus, leptospirosis) Tubular epithelial cells normally do not express costimulatory
Immune-mediated (antitubular basement membrane disease) molecules such as CD80 and CD86, which likely limits their
Drug-induced (antimicrobials, analgesics, lithium, cyclosporine, ability to present antigen under physiologic conditions (30).
Chinese herbs)
Genetic susceptibility may play a factor in antigen expression
Toxins (lead)
Hereditary (cystinosis, hyperoxaluria, Wilson disease) and consequent immune response (15). Other mechanisms,
Metabolic disorders (hypercalcemia, hyperkalemia, hyperuri- such as drugs or infectious agents, may serve as inciting anti-
cosuria) gens of cell-mediated responses targeting the kidney (31,32).
Hematologic disorders (sickle cell disease) In addition, degenerate recognition of peptide antigens by
Miscellaneous (Balkan nephropathy)
autoreactive T cells in autoimmune disease has been reported
Secondary TIN
Glomerular disease (31,33). The potential relevance of such molecular mimicry
Vascular disease in autoimmune T-cell activation to renal immune responses,
Structural disease however, awaits further investigation. There is a large body of
Cystic diseases research focused on analysis of the interaction between effec-
Obstructive disease
tor and suppressor T cells in the inflammatory process in the
Reflux
renal interstitium. However, their definitive role in the patho-
Modified from Rastegar A, Kashgarian M. The clinical spectrum of genesis of TIN is still unclear (9,10).
tubulointerstitial nephritis. Kidney Int 1998;54:313–327.

ANTIBODY-MEDIATED IMMUNITY
as their respective ligands intercellular adhesion module-1 and
vascular cell adhesion molecule-1, is seen in areas of mononu- Antibody-mediated TIN is occasionally seen. When antibod-
clear cell infiltration (19). Further support of cell-mediated ies are present on immunofluorescence investigation they are
events in human disease is derived from the observation of in usually associated with the tubulointerstitial cells, along the
vivo and in vitro activation of lymphocytes isolated from basement membrane or as immune complexes. Anti-TBM
affected patients after repeat exposure to specific inciting agents staining often occurs as part of anti-GBM disease but at times
of TIN (20,21). In view of these observations, the role of cell- it appears to be a primary phenomenon resembling experi-
mediated events in TIN has been extensively studied in murine mental anti-TBM disease. Anti-TBM antibodies can be seen
models of anti-TBM disease and spontaneous TIN (15,22,23). at times in association with drug-induced TIN and more com-
In both of these models, the effector T-cell (Te) lymphocytes monly in the setting of renal transplantation due to the pres-
that differentiate in diseased animals are CD8+ renal tubular ence of foreign antigens in the transplanted kidney (34,35).
antigen–specific T cells. Nephritogenic CD8+ Te cells induce Primary immune deposit–mediated TIN is rare. It seems
renal injury in genetically susceptible animals and adoptively that in such instances the complexes are formed in situ, as cir-
transfer renal disease to naïve hosts (14,15,22,23). culating complexes would have been entrapped in the glomer-
Cell-mediated responses are initiated by T-cell recognition uli. Indeed, in cases in which immune complexes are detected
of relevant antigen presented in the context of appropriate in the interstitium, they are also observed in the glomeruli as in
major histocompatibility complex (MHC) molecules. Class I systemic lupus erythematosus (SLE), immunoglobulin A (IgA)
MHC molecules primarily direct CD8+ T cell responses, and nephropathy, and membranous nephropathy. It is believed that
class II MHC determinants direct CD4+ T cell responses in these disorders immune reactants are delivered to the inter-
(24). In vitro studies show that resident renal cells, such as stitium through the process of glomerular proteinuria. The
renal tubular epithelial cells, glomerular epithelial cells, and presence of interstitial immune complexes might still require
mesangial cells, have the potential to present a variety of anti- cellular immune response genes for the development for com-
gens (25,26). Correlation of in vitro antigen-presenting activ- plete inflammatory reaction. Most studied experimentally is
ity to disease activity in vivo, however, has not been anti-TBM disease. Other animal models for experimental anti-
established (27). It is noteworthy that renal tubular epithelial body- and immune complex–mediated TIN include the Hey-
cells express class II MHC determinants in inflammatory mann nephritis antigen complex in the brush-border disorder
renal disease and in culture when stimulated by proinflamma- (36) and reaction to Tamm-Horsfall protein (37).
tory cytokines such as interferon-gamma and tumor necrosis
factor-α (28,29). Moreover, class II MHC expression on
renal tubular epithelial cells induces in vitro proliferation of LOCAL AND EXTRARENAL ANTIGENS
antigen-specific T-cell clones and hybridomas and promotes
autoimmune renal disease in vivo. These findings suggest that Antigens presented to the immune system may be derived
induced class II MHC expression on renal tubular epithelial from resident cells or arrive at the tubulointerstitial compart-
 43. Tubulointerstitial Nephritis 819

ment from an extrarenal origin. As mentioned previously, mediated TIN. It may also indicate the importance of cor-
resident cells of the tubulointerstitial component and, in par- rection of metabolic acidosis in the prevention of progres-
ticular, tubular epithelial cells have the potential to present sive damage to the kidneys by diminishing ammonia
antigens. In addition, local antigens may be related to production. It is possible that both resident epithelial cells
Tamm-Horsfall protein or uromodulin, which is a glycopro- and infiltrating cells further amplify the damage by express-
tein secreted by the cells of the ascending loop of Henle. The ing chemokines. This subclass of structurally related cyto-
glycoprotein can cause immune deposits at the base of the kines selectively promotes the chemotaxis, adhesion, and
tubular cell and the lymphatic drainage of the ascending activation of leukocytes (58). After obstruction in the
limb and result in binding of neutrophils to the cell-surface mouse, the most potent recruiter of macrophages is mono-
anchored glycoprotein (38,39). In Heymann nephritis, anti- cyte chemoattractant peptide-1, which is expressed in tubu-
bodies reacting to the tubular brush border have been lar epithelium (59). The chemokines induce their effect by
detected, at times associated with interstitial infiltration binding to specific cell-surface receptors on target cells. The
(40,41). Most extensively studied is the antigen in antitubu- various types of leukocytes are activated by different che-
lar basement membrane (anti-TBM) disease, named 3M-1 mokines binding to different receptors. The pharmacologic
(42,43). It was detected both in humans and rodents, show- blockage of chemokines and their receptors may have a
ing extensive polymorphic expression (44,45). potential in controlling the inflammatory response (60). In
Extrarenal antigens may present to the interstitium as humans with ureteropelvic junction obstruction, urinary
isolated antigens or as part of an immune complex. Anti- monocyte chemoattractant peptide-1 is increased and
gens arriving at the tissue like those originally derived from decreases after surgical alleviation of the obstruction (61).
drugs such as penicillin, cephalosporin, and phenytoin,
combine with antibodies and inflammatory cells that ini-
tiate the interstitial disease. Another mechanism by which FIBROGENESIS AND ATROPHY
outside antigens initiate an immune response is by mimick-
ing of epitopes. For instance, some antibodies to Escherichia Interstitial fibrosis is the final common pathway for a vari-
coli cross-react with Tamm-Horsfall protein (46). It is also ety of glomerular and tubular disorders, particularly when
possible that a similar mechanism operates in TIN that fol- associated with massive glomerular proteinuria or the pres-
lows certain viral illnesses (47,48). It has also been hypoth- ence of inflammatory cells in the tubulointerstitial compo-
esized that noninfectious, shared epitopes may instigate the nent (62,63). It seems that both processes induce local
immune process, as might be the case in anti-DNA anti- cytokines that transform and activate several types of resi-
bodies (49,50). dent cells in the tubulointerstitial compartment to produce
Circulating immune deposits that settle in the intersti- new or modified extracellular matrix (64). Some of the acti-
tium can cause TIN. This might be the case in SLE, IgA vated cells probably change phenotype (65–67). It is
nephropathy, and possibly some cases of chronic idiopathic unclear why what is usually a self-limited wound repair
human tubulointerstitial nephritis (51,52). Experimentally, process continues unabated. It is possible that homeostatic
a model of chronic serum sickness disease in the rabbit regulatory mechanisms fail to stop or reverse fibrosis during
results in immune deposits in the interstitium (53). the continuous inflammatory process of chronic TIN.
Tubulointerstitial scars are primarily composed of col-
lagen types I and III, fibronectin, and tenascin (68,69).
CYTOKINES AND AMPLIFICATION OF INJURY Other glycoproteins may participate as well. Early in the
development of a scar, the fibrotic tissue may contain
Events resulting from infiltration of T cells, deposition of monocytes, tubular cells, and fibroblasts (63). It seems that
specific antibodies, or immune complexes that augment the local monocytes release cytokines affecting the activity
inflammation and injury are part of the amplification pro- of their neighboring fibroblasts. The phenotypic expression
cess. These processes include the release of cytokines and of the fibroblast and hence the renal glycoprotein synthe-
proteases from T cells; the attraction and activation of non- sized by it may be related to the type of cytokine stimulat-
specific immune effector cells, including eosinophils and ing the cell (70). Among the strongest morphogenic
macrophages (18), which release various effector products; cytokines driving TIN fibrosis is angiotensin II (71).
and the activation of the complement cascade. Interest- The source of the fibroblasts in the tubulointerstitium is
ingly, the presence of complement in TIN is inconsistent yet unknown. There are several lines of evidence that sug-
and when present is usually associated with deposition of gest they may arise from transformation of resident cells
IgG and immune complexes (53–55) or IgE and eosino- like the tubular epithelium. This mechanism is called
phils (53,55,56). Even more important seems to be the epithelial-mesenchymal transformation (72–74). The trans-
activation of the alternative passway by ammonia in the formation is probably activated by immune-mediated
absence of antibodies (57). This process may be instrumen- mechanisms and various cytokines that result in changes in
tal in the process of progressive local injury in nonimmune- the basement membrane of the tubular epithelial cell. Once
820 VI. Tubular Disease

transformed to a mesenchymal cell, it acquires motility and TABLE 43.2. CLINICAL PRESENTATION OF
becomes responsive to growth factor stimuli (75). Several TUBULOINTERSTITIAL NEPHRITIS
local chemoattractants stimulate the migration of the newly History
formed fibroblasts. These chemoattractants are released by Exposure to toxic substances
macrophages or the fibroblasts themselves (76,77). Fibro- Family history of tubulointerstitial nephritis
blasts secrete transforming growth factor β (TGFβ), which Nephrotoxic drug use
Past or family history of vesicoureteral reflux
amplifies the process (78). Once fibroblasts migrate to their
Recurrent urinary tract infections
new location, they begin to deposit fibronectin matrix, Uveitis
which serves as a skeleton for the deposition of other glyco- Symptoms
proteins. Concomitantly with the transformation process, Abdominal pain
the effect of cytokines on tubular cells can result in their Anorexiaa
Arthralgias
atrophy by disturbing their basement membrane synthesis.
Diarrhea
Additional damage to the tubular cell can be caused by T- Dysuria
cell clones, with cytotoxic activity resulting in tubular cell Edema
destruction and atrophy. Thus, tubular atrophy and inter- Emesisa
stitial fibrosis coexist. Eye tenderness (seen in TINU syndrome)
Fatiguea
Fevera
ACUTE TUBULOINTERSTITIAL NEPHRITIS Flank or loin pain
Headache
Lymphadenopathy
Epidemiology Malaise
Myalgia
Acute TIN accounts for 10 to 25% of reported cases of
Nocturia
acute renal failure (ARF) in adults (3) and up to 7% of chil- Polydipsia
dren with ARF (79). However, in both children and adults, Polyuria
acute TIN may be underreported because many patients Rash
with ARF recover spontaneously after removal of the sus- Sore throata
Weight lossa
pected offending agent, and definitive diagnosis based on a
Signs
renal biopsy is not routinely established (7). Lower reported Abdominal pain
incidences of acute TIN in children than in adults may Arthritis
reflect more common use of nephrotoxic pharmacologic Costovertebral tenderness
agents and greater prevalence of preexisting renal abnor- Edema
Evidence for left ventricular hypertrophy
malities in older patients.
Fevera
Hypertension
Hypertensive retinopathy
Clinical, Laboratory, and
Lacrimation (seen in TINU syndrome)
Radiologic Features Lethargy
Pallora
The clinical manifestations of TIN are variable, and many
Pharyngitis
of them are seen in both acute and chronic TIN (Table Poor growtha
43.2). The severity of renal impairment ranges from mild Rachitic changesa
azotemia and asymptomatic urinary abnormalities to oligu- Rash (may be maculopapular, morbilliform, or urticarial)
ric ARF (80). The nonspecific nature of the clinical find- Volume depletion
ings in TIN emphasizes the need for a renal biopsy to make
TINU, tubulointerstitial nephritis with uveitis.
a definitive diagnosis in questionable cases (4,81). In one aCommon finding in children.

series of 13 children with biopsy-proven acute TIN, only 6

were suspected of having the diagnosis before the procedure
(82). However, in a child suspected to have acute TIN, if one-half of the patients (84). The rash may be maculopapu-
improvement in kidney function is noticed after withhold- lar, morbilliform, or urticarial, and it often is fleeting (84–
ing the suspected offending agents and starting treatment, 87). Arthralgias are seldom a prominent feature (84). Acute
there is no need for a biopsy. TIN associated with infection may present with extrarenal
Systemic manifestations of a hypersensitivity reaction, manifestations of disease, such as fever and sore throat (82).
such as fever, rash, and arthralgias, are variable findings. Nonspecific constitutional symptoms of fatigue, anorexia,
Although these symptoms are more likely to occur in drug- weight loss, nausea and vomiting, and flank pain with mac-
induced acute TIN (79,83), in a series of nine adults with roscopic hematuria may occur (85,87). Oliguria can occur,
acute TIN caused by medications, components of this clas- but 30 to 40% of patients with acute TIN have nonoliguric
sic triad were presenting symptoms in only approximately ARF (3). Hypertension and edema are seldom noted,
 43. Tubulointerstitial Nephritis 821

TABLE 43.3. PATTERNS OF RENAL TUBULAR DYSFUNCTION IN

TUBULOINTERSTITIAL NEPHRITIS
Nephron site Functional defect Clinical manifestation

Proximal tubule Decreased HCO3, PO4, amino acid, uric Fanconi syndrome, hyperchloremic
acid, and glucose reabsorption metabolic acidosis
Loop of Henle Defective NaCl reabsorption, decreased Polyuria (polydipsia), salt wasting,
magnesium reabsorption magnesium losses
Distal tubule Defective NaCl reabsorption and potas- Hyperkalemia, hyperchloremic meta-
sium and hydrogen ion secretion bolic acidosis, salt wasting
Collecting Defective water reabsorption Nephrogenic diabetes insipidus
tubule

Modified from Toto RD. Review: acute tubulointerstitial nephritis. Am J Med Sci 1990;299:392–410.

except in specific drug-induced lesions, such as the neph- TABLE 43.4. SERUM AND URINE ABNORMALITIES
rotic syndrome associated with nonsteroidal antiinflamma- OFTEN FOUND WITH TUBULOINTERSTITIAL
NEPHRITIS (TIN)
tory drugs (NSAIDs) (88) or in TIN secondary to
glomerular disease (8,80). Serum abnormalities
Acute and chronic TIN have quite similar laboratory Anemia
findings (7). Renal tubular epithelial cell damage is the pre- Hemolytic
Nonhemolytica
dominant finding in all forms of TIN, and biochemical
Circulating immune complexes (may be seen in immune-
abnormalities observed in patients with TIN may reflect mediated TIN)
injury to specific nephron segments involved in the inflam- Elevated hepatic enzymes (in patients with associated drug-
matory process (Table 43.3) (89). In general, cortical dam- induced liver damage)
age may affect mainly the proximal and distal tubules, Eosinophiliaa
Hyperchloremic metabolic acidosis (normal anion gap) a
whereas medullary lesions may affect mainly the loop of
Hypernatremia
Henle and the collecting duct. Proximal tubule injury Hypokalemia
results in Fanconi syndrome with impaired urinary reab- Hypoaldosteronism
sorption of glucose, bicarbonate, phosphorus, amino acids, Hypophosphatemia
and uric acid (90). Damage to the loop of Henle may result Hyporeninemia
Hypouricemia
in magnesium and sodium losses. Distal tubular lesions
Increased erythrocyte sedimentation rate a
result in renal tubular acidosis, impaired potassium excre- Increased immunoglobulin E level
tion, and sodium wasting (91). Biochemical abnormalities Increased immunoglobulin G level
in TIN include an elevated serum creatinine concentration Increased blood urea nitrogena
with hyperchloremic metabolic acidosis (3,82). If proximal Increased creatininea
Leukocytosis (with or without eosinophilia)
tubular involvement is prominent, serum phosphorus,
Urine abnormalities
bicarbonate, uric acid, and potassium concentrations may Aminoaciduria
be decreased. Many patients have nonoliguric renal failure, Bacteriuria
a urine concentrating defect, and relatively bland changes Bicarbonaturia
on urine microscopic examination. Eosinophiluria (seen usually in drug-induced TIN)
Glucosuria (in absence of hyperglycemia)
Relevant serologic studies in the investigation of TIN are
Hematuria (usually microscopic)a
listed in Table 43.4. Normochromic, normocytic anemia is Hyposthenuria
often associated with TIN (79,89). Hemolytic anemia has Leukocyturia
also been reported in acute TIN induced by allopurinol, Magnesium wasting
penicillins, and rifampin (4,89). Leukocytosis may occur, Phosphaturia
Proteinuria (usually mild to moderate)a,b
and some but not all patients with drug-induced TIN have
Casts
a peripheral eosinophilia (83,84,87). Drug-induced liver Granular
damage is reflected in elevated hepatic enzymes (92,93). Hyaline
Elevated serum IgE titers have been reported in up to 50% Red blood cells (seen occasionally in drug-induced TIN)
of biopsy-proven cases and is suggestive but not diagnostic White blood cells (seen in infection-mediated acute or chronic
TIN)
of drug-induced TIN (80,84). Anti-DNA antibodies, anti-
Salt-wasting
nuclear antibodies, and complement levels are normal in
most forms of TIN, unless associated with a systemic aCommon finding in children.
autoimmune disorder (3,85). Circulating anti-TBM anti- bCanbe massive with nonsteroidal antiinflammatory drugs and less
bodies are detected on rare occasions (86). commonly with other drugs.
822 VI. Tubular Disease

Urinary abnormalities vary considerably (Table 43.4). tures are unaffected (80,84). Mild mesangial hypercellular-
Microscopic hematuria is commonly detected, and macro- ity or periglomerular inflammation and fibrosis occasionally
scopic hematuria and sterile pyuria also occur (79,87). Mild are seen (82). Cellular infiltrates, primarily of lymphocytes,
to moderate proteinuria (less than 1 g/day) is detected in often are patchy and are variably distributed throughout the
most patients with TIN (82). Nephrotic range proteinuria is kidney (5,84). Monocytes, macrophages, and plasma cells
not characteristic of TIN but has been associated with acute are also commonly noted in affected areas (82,85). Eosino-
TIN induced by certain medications, including NSAIDs, phils are most commonly seen with drug-induced acute
lithium, ampicillin, and rifampin (5,94). Urinary sediment TIN and can comprise up to 10% of the infiltrate (86).
analysis shows granular, hyaline, and white blood cell casts Interstitial granulomas occasionally are seen, particularly in
(5). Red blood cell casts are rarely seen in acute TIN (82). drug-induced or infection-related acute TIN (97). Comple-
Eosinophiluria, defined as greater than 1% of urinary leuko- ment and immunoglobulin deposition is not characteristic
cytes stained positively with Hansel’s or Wright’s stain, is sug- of acute TIN (5,98,99) but has been noted in children with
gestive of acute TIN and has been reported in 50 to 90% of acute TIN because of SLE, syphilis, hepatitis B, shunt
patients with drug-induced acute TIN (84,95). However, nephritis, and some drug-induced lesions (100).
eosinophiluria can be seen in many inflammatory renal dis- Tubular epithelial cell damage varies from minimal his-
eases (96). In a study of 51 patients, eosinophiluria had a tologic changes to frank necrosis (83,98). Most often there
sensitivity of 40% and specificity of 72%, with a positive pre- is flattening of renal tubular cells with atrophy, degenera-
dictive value for TIN of only 38% (96). tion, and loss of the brush border in proximal convoluted
Renal ultrasound examination usually demonstrates nor- tubules (5,8). Renal tubules are commonly dilated, with
mal or enlarged kidneys, depending on the extent of tubuloin- splitting or fracturing of the basolateral membrane. The
terstitial inflammation and edema (3,79). Gallium citrate tubule lumens may contain desquamated cells or blood
scanning may be useful in distinguishing acute TIN from (8,82). In some instances, infiltrating lymphocytes are
other common causes of ARF (84). However, increased uptake observed between tubular epithelial cells (84). Electron
on gallium scan is a relatively nonspecific finding and may be microscopy typically reveals striking renal tubular epithelial
seen in cases of allograft rejection, acute pyelonephritis, and cell mitochondrial damage, cytoplasmic vacuoles, and sig-
severe minimal change nephrotic syndrome (3). A definitive nificantly dilated rough endoplasmic reticulum (81,82,84).
diagnosis of TIN can be made only by renal biopsy (3,4).
Etiology
Pathology
The causes of acute TIN can be grouped into four broad
Typical light microscopy findings in acute TIN consist of categories: medications, infections, immunologic diseases,
tubulointerstitial mononuclear cell infiltration and edema, or idiopathic processes (Table 43.5). As described in the
with varying abnormalities in renal tubular epithelial cells Pathology section of this chapter, all forms of acute TIN
(Fig. 43.1) (80,81). In general, vessels and glomerular struc- have similar pathologic features. Unique features of distinct
forms of acute TIN are described in more detail.

Medications
Medications, rather than infection, are now the leading
cause of acute TIN in children (8). An ever-increasing list of
medications is implicated as causing acute TIN (Table
43.6). Establishing a clear link between a medication and
acute TIN may be difficult, however, because many case
reports of drug-induced acute TIN are in patients who have
received several medications simultaneously. Nevertheless, a
few categories of drugs are consistently associated with acute
TIN in both children and adults: antibiotics, NSAIDs, anti-
convulsants, and diuretics (83,84,87). Although symptoms
of acute TIN caused by a specific drug can occur within
hours or months after starting a medication, they are usually
seen 2 to 3 weeks after starting therapy (4). Antimicrobials
FIGURE 43.1. Acute tubulointerstitial nephritis (TIN). Light and NSAIDs are the most common drugs causing acute
microscopy of idiopathic TIN in a 14-year-old girl. Cortical renal TIN in children (83,87). The incidence of acute TIN
parenchyma showing a normal glomerulus, extensive interstitial
edema, infiltration by lymphocytes and eosinophils, and cellular caused by methicillin, which is no longer used, was 16%
epithelial tubular degenerative changes. (87). The incidence of acute TIN with currently available
 43. Tubulointerstitial Nephritis 823

TABLE 43.5. ETIOLOGIC CLASSIFICATION OF ACUTE ents. Early diagnosis and treatment prevented permanent
TUBULOINTERSTITIAL NEPHRITIS damage (103).
Immune-mediateda
Drug hypersensitivity (see Table 43.6)
β-Lactam antibioticsa
Infections
Other antibiotics In the preantibiotic era, infection, especially streptococ-
Diuretics
Nonsteroidal antiinflammatory drugs
cal, was the predominant cause of acute TIN in children.
Other drugs Councilman’s initial postmortem findings of TIN
Immunologic diseases involved 42 children who succumbed to scarlet fever
Usually associated with glomerulonephritis (104). With the development of effective antimicrobial
Immunoglobulin A nephropathy therapy, the incidence of Streptococcus-associated acute
Membranous glomerulonephritis
Syphilis
TIN has decreased. Other infectious organisms have
Systemic lupus erythematosusa become important causes of acute TIN. These include
Usually not associated with glomerulonephritis unusual bacteria such as Rickettsia species, Yersinia, and
Allograft rejection mycoplasma. Viruses, such as adenovirus and human
Tubulointerstitial nephritis with uveitis syndrome immunodeficiency virus, and parasites are also causes of
Infection-mediateda (see Table 43.7)
Direct infection of renal parenchyma (infectious agents identi-
acute TIN (Table 43.7) (5,105–107). Infections may
fied in the interstitium) induce acute TIN by two distinct processes. Organisms
Reactive (sterile) interstitial nephritis (infectious agents not may directly invade the renal parenchyma, producing
identified in the interstitium) local renal infection and inflammation. This form of acute
Idiopathic TIN may respond to treatment of the underlying infec-
aCommon
tion. Alternatively, organisms may induce “reactive” intra-
cause in children.
renal inflammation without evidence of renal infection.
The latter mechanism is implicated by the observation
penicillins is significantly less than that previously observed that group A streptococcal infections can cause acute TIN
with methicillin. Systemic manifestations of hypersensitivity in children, even with appropriate antibiotic therapy (82).
with fever, rash, and eosinophilia in the setting of ARF can Mechanisms for the renal inflammatory reaction in the
occur in up to 30% of patients with penicillin-induced absence of renal infection are not clearly elucidated but
acute TIN (4). By contrast, these hypersensitivity manifesta- are presumed to be immunologically mediated (82).
tions are not typically associated with NSAID-induced
acute TIN (88). NSAID-induced TIN may be associated
Immunologic Diseases
with minimal change nephrotic syndrome (94). Although
NSAID-induced acute TIN in children occurs less often Immunologic diseases elicit acute TIN in two distinct settings
than in adults, the increasing availability of over-the-counter (Table 43.5). TIN occurs with either a primary glomerular
NSAID preparations for children may result in an increase lesion or, less commonly, as an isolated primary TIN. SLE
in cases in that age group. is the most important cause of acute TIN seen in associa-
Drug-induced acute TIN is an idiosyncratic reaction, tion with glomerulonephritis in children. Tubulointerstitial
and, as such, it is difficult to predict which patients will be immune deposits may be present in up to 60% of SLE
affected. No specific risk factors have been consistently patients who are biopsied and correlate with both severity of
identified. Acute TIN has been reported with various routes interstitial inflammation and degree of functional renal
of administration, including oral, intravenous, intramuscu- impairment (108,109). As is the case with other glomerular
lar, and rectal (83,87,98,101). Duration and the dosage of diseases, severe tubulointerstitial involvement with fibrosis is a
therapy also do not correlate well with the development of poor prognostic indicator for renal function (100). Acute TIN
disease, although several reports suggest that acute TIN is occasionally is seen in children with membranous nephropa-
more common with high-dose therapy (7,87). thy, postinfectious glomerulonephritis, and shunt nephritis
The association of drug-induced acute TIN with fever, (100). IgA nephropathy was associated with significant TIN in
rash, and eosinophilia suggests an underlying allergic reac- 37% of 51 patients (110). At follow-up, patients with renal
tion to administered drugs. Reports of accelerated, recurrent tubular deposits had significantly worse renal function than
TIN on drug rechallenge also implicate drug hypersensitivity those with isolated glomerular findings (110). Immunologic
(101,102). Administration of structurally similar medica- diseases that present with isolated tubulointerstitial involve-
tions may induce cross-reactivity as evidenced by recurrent ment are tubulointerstitial nephritis with uveitis (TINU) syn-
acute TIN after cephalosporin administration in individuals drome, allograft rejection, and rarely SLE (111,112). Allograft
with previous penicillin-induced TIN (87). rejection is discussed in detail in Chapter 75.
Drug-induced acute interstitial nephritis was found to TINU syndrome was first described in 1975 in a report
be a cause for graft dysfunction in kidney transplant recipi- of two adolescent girls who developed ARF with eosino-
824 VI. Tubular Disease

TABLE 43.6. DRUGS ASSOCIATED WITH ACUTE TUBULOINTERSTITIAL NEPHRITIS

Anticonvulsants Cloxacillina Triamterene
Carbamazepinea Flucloxacillin Other drugs
Lamotrigine Methicillina Aldomet
Phenobarbitala Mezlocillin Allopurinol
Phenytoina Nafcillina Amlodipine
Sodium valproatea Oxacillin Amphetamine
Antiinflammatory drugs and analgesics Penicillin Ga Anti-CD4 antibodies
Benoxaprofen Other antibiotics Aspirin
Diclofenac p-Acyclovir Azathioprine
Diflunisal Azithromycin Captopril
Fenoprofen Aztreonam Chlorprothixene
Floctafenine Chloramphenicol Cimetidine
Ibuprofen Ciprofloxacin Clofibrate
Indomethacin Clarithromycin Clozapine
Ketoprofen Clotrimazole Coumadin
Mefenamic acid Erythromycina Crack cocaine
Naproxena Gentamicin Creatine
Niflumic acida Indinavir Cyclosporina
Phenazone Isoniazid Cytosine-Arabinoside
Phenylbutazone Lincomycin Diazepam
Piroxicam Loracarbefa Doxepin
Rofecoxib Nitrofurantoin Ethambutol
Sulfasalazine Norfloxacin Haloperidol
Sulfinpyrazone p-Aminosalicylic acid Heroin
Sulindac Piromidic acid Herbal medicines
Suprofen Polymyxin sulfate Imipramine
Tolmetina Rifampin Interleukin-2
Zomepirac Spiramycin Mesalaminea
β-Lactam antibiotics Sulfonamidesa Omeprazole
Cephalosporins Sulfadiazine Phenazone
Cefaclor Trimethoprim-sulfamethoxazole a Phenindionea
Cefotaxime Tetracyclines Phenylpropanolamine a
Cefoxitin Minocyclinea Propranolol
Cephalexin Vancomycin Propylthiouracil
Cephaloridine Diuretics Quinine
Cephalothin Chlorthalidone Radiographic contrast agents
Cephradine Ethacrynic acid Ranitidine
Penicillins and derivatives Furosemide Recombinant interferon-alpha
Amoxicillin Thiazides Streptokinase
Ampicillina Ticrynafen
Carbenicillin Tienilic acid

aCases reported in children.

philic TIN associated with anterior uveitis and bone mar- Patients usually have anorexia, fever, weight loss, abdomi-
row granulomas (113). Subsequent reports have highlighted nal pain, and polyuria. Eye tenderness may not be evident
the significant adolescent female predominance (111). The at presentation, as uveitis could occur at any time with
pathogenesis of this syndrome remains unclear, and pre- respect to the onset of renal disease (117). Laboratory
liminary evidence suggests roles for both humoral- and abnormalities include an elevated erythrocyte sedimenta-
cell-mediated immune mechanisms. Circulating or depos- tion rate, increased serum IgG levels, azotemia, and non-
ited immune complexes were detected in up to 60% of hemolytic anemia. Urinary abnormalities include proteinuria,
patients (114,115). A significant number of patients, how- glucosuria, and sterile pyuria. The interstitial nephritis
ever, have no evidence of tubulointerstitial immune depos- resolves completely, either spontaneously or after steroid
its on renal biopsy. Analysis of the lymphocytic infiltrates therapy in most cases. In a study of 21 children with acute
on biopsies of patients with TINU demonstrated a pre- TIN, TINU patients required a longer period of time for
dominance of activated memory T-helper cells, thus pro- the renal function to recover in comparison with other eti-
viding further evidence for cell-mediated events (111). A ologies (118). The uveitis often requires multiple steroid
genetic predisposition is suggested by the occurrence of courses and other immunosuppressives, like methotrexate,
TINU in identical twins within the same year (116). azathioprine, and cyclosporin A, and may relapse (100,119).
 43. Tubulointerstitial Nephritis 825

TABLE 43.7. CLASSIFICATION OF by exclusion of the previously described entities and usually
TUBULOINTERSTITIAL NEPHRITIS ASSOCIATED are not associated with hypersensitivity symptoms of fever,
WITH INFECTION
rash, or eosinophilia (3).
Direct infection of renal parenchyma (infectious agents identi-
fied in the interstitium)
Bacteria Treatment and Prognosis
Leptospira spp.a
The initial treatment of acute TIN is primarily supportive,
Mycobacteria
Various species commonly associated with pyelonephritis a with dialysis therapy as indicated (3,4). It is important to
Viruses immediately discontinue all possible offending medications,
Adenovirusa which in many patients might be multiple. When replacing
Cytomegalovirusa the suspected medications, it is important to select medica-
Hantavirusesa
tions that are not potentially cross-reactive (e.g., a cepha-
Polyoma virus (BK type)
Fungi losporin to replace a β-lactam penicillin) or other potentially
Histoplasma nephrotoxic agents (87). In infection-related acute TIN, spe-
Various species commonly associated with pyelonephritis cific treatment of the underlying infection is indicated (82).
Rickettsia The use of corticosteroid therapy for acute TIN remains
Rickettsia diaporica (Q fever)
controversial (7). Anecdotal case reports and uncontrolled
Rickettsia rickettsii
Reactive (sterile) interstitial nephritis (infectious agents not trials with small numbers of patients suggest a therapeutic
identified in the interstitium) benefit (9,79,82). However, prospective controlled studies of
Bacteria corticosteroids or other cytotoxic agents in acute TIN are
Brucella spp. lacking, and it is possible that many reported patients would
Corynebacterium diphtheriae
have recovered merely from the withdrawal of the inciting
Francisella tularensisb
Group A-hemolytic streptococcusa agent. Various steroid treatment regimens for acute TIN have
Legionella pneumophila been reported, primarily in the adult literature (84,123).
Mycoplasma hominis Some children have been treated with daily prednisone at a
Salmonella typhi dosage of 2 mg/kg/day, which is tapered rapidly over 2 to 4
Streptococcus pneumoniaea
weeks (82). Pulse methylprednisolone followed by high-dose
Treponema pallidumb
Yersinia pseudotuberculosisa daily or alternate-day prednisone has also been used in adults
Viruses (123). In idiopathic cases and in those in whom removal of
Epstein-Barr virusa the offending agent do not result in improvement, we use a
Hepatitis B virusb similar protocol of pulse methylprednisolone followed by
Human immunodeficiency virus (HIV)
daily oral prednisone of 2 mg/kg (maximum dose, 80 mg)
Mumpsa
Rubella virusb (togavirus) for 4 weeks, then change to alternate day treatment and taper
Rubeola virus (paramyxovirus) over a period of several months depending on the response.
Parasites Prognosis for recovery of renal function in children with
Ascarisb acute TIN is excellent (8,79,82,87). Most affected patients
Leishmania donovani
recover renal function completely within weeks to months of
Toxoplasma gondiia
Other onset of ARF. The mean renal recovery time of 13 children
Kawasaki disease with acute TIN from various causes was 69.5 ± 34.7 days (82).
All patients had normal serum creatinine and urinalysis at fol-
aCases reported in children. low-up examination 1.5 to 10.0 years after presentation. Of
bIncompletely documented or isolated cases.
Modified from Colvin RB, Fang LST. Interstitial nephritis. In: Tischer
note, the clinical or histologic severity of disease at presenta-
CC, Brenner BM, eds. Renal pathology. Philadelphia: JB Lippincott, tion did not correlate with rate of recovery of renal function.
1994:723–768. Another study of seven children with acute TIN, 42% of who
required dialysis, reported a renal recovery rate of 86% (79). In
two studies of adults with acute TIN, poor prognostic factors
Recent case reports described the association of TINU included the severity of the interstitial inflammation on renal
with granulomatous hepatitis (120), hyperthyroidism biopsy and the duration of ARF (124,125).
(121), and Epstein-Barr virus infection (122).
CHRONIC INTERSTITIAL NEPHRITIS
Idiopathic
Epidemiology
Idiopathic cases of acute TIN are uncommon in childhood.
In a series of 12 children with biopsy-proven acute TIN, Chronic TIN in children occurs primarily in the setting of
only 1 had idiopathic disease (8). These cases are diagnosed obstructive uropathy, vesicoureteral reflux, and inherited
826 VI. Tubular Disease

conditions (3,9). Obstructive uropathy accounts for almost

20% of all children who develop end-stage renal disease
(126). Primary chronic TIN from other causes accounts for
only 2 to 4% of children with chronic renal failure (126).
In a few families, chronic TIN has been described in associ-
ation with cholestatic liver disease (92,93) and in others
with mitochondrial abnormalities (127,128).

Clinical, Laboratory, and

Radiologic Features
Chronic TIN tends to progress more slowly than other forms
of chronic renal diseases, and therefore, affected individuals
often have no clinical evidence of disease until late in the
course of renal insufficiency (3). Many patients have nonspe-
FIGURE 43.2. Chronic tubulointerstitial nephritis (TIN). Light
cific constitutional symptoms characteristic of chronic renal microscopy of idiopathic chronic TIN in a 12-year-old boy. Renal cor-
failure, with weight loss, growth retardation, fatigue, tex showing moderate interstitial fibrosis, tubular atrophy, and tor-
anorexia, vomiting, and occasionally polyuria and polydipsia tuosity. The interstitium is infiltrated by scattered mononuclear
cells. A preserved glomerulus with periglomerular fibrosis is seen.
(129). Hypertension may also occur (130). Patients with the
syndrome of chronic TIN associated with cholestatic liver
disease may also have symptoms of hepatic dysfunction, such
as pruritus and scleral icterus (131). Naturally, in children associated with primary glomerular or vascular disorders,
with chronic TIN, a major manifestation may include especially focal segmental glomerulosclerosis and hemolytic
growth failure. uremic syndrome, is a common histologic finding in
The clinical, laboratory, and radiologic findings in patients with progressive renal disease (133).
chronic TIN are in essence similar to those observed in Chronic TIN in children most commonly is due to
acute TIN. However, as in other cases of chronic kidney obstructive uropathy or high-grade vesicoureteral reflux,
failure, the deterioration in kidney function is insidious, especially when associated with urinary tract infections
and the medical history might extend back to a longer (126). Urinary tract obstruction of only a few weeks’ dura-
period. In contrast to some patients with acute TIN, tion may result in irreversible renal damage (7). Isolated
patients with chronic TIN do not have oliguria; on the vesicoureteral reflux without obstruction might also be
contrary, some may be polyuric. In advanced cases, renal associated with chronic TIN in children. Interstitial dam-
ultrasonography shows small and hyperechogenic kidneys. age and progressive fibrosis in these disorders may result
Skeletal radiographs may show renal osteodystrophy and in from renal immune responses that amplify tubulointersti-
the case of Fanconi syndrome rickets and osteomalacia. tial injury initially induced by high urinary tract pressures
in the setting of infection. Persistent and progressive renal
inflammation and damage can occur, however, despite
Pathology
relief of the obstruction or surgical correction of the vesi-
Histologic findings in chronic TIN are characterized by coureteral reflux (127,134,135).
tubulointerstitial fibrosis with a lymphocytic infiltrate, as
well as tubular atrophy and thickening of the TBM
Treatment and Prognosis
(3,132). When renal damage is associated with high
intratubular pressure, as occurs with vesicoureteral reflux There is currently no known effective therapy for chronic
and urinary tract obstruction, dilated renal tubules are par- TIN. Naturally, when an offending agent is detected, it
ticularly evident (81). In primary chronic TIN, glomeruli should be removed. However, even in such cases the dam-
are often normal until late in the course of disease, when age may be irreversible and its progression self-perpetuat-
periglomerular fibrosis and global sclerosis are seen (Fig. ing. Some experimental data indicate the potential for
43.2) (131,132). pharmacotherapy in stopping or slowing the inflamma-
tory, apoptotic, and fibrotic mechanisms by blocking their
stimulating signals and activity. For instance, in the mouse
Etiology
model of unilateral obstruction, injury to the kidneys
The causes of chronic TIN are summarized in Table 43.8. starts with increased angiotensin II production, which
Of note, acute TIN from any cause can progress to chronic activates TGFβ in a cascade, leading to tubulointerstitial
TIN if the disease process is not abated by removal of the inflammation and fibrosis (59). The use of angiotensin-
inciting agent or steroid therapy. Secondary chronic TIN converting enzyme (ACE) inhibitors in this model dimin-
 43. Tubulointerstitial Nephritis 827

TABLE 43.8. ETIOLOGIC CLASSIFICATION OF CHRONIC INTERSTITIAL NEPHRITIS

Drug-related Systemic lupus erythematosusa
Acetaminophen Antiglomerular basement membrane disease
Aspirin Mixed cryoglobulinemia
cis-Platinum Polyarteritis nodosa
Cyclosporinea Wegener granulomatosis
Lithium Diseases not usually associated with glomerulonephritis
L-Lysine Allograft rejection
Methoxyflurane Chronic active hepatitis
Nitrosoureas (streptozotocin, CCNU, BCNU) Familial immune complex interstitial nephritis
Phenacetin Sjögren syndrome
Phenylbutazone Tubulointerstitial nephritis and uveitis syndrome
Propylthiouracil Radiation-induced (induces cytotoxic reactive oxygen molecules)
Heavy metal–related Metabolic
Arsenic Hypercalcemia
Bismuth Hypercalciuria
Cadmium Hyperphosphatemia
Copper Hyperuricemia
Gold Hyperoxaluria
Iron Hyperparathyroidism
Lead Hypokalemia (potassium-losing nephropathy)
Mercury Miscellaneous
Uranium Anorexia nervosa
Hereditary Balkan nephropathy
Acute intermittent porphyria Hemorrhagic fever
Alport syndrome Syndrome of tubulointerstitial nephritis and chronic cholestatic liver
Cystinosis disease
Lesch-Nyhan syndrome Hypoxic disorders
Medullary cystic disease–Juvenile nephronophthisis complex Sarcoidosis
Medullary sponge kidney Neoplastic
Oxalosis Leukemia
Methylmalonic acidemia Lymphoma
Fabry disease Multiple myeloma
Polycystic kidney disease Urologica
Sickle cell disease Urinary tract obstruction
Wilson disease Calculi
Idiopathic Congenital
Infection-mediated (see Table 43.7) Posterior urethral valvesa
Direct infection of renal parenchyma (chronic or recurrent Prune belly syndromea
acute pyelonephritis) Ureteropelvic junction obstructiona
Reactive (sterile) interstitial nephritis Surgery
Immune-mediated Tumor
Diseases usually associated with glomerulonephritis Vesicoureteral reflux a

aCommon cause in children.

ished transformation of renal cells to interstitial myofibro- of action by which bone morphometric protein-7 provides
blasts and decreased migration of inflammatory cells into a renoprotective effect is yet unknown (59). Several studies
the interstitium. However, ACE inhibitors do not affect have demonstrated the potential of 3-hydroxy-3-methyl-
tubular atrophy (59). glutaryl CoA reductase inhibitors, like simvastatin and flu-
In a recent study in the mouse model of unilateral ure- vastatin, in ameliorating interstitial fibrosis in animal
teral obstruction, injection of exogenous hepatocyte growth models (137,138). Other studies examined the effect of
factor blocked myofibroblast activation and dramatically blocking TGFβ and its mediators, like connective tissue
prevented interstitial fibrosis (136). The study suggested growth factor in tissue fibrosis (139). A different direction
that blockage of the transition of tubular epithelial cells to of investigation is focused on the potential protective effect
myofibroblasts may provide a novel therapeutic approach of nitric oxide in chronic TIN (61,140,141).
in halting fibrosis. In another study on the same animal At the moment, patients with chronic TIN are treated
model, administration of bone morphometric protein-7 with supportive therapy. Whether the use of angiotensin-
prevented tubulointerstitial fibrosis and inhibited tubular converting enzyme inhibitors or 3-hydroxy-3-methylgluta-
atrophy by prevention of apoptosis. The exact mechanism ryl CoA reductase inhibitors in human chronic TIN is
828 VI. Tubular Disease

effective as it is in some glomerular diseases associated with that mediates murine interstitial nephritis. J Clin Invest
proteinuria has yet to be proven. 1991;88:408–416.
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cific forms of chronic TIN, such as juvenile nephronoph- morphic gene products defining both immune response
genes and a restrictive requirement for cytotoxic T cells at
thisis, progression to end-stage renal disease is almost
H-2K. J Exp Med 1982;125:1075–1085.
inevitable (129,130). By contrast, patients with chronic 16. Bender WL, Whelton A, Beschorner WE, et al. Interstitial
TIN in the setting of obstructive uropathy have a more nephritis, proteinuria, and renal failure caused by nonsteroidal
variable clinical course (127,134). anti-inflammatory drugs: immunologic characterization of the
inflammatory infiltrate. Am J Med 1984;76:1006–1012.
17. Boucher A, Droz D, Adafer E, et al. Characterization of
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Supported by the Sam and Helen Kaplan Research Fund in 18. Rosenberg ME, Schendel PB, McCurdy FA, et al. Charac-
Pediatric Nephrology. terization of immune cells in kidneys from patients with
Sjögren’s syndrome. Am J Kidney Dis 1988;11:20–22.
19. Mampaso F, Sanchez-Madrid F, Molina A, et al. Expression
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 S E C T I O N

V I I

SYSTEMIC DISEASE
 44

RENAL VASCULITIS
RUDOLPH P. VALENTINI
WILLIAM E. SMOYER

Renal vasculitis, or inflammation of the blood vessels clinically as glomerulonephritis. The vasculitides involving
involving the kidney, is a process mediated by neutrophilic larger vessels such as classic polyarteritis nodosa (cPAN)
infiltration of vessel walls resulting in fibrinoid necrosis. Its (medium vessels) or Takayasu’s arteritis (TA) (large vessels) are
consequences include compromise of the vascular lumen more apt to result in renal ischemia and hypertension.
and tissue ischemia. Renal vascular involvement is defined as Further characterization of a child with a small vessel vascu-
a process affecting the blood supply to the kidney and its litis manifesting as a pulmonary renal syndrome requires labo-
vessels within. Therefore, this consists of vessels of various ratory testing and in many instances a tissue biopsy. Serologic
sizes such as the aorta, renal artery, renal arterioles, or glo- testing includes C3 and C4, antinuclear antibodies (ANAs),
merular capillaries. Typically, renal manifestations of vascu- anti–double-stranded DNA antibodies, anti-GBM antibodies,
litis include hypertension and glomerulonephritis. antineutrophil cytoplasmic autoantibodies (ANCAs), anti-
Pediatric diseases associated with renal vasculitis may be proteinase 3 (PR3) antibodies, and anti-myeloperoxidase
renal-limited, but are more commonly systemic. Those (MPO) antibodies.
with systemic manifestations may involve other large, A histologic classification scheme uses features of the renal
medium, and small vessels outside of the renovascular sys- biopsy to differentiate disease states. Light microscopy can
tem, such as the subclavian arteries, coronary arteries, alve- point out the presence of a necrotizing glomerulonephritis as
olar capillaries, and postcapillary venules of the dermis. can be seen in the small vessel vasculitides. In addition, the
Depending on the organ systems involved, sinopulmonary, presence of granulomas would point further toward WG or
dermatologic, gastrointestinal (GI), and/or musculoskeletal Churg-Strauss disease (1). Unfortunately, granulomas, when
symptoms may develop. In an effort to afford clinicians an present, are less likely to be seen on a renal biopsy when com-
easier way to differentiate the various forms of systemic vas- pared to a lung biopsy (2,3).
culitis, a few classification schemes have been developed. Immunofluorescence is the most useful part of the renal
Because no one classification scheme is complete, a combi- biopsy when classifying forms of renal vasculitis. Immune
nation of various schemata is typically needed. complex diseases such as HSP and lupus have characteristic
A clinical classification scheme based on the organs glomerular staining patterns. Linear immunoglobulin G
involved can be used to develop a differential diagnosis. For staining along the GBM is characteristic of anti-GBM dis-
example, a child presenting with glomerulonephritis and ease. Minimal or absent immunofluorescence staining is
pulmonary hemorrhage is considered to have a pulmonary seen in the pauciimmune glomerulonephritides, such as
renal syndrome. The differential diagnosis for this clinical WG, MPA, Churg-Strauss disease, and ANCA (+) pauci-
entity includes Wegener granulomatosis (WG), micro- immune crescentic glomerulonephritis. A summary of the
scopic polyangiitis (MPA), anti–glomerular basement above classification systems can be seen in Table 44.2.
membrane (anti-GBM) disease, systemic lupus erythema- This background is used as a foundation on which to
tosus (SLE), and Henoch-Schönlein purpura (HSP) (Table develop a more detailed comparison of these various forms of
44.1). This differential diagnosis can be further narrowed renal vasculitis. The reader should note that anti-GBM anti-
after considering further clinical and laboratory data. body disease (Goodpasture disease) is not always classified
Another vasculitis classification scheme relates to the cali- with vasculitis, although it is an immune-mediated process
ber of the vessel(s) involved. The differential diagnosis can be resulting in a focal necrotizing and crescentic glomerulone-
separated into diseases involving small, medium, or large ves- phritis and pulmonary hemorrhage. For further discussion of
sels. The small vessel vasculitides with renal involvement (e.g., anti-GBM antibody disease, see Chapter 34. The forms of
HSP, SLE, WG, MPA, Churg-Strauss disease, and anti-GBM vasculitis are discussed by the size of the vessel involved
disease) typically affect the glomerular capillaries and manifest beginning with the small vessel vasculitides.
836 VII. Systemic Disease

TABLE 44.1. PULMONARY RENAL Pauciimmune-Mediated Disease

SYNDROMES IN CHILDREN
By definition, pauciimmune vasculitis is a small vessel vascu-
Antineutrophil cytoplasmic autoantibody–associated diseases litis that features minimal or no immune deposits on immu-
Wegener granulomatosis
Microscopic polyangiitis
nofluorescence on a renal biopsy. Most varieties are ANCA
Churg-Strauss syndrome associated, but a small number are ANCA negative (4,5).
Immune complex diseases Based on the Chapel Hill Consensus Conference, ANCA-
Systemic lupus erythematosus associated vasculitis is comprised of three systemic diseases
Henoch-Schönlein purpura and one renal limited disease (1). MPA is an ANCA-associ-
Anti–glomerular basement membrane disease (Goodpasture dis-
ease)
ated systemic vasculitis that often involves the skin, lungs,
Other and kidneys. It is characterized by a positive ANCA test
Legionella infection [perinuclear ANCA (pANCA) or cytoplasmic ANCA
Renal vein thrombosis with pulmonary embolus (cANCA)], absence of airway symptoms, and absence of
granulomas on biopsy. WG is a systemic vasculitis with mul-
tiorgan involvement, including the respiratory tract; is usu-
SMALL VESSEL VASCULITIS ally cANCA positive; and is characterized by the presence of
granulomas on biopsy. Churg-Strauss disease is the third
By definition, the small vessel vasculitides are diseases with a ANCA-associated systemic vasculitis and is characterized by
predilection for involvement of small arteries, arterioles, the presence of granulomas on biopsy and clinical asthma
capillaries, and postcapillary venules. The differential diag- and eosinophilia. The renal limited ANCA (+) vasculitis is
nosis for this disease category is quite extensive. As men- known as pauciimmune crescentic glomerulonephritis. These
tioned earlier, the types of small vessel vasculitis can be four entities are described in greater detail later beginning
further classified using immunopathology. When glomeru- with the granulomatous ANCA (+) systemic vasculitides.
lonephritis is present, the immunofluorescence component
of the renal biopsy reveals the presence or absence of sig-
nificant immunostaining and results in classification as Wegener Granulomatosis
immune complex–mediated disease, anti-GBM disease, or WG is a clinicopathologic entity that is uncommon in chil-
pauciimmune disease. dren. The triad of generalized WG consists of necrotizing
granulomatous lesions of the upper or lower respiratory
Immune Complex–Mediated Disease tract, generalized necrotizing vasculitis involving both
small- and medium-sized vessels, and focal necrotizing glo-
The two most common systemic forms of immune com- merulonephritis (6,7). Regional WG is distinguished from
plex–mediated small vessel vasculitis with renal involve- generalized WG by the absence of renal disease. While the
ment in pediatrics are HSP and SLE. These two topics are largest case series have been reported in adults, scattered
dealt with in Chapters 45 and 46. reports of WG exist in the pediatric literature (8–10). Since
the advent of ANCA testing, the definitions of WG have
TABLE 44.2. RENAL VASCULITIS IN CHILDREN been revised. This has enabled clinicians to better distin-
Systemic diseases with renal involvement
guish it from MPA, a disease with which it shares much in
Large vessel vasculitis common.
Takayasu’s arteritis The definition of WG, as determined by the Chapel Hill
Medium vessel vasculitis Consensus Conference on the Nomenclature of Systemic
Polyarteritis nodosum Vasculitis, requires evidence of granulomatous inflamma-
Kawasaki disease
Small vessel vasculitis
tion (1). This “new” definition is contrasted from that pre-
Immune complex diseases viously used by the National Institute of Health (NIH)
Systemic lupus erythematosus studies of WG patients. At the NIH, patients were required
Henoch-Schönlein purpura to have a clinical history compatible with WG and histo-
Pauciimmune diseases logic evidence of either a vasculitis or granulomatous
Granulomatous
Wegener granulomatosis
inflammation in a typical organ system (10). The absence
Churg-Strauss Disease of an absolute need for evidence of granulomatous inflam-
Non-granulomatous mation in the NIH studies suggests that their WG patient
Microscopic polyangiitis population also included patients who are today classified
Anti–glomerular basement membrane disease as MPA. This distinction makes comparison of older stud-
Renal-limited disease
Antineutrophil cytoplasmic autoantibodies (+) pauciimmune
ies to more modern ones difficult.
crescentic glomerulonephritis This section focuses on renal manifestations of WG and
their treatment. The extrarenal symptoms are discussed
 44. Renal Vasculitis 837

A B
FIGURE 44.1. A: A 10-year-old boy with a history of fever, rash, and arthritis presented with
hemoptysis accompanied by hematuria and proteinuria. His c-antineutrophil cytoplasmic autoan-
tibody was positive at 1:512. Chest x-ray revealed opacification of the middle and lower lung
fields on the right side. B: Ten weeks later, a follow-up chest x-ray showed complete resolution of
the pulmonary infiltrates.

only briefly, as more comprehensive reports exist in the oto- monary involvement in WG can range from asymptomatic
laryngology and ophthalmology literature (12,13). to severe and fulminant. Pulmonary infiltrates (61%), pul-
From the outset of its initial descriptions in the litera- monary nodules (43%), and hemoptysis (26%) were the
ture, the prognosis of patients afflicted with WG was grim. most common lung abnormalities. Radiographic abnor-
Untreated adults with WG survived an average of only 5 malities may occur in the absence of symptoms. In its most
months, with 82% succumbing to the disease within the severe form, WG may present as life-threatening diffuse
first year and 90% by the second year (14). Corticosteroid alveolar hemorrhage, which is often accompanied by pro-
treatment only increased the mean survival time to 12.5 gressive glomerulonephritis (Fig. 44.1).
months. The advent of cytotoxic therapy for WG, however, Constitutional symptoms at disease onset included fever
resulted in markedly improved patient survival. At a mean and weight loss. Other systemic symptoms at presentation
follow-up of 51 months, Fauci et al. reported a patient sur- included arthalgia/arthritis in approximately one-third of
vival rate of 88% in 85 patients with WG (2). patients and rash in 9%. Eye disease was common and
The most comprehensive report on children with WG included dacrocystitis, proptosis, eye pain, and episcleritis.
was published from the NIH (8). This study did not The neurologic and cardiovascular systems can be
include any data on ANCA testing but described the clini- involved as well. The adult literature states that 4% of
cal presentation, clinical course, and morbidity of children patients with WG present with nervous system involve-
classified as having WG. Affected children were predomi- ment, but it eventually develops in 10 to 34% (15). A
nantly adolescent (mean age of onset, 15.4 years) and peripheral neuropathy such as mononeuritis multiplex has
female (16 of 23 patients). There was a median of 8 months been the most frequent neurologic manifestation described
from onset of symptoms until a definitive diagnosis was in adults with WG. Cardiac involvement is rarely detected
secured. Clinical features at presentation were largely sino- antemortem but includes pericarditis and coronary arteritis
pulmonary, followed later by renal symptoms. in 10 to 20% of cases. Necrotizing vasculitis of the coro-
Children with WG usually sought medical attention for nary vessels can result in a myocardial infarction or sudden
symptoms related to the upper and lower respiratory tract. death (15).
Ear, nose, and throat pathology was present in 87% of In this same large pediatric WG series, glomerulonephri-
patients, with sinusitis occurring most commonly (61%) tis was present in only 9% of children at disease presenta-
(8). Large numbers of children developed large airway dis- tion, although it ultimately developed in 61% during the
ease early on and subglottic stenosis later in their courses. course of their disease (8). Hematuria, proteinuria, and
The 48% of pediatric patients with subglottic stenosis far renal insufficiency were common, whereas hypertension
exceeded the 10% occurrence reported in adults (11). Pul- and gross hematuria were unusual. Fifty-seven percent of
838 VII. Systemic Disease

children with renal involvement developed chronic renal monocytes. A cytoplasmic staining pattern on indirect immu-
insufficiency, and 14% required dialysis. nofluorescence (cANCA) is characteristic of WG. More spe-
The incidence of WG in children is difficult to deter- cific antibody assays, such as enzyme-linked immunosorbent
mine, but it is known to occur much less frequently than in assay (ELISA), have determined that PR3 is the antigen to
adults. According to the 1999 United States Renal Data Sys- which the cANCA in WG is most often directed. Although
tems Annual Data Report, WG was the underlying cause of the cANCA test in WG is not specific for PR3, in patients
end-stage renal disease (ESRD) in 40 pediatric patients (age with vasculitis approximately 90% of cytoplasmic ANCA are
younger than 20) over 5 years (1993 to 1997). The median PR3-ANCA (4). It should be noted that neither cANCA nor
age of these patients was 16 years. Forty-eight percent were anti-PR3 antibodies are specific for WG, as they are found in
male and 82% were white (16). other forms of vasculitis, including MPA. Therefore, it is gen-
erally recommended that both indirect immunofluorescence
ANCA testing and more specific ANCA testing be done in
Pathogenesis
the clinical setting of vasculitis.
Necrotizing vasculitis of small arteries and veins together with The likelihood of obtaining a positive ANCA test in
granuloma formation are the hallmark lesions of WG. Granu- patients with WG varies depending on severity and activity
lomatous necrotizing lesions are most commonly found in the of disease. Circulating cANCA are detected in 90% of
respiratory tract. Lung involvement may include bilateral nod- patients with active, generalized (sinopulmonary and renal)
ular cavitary infiltrates (15). The renal biopsy lesion is that of a WG and also in 40 to 70% of those with active, regional
pauciimmune-necrotizing and crescentic glomerulonephritis. (sinopulmonary) disease. In contrast, approximately 30 to
The pathogenesis of WG is still unknown. The role of 40% of patients in remission still have a positive cANCA
ANCAs has not yet been determined. An autoimmune (15). This emphasizes the need to treat patients based on
mechanism seems likely, but direct evidence does not yet their clinical findings rather than just the ANCA test.
exist in vivo. It is known that in vitro neutrophils from
patients with active WG express PR3 on their surface, thus
Differential Diagnosis
increasing their accessibility to circulating PR3 antibodies.
Furthermore, “primed” neutrophils from patients with WG The differential diagnosis for regional WG includes acute
have increased basal superoxide anion production, which infectious processes that lead to sinopulmonary symptoms
enhances their inflammatory potential (17). In addition, in alone. The differential diagnosis for patients with glomeru-
vitro tests have shown that neutrophils activated by ANCA lonephritis and pulmonary hemorrhage (pulmonary-renal
are retained within the vessel longer owing to increased syndrome) includes anti-GBM disease, SLE, HSP, and the
endothelial adhesion and reduced transendothelial migra- other ANCA-associated diseases such as MPA and Churg-
tion, thereby increasing their ability to induce endothelial Strauss disease. Other rarer causes are listed in Table 44.1.
injury (18). Finally, ANCA induces endothelial injury in Those with skin involvement, abdominal pain, arthralgias,
vitro through the activation of neutrophils that release reac- and glomerulonephritis could have HSP, SLE, or an
tive oxygen radicals and inflammatory cytokines and pro- ANCA-associated disease.
teases (reviewed in 18). Respiratory infections may be part
of the inciting event for both the initial onset of disease, as
Workup
well as for relapses. There is also an increased association of
WG and the HLA-B8 and HLA-DR2 gene loci (19,20). A routine evaluation should start with a complete blood
count, electrolytes, blood urea nitrogen, creatinine, albu-
min, urinalysis, Westergren sedimentation rate, and chest
Laboratory Features
radiograph. Depending on the clinical indication, indirect
General immunofluorescence ANCA and ELISA ANCA testing for
Nonspecific laboratory abnormalities in WG include ane- anti-PR3 and anti-MPO antibodies, ANA, C3, C4, and
mia, thrombocytosis, and leukocytosis in 30 to 60% of anti-GBM antibody, should be performed. Consideration
adults. Marked eosinophilia is rare. Rheumatoid factor is should also be given to obtaining computed tomography of
positive in a low titer in two-thirds of patients, whereas ANA the sinuses and orbits.
is present in 10 to 20% of patients. Serum complement A renal biopsy is essential for proper diagnosis if hematuria,
levels are normal or increased. Westergren erythrocyte sedi- proteinuria, or azotemia is present. It allows the clinician to
mentation rate and C-reactive protein are elevated in 90% of classify the glomerulonephritis as immune complex mediated,
patients with active and generalized disease (15). anti-GBM disease, or pauciimmune. In many centers, the
biopsy results return more rapidly than immunologic serolo-
Antineutrophil Cytoplasmic Autoantibodies gies, thereby allowing a more prompt diagnosis. Moreover,
The most specific serologic test for WG is autoantibodies approximately 10% of patients with WG are ANCA negative,
directed against cytoplasmic constituents of neutrophils and so serologic testing cannot be relied on exclusively (4). It
 44. Renal Vasculitis 839

ophilia); and a vasculitic phase (with multiorgan involve-

ment) (26). Six criteria were developed by the American
College of Rheumatology in an effort to standardize the
diagnosis of CSS for study purposes. The criteria included
asthma, eosinophilia (greater than 10% or absolute eosino-
phil count greater than 1500), mononeuropathy or poly-
neuropathy, histologic evidence of a vasculitis, paranasal
sinusitis, and nonfixed pulmonary infiltrates on chest x-ray.
Two adult studies have helped characterize this disorder
(3,25). Asthma was present at the time of presentation in
nearly all cases. Mononeuritis multiplex was present in 44 to
77%; constitutional symptoms (weight loss, fever, and myal-
gias), sinusitis, and allergic rhinitis all occurred in approxi-
mately 60% of cases. Skin involvement occurred in 50 to
70% of cases, with palpable purpura being the most com-
FIGURE 44.2. Segmental necrotizing and crescentic glomerulo-
nephritis. (Periodic acid-Schiff stain ×125.) mon manifestation. Cardiac involvement occurred at
presentation in 12.5 to 28.1%, and existed as pericardial
effusion, pericarditis, myocarditis, ischemic cardiomyopathy,
should be noted that, unlike a biopsy of the lungs or sinuses, a and conduction abnormalities. The GI tract was involved in
renal biopsy in a patient with WG rarely shows the presence of 31 to 38% of patients. Abdominal pain was the most com-
granulomas (2,15). A kidney biopsy typically shows a pauciim- mon symptom; more severe involvement included intestinal
mune-necrotizing and crescentic glomerulonephritis. Immu- perforation, GI bleeding, and pancreatitis.
nofluorescence shows scant immunostaining and few, if any, Renal involvement (proteinuria, glomerulonephritis,
electron-dense deposits on electron microscopy (21). A renal hypertension, renal insufficiency, and renal infarct) was
biopsy documenting the focal, necrotizing, and crescentic glo- evident in 26% of cases of CSS in one study. A second
merulonephritis of WG is shown in Figure 44.2. study reported that 12.5% of CSS patients had renal
In a patient with minimal renal disease, another tissue involvement that included glomerulonephritis, renal insuf-
biopsy site may be needed. In the presence of nodules or ficiency, and hematuria. Hypertension was present in 16%
infiltrates on chest radiograph or computed tomography, of patients (3,25).
lung biopsy is often diagnostic, with patchy necrosis, granu- Laboratory findings consisted of leukocytosis with eosin-
lomatous inflammation, and vasculitis seen. In the setting of ophilia, and elevated erythrocyte sedimentation rate was
pulmonary hemorrhage, surgical lung biopsy is much riskier. present in 80 to 100% of cases, ANA greater than 1:160 was
Fiberoptic bronchoscopy with bronchoalveolar lavage can present in 11 to 15%, rheumatoid factor in 24% of patients,
confirm the presence of blood and/or hemosiderin-laden and less than 1% had a positive anti–double-stranded DNA
macrophages. Stains and cultures can be obtained to rule out with clinical manifestations of lupus (3,25). ANCA testing was
infection (15,22). positive in 48% of sera from CSS patients using indirect
immunofluorescence and was pANCA in the majority of cases
(3). ELISA revealed ANCA specificity toward MPO in 10 of
Churg-Strauss Syndrome
11 cases (3). In another study, IIF ANCA were negative in
Churg-Strauss syndrome (CSS) is a systemic necrotizing eight patients with CSS who were in remission and in 78%
vasculitis with hypereosinophilia and extravascular granulo- who were positive during the acute phase of illness (25). It was
mas that occurs almost exclusively in patients with asthma also noted that only 50% of patients had a positive ANCA in
(23). It is also characterized as a pauciimmune small vessel association with a clinical relapse. Chest x-rays revealed patchy
vasculitis, being distinguished from MPA and WG by the infiltrates in 38 to 75% (3,25).
presence of granulomatous inflammation and asthma, Histologic findings included necrotizing vasculitis (64%),
respectively (24). It is a systemic vasculitis that affects the vascular and perivascular eosinophil infiltrates (55%), and
lungs, skin, peripheral nerves, GI tract, heart, and kidneys extravascular granulomas (15%) in one study (25). Because
(25). Unlike WG, CSS is extremely uncommon in chil- extravascular granulomas are only infrequently seen on
dren, with only an occasional case report in the literature biopsy specimens, it is recommended that this feature of the
and no pediatric case series. As a result, most information disease not be required to make the diagnosis. Renal biopsy
presented is adult data used in an effort to prepare pediatri- specimens have shown segmental proliferative glomerulone-
cians to recognize the rare patient with this disorder. phritis in some and necrotizing vasculitis with crescents in
It has been postulated that CSS occurs in three clinical others; immunofluorescence has been negative (3,25,27).
phases: a prodromal, allergic phase (asthma and/or allergic Treatment consisted of prednisone alone or usually in
rhinitis); an eosinophilic phase (tissue and peripheral eosin- accompaniment with cyclophosphamide (CYP). CYP treat-
840 VII. Systemic Disease

ment lasted 1.0 to 1.5 years and was most often given death of 8.6. Other risk factors of statistical significance
orally. Plasma exchange has been studied and does not included increased mortality when cANCA pattern was
appear to have additional benefit to that of corticosteroids present (as compared to pANCA; RR, 3.8). A protective
and CYP (28). effect of CYP treatment was seen when compared to corti-
Clinical outcome in adults was favorable. In one study costeroids alone (RR, 0.18; p = .012). Predictors of need for
of 32 patients, the patient survival was 94% at 1 year and renal replacement therapy were statistically associated with
90% at 5 years. Four deaths occurred—three directly serum creatinine at the time of study entry and African
related to vasculitis (intestinal perforation in two patients American race (29). This study thus clearly highlighted the
and severe cardiac, GI, and renal involvement) and one potential morbidity and mortality of this disease.
related to a nosocomial infection. Remissions occurred in In another adult study using Chapel Hill criteria,
all patients, and relapses occurred in 28% of patients; patients with WG (N = 56) were compared to those with
approximately one-half of these relapses occurring in the MPA (N = 67). ANCA testing on patients with WG
first year of therapy (25). Long-term prognosis in another revealed 87% were PR3 (+), 11% MPO (+), and 2% ANCA
study correlated with myocardial involvement, proteinuria negative. Those with MPA showed that 39% were PR3 (+),
greater than 1 g/24 hr, and severe GI tract involvement 56% MPO (+), and 5% ANCA negative. Patients with WG
including GI hemorrhage, intestinal perforation, pancreati- were more likely to have upper respiratory and pulmonary
tis, or laparotomy (3). disease. Relapses and renal outcomes were similar in both
groups (30).
Finally, the French Vasculitis Study Group conducted a
Microscopic Polyangiitis
recent retrospective study of 85 adults meeting Chapel Hill
The distinction between MPA and the other ANCA-associ- criteria for MPA. ANCA testing was positive in 75%; 65%
ated diseases has been discussed. In brief, it is a nongranulo- were pANCA (+) and 10% were cANCA (+). Constitutional
matous, multisystem, pauciimmune, small vessel vasculitis symptoms were prevalent with fever in 55% and anorexia or
without upper airway involvement. There is significant weight loss in 73% of patients. Renal disease was present in
overlap with WG in terms of organ system involvement. It 79%, skin involvement in 62% (purpura in 41%), and
is associated with ANCA in more than 80% of patients. mononeuritis multiplex in 58%. Deaths occurred in nearly
pANCA on indirect immunofluorescence is most common one-third of patients followed 70 months, most owing to
and more specifically on MPO. Owing to the small number vasculitis or infection (31).
of ANCA-associated diseases reported in pediatrics, most In another study looking at causes of pulmonary renal
specifics are derived from adult studies. As previously men- syndromes in adult patients, serologic testing found that 48
tioned, because the Chapel Hill Conference has set stricter of 88 patients were ANCA positive (PR3—19 patients;
diagnostic criteria, future pediatric studies are likely to MPO—29 patients), 6 patients had exclusively anti-GBM
attempt to more carefully distinguish the various ANCA- disease, 7 others had both ANCA and anti-GBM antibod-
associated small vessel vasculitides. ies, and 27 patients had neither antibody. The authors
MPA can often mimic cPAN histologically, as both dis- noted that only eight of these patients had clinical or histo-
eases are capable of producing a necrotizing arteritis. The key logic evidence to support a diagnosis of WG (32). By
distinction is in the size of the vessels involved. Because MPA Chapel Hill criteria, then, MPA was the most common
is a small vessel vasculitis, it is capable of affecting arterioles, identifiable cause of pulmonary renal syndrome in adults.
capillaries, and postcapillary venules. This clinically results in Pediatric reports of this entity are rare. Similar to adults,
alveolar capillaritis (pulmonary hemorrhage), glomerular pediatric patients can present with life-threatening pulmo-
capillary inflammation (glomerulonephritis), and inflamma- nary hemorrhage as part of the pulmonary renal syndrome.
tion of the dermal postcapillary venules (leukocytoclastic In addition to these organ systems, a recent case report was
angiitis resulting in purpura). By definition, MPA can affect published of an adolescent female with a pANCA (+)
medium vessels, but cPAN is restricted from affecting vessels pauciimmune crescentic glomerulonephritis who presented
smaller than this size (1, reviewed in 4). in renal failure with cerebral vasculitis (seizures with multi-
A study from Chapel Hill of 107 patients with necroti- focal ischemic defects on magnetic resonance imaging).
zing glomerulonephritis who met criteria for MPA was fol- The CNS manifestations of this patient’s disease responded
lowed prospectively for an average of 2.5 years. Indirect to CYP and plasma exchange (33).
immunofluorescence revealed that 65% were pANCA (+) In summary, the prognosis of MPA appears similar to
and 35% cANCA (+) are ANCA positive. Twelve disease- WG. Although granulomatous inflammation does not
related deaths occurred, and 46 patients reached ESRD. occur, it is often the sinopulmonary symptoms of WG that
The patients had pulmonary involvement in 36% of cases result in patients seeking medical attention. The relative
and cutaneous involvement in 12%. Mortality risk factors reduction of these symptoms means patients with MPA
were analyzed and revealed that those patients presenting may be more apt to present with advanced disease. Because
with pulmonary hemorrhage had a relative risk (RR) of patients with advanced disease tend to have a poor out-
 44. Renal Vasculitis 841

come, a high index of suspicion for MPA (ANCA disease) nosis (38). Prednisone is given typically as 1 to 2 mg/kg/
is needed to minimize long-term morbidity. day initially divided b.i.d., which is continued for 1 to 3
months before being tapered. Some centers taper to alter-
nate day prednisone at a dose of 1 to 2 mg/kg/q.o.d. after
Treatment of Antineutrophil Cytoplasmic
2 months of treatment, while others slowly reduce the
Autoantibody–Associated
dose of steroids, which are continued on a daily basis.
Glomerulonephritis and Vasculitis—
With regard to the CYP dose, those centers giving
Wegener Granulomatosis, Microscopic
monthly IV CYP give an initial dose of 500 to 600 mg/
Polyangiitis, and Churg Strauss Disease
m2/dose with a nadir white blood cell count 10 to 14 days
The mainstay of treatment for ANCA-associated diseases later being used to determine the next dose. Dose
includes a combination of corticosteroids and cytotoxic increases in increments of 250 mg/m2/dose are then used
agents. Because the dosages and the manner of administra- up to a maximum dose of 1000 mg/m2/dose. Oral CYP is
tion can vary according to the stage of treatment, the treat- given at a starting dose of 2 mg/kg/day (maximum dose of
ment section is separated into three parts: acute supportive 150 mg). Dosage reductions are made based on the pres-
treatment, induction therapy, and maintenance therapy. ence of leukopenia/neutropenia on complete blood cell
counts, monitored weekly.
Acute Supportive Treatment
Maintenance Immunosuppressive Therapy
From an immunosuppressive standpoint, most centers treat
adults and children with ANCA diseases with either oral This period has been arbitrarily defined as the period that
prednisone or intravenous (IV) methylprednisolone. Pulse follows the first 6 months of treatment. The optimal dura-
methylprednisolone at a dose of 7 to 15 mg/kg/dose is tion of treatment remains debatable. Many adult centers are
often given in three successive daily doses followed by daily now stopping corticosteroids 3 to 4 months into therapy and
prednisone at 1 to 2 mg/kg/day (4,28,34–38). CYP is given discontinuing all treatment as early as 6 months (42). Other
in one of two ways. Many centers prefer oral CYP to induce centers use the 6-month cutoff as a time to transition
a remission and optimally reduce the incidence of relapses patients to a less toxic regimen and substitute azathioprine
(30,39). Others prefer IV CYP, which is as efficacious at (AZA) for CYP at this point. Rottem et al. chose to treat
inducing a remission as the oral preparation (4,28,34). IV children with oral CYP for 1 year after a remission was
CYP can be given safely with aggressive hydration along achieved before reducing the dosage by 25 mg every 2
with sodium 2-mercaptoethane sulfonate (MESNA) to months as tolerated (8). Their median treatment time with
minimize the risk of hemorrhagic cystitis. In addition, the oral CYP was 28 months. In our cohort of children with
cumulative dose tends to be much lower with the IV prepa- ANCA-associated vasculitis and glomerulonephritis, we
ration, which should theoretically result in a reduction in chose to transition patients to AZA after an approximate 6-
dose-related, long-term malignancy risks. In our pediatric month treatment period with IV CYP. A select group of
cohort of ANCA (+) glomerulonephritis and vasculitis, patients who had either a poor response to IV CYP or life-
those children with a history of pulmonary hemorrhage threatening disease at presentation were treated instead with
(defined as life-threatening disease) were treated with oral oral CYP, which was continued for 1 year. Those treated with
CYP for fear of the consequences of a catastrophic relapse. oral CYP were changed at 1 year to either AZA or metho-
All others were treated with IV CYP, except for one child trexate (MTX) in an effort to provide sufficient immunosup-
who was changed from IV to oral due to lack of response to pressive therapy while avoiding long-term use of CYP (38).
IV (38).
Other empiric treatments that have been tried but have
Newer Therapeutic Regimens
not been well studied include plasma exchange and IV
immunoglobulin (IVIG) (28,36,40). The former treatment MTX has been used as a substitute for CYP during the
has the theoretical advantage of removing a circulating induction stage of WG treatment of children and adults
agent that may be causative, whereas the latter may be (9,43). This agent appears to be reasonably effective (71%
immunomodulating (41). Additional supportive treat- remission rate) if selectively used in patients with non–life-
ments such as oxygen, assisted ventilation, transfusion, threatening forms of WG and when used in conjunction
extracorporeal membrane oxygenation, and dialysis have with corticosteroids. MTX dosages in WG range from 0.15
been used as life-saving measures. to 0.30 mg/kg/wk in adults, to as high as 0.6 mg/kg/wk in
children (9,11). Although AZA has been proven to be infe-
rior to CYP during the induction phase of treatment, it may
Induction Immunosuppressive Therapy
have its greatest role in “maintaining” a remission state, as has
This phase of treatment has been arbitrarily defined as been discussed (2,38). Its main advantage is that it is far less
that occurring between 2 weeks and 6 months after diag- toxic than CYP and is usually well tolerated.
842 VII. Systemic Disease

Mycophenolate mofetil (MMF) is a newer antiprolifera- glottic stenosis (35%), which necessitated subglottic dilata-
tive immunosuppressive medication that possesses a rela- tion in two patients and tracheostomy in six others. Renal
tively favorable toxicity profile and a lymphocyte selective insufficiency was present in approximately one-third of
mode of action, through inhibition of IMPDH (inosine patients. Treatment-induced morbidity included cystitis
monophosphate dehydrogenase). In a study of 11 adults (50%) and infertility (28%). Steroid side effects included
with ANCA-associated systemic vasculitis (WG = 9, MPA cataracts and aseptic necrosis. Infections requiring hospital-
= 2), MMF was substituted for CYP during the mainte- ization and IV antibiotics occurred in 43% of patients. The
nance phase of treatment and used with corticosteroids to frequency of infections correlated with the degree of immu-
maintain a remission in 10 of 11 patients followed for 15 nosuppression. The lowest rate occurred while off immuno-
months (44). No pediatric reports of MMF usage in WG suppression and increased twofold with steroids alone and
exist at this time. Certainly, larger studies are needed during increased twelvefold during combined steroid and CYP
the maintenance phase to determine whether MMF will therapy (8). Although no malignancies were reported,
prove efficacious. longer-term follow-up is clearly needed. Two pediatric
deaths were reported (8.7%), occurring 2.2 and 15 years
after disease onset. Adult studies have reported a marked
Treatment Summary
increase in bladder carcinomas (11- to 33-fold increased
At the present time, our typical approach to a patient with risk) after successful treatment of WG with CYP for at least
ANCA (+) vasculitis and glomerulonephritis is to treat with 1 year (11,30,46). The risk of bladder carcinoma is higher
prednisone (1 to 2 mg/kg/day; maximum, 80 mg) for 1 to in patients having had hemorrhagic cystitis, again making a
2 months before tapering to alternate day dosing schedule. case for the use of IV CYP in whom this complication can
In addition, CYP is given for 6 to 12 months before chang- be largely eliminated by using IV hydration and MESNA.
ing over to AZA. The decision on the duration of CYP is Outcomes from other more recent studies have been
related to the severity of disease at presentation, with slightly better, but patient selection criteria and treatment
patients with non–life-threatening disease typically receiv- protocols have been modified. Of those pediatric cases with
ing monthly IV CYP for 6 months in conjunction with ANCA-associated vasculitis and glomerulonephritis, a cumu-
corticosteroids. If in remission, the patient is then transi- lative incidence of ESRD of 30% has occurred in 20 patients
tioned to AZA at 2 mg/kg/day while steroids continue to from six clinical reports (9,35,38,47–49). Because these
be tapered slowly. Those patients with pulmonary hemor- reports are of children with coexisting glomerulonephritis, all
rhage (life-threatening disease) are treated with oral CYP patients had renal disease at presentation. Those developing
for 1 year in conjunction with corticosteroids. At that ESRD typically presented with very advanced disease. Seven
point, AZA is again substituted for CYP if the patient is in of these patients were reported from a single center, and a
remission. Remission is defined as lack of symptoms, stable lower incidence of ESRD (14%) was found. Of the remain-
renal function, benign urinalysis (except for some pro- ing 13 patients, the incidence of ESRD was 38%.
teinuria), and a stable erythrocyte sedimentation rate. A Until recently, children with ANCA (+) glomerulone-
repeat renal biopsy looking for histologic confirmation of phritis were believed to have an ESRD risk of 30%. This
inactive disease is usually performed before transitioning a figure was corroborated by a large recent retrospective
patient off of CYP. series of 31 pediatric patients with ANCA (+) glomerulo-
Trimethoprim/sulfamethoxazole has become a mainstay nephritis conducted by members of the Japanese Society
of WG treatment, as it can be used as prophylaxis for Pneu- for Pediatric Nephrology. All patients were ANCA (+) and
mocystis carinii pneumonia, which occurred in approxi- had renal biopsy–proven pauciimmune necrotizing and
mately 4% of adult WG patients from the NIH (11). An crescentic glomerulonephritis. Ten had renal-limited,
additional advantage is its efficacy in reducing respiratory necrotizing crescentic glomerulonephritis, and 21 (68%)
relapses in adults with WG, a fact that may be secondary to were classified as MPA. Twenty-six patients (84%) received
treatment of nasal carriage of Staphylococcus aureus, which treatment with corticosteroids and CYP, and five patients
was associated with serious sinopulmonary infections in the with corticosteroids alone; 84% of patients achieved remis-
early NIH studies (2,45). sion, and 39% of responders relapsed in a median of 24
months. Nine of 31 patients (29%) progressed to ESRD;
an additional 6 (19.4%) had reduced renal function,
Outcome of Antineutrophil Cytoplasmic
whereas nearly one-half (48.4%) had normal renal func-
Autoantibody-Associated Vasculitis
tion at a median follow-up of 42 months. Life-table analy-
and Glomerulonephritis
sis showed 75% renal survival at 39 months. Patients who
Despite high remission rates in childhood WG (87%), subsequently developed ESRD (N = 9) had significantly
relapses were frequent (53%) (7). In addition, morbidity higher average peak serum creatinine levels and more
from either the disease or its treatment was extremely high chronic pathologic lesions at diagnosis compared with
(87%). Nasal deformity was common (48%), as was sub- patients with favorable renal outcome (N = 15) (50).
 44. Renal Vasculitis 843

With regard to renal transplantation, disease recurrence adults and children (57–59). Classic PAN is a multisystem
has occurred and is estimated to be approximately 17%, disease that can lead to GI, nervous system, musculoskeletal
with the time to relapse at an average of 31 months (51). system, cutaneous, and renal involvement. Cutaneous fea-
Disease recurrence has been successfully treated with CYP tures include livedo reticularis or inflammatory nodules, with
(52). The presence of a positive ANCA test, however, is not or without gangrene. Musculoskeletal symptoms consist of
a contraindication to transplantation, which has occurred severe myalgias and/or arthritis/arthralgias. Nervous system
as early as 6 months after the development of ESRD (53). involvement includes both peripheral neuropathy (mononeu-
ritis multiplex) and encephalopathy. GI symptoms include
abdominal pain and blood in the stool. Renovascular hyper-
Conclusions
tension occurs in approximately one-third of adults; renal
WG and other forms of ANCA-associated small vessel vascu- involvement is usually flank pain and hematuria and, more
litis continue to be diagnosed in the pediatric population. rarely, retroperitoneal hemorrhage from aneurysmal rupture
The renal prognosis is difficult to estimate when comparing (54,60,61). Hypertension and renal involvement were present
patients with WG who have mild if any renal disease at pre- in 65% of patients in one pediatric study, but this study pre-
sentation versus those who present to pediatric nephrology dated Chapel Hill criteria and ANCA testing and almost cer-
units with advanced renal failure. The largest report of chil- tainly included a mixture of pediatric patients now classified
dren with WG, in which only 9% presented with renal dis- as having cPAN and MPA (57).
ease, estimated that 14% went on to ESRD (8). This is Renal involvement in cPAN, as defined by Chapel Hill cri-
contrasted to published reports of children presenting with teria, was analyzed in an adult patient cohort from Kuwait.
renal disease to a pediatric nephrology center, where the inci- Chronic renal failure with (38%) or without proteinuria
dence of ESRD ranges from 14 to 60%, with the largest (31%) was frequently seen. Less frequently seen were neph-
series reporting 29%. The speed with which the children are rotic syndrome (6%) and rapidly progressive renal failure (6%)
diagnosed and treated is likely to affect both the short- and (62). A recent pediatric case report of severe renal impairment
long-term outcomes. The long-term outcome can be further in a child with cPAN demonstrated the need to use histology
improved by finding less toxic treatment regimens capable of to discern necrotizing glomerulonephritis versus necrotizing
achieving disease remission with fewer side effects. Long- arteritis of the renal vascular bed (56). Either form of vasculitis
term follow-up of these patients is necessary, for even those is capable of inducing renal failure; the latter form, although
in sustained remission have the potential to relapse. less common, occurs through renal ischemia.
The diagnosis of cPAN is often made with the aid of a
kidney biopsy, in which a necrotizing vasculitis without a
POLYARTERITIS NODOSA crescentic or necrotizing glomerulonephritis is seen. Angiog-
raphy of vessels involving the kidney or intestinal vessels
cPAN is a necrotizing vasculitis of medium-sized vessels often shows aneurysms. The high-resolution technique of
without involvement of arterioles, capillaries, and venules. selective renal angiography is often required to detect very
These medium-sized vessels are defined as main visceral small peripheral aneurysms (63). The interlobar and arcuate
arteries, such as the coronary, hepatic, mesenteric, and renal arteries are most commonly involved, with the main renal
arteries. Focal-necrotizing injury to the vessel wall leads to artery occurring on rare occasions (54). Although aneu-
aneurysm formation. The clinical features are tissue infarc- rysms in the medium-sized renal arteries are seen in cPAN,
tion, hemorrhage, and organ dysfunction (54). It is impor- they are not specific for this entity as they can be seen in TA,
tant that cPAN be distinguished from MPA, a disease with Kawasaki disease (KD), WG, MPA, and Churg-Strauss dis-
which it is often confused. The confusion dates back to the ease (54). Aneurysms of the renal vessels can be seen on an
early descriptions of polyarteritis nodosa when it was arteriogram done in a child presenting with hypertension
believed to occur as two types: polyarteritis with extraglom- who was diagnosed with cPAN (Fig. 44.3).
erular vasculitis (cPAN) and that with glomerulonephritis, Treatment consists of corticosteroids and usually an
which they named microscopic polyarteritis (55). These two alkylating agent such as CYP or AZA (54,56,59). Plasma
types of PAN were redefined by the Chapel Hill Consensus exchange has not proven beneficial in this disease (64). The
Conference. In short, cPAN is a medium vessel vasculitis, prognosis of this disease has been examined in adults, and it
which is limited to medium vessels. This distinguishes was found that patients with more than 1 g of protein
cPAN from patients with MPA whose necrotizing vasculitis excretion in 24 hours, renal insufficiency, cardiomyopathy,
is usually limited to small vessels but can overlap into the CNS involvement, or severe GI involvement had a worse
medium vessels (1). This means a patient presenting with a prognosis (65). Prognosis in children appears to be improv-
systemic vasculitis whose renal involvement is manifested as ing with more aggressive immunosuppressive treatment. A
glomerulonephritis cannot be classified as cPAN. recent series of 11 children from Saudi Arabia reported
Unlike patients with MPA, cPAN is typically ANCA nega- 100% survival. All of these patients were treated with ster-
tive (56). It has been associated with hepatitis B infection in oids, and nine children also received cytotoxic agents (66).
844 VII. Systemic Disease

The diagnostic criteria for KD includes six signs and

symptoms: fever of 5 days or more; bilateral nonpurulent,
conjunctival injection; mucous membrane changes of the
upper respiratory tract (injected pharynx, injected, fissured
lips, and strawberry tongue); peripheral extremity changes
(peripheral edema and erythema, and periungual desqua-
mation); polymorphous rash; and cervical lymphadenopa-
thy (at least one node more than 1.5 cm in diameter) (72).
Typical cases have fever of 5 days or more and four of the
five criteria without any other known disease process. Atyp-
ical cases are characterized by coronary aneurysms and at
least four other criteria (73).
KD is a systemic medium vessel vasculitis, and its pri-
mary morbidity involves the coronary arteries, in which cor-
onary artery aneurysms have been reported to occur in 20 to
25% of untreated children (74). Renovascular involvement
also occurs, albeit less commonly, and typically involves the
interlobar arteries of the kidney (75). Recently, a previously
normotensive child was reported to develop renovascular
hypertension 6 months after an episode of KD. Arteriogra-
phy revealed unilateral ostial stenosis of the main renal
artery (76).
Renal parenchymal involvement has also been noted in
FIGURE 44.3. Renal arteriogram of a 5-year-old child present- KD. Veiga et al. reported a child with acute renal failure
ing with hypertension. Selective injection of left renal artery associated with biopsy-proven acute interstitial nephritis
showed small aneurysms arising from the interlobar and inter-
lobular arteries (arrows). This study aided in the diagnosis of during the acute phase of illness (77). Nardi et al. performed
classic polyarteritis nodosa in this child. ultrasonography in 7 children with KD. They found a cor-
relation between enlarged, echogenic kidneys with increased
corticomedullary differentiation in affected children and
This is contrasted to earlier reports in which cytotoxic abnormal renal function, hematuria, proteinuria, and pyuria
immunosuppressive treatment was used more sparingly and (78). Lande et al. reported a patient with KD who presented
mortality was as high as 16% (57,67). with acute renal failure requiring 1 week of hemodialysis
before renal function normalized (79).
Laboratory findings in KD include leukocytosis, throm-
KAWASAKI DISEASE bocytosis, sterile pyuria, and pleocytosis of the cerebrospi-
nal fluid. ANCAs and antiendothelial cell antibodies have
Tomisaku Kawasaki’s initial report in English of 50 Japanese been reported to be present in KD (80,81). Two subse-
children with an illness characterized by fever, rash, conjunc- quent studies using control patients with nonspecific febrile
tival injection, swelling of the hands and feet, and cervical illnesses revealed that neither ANCA nor antiendothelial
lymphadenopathy appeared in 1974 (68). This illness would cell antibodies were of diagnostic value when attempting to
later become known as Kawasaki disease or mucocutaneous distinguish early KD from other childhood illnesses and,
lymph node syndrome. Coronary artery vasculitis is the most therefore, were unlikely to be involved in the pathogenesis
important feature of this disease. KD is the second most of KD (82,83). Because of the high incidence of coronary
common form of vasculitis in childhood, behind HSP (69). aneurysms in KD, echocardiography and ECG are recom-
This disease occurs worldwide but is much more common in mended for all patients at the time of diagnosis (69).
Japan where the finest epidemiologic studies exist. This dis- After diagnosis of KD is made, the standard of care is
ease is 1.3 times more common in males than females (70). IVIG and high-dose aspirin, which have been shown to
In Japan, KD incidence rates peak at 3 to 5 months of age in reduce the risk of coronary aneurysms to less than 5%
females, with two peaks occurring in males—at 3 to 5 (84,85). A single dose of 2 g/kg of IVIG is superior to lower
months and a second peak at 9 to 11 months of age. This doses (400 mg/kg/dose) given on 4 consecutive days (85).
recent survey also revealed that 82% of cases occurred in IVIG treatment is ideally administered within the first 10
children younger than 4 years of age (70). It is also the most days of illness (85,86). It should be noted that IVIG can be
common cause of acquired heart disease in children in the given beyond this 10-day window, and failure to respond to
United States, the United Kingdom, and Japan (69,71). An single-dose IVIG may necessitate administration of a second
infectious etiology is suspected but is as yet unproven. dose later if the patient remains symptomatic (87). Aspirin
 44. Renal Vasculitis 845

dosing is recommended at 30 to 50 mg/kg/day in four present with symptoms of claudication, abdominal pain,
divided doses until fever resolves, then 2 to 5 mg/kg/day for and/or ischemic changes to the hands. Pediatric patients
a minimum of 6 weeks (69). The role of corticosteroids in more often present with symptoms attributed to hyperten-
KD is controversial but, in general, their use is discouraged. sion and its complications, namely hypertensive encephalo-
Long-term outcome of KD largely depends on the pres- pathy and congestive heart failure (93). Other features of TA
ence of coronary artery aneurysms. Follow-up echocardiog- include dermatologic manifestations (erythema nodosum
raphy is recommended at 10 to 14 days and again in 6 to 8 and pyoderma gangrenosum of the lower extremities in some
weeks in an effort to closely monitor for the development cases) and symptoms attributable to compromised cerebral
of aneurysms or follow up the status of previously diag- blood flow (dizziness, headaches, syncope, and stroke).
nosed aneurysms. It is recommended to discontinue aspirin Hemoptysis, shortness of breath, and pulmonary hyperten-
in those without aneurysms 6 weeks into the illness. Most sion can occur due to pulmonary arterial involvement, and
mild coronary artery aneurysms (3 to 4 mm) regress within angina can occur due to compromised coronary blood flow
2 years, but those who have giant aneurysms (greater than 8 attributable to either aortic or coronary arteritis. Signs of dis-
mm) are unlikely to resolve. Persistent aneurysms are prone ease at presentation include hypertension in approximately
to stenosis, which can lead to thrombosis, myocardial 50% of cases, asymmetrical pulses, and bruits over the sub-
infarction, or death (69). Indeed, KD is the most common clavian, brachial, and abdominal vessels (94,95).
cause of myocardial infarction in childhood (54). Laboratory findings include anemia, elevated erythro-
The overall prognosis for KD, however, is quite good. cyte sedimentation rate, and elevated C-reactive protein
Those with giant aneurysms require the closest follow-up, (93,95). Serologic markers, including ANA and ANCA, are
but it is recommended that all children with KD receive typically negative. Arteriography often shows involvement
lifelong follow-up (69). of the aortic arch, its branches, and/or the distal aorta and
its branches (95) (Fig. 44.4).
Treatment of TA has consisted of corticosteroids initially in
TAKAYASU’S ARTERITIS most settings. Steroid-resistant cases have been treated with
CYP (96). More recently, MTX has been used in conjunction
TA is the most common large vessel vasculitis in the pediat- with corticosteroids and resulted in a higher remission rate and
ric age group. It is a granulomatous inflammatory condi- lower maintenance steroid dosage compared to historical con-
tion that usually begins in the subclavian vessels and
progressively involves the carotids, aorta, and possibly the
renal arteries. It is known as the pulseless disease, owing to its
involvement with the aortic arch and subclavian vessels
resulting in arterial narrowing and decreased brachial pulses
(54,88). Six diagnostic criteria have been established and
include age of onset of 40 years or less, claudication of an
extremity, decreased brachial pulse, greater than 10 mm Hg
difference in systolic blood pressure between arms, bruit
over the subclavian artery, and narrowing or occlusion of
the aorta, its primary branches or major arteries of the
extremities not caused by atherosclerosis, fibromuscular
dysplasia, or other causes (89). The diagnosis is most likely
when three of these six criteria are met.
Hypertension is the most common renal manifestation
and can be attributed to coarctation of the aorta and, less
often, renal artery stenosis (90,91). Another renal manifes-
tation is ischemic kidney disease (92). TA occurs predomi-
nantly in females. The female to male ratio is 9:1, with
most cases being diagnosed between 10 and 20 years of age
(54). It is more frequently seen in the Japanese population
but does occur worldwide. The pathogenesis of this disor-
der is poorly understood.
Clinical features are divided into a prepulseless phase and FIGURE 44.4. Aortogram obtained at cardiac catheterization in
a pulseless phase. Constitutional symptoms (fever, malaise, a child with Takayasu’s arteritis. The small arrow indicates the
headache, myalgias, arthralgias, and weight loss) occur dur- catheter within the descending aorta. The large arrow indicates
an abrupt tapering of the descending aorta with extensive col-
ing the prepulseless phase. As arterial involvement advances, lateral vessels. (Courtesy of Dr. Thomas J. Forbes, Pediatric Cardi-
end-organ–ischemic symptoms occur (93). Adult patients ology, Children’s Hospital of Michigan, Detroit, Michigan.)
846 VII. Systemic Disease

trols (97). Strategies to manage severe hypertension in child- There is one report of two children on PTU who subse-
hood TA have included the use of vasodilators, beta blockers, quently developed pauciimmune-necrotizing and crescentic
and diuretics. Angiotensin-converting enzyme inhibitors have glomerulonephritis associated with ANCA (+) serology.
been used but must be used with great caution in those with Both patients had hyperthyroidism and were exposed to
renovascular involvement due to the risk of precipitating acute PTU for 34 months and 1 month, respectively. Indirect
renal failure (90). Percutaneous transluminal angioplasty for immunofluorescence demonstrated pANCA, and ELISA
treatment of stenosis of the descending thoracic and/or demonstrated antibodies to MPO (101). Both patients
abdominal aorta has been used with moderate success (98). were treated with corticosteroids and CYP, with one patient
The long-term prognosis of TA is generally quite good, with 5- improving and the other developing ESRD.
year survival rates of approximately 95% (95,97). A recent report of seven children from Japan described
an additional cohort of children with PTU-associated
ANCA (+) glomerulonephritis. All patients were positive
DRUG-ASSOCIATED VASCULITIS for MPO-ANCA, and had biopsy-proven pauciimmune-
necrotizing and crescentic glomerulonephritis. They were a
Several drugs have been associated with ANCA positive vascu- mean age of 14 years and were exposed to PTU for a mean
litis, with hydralazine and propylthiouracil (PTU) being the of 37 months (102). Treatment consisted of corticosteroids
two most commonly reported (99). In one study, the authors in all patients, with CYP added in two. Only one of seven
analyzed the 30 patients with the highest titers of anti-MPO patients presented with rapidly progressive glomerulone-
antibodies from a pool of 250 patients with ANCA-positive phritis. Nearly 5-year mean follow-up showed that renal
vasculitis and anti-MPO antibodies. Of these 30 patients, 10 function was normal in all patients; as such, the authors
patients (33%) were exposed to hydralazine and 3 patients concluded that PTU-associated ANCA (+) glomerulone-
(10%) to PTU. An additional 17% were exposed to other phritis has a better prognosis than nondrug-induced
agents such as penicillamine, allopurinol, or sulfasalazine (99). ANCA (+) glomerulonephritis, such as WG and MPA.
Patients in the hydralazine group had multisystem disease In light of the previously mentioned reports, it behooves
with renal, sinopulmonary, and dermatologic manifestations. the caregiver to take a detailed medication exposure history
Nine of ten patients had glomerulonephritis, and all five biop- in all patients with vasculitis. Although hydralazine and
sied patients had pauciimmune necrotizing and crescentic glo- PTU are the most commonly associated agents, isolated
merulonephritis. Pulmonary abnormalities included reports of a number of other medications associated with
pulmonary infiltrates (five patients), hemoptysis (four vasculitis currently exist (penicillamine, allopurinol, and
patients), and alveolar hemorrhage with granulomatous minocycline), and additional medications ar