http://www.kidney-international.

org original article

& 2009 International Society of Nephrology

Urinary neutrophil gelatinase-associated lipocalin

levels reflect damage to glomeruli, proximal tubules,
and distal nephrons
Takashige Kuwabara1, Kiyoshi Mori1, Masashi Mukoyama1, Masato Kasahara1,2, Hideki Yokoi1,
Yoko Saito1, Tetsuro Yoshioka1, Yoshihisa Ogawa1, Hirotaka Imamaki1, Toru Kusakabe1, Ken Ebihara1,
Mitsugu Omata3, Noriko Satoh4, Akira Sugawara5, Jonathan Barasch6 and Kazuwa Nakao1
1
Department of Medicine and Clinical Science, Graduate School of Medicine, Kyoto University, Kyoto, Japan; 2Division of Nephrology,
Kobe City General Hospital, Kobe, Japan; 3Biomedical Research Laboratories, Asubio Pharma, Osaka, Japan; 4Clinical Research Institute
for Endocrine Metabolic Diseases, National Hospital Organization, Kyoto Medical Center, Kyoto, Japan; 5Department of Nephrology,
Kyoto Medical Center, Kyoto, Japan and 6Department of Medicine, College of Physicians and Surgeons, Columbia University, New York,
New York, USA

Urinary neutrophil gelatinase-associated lipocalin (Ngal or Neutrophil gelatinase-associated lipocalin (Ngal) is a differ-
lipocalin 2) is a very early and sensitive biomarker of kidney entiation inducer for epithelia in embryonic kidney, whose
injury. Here we determined the origin and time course of expression is dramatically increased in acute kidney injury
Ngal appearance in several experimental and clinically (AKI).1–5 Ngal exerts a spectrum of iron-dependent biological
relevant renal diseases. Urinary Ngal levels were found to be activities,1–4,6 and administration of Ngal protein mitigates
markedly increased in lipoatrophic- and streptozotocin- renal injury in mice, suggesting that functional consequence
induced mouse models of diabetic nephropathy. In the latter of Ngal upregulation is renoprotection.2 Ngal mRNA levels in
mice, the angiotensin receptor blocker candesartan the kidney are increased as much as by 1000-fold during renal
dramatically decreased urinary Ngal excretion. The ischemia–reperfusion injury in mice.2,7 Ngal protein starts to
reabsorption of Ngal by the proximal tubule was severely accumulate within a few hours in the blood and urine during
reduced in streptozotocin-induced diabetic mice, but AKI.8–11 These characteristics of Ngal have made it a
upregulation of its mRNA and protein in the kidney was promising biomarker of AKI that is found in the blood and
negligible, compared to those of control mice, suggesting urine.1,7,12–15 Furthermore, several studies reported that
that increased urinary Ngal was mainly due to impaired renal serum and urinary Ngal levels are elevated proportionally to
reabsorption. In the mouse model of unilateral ureteral the extent of renal damage in chronic kidney disease,16,17 but
obstruction, Ngal protein synthesis was dramatically the source and the time course of urinary Ngal concentrations
increased in the dilated thick ascending limb of Henle and N are largely unknown. In this study, we investigated urinary
was found in the urine present in the swollen pelvis of the Ngal levels in four types of renal damage caused by distinct
ligated kidney. Five patients with nephrotic syndrome or mechanism: nephrotic syndrome caused by glomerular
interstitial nephritis had markedly elevated urinary Ngal disorders, diabetic nephropathy, obstructive nephropathy,
levels at presentation, but these decreased in response to and interstitial nephritis. We also examined whether measure-
treatment. Our study shows that the urinary Ngal level may ment of urinary Ngal is useful for the monitoring of renal
be useful for monitoring the status and treatment of diverse damage in mice or humans during the treatment course.
renal diseases reflecting defects in glomerular filtration
barrier, proximal tubule reabsorption, and distal nephrons. RESULTS
Kidney International (2009) 75, 285–294; doi:10.1038/ki.2008.499; Urinary Ngal excretion is proportional to albumin excretion
published online 1 October 2008 in mouse models of diabetic nephropathy
KEYWORDS: diabetic nephropathy; nephrotic syndrome; obstructive
As a model of diabetic nephropathy, we first examined
nephropathy; acute renal failure; albuminuria urinary Ngal concentrations in A-ZIP/F-1 transgenic mice,
which are characterized with lipoatrophic diabetes, fatty liver,
hyperlipidemia, severe insulin resistance, and massive
Correspondence: Kiyoshi Mori, Department of Medicine and Clinical
proteinuria.18–20 Urinary Ngal excretion in A-ZIP/F-1 mice
Science, Graduate School of Medicine, Kyoto University, 54 Shogoin Kawa- at 10 months of age was much larger than that in control
hara-cho, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: keyem@kuhp.kyoto-u.ac.jp FVB/N mice (Figure 1). By Western blot, we observed 30 and
Received 8 April 2008; revised 14 July 2008; accepted 12 August 2008; 25 kDa bands with Ngal immunoreactivity, and the larger
published online 1 October 2008 band was found only in the urine from A-ZIP/F-1 mice and

Kidney International (2009) 75, 285–294 285

original article T Kuwabara et al.: Source and changes of urinary Ngal in renal injury

a Standard Control
rNgal FVB/N A-ZIP/F-1

1 ng 0.2 ng #1 #2 #3 #4

uNgal 30 kDa
21 kDa 25 kDa

b 1000 c 30,000
uNgal/uCr (ng/mgCr)

uAlb/uCr (µg/mgCr)
800
20,000
600
10,000
400

5000
200

0 0
#1 #2 #3 #4 #1 #2 #3 #4
Control A-ZIP/F-1 Control A-ZIP/F-1
FVB/N FVB/N

d Standard Non-
rNgal STZ STZ 8W
1 ng 0.2 ng #1 #2 #3 #4 #5

uNgal 25 kDa
21 kDa

e
600
uAlb/uCr (µg/mgCr)

400

200

0
#1 #2 #3 #4 #5
Non- STZ 8W
STZ
Figure 1 | Urinary Ngal and albumin excretion in two models of diabetic nephropathy. (a–c) A-ZIP/F-1 diabetic mice at 10 months of
age and (d, e) diabetic mice at 8 weeks after streptozotocin (STZ) injection. (a, d) Western blot of urine (25 ml each) in individual mice and
(b, c, e) urinary levels of Ngal and albumin (Alb) normalized by creatinine (Cr) are shown. (b) A-ZIP/F-1 mice, including no. 1, excreted
much larger volumes of urine than control FVB/N mice. In three control FVB/N mice, mean urinary Ngal/Cr ratio (±s.e.) was 42±24 ng per
mgCr. uNgal, urinary Ngal; rNgal, recombinant Ngal.

not in the urine or tissues from normal, diabetic, or Ngal and albumin excretion was highly variable among
obstructed kidneys of mice with C57BL/6 background (see different mice, but urinary albumin levels and log transfor-
below). The larger protein may have heavier glycosylation mation of Ngal levels showed a close linear correlation
than the smaller protein.21 When the amounts of two throughout the course of 12-week observation period.
proteins were added, mice with larger urinary Ngal levels
tended to have larger urinary albumin levels. Elevation of urinary Ngal excretion in STZ mice is not caused
Next, we studied streptozotocin (STZ)-induced diabetes, by renal synthesis but by reabsorption defect, and treatment
which manifests with insulin deficiency and microalbumi- with angiotensin receptor blocker reduces urinary Ngal levels
nuria. In STZ mice, urinary albumin excretion increased To examine whether local expression of Ngal is increased in
gradually and, after 8 weeks, reached 7.8-fold of the level STZ mouse kidneys, we studied expression levels of Ngal
before STZ injection (Figure 2). On the other hand, urinary protein in the whole kidney preparation of STZ and non-STZ
Ngal levels were elevated by 77-fold at 8 weeks. The extent of control mice and found no significant difference at 8 weeks

286 Kidney International (2009) 75, 285–294

T Kuwabara et al.: Source and changes of urinary Ngal in renal injury original article

× 75 Standard
a 7000 × 77
Non-STZ STZ 8W
rNgal (ng)
6000 uNgal/uCr 5 1 0.2
uNgal/uCr (µg/mgCr)

uAlb/uCr
5000
Serum
#
4000

3000 Whole

uAlb/uCr (µg/mgCr)
1000 kidney
2000 × 16

× 7.8 × 9.6 500

1000 × 4.4
Liver
# #
#
0 0
Pre- STZ STZ STZ P <0.001
STZ 4W 8W 12W
100
b

Ngal/GAPDH mRNA ratio

10,000
80
uNgal/uCr (ng/mgCr)

1000
60

100
pre STZ 40
STZ 4W
10 STZ 8W
20 P <0.01 N.S.
STZ 12W

1
0
0 100 200 300 400 500
uAlb/uCr (µg/mgCr) Non-STZ STZ Non-STZ STZ UUO
Whole Liver Whole
Figure 2 | Time course and correlation of urinary Ngal and kidney kidney
albumin excretion in streptozotocin (STZ)-induced diabetic
mice. (a) Urinary Ngal (uNgal) and albumin (uAlb) levels Figure 3 | Ngal protein and mRNA expression in the serum,
normalized by urinary creatinine (uCr) were examined before and kidney, and liver of STZ mice. (a) Western blot of serum, whole
at 4, 8, and 12 weeks after STZ injection (mean±s.e.). #, Po0.05 kidney, and liver at 8 weeks after induction of diabetes. Mice
versus pre-STZ. Elevation of urinary Ngal levels was significant at without STZ injection served as control (non-STZ). Equal amounts
4, 8, and 12 weeks if analyzed after log transformation. (b) of serum (15 ml) and protein (30 mg) of whole kidney and liver
Correlation between uNgal/uCr and uAlb/uCr; r ¼ 0.86, Po0.001, were separated by electrophoresis; rNgal, recombinant Ngal. (b)
n ¼ 19. Ngal mRNA expression levels were measured using real-time PCR
and normalized by GAPDH expression (n ¼ 4). The mean Ngal/
GAPDH level in non-STZ whole kidney was arbitrarily defined as
1.0. The whole kidneys at 1 day after unilateral ureteral
obstruction (UUO) were also examined as a positive control. NS,
after STZ treatment (Figure 3). We did not find significant not significant.
alteration of Ngal protein expression in the livers, either.
Of note, serum Ngal levels in STZ mice were significantly
lower than those in non-STZ mice (23±5 versus 111±22 ng/
ml, n ¼ 3–4, Po0.01). We measured Ngal mRNA expression the urine and reabsorption of labeled Ngal was reduced by
levels in the kidneys and livers of STZ mice, but they 47% (Po0.01).
were increased only marginally compared to control mice. As treatment of diabetic nephropathy with angiotensin
As a positive control, Ngal mRNA expression was increased receptor blocker (ARB) reduces proteinuria and ameliorates
by 100-fold in obstructed kidneys after 1 day of ureter renal injury,22 we gave the ARB candesartan to STZ mice
ligation (Po0.001). These findings suggested that dramatic through drinking water at 10 mg/kg/day (Figure 5). After 1
(nearly 80-fold) increase of urinary Ngal excretion in STZ week, urinary albumin levels were decreased by 14%
mice cannot be explained by augmentation of Ngal (Po0.05) and urinary Ngal levels were decreased by 77%
protein synthesis in the kidney, and led us to investigate (Po0.05). Serum Ngal levels were not altered by candesartan
tubular reabsorption of Ngal. Injection of histidine-tagged or (20±6 ng/ml). The dose of candesartan used was a
Alexa Fluor 546-labeled Ngal (21 kDa in size) in the subdepressor dose, and did not significantly affect body
peritoneum of non-STZ mice resulted in glomerular filtra- weights, blood glucose, urea nitrogen and creatinine levels
tion and efficient reabsorption of Ngal at the proximal (Table 1). Through these findings, we conclude that increased
tubules from the apical side, thus no exogenous Ngal was urinary Ngal excretion in STZ mice was caused mainly by
detected in the urine (Figure 4). In STZ mice, on the other reabsorption defect and treatment with candesartan partially
hand, substantial amount of exogenous Ngal was excreted in normalized urinary Ngal levels.

Kidney International (2009) 75, 285–294 287

original article T Kuwabara et al.: Source and changes of urinary Ngal in renal injury

a Injection of His-tagged Ngal urinary Ngal levels in human subjects. Case 1 was a 68-year-
+ + – old woman with biopsy-proven minimal change disease. She
Standard had nephrotic range proteinuria (14 g/day) and gained body
rNgal Non-STZ STZ STZ
weight by 15 kg (from 62 to 77 kg) within 3 weeks. She was
1 ng 0.2 ng treated with i.v. steroid pulse (methyl prednisolone 1 g  3
Anti-Ngal 1 days), followed with oral prednisolone (beginning with
2
(total) 35 mg/day), and with three courses of hemodialysis on days
Anti-His-Tag
8, 9, and 12 after admission (Figure 6). Her proteinuria,
(exogenous) edema, and azotemia resolved gradually. Concomitantly,
urinary and serum Ngal levels decreased during the
b treatment, but reduction was much faster for urinary Ngal
G levels. There was a temporal elevation of urinary Ngal levels
G
on day 21, which might reflect the reappearance of oligouria
G or proteinuria.
G
Case 2 was a 26-year-old woman, who was diagnosed
to have membranous-type lupus nephritis (ISN/RPS
G class V). She was treated with two courses of i.v. steroid
No Alexa injection Non-STZ
pulse, followed with oral prednisolone (Figure 6). Her
urinary levels of protein and Ngal were decreased sharply
within 17 days.
G Case 3 was a 55-year-old woman with clinically diagnosed
lupus nephritis (no biopsy). She had been treated with two
G
G courses of i.v. steroid pulse (1 g  3 days) and oral
prednisolone had been tapered from 50 to 35 mg/day, before
Non-STZ G STZ changing the hospitals to ours. She was given i.v. cyclo-
phosphamide pulse (0.4 g, once, 6 days after admission) and
c P<0.01
the immunosuppressant mizoribine (Asahi Kasei Pharma,
Positive area of Alexa 546

Osaka, Japan; 100 mg/day). Urinary excretion of protein and

100
Ngal decreased slowly (Supplementary Figure S1). Serum
(% of non-STZ)

creatinine levels were constant throughout the course

(0.4–0.5 mg/100 ml).
50 Case 4 was a 69-year-old man, who suffered from rapidly
progressing, crescentic glomerulonephritis accompanied
with moderate tubulointerstitial damage. His serum con-
0
tained myeloperoxidase-type antineutrophil cytoplasmic
Non Alexa Non-STZ STZ antibody (MPO-ANCA, 138 EU). The maximum serum
Figure 4 | Urinary excretion and tubular reabsorption of
creatinine level was 5.6 mg/100 ml, and the proteinuria and
exogenously administered Ngal in STZ mice. (a) Western blot azotemia responded slowly to treatment containing oral
of urinary Ngal detected either with anti-Ngal or with anti-His-tag and i.v. steroid and i.v. cyclophosphamide (Supplementary
antibody at 8 weeks after induction of diabetes. Urine samples Figure S1). Macrohematuria was observed, peaking at 5 days
were collected for 12 h after His-tagged Ngal injection (i.p.) and
25 ml aliquots of them were separated by electrophoresis. Of note, after cyclophosphamide administration. After 10 months,
STZ mice excreted much more diluted urine compared to non-STZ he showed signs of recurrence which were worsening in
control mice. 1, endogenous Ngal (25 kDa, glycosylated); 2, His- proteinuria, hematuria, azotemia, and MPO-ANCA titer
tagged Ngal (21 kDa, unglycosylated). (b) Alexa Fluor 546-labeled (from o10 to 37 EU). We observed reelevation of urinary
Ngal was injected into STZ mice and kidneys were examined
30 min later. Arrows indicate Ngal protein distribution (in orange), Ngal levels during acute worsening of nephritis. Cyclosporine
which was homogeneous in non-STZ but was irregular and sparse A (75 mg/day before breakfast) was added at 13 months,
in STZ mice. Top right panel shows selected area (in green) of which appeared to support reduction of above mentioned
positive fluorescence by computer software. G, glomeruli.
signs.
Magnification,  20. (c) Quantitation of exogenous Ngal uptake in
kidneys of STZ and non-STZ mice (n ¼ 4). To understand renal localization of Ngal protein in
nephrotic patients, we carried out immunofluorescence study
of renal biopsy samples and found close colocalization of
Urinary Ngal levels are highly elevated in human cases of signals of Ngal and albumin at the apical side of tubules. The
nephrotic syndrome and are decreased in response to results for case 1 are shown in Figure 7. These findings are
treatment consistent with those in animal experiments shown above
As cases of glomerular disorders with nephrotic syndrome, (Figure 4), indicating highly active reabsorption of Ngal from
we investigated the clinical courses and changes in serum and glomerular filtrate.

288 Kidney International (2009) 75, 285–294

T Kuwabara et al.: Source and changes of urinary Ngal in renal injury original article

P<0.05
P<0.05
P<0.05 250
1600

uAlb/uCr (µg per mgCr)

uNgal/uCr (ng per mgCr)
200
1200
150
800
100
400 Candesartan
50
Vehicle
0 0
Before After Before After Pre STZ STZ Candesartan
STZ 4W 8W 1W
Vehicle Candesartan

Figure 5 | Reduction of urinary Ngal and albumin excretion by candesartan in STZ mice. (a) Urinary Ngal (uNgal) and (b) albumin (uAlb)
levels at 4 and 8 weeks after STZ injection and after one more week with candesartan (10 mg/kg/day, orally) or vehicle treatment (n ¼ 4).

Table 1 | Blood glucose, urea nitrogen, creatinine levels, body weight, and blood pressure in STZ diabetic mice before and after
candesartan treatment
Vehicle Candesartan
Before After Before After
Blood glucose (mg/100 ml) 598±2 593±6 600±3 591±5
HbA1c (%) 11.7±0.5 ND 12.5±0.2 ND
Blood urea nitrogen (mg/100 ml) ND 50±2 ND 54±1
Serum creatinine (mg/100 ml) ND 0.13±0.01 ND 0.11±0.01
Body weight (g) 23.8±0.9 23.5±0.7 22.4±0.5 22.8±0.5
Systolic blood pressure (mm Hg) 104±2 105±0.9 103±1 100±1
Diastolic blood pressure (mm Hg) 55±2 56±1 50±1 49±2
Treatment with candesartan did not significantly alter these parameters. Blood urea nitrogen and serum creatinine levels in non-STZ control mice were 23±3 and
0.09±0.02 mg/100 ml, respectively. Blood was drawn when mice were fed ad libitum. ND, not determined.

In mice with obstructive nephropathy, Ngal protein is using polyclonal anti-Ngal antibody may have been generated
specifically located in the distal nephrons in the obstructed by lysosomal proteolysis of Ngal in the tubules.2 Ngal mRNA
side by local synthesis, whereas it is confined to the proximal expression was elevated by 100-fold in the obstructed kidneys
tubules in the contralateral side of the kidneys by (Figure 3), but it was only elevated by threefold in the
reabsorption
contralateral kidneys at day 1 (data not shown). These
As a model of post-renal kidney injury, we investigated mice
findings indicate that Ngal was synthesized de novo in distal
with unilateral ureteral obstruction (UUO), in which distal
nephrons of obstructed kidneys, although it was highly but
nephrons are primarily affected, and studied the changes of
transiently accumulated in the serum, filtrated and reab-
Ngal protein levels in the obstructed and nonobstructed sides
sorbed in the contralateral kidneys.
of the kidneys, serum, and urine (Figures 7 and 8). We also
determined the renal distribution of Ngal by immunohis- In a case with interstitial nephritis, urinary Ngal levels
tochemistry along with nephron segment markers: aquaporin decreased more rapidly than classic markers of tubular injury
(AQP) 1 for proximal tubules, Tamm-Horsfall protein (THP) Next, we investigated whether urinary Ngal is useful for
for thick ascending limbs of Henle, and AQP 2 for collecting evaluation of renal disorder with low-level proteinuria. Case
ducts (Figure 7; Figures S2–S4). In the UUO kidneys, after 1 5 was a 25-year-old man, who was admitted to our hospital,
day of ureter ligation, Ngal protein was expressed exclusively presenting with general malaise, proteinuria (0.4 g/day), renal
in thick ascending limbs of Henle, which was also expressing glucosuria and mild azotemia. He had taken an over-the-
THP and was prominently dilated, suggesting that Ngal was counter cold medicine 6 weeks earlier, and was positive in
synthesized in damaged epithelia. By striking contrast, in the lymphocyte stimulation test for the drug. Renal biopsy
contralateral kidneys, Ngal protein was confined to the apical revealed subacute interstitial nephritis with minor glomerular
side of aquaporin 1 þ proximal tubules. Ngal protein levels in lesions. His signs and symptoms resolved by oral and i.v.
the obstructed kidneys and in the urine from the dilated prednisolone treatment. Table 2 and Figure S5 summarize the
pelvis were continuously elevated for 2 weeks, whereas those clinical course and changes in urinary biomarkers. The
in the nonobstructed kidneys and in the serum peaked at day time to 50% reduction of urinary markers was in the
1 and decreased gradually. A smaller (17 kDa) fragment order of Ngal p (total) proteinoa1- and b2-micro-
detected in the kidneys (but not in the urine and serum) globulins (classic markers of tubular proteinuria)

Kidney International (2009) 75, 285–294 289

original article T Kuwabara et al.: Source and changes of urinary Ngal in renal injury

(Case 1, MCD) mPSL 1g×3 (Case 2, LN) mPSL 0.5g×3 mPSL 0.5g×3

PSL 35 mg 30 mg PSL 50 mg 45 40 30
1200 400
Hemodialysis
uNgal
8 4
uNgal/uCr
1000
uNgal/uCr (µg/gCr)

uNgal/uCr (µg/gCr)
BUN (mg/100 ml)

300

sCr (mg/100 ml)

sCr (mg/100 ml)
uNgal 3
uNgal (ng/ml)

sNgal (ng/ml)

800 6

uNgal (ng/ml)

sNgal (ng/ml)
600 200
4 2
sNgal
400 sCr
uNgal/uCr 100
sNgal
2 1
200 sCr
BUN

0 0 0 0
0 10 20 30 0 10 20 30 40 50
Days Days

15 uProt 2000 uProt

Anti-DNA Ab (U/ml)
8 40

uVolume (ml/day)

uProt (g/day)
uProt (g/day)

1500 6 30
10
1000 4 Anti-DNA Ab 20
5
uVolume 500 2 10

0 0 0 0
0 10 20 30 0 10 20 30 40 50
Days Days

Figure 6 | Clinical course of 2 cases with nephrotic syndrome. (a) minimal change disease (case 1, MCD) and (b) lupus nephritis (case 2,
LN). mPSL, methyl prednisolone; PSL, prednisolone; uProt, urinary protein excretion; sNgal, serum Ngal; uNgal, urinary Ngal; BUN, blood urea
nitrogen; sCr, serum creatinine; uNgal/uCr, urinary Ngal normalized by urinary creatinine; Ab, antibody.

oN-acetyl-b-D-glucosaminidase. The fold-change during Cross-sectional studies published so far have elucidated
treatment was also largest in urinary Ngal, suggesting that that urinary Ngal levels show certain correlation with urinary
urinary Ngal may be useful in monitoring the activity of protein levels.16,23 To our knowledge, this is the first human
nonglomerular renal disorders. report to show very rapid and simultaneous reduction of
urinary Ngal and protein concentrations by medical inter-
DISCUSSION vention. Surprisingly, the time course of urinary Ngal levels
In the present study, we investigated urinary Ngal concentra- was associated to that of urinary protein levels not only in
tions in patients with nephrotic syndrome (caused by acute diabetic nephropathy and minimal change disease but also in
and severe glomerular disorders) and interstitial nephritis crescentic glomerulonephritis and interstitial nephritis. In the
and in mouse models of diabetic or obstructive nephropathy latter disorders, treatment with steroid and immunosuppres-
and found that the levels were unequivocally elevated (over sant may have ameliorated Ngal reabsorption impairment
10-fold of control). In diabetic mice induced by STZ (as a and epithelial Ngal synthesis at the same time. The present
model of slowly progressive chronic kidney disease), urinary findings suggest that urinary Ngal may be useful in the
Ngal appeared to derive mostly from impaired reabsorption monitoring of disease activity and treatment efficacy. Of
in proximal tubules. In obstructed kidneys (as a model of note, we cannot overgeneralize the findings in this study to
post-renal AKI), Ngal was highly expressed in distal nephrons all renal disorders, especially because we did not examine
and accumulated in the urine collected from the pelvis. patients with severe, acute tubular necrosis, for instance,
Therefore, STZ-diabetic and obstructed kidneys are the two caused by renal ischemia or nephrotoxins (in which Ngal is
extreme examples in which the primary source of urinary abundantly synthesized by renal epithelia).2
Ngal is glomerular filtrate and renal synthesis, respectively. In In diabetic nephropathy, albumin excretion is increased by
human renal disorders, urinary Ngal should be a mixture of leakage from glomerular filtration barrier.24 On the other
these two major components. These findings indicate that in hand, a number of reports elucidated the involvement of
a variety of kidney diseases, urinary Ngal is a biomarker that tubular dysfunction as a cause of albuminuria.24–27 The size of
can reflect damage in glomeruli, proximal tubules and distal Ngal protein (25 kDa) is smaller than albumin and, in normal
nephrons. conditions, Ngal is rapidly filtered by glomeruli and

290 Kidney International (2009) 75, 285–294

T Kuwabara et al.: Source and changes of urinary Ngal in renal injury original article

a b a Day
0 1 2 3 7 10 14

UUO urine ND

25 kDa
UUO kidney
Albumin in MCD Ngal in MCD 17 kDa

c d
UUN kidney

Serum
Ngal in UUN Ngal in UUO
e f b
1000

UUO kidney (µg/g protein)

UUN kidney (µg/g protein)
UUO urine
2000

UUO urine (ng/ml)

750
THP in UUO

Serum (ng/ml)
AQP1 in UUN

Figure 7 | Localization of Ngal protein expression in human 500 UUO kidney

and mouse kidneys. Immunofluorescence of (a, green) albumin
1000
and (b, red) Ngal in a patient with minimal change disease (MCD,
magnification  40). Arrows, colocalization of signals; asterisks,
tubular lumen. Immunohistochemistry of (c, d) Ngal, (e) aquaporin 250 Serum
1 (AQP1), and (f) Tamm-Horsfall protein (THP) in the mouse kidney UUN kidney
treated with (d, f) unilateral ureteral obstruction (UUO) and in the
(c, e) contralateral kidney (UUN) at 1 day after operation (original 0 0
magnification,  20). Serial sections were analyzed. 0 0 10 15
DAYS

Figure 8 | Ngal protein levels in UUO, UUN, serum, and urine

in mice with kidney obstruction. (a) Western blot analysis of
reabsorbed very efficiently by proximal tubules, leaving only Ngal protein. ND, not determined. (b) The mean Ngal
concentrations in UUO, UUN, serum, and urine (collected from
0.1–0.2% in the urine.2 By analyzing renal handling of dilated pelvis of the UUO side using a needle and syringe).
exogenously injected Ngal, the present study emphasized the Elevation of Ngal concentrations were statistically significant at
existence of tubular reabsorption impairment in diabetic day 1 in UUO or UUN kidneys and serum, and at day 3 in urine
(Po0.01 by unpaired t-test, n ¼ 3) compared to day 0 (no
nephropathy.28 In reverse, cellular stress in the proximal
operation).
tubules may cause deterioration in diabetic nephropathy, as
it was reported that transgenic overexpression of catalase in the
proximal tubules of mice attenuated development of hyper- mice (which is 77-fold of control mice).33 Hyperfiltration is
tension and albuminuria associating diabetic nephropathy.29 reported in rodents given STZ.32 In a previous report,
Treatment of STZ-diabetic mice with ARB signi- threefold elevation in creatinine clearance was observed in
ficantly reduced urinary excretion of both Ngal and albumin. C57BL/6 mice after treatment with STZ, but serum creatinine
Two major scenarios can be proposed. First, ARB directly levels were not significantly decreased, likely due to
improved reabsorption efficiency in the proximal tubules, concomitant osmotic diuresis and dehydration.34 In the
which may be mediated by increased peritubular capillary present study, hyperfiltration may have similarly occurred in
blood flow30 and by amelioration of oxidative stress.31 STZ-diabetic mice. Growth arrest and leanness of STZ-
Second, ARB reduced intraglomerular hypertension and diabetic mice may be partly related to selective reduction of
hyperfiltration,32 reduced albumin leakage from glomeruli, serum Ngal levels (but not serum creatinine and BUN levels)
and the total amount of the ligands for the common in the present study, as obesity has been shown to be
scavenger receptor megalin was decreased in the tubular associated with elevated circulating Ngal levels.35
lumen. This may increase the ratio of Ngal and albumin We also observed abundant urinary Ngal excretion in 4
endocytosed at proximal tubules. patients with nephrotic syndrome. The mechanism may involve
Here we used high-dose STZ to induce a model of diabetic direct competition between Ngal and albumin at the surface of
nephropathy. Direct toxicity of STZ to proximal tubules may megalin molecule for receptor-mediated endocytosis26,36 and
have exaggerated the elevation of urinary Ngal levels in these also general malfunctioning of proximal tubules because of

Kidney International (2009) 75, 285–294 291

original article T Kuwabara et al.: Source and changes of urinary Ngal in renal injury

Table 2 | Changes of urinary biomarker levels in case 5 (interstitial nephritis)

Days after admission 9 37 Fold differencea Normal value
uNgal/uCr (mg/gCr) 256.6 25.4 10.1 o10 mg/lb
uProt/uCr (g/gCr) 0.381 0.052 7.3 o0.15 g/day
ub2MG/uCr (mg/gCr) 18.2 2.8 6.5 o0.3 mg/l
ua1MG/uCr (mg/gCr) 46.4 17.2 2.7 o15 mg/l
sNgal (mg/l) 224 90 2.5 o106 mg/lb
uNAG/uCr (U/gCr) 21.1 8.7 2.4 o7 U/l
BUN (mg/100 ml) 15 11 1.4 o22 mg/100 ml
sCr (mg/100 ml) 1.2 1.0 1.2 o1.1 mg/100 mlc
MG, microglobulin; NAG, N-acetyl-b-D-glucosaminidase; Prot, protein; u and sNgal, urinary and serum Ngal; uCr, urinary creatinine.
a
Fold difference between days 9 and 37.
b
Value suggested by ELISA kit supplier.
c
o1.1 for man and o0.8 for woman.

protein overload. Furthermore, a fraction of urinary Ngal may Analyzer (Bayer Medical, Tokyo, Japan), respectively. Mice were
originate from renal synthesis in addition to reabsorption killed under pentobarbital anesthesia before organ collection.
defect, but Ngal gene expression in human samples was not Prodrug of candesartan, candesartan cilexetil (TCV-116; Takeda
investigated in this study. The time required for urinary Ngal Chemical Industries, Osaka, Japan), was initially dissolved at 10 mg/
ml in a solution containing 16% polyethylene glycol no. 300 (vol/
reduction was variable among cases: ranging from 2 weeks
vol; Nacalai, Kyoto, Japan), 16% ethanol (Nacalai), and 0.7 M
(cases 1 and 2) to more than a month (cases 3 and 4).
Na2CO3, and further diluted in drinking water to be given at a final
In a case with interstitial nephritis, urinary Ngal showed dose of 10 mg/kg/day. This treatment method38 gave less blood
the largest fold increase and the quickest response to steroid pressure lowering effects compared to gavage administration as
treatment in comparison to other urinary biomarkers: total previously described.39 For UUO, mice were anesthetized with
protein, a1- and b2-microglobulins and N-acetyl-b-D- pentobarbital, the left kidney was exposed by midline incision, and
glucosaminidase. These findings suggest that urinary Ngal the left ureter was ligated with 4–0 silk at two points.40 Mice were
might be particularly useful in the evaluation of kidney killed 1–14 days after the operation.
recovery in patients with low-grade proteinuria.
To summarize, urinary Ngal is a rapid biomarker of Recombinant Ngal injection and detection
kidney injury and recovery showing a large fold-increase or To investigate renal reabsorption of Ngal, 200 mg of 6  histidine-
decrease during clinical course of various renal disorders. tagged (at the C terminus) or 60 mg of Alexa Fluor 546 (Molecular
Probes, Eugene, OR, USA)-labeled recombinant mouse Ngal
Proteinuria seems to be one of important factors affecting
(expressed in BL21 strain of Escherichia coli)2 was injected into
Ngal’uria.
the peritoneum of mice. Urine samples were collected for 12 h after
His-tagged Ngal injection. Urinary excretion of administered
His-tagged Ngal was evaluated by Western blot analysis described
MATERIALS AND METHODS below with anti-His antibody (MBL, Nagoya, Japan) or with goat
Animal experiments polyclonal anti-mouse Ngal antibody (R&D Systems, Minneapolis,
All animal experiments were conducted in accordance with our MN, USA). Kidneys were harvested 30 min after Alexa Fluor
institutional guidelines for animal research. Male A-ZIP/F-1 546-labeled Ngal injection, snap frozen, sliced at 10 mm thickness
heterozygous transgenic mice and control FVB/N littermates were and examined by a fluorescence microscope (IX81-PAFM; Olympus,
used at 10 months of age, when A-ZIP/F-1 mice exhibit diabetic Tokyo, Japan). The signal-positive areas were measured using
nephropathy with massive proteinuria.18–20 Other animal experi- MetaMorph 7.5 software (Molecular Devices, Downingtown, PA, USA).
ments were carried out with male C57BL/6J mice (Japan Clea,
Tokyo, Japan) starting at 8 weeks of age, when they weighted
Patients and measurement of human Ngal
21–23 g. Diabetes was induced by intraperitoneal injection of STZ
Patients who admitted to Kyoto University Hospital for the
(180 mg/kg of body weight; Sigma, St. Louis, MO, USA) in citrate
diagnosis and treatment of renal disorders were enrolled under
buffer (pH 4.6) and control mice received only citrate buffer. Blood
informed consent. This study was approved by the ethical
pressure was measured by the indirect tail-cuff method with MK-
committee on human research of Kyoto University Graduate School
2000ST (Muromachi Kikai, Tokyo, Japan). Urine samples were
of Medicine. Ngal concentrations in the human serum and urine
collected with metabolic cages. Urinary albumin was measured with
were determined by sandwich ELISA (AntibodyShop, Gentofte,
murine albumin ELISA (Exocell, Philadelphia, PA, USA). Serum and
Denmark) usually after 1000- and 250-fold dilution, respectively.
urinary creatinine levels were assayed by the enzymatic method
(SRL, Tokyo, Japan). This method gives reliable measurement when
compared to high-performance liquid chromatography method, Western blot analysis
even in low concentration materials, and performs much better than Urine, serum, and proteins extracted from organs were separated by
Jaffe’s colorimetric method.37 Blood glucose and HbA1c levels were SDS–polyacrylamide gel electrophoresis, transferred onto polyviny-
determined in tail vein blood at ad libitum-fed conditions using lidene difluoride membranes, incubated with primary antibody and
Glutest Ace (Sanwa Kagaku, Nagoya, Japan) and DCA2000 þ detected with peroxidase-conjugated secondary antibody and

292 Kidney International (2009) 75, 285–294

T Kuwabara et al.: Source and changes of urinary Ngal in renal injury original article

chemiluminescence. Serum was passed through 100-kDa cutoff Metabolic Disorders, Kanae Foundation for the Promotion of Medical
membrane (Microcon YM-100; Millipore, Bedford, MA, USA) to Science, Kurozumi Medical Foundation, Takeda Science Foundation,
remove immunoglobulins before analysis.2 The amount of Ngal Smoking Research Foundation, Salt Science Research Foundation,
protein was measured by densitometry. Known amounts of and by Grant-in-Aid for Scientific Research of Japan Society for the
Promotion of Science.
recombinant mouse Ngal protein were used as standards.2

Real-time reverse transcription PCR SUPPLEMENTARY MATERIAL

Total RNA was extracted from mouse kidneys and livers with TRIzol Figure S1. Clinical course of cases with (a) lupus nephritis (case 3, LN)
reagent (Invitrogen, Carlsbad, CA, USA) and cDNA in each sample and (b) crescentic glomerulonephritis (case 4, CrescGN).
was synthesized by High Capacity cDNA Reverse Transcription Kit Figure S2. Low-power fields showing expression of Ngal and
nephron markers in unilateral ureteral obstruction (UUO) and
(Applied Biosystems, Foster City, CA, USA). The mRNA levels of
contralateral kidneys.
Ngal were determined using Premix Ex Taq (Takara Bio, Otsu, Figure S3. High-power fields of cortex showing expression of Ngal
Japan) and ABI Prism 7300 Sequence Detector with the following and nephron markers in UUO and contralateral kidneys.
primers and probe: Ngal forward, 50 -ggcagctttacgatgtacagca-30 ; Ngal Figure S4. High-power fields of medulla showing expression of Ngal
reverse, 50 -tctgatccagtagcgacagcc-30 ; Ngal probe, 50 -FAM-catcctggt- and nephron markers in UUO and contralateral kidneys.
cagggaccaggaccag-TAMRA-30 . Expression levels of Ngal were Figure S5. Clinical course of a case with drug-induced interstitial
normalized by glyceraldehyde-3-phosphate dehydrogenase (internal nephritis (case 5, IntN).
control) levels, whose primer–probe set was purchased from Applied Supplementary material is linked to the online version of the paper at
Biosystems.41 Standard curve was made by serial dilution of cDNA http://www.nature.com/ki
from UUO kidneys.
REFERENCES
1. Mori K, Nakao K. Neutrophil gelatinase-associated lipocalin as the real-
Immunofluorescence of Ngal and albumin time indicator of active kidney damage. Kidney Int 2007; 71: 967–970.
Snap-frozen human biopsy samples were sliced at 3 mm thickness, 2. Mori K, Lee HT, Rapoport D et al. Endocytic delivery of
fixed with acetone, and incubated with a solution containing both lipocalin–siderophore–iron complex rescues the kidney from
fluorescein isothiocyanate-labeled rabbit anti-human albumin anti- ischemia–reperfusion injury. J Clin Invest 2005; 115: 610–621.
3. Barasch J, Mori K. Cell biology: iron thievery. Nature 2004; 432: 811–813.
body (Dako, Glostrup, Denmark) and goat polyclonal anti-human 4. Yang J, Goetz D, Li JY et al. An iron delivery pathway mediated by a
Ngal antibody (R&D Systems), followed by incubation with lipocalin. Mol Cell 2002; 10: 1045–1056.
TexasRed-labeled anti-goat IgG (Jackson Immunoresearch, West 5. Mori K, Yang J, Barasch J. Ureteric bud controls multiple steps in the
Grove, PA, USA). The spillover of each signal was negligible. conversion of mesenchyme to epithelia. Semin Cell Dev Biol 2003; 14:
209–216.
6. Flo TH, Smith KD, Sato S et al. Lipocalin 2 mediates an innate immune
Immunohistochemistry of Ngal and nephron markers response to bacterial infection by sequestrating iron. Nature 2004; 432:
Mouse kidneys were fixed in 4% paraformaldehyde at 4 1C for 12 h 917–921.
7. Schmidt-Ott KM, Mori K, Li JY et al. Dual action of neutrophil gelatinase-
and embedded in paraffin. Renal sections of 4 mm were deparaffined, associated lipocalin. J Am Soc Nephrol 2007; 18: 407–413.
hydrated, and incubated with 0.3% hydrogen peroxide. Antigen 8. Mishra J, Dent C, Tarabishi R et al. Neutrophil gelatinase-associated
retrieval was performed by 0.05 mol/l citrate buffer (pH 6.0) for lipocalin (NGAL) as a biomarker for acute renal injury after cardiac
10 min in a water bath heated at 100 1C (for Ngal, AQP1 and AQP2), surgery. Lancet 2005; 365: 1231–1238.
9. Mishra J, Ma Q, Prada A et al. Identification of neutrophil gelatinase-
or in a microwave oven (for THP). After cooling, the sections were associated lipocalin as a novel early urinary biomarker for ischemic renal
incubated with 10% normal donkey or goat serum, followed by goat injury. J Am Soc Nephrol 2003; 14: 2534–2543.
anti-mouse Ngal (1:300; R&D Systems), rabbit anti-AQP1 and 10. Wagener G, Jan M, Kim M et al. Association between increases
AQP2 (1:200; Chemicon International, Temecula, CA, USA), or in urinary neutrophil gelatinase-associated lipocalin and acute renal
dysfunction after adult cardiac surgery. Anesthesiology 2006; 105:
rabbit anti-THP antibodies (1:200; Biomedical Technologies, 485–491.
Stoughton, MA, USA). Primary antibodies were visualized with 11. Bennett M, Dent CL, Ma Q et al. Urine NGAL predicts severity of acute
horseradish peroxidase-conjugated secondary antibodies and 3,3- kidney injury after cardiac surgery: a prospective study. Clin J Am Soc
diaminobenzidine tetrahydrochloride. Nuclei were counterstained Nephrol 2008; 3: 665–673.
12. Devarajan P. Emerging biomarkers of acute kidney injury. Contrib Nephrol
with hematoxylin. 2007; 156: 203–212.
13. Coca SG, Yalavarthy R, Concato J et al. Biomarkers for the diagnosis and
risk stratification of acute kidney injury: a systematic review. Kidney Int
Statistical analysis
2008; 73: 1008–1016.
Results are expressed as mean±s.e. Student’s t-test was carried out 14. Waikar SS, Bonventre JV. Biomarkers for the diagnosis of acute kidney
to compare two groups. Statistical significance was defined as injury. Curr Opin Nephrol Hypertens 2007; 16: 557–564.
Po0.05. 15. Nickolas TL, O’Rourke MJ, Yang J et al. Sensitivity and specificity of a
single emergency department measurement of urinary neutrophil
gelatinase-associated lipocalin for diagnosing acute kidney injury. Ann
DISCLOSURE Intern Med 2008; 148: 810–819.
All the authors declared no competing interests. 16. Ding H, He Y, Li K et al. Urinary neutrophil gelatinase-associated lipocalin
(NGAL) is an early biomarker for renal tubulointerstitial injury in IgA
nephropathy. Clin Immunol 2007; 123: 227–234.
ACKNOWLEDGMENTS 17. Mitsnefes MM, Kathman TS, Mishra J et al. Serum neutrophil
The authors are grateful to Dr O. Gavrilova and Dr M.L. Reitman gelatinase-associated lipocalin as a marker of renal function in
children with chronic kidney disease. Pediatr Nephrol 2007; 22:
(Diabetes Branch, NIDDKD, National Institutes of Health, Bethesda,
101–108.
MD, USA) for kindly providing A-ZIP/F-1 mice. We also acknowledge 18. Suganami T, Mukoyama M, Mori K et al. Prevention and reversal of renal
Ms Y. Ogawa, A. Yamamoto and other lab members for assistance. injury by leptin in a new mouse model of diabetic nephropathy. FASEB J
This work was supported by Astellas Foundation for Research on 2005; 19: 127–129.

Kidney International (2009) 75, 285–294 293

original article T Kuwabara et al.: Source and changes of urinary Ngal in renal injury

19. Moitra J, Mason MM, Olive M et al. Life without white fat: a transgenic 31. Onozato ML, Tojo A, Goto A et al. Oxidative stress and nitric oxide
mouse. Genes Dev 1998; 12: 3168–3181. synthase in rat diabetic nephropathy: effects of ACEI and ARB. Kidney Int
20. Ebihara K, Ogawa Y, Masuzaki H et al. Transgenic overexpression of leptin 2002; 61: 186–194.
rescues insulin resistance and diabetes in a mouse model of lipoatrophic 32. Zatz R, Dunn BR, Meyer TW et al. Prevention of diabetic glomerulopathy
diabetes. Diabetes 2001; 50: 1440–1448. by pharmacological amelioration of glomerular capillary hypertension.
21. Kjeldsen L, Johnsen AH, Sengeløv H et al. Isolation and primary structure J Clin Invest 1986; 77: 1925–1930.
of NGAL, a novel protein associated with human neutrophil gelatinase. J 33. Breyer MD, Böttinger E, Brosius III FC et al. Mouse models of diabetic
Biol Chem 1993; 268: 10425–10432. nephropathy. J Am Soc Nephrol 2005; 16: 27–45.
22. Brenner BM, Cooper ME, de Zeeuw D et al. RENAAL Study Investigators. 34. Ichinose K, Maeshima Y, Yamamoto Y et al. Antiangiogenic
Effects of losartan on renal and cardiovascular outcomes in patients with endostatin peptide ameliorates renal alterations in the early stage
type 2 diabetes and nephropathy. N Engl J Med 2001; 345: 861–869. of a type 1 diabetic nephropathy model. Diabetes 2005; 54:
23. Bolignano D, Coppolino G, Campo S et al. Urinary neutrophil gelatinase- 2891–2903.
associated lipocalin (NGAL) is associated with severity of renal disease in 35. Yan QW, Yang Q, Mody N et al. The adipokine lipocalin 2 is regulated
proteinuric patients. Nephrol Dial Transplant 2008; 23: 414–416. by obesity and promotes insulin resistance. Diabetes 2007; 56:
24. Tucker BJ, Rasch R, Blantz RC. Glomerular filtration and tubular 2533–2540.
reabsorption of albumin in preproteinuric and proteinuric diabetic rats. 36. Hvidberg V, Jacobsen C, Strong RK et al. The endocytic receptor megalin
J Clin Invest 1993; 92: 686–694. binds the iron transporting neutrophil-gelatinase-associated lipocalin
25. Tojo A, Onozato ML, Kurihara H et al. Angiotensin II blockade restores with high affinity and mediates its cellular uptake. FEBS Lett 2005; 579:
albumin reabsorption in the proximal tubules of diabetic rats. Hypertens 773–777.
Res 2003; 26: 413–419. 37. Keppler A, Gretz N, Schmidt R et al. Plasma creatinine determination in
26. Birn H, Christensen EI. Renal albumin absorption in physiology and mice and rats: an enzymatic method compares favorably with a high-
pathology. Kidney Int 2006; 69: 440–449. performance liquid chromatography assay. Kidney Int 2007; 71: 74–78.
27. Osicka TM, Houlihan CA, Chan JG et al. Albuminuria in patients with type 38. Kosugi R, Shioi T, Watanabe-Maeda K et al. Angiotensin II receptor
1 diabetes is directly linked to changes in the lysosome-mediated antagonist attenuates expression of aging markers in diabetic mouse
degradation of albumin during renal passage. Diabetes 2000; 49: heart. Circ J 2006; 70: 482–488.
1579–1584. 39. Shao J, Iwashita N, Ikeda F et al. Beneficial effects of candesartan, an
28. Chouinard S, Viau C. Reversibility of renal tubular dysfunction in angiotensin II type 1 receptor blocker, on beta-cell function and
streptozotocin-induced diabetes in the rat. Can J Physiol Pharmacol 1992; 70: morphology in db/db mice. Biochem Biophys Res Commun 2006; 344:
977–982. 1224–1233.
29. Brezniceanu ML, Liu F, Wei CC et al. Attenuation of interstitial fibrosis and 40. Nagae T, Mori K, Mukoyama M et al. Adrenomedullin inhibits connective
tubular apoptosis in db/db transgenic mice overexpressing catalase in tissue growth factor expression, extracellular signal-regulated kinase
renal proximal tubular cells. Diabetes 2008; 57: 451–459. activation and renal fibrosis. Kidney Int 2008; 74: 70–80.
30. Manotham K, Tanaka T, Matsumoto M et al. Evidence of tubular hypoxia 41. Yokoi H, Mukoyama M, Mori K et al. Overexpression of connective tissue
in the early phase in the remnant kidney model. J Am Soc Nephrol 2004; growth factor in podocytes deteriorates diabetic nephropathy in mice.
15: 1277–1288. Kidney Int 2008; 73: 446–455.

294 Kidney International (2009) 75, 285–294