RENAL FUNCTION TESTS

J. Okoboi
Department of Biochemistry, SUN
By the end of the lecture you should be able to:
• List the functions of the glomerulus
• Explain tubular function
• Discuss the normal and abnormal constituents of urine
• Explain the biochemical basis of clearance test of renal function
• Describe kidney function tests that measure tubular functioning
Functions of the kidney
• Excretion of metabolic waste products
• Maintenance of body water, pH and electrolyte balance
• Production of calcitriol and erythropoietin
• Decrease in kidney function is usually due to reduction in the
performance of nephrons
• The nephron is the functional unit; it consists of Bowman’s capsule,
PCT, Loop of Henle, DCT, collecting ducts
Renal function tests
1. Tests to screen for kidney disease
• Complete urinalysis
• Plasma urea and creatinine
• Plasma electrolytes
2. Tests to assess renal function
a) To assess glomerular function
• Glomerular filtration rate
• Clearance tests
• Glomerular permeability
• Proteinuria
Renal function tests
B) Tests to assess tubular function
• Reabsorption studies
• Secretion tests
• Concentration and dilution tests
• Renal acidification
Glomerular function
• Ultrafiltration of blood occurs in the Bowman's capsule,
retains cells and proteins in blood
• Hb (Mol wt-64.6kD) is passed through while albumin (69kD)
is retained
• Appearance of albumin in urine is the earliest manifestation
of abnormal glomerular function
Glomerular filtration rate
• GFR is 120-125mL per liter per minute in a 70Kg man
• Daily, glomerular filtrate volume is 170-180L, of which only 1.7L are
excreted in urine
• GFR is decreased when BP is below 80mmHg
• Reduced GFR is observed in obstruction to renal flow (calculi,
enlarged prostate, age)
Functions of tubules

• PCT -70% of H2O, Na+, Cl-, and

100% of glucose, aa, and K+ are
reabsorbed
• Urea, phosphate, and Ca
particularly reabsorbed
• Major processes in tubules are
absorption or secretion
 Renal threshold and tubular maximum
• Threshold substances-Compounds whose excretion in urine is dependent on
its blood levels
• At normal or low plasma levels, they are completely reabsorbed and not
excreted in urine
• Tubular re-absorptive capacity is saturated when plasma levels are elevated
leading to urinary excretion
• The renal threshold of any substance is its concentration in plasma beyond
which it is excreted in urine.
• Maximum re-absorptive capacity of a substance is known as the tubular
maximum, tm.
• E.g. for glucose; Renal threshold-180mg/dl, Tm-375mg/min
• glucose appears in urine when [glucose] in blood is > 180mg/dl, in the
kidney, all glucose molecules above 375mg are excreted
Reabsorption of solutes in tubules
• Sodium
• Reabsorbed in the PCT by a co-transport mechanism (accompanied
with glucose, and amino acids)
• Reabsorption coupled with Na+-K+ ATP-ase
• Passive transport of equivalent amount of Cl-
• Co-transport of glucose is inhibited by ouabain and phlorhizin
• Sodium-pi co-transport system is inhibited by PTH and facilitated by
calcitriol
• PCT also contains Na+-H+ exchange system Na+ is reabsorbed in
exchange for H+. (occurs in acidosis)
• When H+ is conserved, Na+ and K+ are exchanges
Reabsorption of solutes in tubules
• Calcium
• 90% of Ca2+ is released from the glomerular filtrate
• Regulation of calcium balance is achieved at the DCT
• Calcium reabsorption is regulated by PTH and vitamin D
• Uric acid
• Almost completely reabsorbed in the PCT
• Absorption is both active and passive
• Probenecid is a drug secreted in the tubules, and competes with
uric acid for reabsorption (it increases excretion of uric acid-
uricosuric)
Reabsorption of solutes in tubules
• Urea
• Freely filtered in the glomerulus
• About 40% is reabsorbed actively by tubules
• Rate of reabsorption inversely proportional with
tubular function (Blood urea levels elevated in low
renal failure)
• Urinary [urea] 70 times [urea] in plasma
• Urea forms 80% of total urinary solutes
Reabsorption of solutes in tubules
• Creatinine
• Filtered by the Bowman’s capsule
• Neither reabsorbed nor secreted
• Urinary [creatinine] is 70 times [creatinine] in plasma.
• Potassium
• 70% of K+ in the glomerular filtrate is reabsorbed by the PCT
• Net secretion of K+ takes place in the DCT in exchange for
Na+ reabsorption
Reabsorption of water
• Osmolality of urine varies between 60-1200mosmol/kg. (specific gravity 1.003-
1.0320
• GFR is about 125ml/min.
• In the PCT,
• Most of the Na+, Cl- and HCO3- are reabsorbed.
• Water has to move along with solutes to maintains osmolality
• This is called obligatory reabsorption of water
• In the loop of Henley,
• flow rate is 25ml min-1.
• reabsorption of Na+
• Reabsorption of water is slow,
• Urine at this stage is hypotonic
 Reabsorption of water…
• In DCT, flow rate reduces flow rate is 16ml min-1 .
• Reabsorption of water is influenced by ADH, whose
secretion is controlled by hypothalamic osmo-receptors
• Collecting ducts;
• Flow rate -1mlmin-1
• Urine is hypertonic
Abnormal constituents of urine
• Parameters checked when reporting on a urine sample include
• Physical characteristics;
• Volume
• Average daily output-1.5l per day
• May be increased in excess water intake or diuretic therapy, DM, chronic renal
disease
• May be decreased in excessive sweating, dehydration, edema, kidney damage
• Summary;
• normal 1.5l in 24 hrs,
• oliguria <400ml,
• anuria <100ml,
• polyuria > 3L
Physical characteristics…
• Appearance
Physical characteristics…
• Odor
• Normal urine-faintly aromatic due to presence of volatile organic acids
• Urine in DKA may have a fruity smell due to presence of acetone
• Color
• Normal-amber yellow due to pigment urochrome
• Excessive yellow urine is produced in jaundiced patients
• Refer to table on appearance
• Specific gravity
• normal 1.015-1.025,
• Decreased in excessive water intake, chronic nephritis, diabetes insipidus
• Increased in DM, nephrosis, and excessive perspiration
Chemical X-tics of urine
• Reaction to litmus
• Normal pH 5.5-6.5
• Protein rich diet-lead to production of sulphuric and phosphoric acids from aa
break down making urine acidic
• Vegetable rich diet-urine is alkaline due to conversion of organic acids in
vegetables (citric and tartaric) to bicarbonate
• Proteins
• Proteinuria is an index of renal disease
• Normal urine
• Very low [protein], undetectable
• These proteins are secreted by tubular epithelial cells
• Proteinuria
• Commonly assessed by heat and acetic acid test
• Nowadays a dipstick is used
• Establishes renal disease, nature of renal disease, degree of kidney dysfunction
Chemical X-tics of urine…
• Blood
• Hematuria is presence of blood in urine
• Seen in nephritis, post renal hemorrhage
• Hemoglobinuria occurs due to abnormal hemolysis
• Rapid test of blood in urine-Occultest tablets and Hemastix strips
• Reducing sugars- glycosuria
• Benedicts test used to quantify sugar
• 0.5g/100ml-green
• 1g/100ml-yellow
• 1.5g/100ml-orange
• 2g/100ml-red
• Benedict’s reagent my be oxidized by many other agents
• Dipstick is used in place of Benedict’s test
 Chemical X-tics of urine…
• Ketone bodies
• Include acetoacetic acid, betahydroxybutyric acid, acetone
• Ketonuria is seed in DM and starvation, persistent vomiting, Von Gierkes
disease and in alkalosis
• Ketone bodies are analyzed using Rothera’s test
• Nowadays, Ketosfix strips are used
• Bile salts
• Present in urine in the early phase of obstructive jaundice
• Identified by Hay’s test
• Bile pigments
• Appears in urine in obstructive jaundice
• Detected by Fouchet’s test
Chemical X-tics of urine…

• Urobilinogen
• Urobilinogen is oxidized to urobilin causing the deepening colour of urine on
standing
• In hepatocellular jaundice, there is no urobilinogen in urine
• Appearance of urobilinogen in urine is an earliest sign of recovery
• In normal urine, there is traces of undetectable urobilinogen
• Identified by Ehrlich test or Schesinger’s test
• It is increased in hemolytic jaundice, testing positive
1. Markers of glomerular filtration rate
• GFR-the volume of blood filtered by the glomerulus in a unit time
• Clearance tests are used to estimate GFR
• A decrease in renal function is due to loss of functional nephrons, not an
individual nephron
• Usually kidney damage occurs before GFR is decreased.
• Clearance is defined as the amount of blood completely cleared of a substance
per unit time
• Unit-mL of plasma min-1
• It is the amount plasma that must have passed through the glomeruli in
1 min with complete removal of the substance

𝑚𝑔 𝑜𝑓 𝑎 𝑠𝑢𝑏𝑠𝑡𝑎𝑛𝑐𝑒 𝑒𝑥𝑐𝑟𝑒𝑡𝑒𝑑 𝑝𝑒𝑟 𝑚𝑖𝑛𝑢𝑡𝑒

𝐶𝑙𝑒𝑎𝑟𝑎𝑛𝑐𝑒 =
𝑚𝑔 𝑜𝑓 𝑎 𝑠𝑢𝑏𝑠𝑡𝑎𝑛𝑐𝑒 𝑝𝑒𝑟 𝑚𝑙 𝑜𝑓 𝑝𝑙𝑎𝑠𝑚𝑎
Markers of glomerular filtration rate…
𝑈𝑥𝑉
•𝐶=
𝑃
• U=concentration of the substance in urine
• P=concentration of the substance in plasma
• V= ml of urine excreted per minute
• Units-mlmin-1
• NB:
1. If the substance is freely filtered across the capillary wall and is neither secreted nor
absorbed, clearance is equal to GFR. Such a substance is an ideal marker
2. For a substance reabsorbed by the tubules, clearance < GFR
3. If a substance ix excreted by the tubules, clearance >GFR
• Endogenous clearance markers are urea & creatinine, Creatinine is an ideal
marker
 a) Creatinine clearance test
• Cr is a waste product from creatine phosphate, produced in a
spontaneous non-enzymatic reaction
• Levels depend on total muscle mass of the body
• Women and children excrete less Cr than men (due to muscle mass)
• 98% of Creatine pool is in the muscle where 1.6% is converted to
creatinine per day which is rapidly excreted
• Since,
1. its is continuously produced, blood levels don’t fluctuate a lot, hence it
is a vital marker for clearance
2. levels are constant in a particular person, urine output of Cr can be
used to assess a patient’s 24hr urine output
 Creatinine clearance test
• Reference values for Cr.
• Adult males 0.7-1.4mg/dl
• Adult females 0.6-1.5mg/dl
• Children 0.4-1.3mg/dl
• The kidney reserve is such that 50% of kidney function must be lost before
there is a rise in [Cr] in blood
• Cr levels >1.5mg/dl indicate impairment of renal function
• Decreased Cr clearance indicated reduced GFR
• Clearance up to 75% indicates adequate renal function
 Procedure for Cr estimation
• 500ml of water is given to a patient to promote urine flow
• After 30 mins, a patient is asked to urinate and the urine is discarded
• After 60 mins, a patient urinates and urine is collected, vol of urine is
measured
• Blood is obtained
• Blood and urine [Cr] is obtained using Jaffe’s test (or an enzyme specific
reaction)
𝑈𝑋𝑉
• 𝑈𝑛𝑐𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑐𝑙𝑒𝑎𝑟𝑎𝑛𝑐𝑒 =
𝑃
• U=[Cr] in urine
• P=[Cr] in plasma
• V=urine flow in ml/min
Procedure for Cr estimation…
• Estimated GFR (eGFR)
• It is a useful way to estimate serum Cr without timed urine
collection
• Uses Cockcroft –Gault equation,
𝐴𝑔𝑒 𝑦𝑟 𝑋𝑤𝑒𝑖𝑔ℎ𝑡(𝑘𝑔)
• 𝐶𝑟 = ( )x0.85 in females
72𝑋 𝐶𝑟 𝑝𝑙𝑎𝑠𝑚𝑎
• Creatinine coefficient
• It is urinary creatinine expressed in mg/kg of body weight
• Normal
• 20-28mg/kg-males
• 15-21mg/kg-females
• It is elevated in muscle dystrophy
Chronic kidney disease (CKD)
• Indicated by GFR <60ml/min/1.73m2 for 3 months or more
• Graded using GFR calculated from serum creatinine and MDRD
equation
• Risk factors: DM hypertension, glomerular nephritis, UTI etc
• Markers; serum creatinine, eGFR, microalbumen, cysteine C
• eGFR=186 × [serum creatinine (mg/dl)]−1.154 × (age)−0.203 × (0.742 if female)
Acute kidney disease
• An abrupt kidney dysfunction due to renal or tubular cell injury
• It is characterized by a rise in serum creatinine and low urine output
 b) Cysteine-C as a filtration biomarker
• Serum cysteine C is useful in detection of early kidney disease
which is usually missed by Cr
• It is a 13KD(120aa) non glycosylated protein
• It is highly concentrated in bio-fliud (saliva, breast milk, tears and semen)
• Normal plasma levels 0.8-1.2mg/l
• Cr is the most widely used biomarker of kidney function however,
• It is inaccurate in detecting mild renal impairment.
• The tubular secretion contributes approximately 20% of the total Cr
excretion by the kidney, & this increases as GFR decreases.
• Serum Creatinine does not increase until the GFR is moderately
decreased
Cysteine-C as a filtration biomarker…

• It is freely filtered by the glomeruli and is produced at a constant rate

• It is completely reabsorbed but degraded in tubules
• Blood levels not dependent on age, sex, muscle mass, inflammation
c) Urea clearance test
• Urea clearance is less than GFR because urea is partially
reabsorbed.
• It is the number of ml of blood, which contains the urea excreted
in a minute by the kidneys
• Procedure
• A patient is allowed to take a normal breakfast,
• A patient is given water and allowed to urinate. The urine is discarded
• After 1 hour, the patient completely empties the bladder and the volume
of urine is measured
• Urine urea is estimated
• A blood sample is taken and blood urea is estimated
Urea clearance test…

𝑼𝒙𝑽
• 𝑼𝒓𝒆𝒂 𝒄𝒍𝒆𝒂𝒓𝒂𝒏𝒄𝒆 = ,
𝑷
• U=mg of urea per ml of urine
• P=mg of urea per ml of plasma
• V=ml of urine excreted per minute
• This is called maximum urea clearance
• Normal-75ml/min
 Standard urea clearance
• The clearance value is decreased when v is less than 2ml/min.
• Standard urea clearance is then used,
𝑼𝒙√𝑽
• 𝑺𝒄𝒍𝒆𝒂𝒓𝒆𝒏𝒄𝒆 = ’
𝑷
• Normal value- 54ml/min
• Interpretations
• A value below 75% of the normal is abnormal
• Clearance value may be abnormal even when plasma urea levels are within range
• Plasma urea levels start to rise when the value falls below 50% of the normal
Standard urea clearance …

• Urea is reabsorbed from the tubules therefore tubular function

affects urea levels
• Hence creatinine clearance is more preferred
• Urea is freely filtered by the glomerulus but reabsorbed in the PCT
and DCT
• Hence urea clearance is usually less that GFR
Blood urea
• Normal serum urea is 20-40mg/dl
• Interpretation of blood urea values
• Urea is the end product of protein
metabolism
• Serum concentration increases
with age e.g.;
• A value of 40mg/dl is suspicious in
individuals <25yrs of age but
normal for a 60 yrs. old
 Blood urea
• Measured as blood urea nitrogen • Serum urea is increased in all
(BUN) forms of kidney disease
• Molecular weight of urea is 60 • Acute glomerular nephritis- can
• 1 molecule of urea contains 28 gram be as high as 300mg/dl
of nitrogen.
• Thus a serum concentration of 28 • Early stage of nephritis-usually
mg/dL of BUN =60 mg/dL of urea or normal but increases with
10 mmol/L of urea. progression
• BUN can be converted into urea by • Decreased levels observed in
multiplying the figure by 2.14.
late pregnancy, starvation,
protein deficient diet, hepatic
failure
d) Inulin clearance
• Inulin is a polysaccharide of fructose, not appreciably metabolized in
the body
• It is freely filtered, neither secreted nor reabsorbed by the tubules
• Inulin clearance is hence a measure of GFR
• Normal-125ml/min
• Procedure:
• 100ml of a sterile 10% solution of inulin is given as a slow intravenous drip in
2hrs
• Urine is collected totally in that period
• Blood sample is collected midway the test
• Inulin is estimated by a reaction with resorcinol giving a red color
Inulin clearance…
• Estimating urea reabsorption using inulin clearance
• Inulin clearance =125ml/min/
• Urea clearance = 75ml/min
𝑖𝑛𝑢𝑙𝑖𝑛 𝑐𝑙𝑒𝑎𝑟𝑎𝑛𝑐𝑒−𝑢𝑟𝑒𝑎 𝑐𝑙𝑒𝑎𝑟𝑎𝑛𝑐𝑒 125−75
• = =0.4
𝐺𝐹𝑅 125

• i.e, 40% of urea present in the glomerular filtrate is reabsorbed in the

tubules
• This is not a routine procedure
2. Markers of glomerular permeability
• Glomerulus acts as a semipermeable filter for blood passing through
its capillaries
• Macromolecules are restricted due to size, charge & shape
• Molecules smaller than 5KD (e.g. urea, glucose, Cr, & electrolytes) are
freely filtered by the glomerulus
• Albumin is retained while hemoglobin is filtered and excreted
• Lower molecular mass proteins are freely filtered, reabsorbed and
catabolized by renal tubular cells
• (normal urinary protein excretion is <150mg in 24 hrs)
• Appearance of proteins e.g. albumin indicates increased permeability
a) Proteinuria
Can be of 3-types
Glomerular proteinuria
Increase in filtered load due to glomerular damage & vascular
permeability
Overflow proteinuria-
Increased circulation of low molecular weight proteins
Tubular proteinuria-
Decrease in re-absorptive capacity due to tubular damage
Proteinuria…
i) Glomerular proteinuria
• Kidney glomerulus is impermeable to substances of
Mw>60,000
• Hence plasma proteins are absent in urine
• Damaged glomeruli become more permeable and
proteins are present
• Small molecules of albumin will pass through more than
other plasma proteins
• Early morning urine is used to for measurement of protein
Proteinuria…
• Interpretation
• Detection limit with Dipstick is 200–300 mg/L.
• 300 mg/day = Benign proteinuria
• 300 mg – 1000 mg = Pathological proteinuria
• > 1000 mg/day = Glomerular proteinuria
• Micro-albuminuria is an example of glomerular proteinuria
• Also called minimal albuminuria or paucialbuminuria
• Identified when small quantity of albumin (30-300mg/day) is
seen in urine
• It is an indicator of nephropathy in patients with DM &
hypertension (need this screening at least once a year)
Proteinuria…
• Micro-albuminuria…
• Expressed in albumin: creatinine ratio
Normal values Males <23mg/g of creatinine
Females <32mg/g of creatinine
Confirmed by overnight urine collection and measurement o albumin in
urine& calculation of albumin excretion rate
A value >20µg/min confirms microalbuminuria
NB: Administration of CAngiotensin-converting enzyme) ACE inhibitors
decreases the rate of microalbuminuria
Proteinuria…
II) Overflow proteinuria
• When small MW proteins are increased in blood, they overflow to
urine, e.g Hemoglobin Mw-67,000 can pass through normal glomeruli
hence exist in free form (haemolytic conditions)
• Hemoglobin can appear in urine-hemoglobinuria in hemolysis
• Myoglobinuria (presence of myoglobin in urine) is seen following
muscle injury
Proteinuria…
• E.g Bence-Jones proteinuria,
• Seen in 20% of cases of multiple myeloma
• Light chains of immunoglobulins are produced abnormally
• Being small proteins they are excreted in urine
• They are called Bence-Jones proteins
• They precipitate when urine is heated at 45oC, maximum
precipitation at 60oC and dissolve at 80oC and form a clear
solution at 100oC. The precipitate reforms on cooling
Proteinuria
iii) Tubular proteinuria
• Occurs when functional nephrons are reduced, GFR is reduced and
the remaining nephrons are overworked
• Tubular reabsorption mechanism is impaired, low Mw proteins
appear in urine
• Markers -retinol binding protein & alpha-1 macroglobulin
• Both are synthesize in the liver and rapidly removed by the
glomerulus
Reading Assignment
• 1. make short notes on each of the following
a) Azotemia
b) Nephron Loss Proteinuria
c) Urogenic Proteinuria
References
• DM Vasudevan et a al (2013); Textbook of Biochemistry for Medical
students, Chapter 27. Jaypee Brothers Medical Publishers (P) Ltd,
London