610 The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 3 March 2005
Endocytic delivery of lipocalin-siderophore-
iron complex rescues the kidney from
Kiyoshi Mori,1 H. Thomas Lee,2 Dana Rapoport,1 Ian R. Drexler,1 Kirk Foster,3 Jun Yang,1
Kai M. Schmidt-Ott,1 Xia Chen,1 Jau Yi Li,1 Stacey Weiss,1 Jaya Mishra,4 Faisal H. Cheema,5
Glenn Markowitz,3 Takayoshi Suganami,6 Kazutomo Sawai,6 Masashi Mukoyama,6
Cheryl Kunis,1 Vivette D’Agati,3 Prasad Devarajan,4 and Jonathan Barasch1
1Department of Medicine, 2Department of Anesthesiology, and 3Department of Pathology, College of Physicians and Surgeons, Columbia University,
New York, New York, USA. 4Department of Pediatrics, Nephrology and Hypertension, Cincinnati Children’s Hospital, Cincinnati, Ohio, USA.
5Department of Surgery, College of Physicians and Surgeons, Columbia University, New York, New York, USA.
6Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, Japan.
Neutrophil gelatinase–associated lipocalin (Ngal), also known as siderocalin, forms a complex with iron-
binding siderophores (Ngal:siderophore:Fe). This complex converts renal progenitors into epithelial tubules.
In this study, we tested the hypothesis that Ngal:siderophore:Fe protects adult kidney epithelial cells or
accelerates their recovery from damage. Using a mouse model of severe renal failure, ischemia-reperfusion
injury, we show that a single dose of Ngal (10 μg), introduced during the initial phase of the disease, dramatically
protects the kidney and mitigates azotemia. Ngal activity depends on delivery of the protein and its siderophore
to the proximal tubule. Iron must also be delivered, since blockade of the siderophore with gallium inhibits
the rescue from ischemia. The Ngal:siderophore:Fe complex upregulates heme oxygenase-1, a protective
enzyme, preserves proximal tubule N-cadherin, and inhibits cell death. Because mouse urine contains an Ngal-
dependent siderophore-like activity, endogenous Ngal might also play a protective role. Indeed, Ngal is highly
accumulated in the human kidney cortical tubules and in the blood and urine after nephrotoxic and ischemic
injury. We reveal what we believe to be a novel pathway of iron traffic that is activated in human and mouse
renal diseases, and it provides a unique method for their treatment.
Acute tubular necrosis (ATN) is a syndrome characterized by loss
of function and death of the proximal tubule of the kidney (1–3).
The syndrome is induced by the accumulation of low–molecular
weight molecules such as pharmaceuticals, by proteins filtered
from the glomerulus, or by hypoxia followed by reperfusion.
A mechanism that may underlie each of these inciting factors is
mislocalized iron. Unbound iron can catalyze the conversion of
H2O2 to OH and OH– (the Haber-Weiss reaction) or form reactive
ferryl or perferryl species (4). These ions mutagenize many types of
molecules, including lipids, nucleotides, and the DNA backbone
(5, 6). Catalytic iron in urine or blood and peroxidized lipids have
been documented in acute renal failure mediated by hemoglobin
and myoglobin (7), chemotherapy (cisplatin, ref. 8; doxorubicin,
ref. 9), ischemia-reperfusion (10, 11), transplant ischemia (12),
and proteinuria induced tubular damage (13). Preloading animals
with iron (14) worsens the disease, and, conversely, chelating iron
with deferoxamine (DFO) (8, 15–19) or bacterial siderophores (20)
blunts the damage. Iron-catalyzed damage is thought to be one of
the earliest events in kidney dysfunction and is likely to be impor-
tant in other organs, including the heart (20) and the liver (21).
Cells acquire iron from carrier proteins (transferrin) or from
cell surface iron transporters (divalent metal transporter) (1, 22).
Intracellular iron is controlled by the actions of the iron-respon-
sive proteins (IRPs 1 and 2) (23–26), the ferritin complex (27–30),
and heme oxygenase-1 (HO-1) (31). Because IRPs are modulated by
hypoxia (32), oxidative stress (33, 34), and phosphorylation (35),
changes in their activity may play an important role in ischemic
disease, particularly by regulating the expression of ferritin (36–38).
However, few other aspects of iron trafficking, storage, or metabo-
lism are known in ischemic cells or in other types of tissue damage,
despite the primacy of catalytic iron in their pathogenesis.
We recently identified a protein that induced the conversion
of rat kidney progenitors into epithelia, tubules, and complete
nephrons. The protein is called neutrophil gelatinase–associated
lipocalin (Ngal) or lipocalin 2 (39), a member of the lipocalin
superfamily. These proteins are composed of 8 β-strands that form
a β-barrel enclosing a calyx (40). The calyx binds and transports
low–molecular weight chemicals. The best evidence for Ngal’s
ligand comes from crystallographic studies that demonstrated a
bacterial siderophore (enterochelin) in the calyx (41). Ngal binds
the siderophore with high affinity (0.4 nM), and the siderophore
traps iron with high affinity (10–49 M) (42). The stoichiometry of
protein:siderophore:Fe is 1:1:1, as demonstrated by binding stud-
ies and x-ray crystallography (41). When the siderophore was load-
ed with iron, the Ngal complex could donate iron to cell lines and
to embryonic mesenchyme in vitro, and when the siderophore was
iron free, the Ngal complex could chelate iron (39, 43). In many
types of cells, Ngal trafficked to a late endosomal compartment
Nonstandard abbreviations used: ATN, acute tubular necrosis; DFO, deferoxamine;
HO-1, heme oxygenase-1; IRE, iron-responsive element; Ngal, neutrophil gelatinase-
associated lipocalin; RBP, retinol-binding protein.
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J. Clin. Invest. 115:610–621 (2005).
The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 3 March 2005
that differed from the transferrin compartment (39). Because Ngal
is the first mammalian protein found to bind and transport a bac-
terial siderophore, it has been renamed siderocalin (41).
The current work was prompted by our recent finding that
Ngal is one of the most highly expressed messages and proteins
in the mouse kidney after ischemia-reperfusion injury (ATN) (44,
45). Here we find that Ngal protein accumulates in the proximal
tubule of human kidneys after ischemic and toxic ATN, just as in
the mouse. To determine whether Ngal is protective, we injected
mice with microgram quantities of the protein and found dramat-
ic preservation of kidney histology, as well as normalized serum
creatinine. The mechanism of protection required the delivery of
siderophore:Fe to the proximal tubule.
Expression of Ngal in ATN of the human. Acute renal failure in humans
was marked by log-order elevations in the concentration of serum
and urinary Ngal protein. Compared with the Ngal concentration in
normal serum (21 ng/ml geometric mean; n = 5) and normal urine
(22 ng/ml; n = 10), serum Ngal was elevated 7.3-fold (146 ng/ml;
P < 0.05; Figure 1) and urinary Ngal was elevated 25-fold (557 ng/ml;
P < 0.001) in our patients with ATN, the most typical form of acute
renal failure. Patients with ATN associated with bacterial infection
tended to have the highest levels of serum Ngal (331 ng/ml) and uri-
nary Ngal (2,786 ng/ml), but this was not statistically different from
ATN without infection. To determine whether Ngal expression cor-
related with the extent of acute renal impairment, we used simple
regression analysis after log transformation of Ngal levels. We found
that both serum Ngal (r = 0.64, n = 32) and urinary Ngal (r = 0.68,
n = 38), as well as urinary Ngal normalized for urinary creatinine
(r = 0.67, n = 36), were highly correlated with serum creatinine levels
(P < 0.0001 each). In comparison, patients with chronic renal failure
had less prominent elevations in serum Ngal (49 ng/ml; n = 10)
and urinary Ngal (119 ng/ml; n = 9), and these values were not pro-
portional to serum creatinine. These data correlate Ngal expression
with acute kidney damage, implicating the kidney as the major
source of serum and urinary Ngal. Indeed, in several cases of severe
renal failure, the fractional excretion of Ngal (the clearance of Ngal,
Ngal expression in ATN of human (A–D) and mouse (E). (A) Monoclonal anti–human Ngal (Mo) and polyclonal anti–mouse Ngal (Po) antibodies
recognized recombinant (21-kDa) and native (25-kDa) human and mouse Ngal. Occasionally, higher–molecular weight species (approximately
35 kDa and 66 kDa) were present in recombinant and native preparations; these might represent dimers and trimers of Ngal. A standard curve
was constructed with 25, 5, 1, and 0.2 ng recombinant proteins on nonreducing gels. Human urine samples (0.1–20 μl) from patients with ATN
showed high levels of Ngal, whereas samples from patients with chronic renal failure (CRF), patients with liver cirrhosis, hemochromatosis, or
pancreatic carcinoma but lacking a renal diagnosis (Others), or normal subjects (Normal) had low levels of Ngal. (B) Similar data were obtained
from human serum. (C and D) Geometric means (bar ± SD) of urinary (C) and serum (D) Ngal were compared in normal, CRF, and ATN groups.
ATN was further divided into sepsis and nonsepsis. *P < 0.05, **P < 0.01, ***P < 0.001 vs. normal. (E) Mouse ATN was also associated with
elevated urinary Ngal. The renal pedicle was cross-clamped for 30 minutes, and urine was collected at 24 hours of reperfusion and analyzed
by immunoblot (5 μl/lane).
612 The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 3 March 2005
normalized for the clearance of creatinine) was greater than 100%,
demonstrating that urinary Ngal derived from local synthesis, rath-
er than only from filtration from the blood.
To visualize Ngal protein, we immunostained human samples
with affinity-purified anti-Ngal polyclonal antibody (Figure 2). The
normal kidney demonstrated weak staining in the distal tubular
epithelia (mean 10% of cortical area) and in the medullary collect-
ing ducts, implicating these tubules as the site of synthesis in the
normal kidney. Only rare staining of glomerular parietal epithelial
cells, but not other glomerular cells, was also identified. Proximal
tubules were entirely negative in normal kidneys. In contrast, near-
ly 50% of cortical tubules, including proximal tubules, were Ngal+
in ischemic or nephrotoxin-damaged kidneys (Figure 2). Prolifera-
tive glomerulopathies also generated Ngal+ cortical tubules, but
the intensity of staining and the percentage of the cortical paren-
chyma that were affected were much less than in ischemic disease
(Ngal+ cortical parenchyma: 20% in minimal-change disease, 40%
in diabetic nephropathy, 50% in anti-neutrophil cytoplasmic anti-
body–associated glomerulonephritis, and 65% in anti–glomerular
basement membrane disease); in these cases Ngal perhaps reflects
a mild degree of tubular damage by proteinuria or hemodynamic
change. Those tubular cells displaying the most obvious features of
cell injury, including simplification and enlarged reparative nuclei
with prominent nucleoli, had the most intense staining. Tubular
cells with less derangement had much less staining. These data
demonstrate de novo and widespread Ngal reactivity in cortical
tubules of different renal diseases, suggesting that Ngal is a com-
mon and sensitive response to tubular injury.
Exogenous Ngal rescues the mouse proximal tubule from ATN. To exam-
ine the functional significance of Ngal expression in renal isch-
emia, we first reproduced a common model of renal damage (44,
46) but used mice. The renal pedicle was clamped for 30 minutes,
and the contralateral kidney was removed. Twenty-four hours after
reperfusion, the plasma creatinine rose from 0.41 ± 0.10 mg/dl
(n = 4) to 3.16 ± 0.17 mg/dl (n = 8, P < 0.001), and Ngal message and
protein were intensely expressed. Ngal message rose approximately
1,000-fold, reducing the threshold for detection by real-time RT-
PCR from 17.7 ± 0.9 cycles in sham-operated kidneys to 7.5 ± 0.4
cycles in ischemic kidneys (normalized to β-actin, ΔCT; n = 4 each,
P < 0.0001). Ngal protein rose 1,000-fold in the urine (40 μg/ml in
ATN compared with 40 ng/ml in the sham-operated and normal
mouse; Figure 1E) and 300-fold in the blood (30 μg/ml in ATN
compared with 100 ng/ml in the sham-operated mouse) and was
elevated close to 100-fold in kidney extracts (73 ± 7 μg/g compared
with <1 μg/g kidney wet weight in sham-operated kidneys; n = 3
each, P < 0.05). Renal Ngal protein correlated well with the duration
Prior work showed that Ngal appeared in the urine a number of
hours after ischemia-reperfusion injury (44). To determine whether
Ngal protein was protective, we introduced Ngal systemically (1–300
μg by s.c. or i.p. injection) in the early stages of ATN. Introduction of
100 μg Ngal 15 minutes before clamping blocked the rise in plasma
creatinine measured 24 hours after reperfusion (1.18 ± 0.18 mg/dl
in Ngal-treated mice, n = 7, compared with 3.16 ± 0.17 mg/dl in
untreated animals, n = 8; P < 0.001). Similar data were obtained for
dosages ranging from 10 to 300 μg of Ngal, but 1 μg Ngal was not
protective (creatinine 3.09 ± 0.11 mg/dl; n = 3). Introduction of Ngal
1 hour after reperfusion also blocked the azotemia (creatinine
1.60 ± 0.28 mg/dl; n = 3, P < 0.001), but to a lesser degree than pre-
treatment with Ngal. In contrast to these studies, treatment with
Ngal 2 hours after ischemia had no protective effect (creatinine
3.12 ± 0.35 mg/dl; n = 3). The data were confirmed by measure-
ment of the blood urea nitrogen (data not shown).
The protective activity of Ngal was equally measurable by histol-
ogy (Figure 3): rather than necrotic tubules and luminal debris,
normal epithelial morphology was preserved in the S1 and S2 seg-
ments of the proximal tubule. The S3 segment in the outer stripe
of the outer medulla, however, was less protected by injection of
Ngal, but tubular casts were also less evident at this site (Figure
3B). These observations were supported by scoring of the sections
on the Jablonski scale (47) (Figure 3C).
Correlates of ischemia-reperfusion injury. Because the trafficking and
metabolism of the cadherins are rapidly affected by ischemia (48),
and because Ngal acts as an inducer of E-cadherin in rat embryonic
metanephric mesenchyme (39), we hypothesized that Ngal rescues
Ngal is expressed in cortical tubules in human acute renal failure. Ngal
was detected with affinity-purified polyclonal antibody. (A) The nor-
mal kidney had little staining for Ngal. (B and C) At high power, focal
staining of distal tubule cells (occupying 10% of the cortical area) and
collecting ducts was found. There was no staining of proximal tubules.
(D–I) Ischemic ATN caused by sepsis (D), by hypovolemia due to
vomiting and diarrhea (E), or by heart failure (F), or nephrotoxic ATN
caused by bisphosphonate (G), by cephalosporin (H), or by hemo-
globinuria (I), produced intense staining of nearly 50% of the cortical
tubules. Staining was heterogeneous and most intense in epithelial
cells that displayed histologic features of cell injury, including simplifi-
cation and enlarged reparative nuclei and prominent nucleoli. (J and
K) In glomerular disease, Ngal was weakly expressed by crescents (J)
and the proximal tubules of nephrosis (K). Scale bars: A, D, G, and K,
11 μm; B, C, E, F, and H–J, 5 μm.
The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 3 March 2005
cadherin expression in the ischemic kidney. To test this hypothesis,
we first confirmed that, whereas E-cadherin could be detected at
a low level in the mouse proximal tubule by immunofluorescence,
N-cadherin was specific to the proximal tubule and appeared to be
its major cadherin (46) (Figure 4). N-cadherin is known to be pro-
cessed by caspases, by γ-secretase, and by MMPs, which generate
30- to 40-kDa cytoplasmic fragments that are potentially impor-
tant signaling molecules that modulate CREB signaling (49). We
found that, after ischemia-reperfusion, N-cadherin was degraded
to fragments (Figure 4, A and B). In some animals degradation of
the protein could be detected within 6 hours of reperfusion, and
by 24 hours, both N-cadherin immunofluorescence and expression
of the full-length protein were nearly abolished. In contrast, pre-
treatment with Ngal preserved N-cadherin immunofluorescence,
enhanced the expression of full-length N-cadherin, and reduced
the appearance of its fragment at 6 hours (in some animals) or 24
hours of reperfusion. E-cadherin, on the other hand, was highly
expressed in the distal tubule and collecting duct and was much
less affected by ischemia and by Ngal treatment. Similarly, metal-
induced nephrotoxic ATN triggered the degradation of N-cadherin
but not E-cadherin (50, 51). Perhaps Ngal directly modulates
N-cadherin processing, but an indirect effect subsequent to the
preservation of tubular morphology by Ngal is more likely.
Because the expression of Ngal correlates with ischemic damage
(44), we examined the expression of endogenous Ngal mRNA after
treatment with exogenous Ngal protein. We found that treatment
of ischemic animals with Ngal (100 μg) reduced the increase in
endogenous Ngal RNA by 72% ± 16% (n = 5, P < 0.01) at 24 hours
of reperfusion as measured by real-time RT-PCR. In addition, the
injection of Ngal reduced the appearance of Ngal protein in the
kidney by 60% ± 10% (ischemia 73 ± 7 μg/g, Ngal-treated ischemia
29 ± 7 μg/g; n = 3 each, P < 0.01) as measured by immunoblot.
Because disruption of the proximal cell results in apoptotic cell
death, we next examined the effect of Ngal on cell viability (Figure
4, C and D). Twenty-four hours after reperfusion, we counted the
percentage of tubules with at least 1 TUNEL+ tubular cell. Isch-
emic kidneys showed that 11.5% ± 0.6% (n = 4) of cortical tubules
contained TUNEL+ cells, but after treatment with Ngal, the per-
centage of positive tubules fell to 2.9% ± 0.9% (n = 7, P < 0.001).
For comparison, 0.5% ± 0.3% of cortical tubules had TUNEL+ cells
in sham-operated kidneys. Similarly, we evaluated the uptake of
BrdU, a cell proliferation marker. We counted the percentage of
cortical tubules with at least 1 BrdU+ tubular cell. Twenty-four
hours after the insult, ischemic cortical tubules contained rare
BrdU+ cells (1.9% ± 0.3% of tubules; n = 3), while ischemic kidneys
pretreated with Ngal had a small but significant increase in posi-
tive cells (3.9% ± 0.5% of tubules; n = 4, P < 0.05). For comparison,
3.7% ± 0.7% of cortical tubules had BrdU+ cells in sham-operated
kidneys. Hence, rescue by Ngal reduced apoptosis of cortical cells
and either stimulated compensatory tubular cell proliferation or
else preserved tubular cells with proliferation potential.
Mechanism of rescue from ATN: Ngal targets the proximal tubule. To
determine the mechanism by which Ngal protects the proximal
tubule from ischemic damage, we first studied the distribution of
exogenous Ngal after an i.p. or s.c. injection. Ngal was found in
the urine within 10 minutes of injection (10 or 100 μg), suggest-
ing that the protein was rapidly cleared by the kidney (Figure 5).
However, only 0.1–0.2% of the injected Ngal was recovered in the
urine in the first hour. To better follow trafficking, we used fluo-
rescent conjugates of Ngal. Both fluorescein-labeled and Alexa-
labeled Ngal localized to large vesicles in the subapical domain of
the cortical proximal tubule (S1 and S2 segments of the nephron)
by 1 hour (Figure 6), but not to other segments of the tubule. To
determine whether these organelles were lysosomes, we labeled
proximal tubular lysosomes with fluorescein-dextran (43 kDa) the
day before administration of Alexa 568–Ngal (52). One hour after
injection of Ngal, 33% of the Ngal vesicles also contained dextran
(Figure 6B). In addition, many of these vesicles costained with the
lysosomal marker LAMP1 (data not shown). We obtained similar
data by injecting 125I-Ngal (Figure 6C), which showed that the full-
Rescue of mouse ATN by Ngal. (A) Holo-Ngal (100 μg) was injected
into the peritoneum 15 minutes before renal pedicle cross-clamp and
30 minutes of ischemia. Kidneys were harvested after 24 hours of
reperfusion for H&E staining. The ischemic kidneys (ATN) demonstrat-
ed loss of tubular nuclei (ATN, bottom) as well as the presence of corti-
cal and medullary intratubular casts (ATN, middle). In contrast, Ngal
pretreatment resulted in preservation of cortical tubules (ATN+Ngal,
bottom) and reduced cortical-medullary casts (ATN+Ngal, middle).
(B) PAS staining highlighted the luminal casts in the ischemic kidney
(ATN) as well as the rescue of cortical tubules by pretreatment with
Ngal (ATN+Ngal). (C) Area of proximal convoluted tubular necrosis
was evaluated by the Jablonski scale, which demonstrated rescue of
the ischemic cortex by Ngal (0: no necrosis; 1: isolated necrotic cells;
2: focal necrosis in inner cortex; 3: diffuse necrosis in inner cortex; 4:
necrosis involving whole cortex). *P < 0.05, **P < 0.01 vs. untreated
ischemic kidneys. Scale bars: A, top row, 800 μm; middle row, 24 μm;
bottom row, 11 μm; B, top row, 800 μm; bottom row, 24 μm.
614 The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 3 March 2005
length protein was rapidly cleared from the blood and located in
the kidney by the 1-hour time point (for example, the kidney had
13-fold more 125I-Ngal than the liver, per milligram protein). Near-
ly identical data were previously reported with human Ngal, which
rapidly cleared the circulation (t1/2 = 10 minutes) and located in
the kidney (the kidney had 12-fold more human 125I-Ngal than
the liver, per milligram protein) (53). The kidney-localized protein
was trichloroacetic acid (TCA) precipitable (70%) and was com-
posed of both full-length Ngal and a specific 14-kDa degradation
product. These species persisted and were only slowly lost from
the kidney. In contrast, the plasma and, particularly, the urine con-
tained mostly low–molecular weight, TCA-soluble 125I fragments
(35% and 20% TCA precipitable, respectively; Figure 6).
These data show that full-length Ngal is rapidly cleared by the
proximal tubule, where it traffics to lysosomes and degrades to a
14-kDa fragment. It is likely that the endogenous protein (low levels
of serum Ngal) traffics in a similar manner, because there is very
little urinary Ngal in normal mouse or human urine, despite the fact
that it is freely filtered from the circulation (human: filtered load =
21 ng/ml × GFR, whereas urinary Ngal = 22 ng/ml; mouse: filtered
load = 100 ng/ml × GFR, whereas urinary Ngal = 40 ng/ml).
Rescue of the proximal tubule from ATN requires siderophore:Fe.
X-ray crystallography and atomic absorption as well as biochemi-
cal studies have demonstrated that Ngal cloned in XL1-Blue bac-
teria contains a siderophore called enterochelin, and that the
siderophore carries iron in a 1:1 stoichiometry (41, 42). To deter-
mine whether Ngal can deliver iron to the proximal tubule, we
prepared 55Fe-loaded Ngal by incubating iron-free Ngal:entero-
chelin with 55Fe at a 1:1 stoichiometry (Ngal:enterochelin:55Fe).
One hour after injecting this labeled protein (10 μg i.p.), we
recovered the majority of 55Fe in the kidney (55%), but only trace
amounts in the plasma (4.3%), urine (0.6%), liver (2.4%,), and
spleen (0.2%). To determine the location of the 55Fe in the kidney,
we performed radioautography and found 55Fe in the proximal
tubule, particularly along the apical surface, beneath the brush
border (Figure 6E; Table 1). In contrast, 55Fe was not found in the
medulla (Figure 6D). These data show that both the Ngal protein
and its ligand, iron, can be captured by the proximal tubule when
the complex is given exogenously. It should be noted that the
distribution of Ngal:enterochelin:55Fe was quite different from
the distribution of non–protein-bound 55Fe citrate (where kidney
recovery was only 2.8%; ref. 54).
To determine the role of iron delivery in renal protection, we
compared iron-loaded and iron-free Ngal. Ngal cloned in XL1-
Blue contains enterochelin and is iron loaded, and this form of
Ngal protected the kidney (holo-Ngal; Figure 7A). In contrast,
Ngal cloned in BL21 bacteria does not contain enterochelin
and is not iron loaded (apo-Ngal) (41), and it only partially pro-
tected the kidney; this suggests that the siderophore:Fe was the
critical factor (Figure 7A). To test this hypothesis further, we
Correlates of ATN. Kidneys were harvested 24 hours after
reperfusion. (A) N-cadherin staining was nearly abolished by
ischemia-reperfusion (ATN), but it predominated apical cell-
cell junctions after treatment (100 μg holo-Ngal; ATN+Ngal).
(B) Full-length N-cadherin (130 kDa) was rescued by Ngal.
Note the presence of N-cadherin fragments (28 kDa) in
ischemia-reperfusion and sham-treated animals, but their
suppression in Ngal-treated animals (arrow). In contrast,
there was little change in the level of E-cadherin protein.
GAPDH (38 kDa) was the loading control. (C) Tubules with
TUNEL+ apoptotic cells (green fluorescence in ischemia-
reperfusion injury; I/R) were reduced by pretreatment with
Ngal (I/R+Ngal). TO-PRO-l was the nuclear counterstain
(red) for the same field. (D) Percentage of tubules contain-
ing at least 1 apoptotic nucleus. Ischemia-reperfusion injury
increased the number of positive tubules 22-fold, and Ngal
reduced the activity fourfold. *P < 0.001 vs. I/R. (E) Ngal
upregulated HO-1 (32 kDa) expression in ischemic kidneys.
Kidneys harvested 24 hours after ischemia-reperfusion
(ATN) expressed HO-1, but when animals were treated with
Ngal, HO-1 expression was enhanced (ATN+Ngal). Purified
HO-1 (HO-1) and rat cortex are included for comparison.
GAPDH was the loading control. Scale bars: A and C, 9 μm;
A, inset, 4.6 μm.
Clearance of Ngal. Ngal (100 μg) was introduced into the peritoneum,
and serum (5 μl) and urine (1 μl) samples collected after the time indi-
cated were analyzed by immunoblot.
The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 3 March 2005
reconstituted apo-Ngal (from BL21) with iron-saturated entero-
chelin (red protein, Figure 7B). In contrast to apo-Ngal, Ngal:
enterochelin:Fe enhanced the protection of the kidney and blunt-
ed the rise in serum creatinine. These data suggest that Ngal pro-
vides protection against ischemia by delivering siderophore:Fe
to proximal tubules.
Because the iron-loaded form of Ngal contains 2 ligands (iron
and enterochelin), we wished to identify the effective molecule
by designing an experiment whereby Ngal:enterochelin could be
delivered to the kidney without iron. However, we realized that
iron-free Ngal:enterochelin is not likely to arrive in the proximal
tubule without iron, because of the great avidity of the siderophore
(10–49 M) (41), which allows it to strip iron from transferrin (55).
Furthermore, when we loaded a mouse with 55FeCl and then intro-
duced iron-free Ngal:enterochelin (100 μg/0.05 ml PBS) 5 and 35
minutes after the dose of iron, we found that the recovery of 55Fe at
65 minutes increased 3-fold (percentage initial dose) from the kid-
ney and urine and decreased threefold from the liver and spleen,
compared with that in mice receiving PBS. These data indicate
that Ngal:enterochelin and Ngal:enterochelin:Fe are equivalent
after introduction in vivo, supporting the view that Ngal:sidero-
phore acts as an iron delivery protein. Indeed, we found that the
iron-free form of Ngal:enterochelin (white protein, Figure 7B) was
as effective in vivo as the iron-loaded form (Figure 7A).
To test the role of Ngal ligands further, we prepared gallium-
complexed Ngal. Because gallium is a metal+3 that occupies iron-
binding sites with high affinity, including enterochelin, but can-
not undergo redox reactions typical of iron, gallium acts as an
iron antagonist. In contrast to the iron complex, mice treated 15
minutes before ischemia with Ngal:enterochelin:gallium were not
protected (creatinine 3.17 ± 0.1 mg/dl; n = 4; Figure 7A). These data
suggest that not only does Ngal transport iron, but iron transport
is necessary for its activity.
It should be noted that protein delivery to the proximal tubule
itself is not likely to be the mechanism of protection, because a
second lipocalin, retinol-loaded retinol-binding protein (RBP),
which is also captured by the proximal tubule and degraded in
lysosomes, was ineffective (Figure 7A). Furthermore, free entero-
chelin and the iron chelator DFO were also ineffective compared
with Ngal:enterochelin, even at higher molar doses.
Clearance of Ngal by the proximal tubule. (A)
Fluorescent Ngal (100 μg, labeled with Alexa
568) was introduced into the peritoneum,
and after 1 hour the kidney was harvested
and sectioned. Fluorescent Ngal was local-
ized to large vesicles in the proximal tubule
(bottom panel) but not in the glomerulus (G)
or medulla (small top panels). Uncoupled
dye did not label the kidney (Alexa 568
only). (B) Alexa 568–Ngal colocalized with
FITC-dextran in S1 and S2 segments of the
proximal tubule. Dextran was introduced into
the animal 1 day before the Ngal injection in
order to label lysosomes. (C) 125I-Ngal was
introduced into mice, and the samples were
assayed by SDS-PAGE. Lanes were load-
ed with 1,000 cpm each. The lane marked
“Ngal” shows the initial preparation of
125I-Ngal. Subsequent lanes of urine or blood
show loss of signal, suggesting degradation
to small fragments. However, full-length Ngal
and a 14-kDa fragment of Ngal were found
in the kidney 1 and 5 hours after injection.
(D and E) Radioautograph of kidney 1 hour
after i.p. injection of 55Fe-loaded Ngal:sid-
erophore. Radioactivity was not found in the
medulla (D). In contrast, radioactive decay
was found in the cortex and was associated
with the apical zones of proximal tubule cells
(E). Original magnification, A and B, ×40; A,
inset, ×10. Scale bar: D and E, 2 μm.
Subcellular localization of iron in the kidney cortex 1 hour after
i.p. injection of Ngal:siderophore:55Fe complex
χ2 = 21.2, P = 0.0017
ATotal silver grains, 2,601; Btotal point count, 999; Cpercent grains/
percent point count.
616 The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 3 March 2005
Ngal upregulates HO-1 in ATN. A number of studies have identi-
fied HO-1 as a critical regulator of the proximal tubule viability
in renal ischemia. HO is necessary for recovery from ATN (56–58),
and its level of expression is directly correlated with the rescue of
tissue damage. We found that ischemia-reperfusion enhanced the
expression of HO-1, but when mice were treated with Ngal (10–100
μg), the enzyme was further upregulated 5- to 10-fold by 24 hours
after reperfusion (Figure 4E). To determine whether holo-Ngal
itself, or holo-Ngal in the setting of renal ischemia, induced HO-1,
we injected normal mice with increasing doses of Ngal and found
upregulation of the protein. However, the expression of HO-1 after
Ngal injection was much less than in Ngal-treated ischemic kidneys,
which indicates that Ngal synergizes with other activators of HO-1.
To determine whether HO-1 activity was required for Ngal activ-
ity, we used the well-known inhibitor zinc protoporphyrin IX (see
Methods). We found that, while Ngal:enterochelin:Fe (40 μg) pro-
tected the kidney from ischemia-reperfusion damage (creatinine =
0.91 ± 0.16 mg/dl; n = 3), injection of the HO inhibitor blocked the
effect (creatinine = 2.74 ± 0.45 mg/dl; n = 5, P < 0.05). Hence, HO
activity is required for Ngal-mediated protection.
A urine siderophore? The actions of endogenous Ngal in vivo
might differ from its pharmacological effects, because the criti-
cal siderophore is a bacterial product. Low–molecular weight
factors that transport iron, however, have been suggested by a
variety of studies (59–61). These molecules may include citrate
and related compounds, but also iron-transporting activities that
have a molecular weight in the range of 1,000 Da. To determine
whether a cofactor for Ngal is present in the urine, we mixed apo-
Ngal with urine samples from normal mice. While neither apo-
Ngal diluted in Tris buffer (Figure 8A) nor the low–molecular
weight components of the urine (<3,000 Da) trapped 55Fe above a
10,000-Da cutoff filter (Figure 8B), incubation of Ngal with urine
(<3,000 Da) permitted the retention of 55Fe. The capture of iron
by Ngal was inhibited by 1,000-fold unlabeled iron citrate, and
more powerfully by a 50-fold concentration of the iron-saturated
enterochelin (Figure 8B, Sid:Fe). The capture of iron was satu-
Rescue of ATN by Ngal. (A) Plasma creatinine in mice subjected to 30 minutes of ischemia followed by 24 hours of reperfusion. The first panel
shows that holo-Ngal (≥10 μg) from XL1-Blue bacteria (containing siderophore and iron) rescued renal function when introduced 15 minutes
before ischemia or within 1 hour after ischemia (+ 1 h). However, Ngal was ineffective when administered later (+ 2 h). The second panel
shows that apo-Ngal from BL21 bacteria (siderophore free) was minimally active, but that, when loaded with a siderophore (enterochelin), the
protein was protective: both iron-free (apo-Ngal:Sid) and iron-loaded siderophores (apo-Ngal:Sid:Fe) had a protective effect. In comparison,
the gallium-loaded complex (apo-Ngal:Sid:gallium) was ineffective, as was a single dose of the iron chelator DFO or the free siderophore
(Sid). Retinol-binding protein (RBP), a lipocalin that is also filtered and reabsorbed by the proximal tubule, was ineffective. *P < 0.001 vs. ATN.
#P < 0.01 vs. apo-Ngal:Sid (10 μg). The numbers in parentheses show the number of animals analyzed. (B) Preparations of Ngal. Ngal:Sid
contains enterochelin, but not iron. Ngal:Sid:Fe contains enterochelin and iron.
Iron-binding cofactor in urine. (A) Buffer was mixed with 55Fe (No
protein, light blue) and with apo-Ngal (dark blue), apo-Ngal plus sid-
erophore (yellow), or apo-Ngal plus siderophore plus unlabeled iron
(pink). The samples were then washed 3 times on a 10-kDa filter, and
small aliquots were measured for retention of radioactivity (Washes
1–3). After 48 hours at 4°C the samples were washed again (Wash
4 + 48 h). Note the retention of 55Fe by apo-Ngal plus siderophore
but not by apo-Ngal alone or apo-Ngal ligated by the iron-saturated
siderophore, which demonstrates that an unsaturated siderophore is
required for retention of 55Fe by Ngal. 55Fe binding to apo-Ngal plus
siderophore was stable for 48 hours. (B) Urine (<3,000 Da) was mixed
with 55Fe and with apo-Ngal, as indicated, and then washed 3 times on
a 10-kDa filter. While urine (<3,000 Da) or apo-Ngal in buffer did not
retain 55Fe on a 10-kDa filter, apo-Ngal plus urine retained 55Fe. 55Fe
retention was blocked by the addition of excess iron citrate (Fe) or of
iron-saturated enterochelin (Sid:Fe).
The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 3 March 2005
rable by increasing doses of urine. These findings suggest that
mouse urine contains a low–molecular weight cofactor that per-
mits Ngal-iron interactions. Because the endogenous factor is
competitive with the bacterial siderophore, which binds the Ngal
calyx with high affinity (0.4 nM) (41), it appears that both bacte-
rial and mammalian factors occupy the same binding pocket of
the lipocalin. However, the chemical structure of the mammalian
factor likely differs from that of enterochelin, since, whereas the
bacterial siderophore was extractable in ethyl acetate, the mam-
malian factor was more polar and remained in the aqueous phase.
Unlike simple salts, the factor was also soluble in methanol and
could be eluted from silica resin with methanol.
We showed that Ngal is a renal lipocalin that is highly accumu-
lated in the human and mouse proximal tubule during ATN. The
synthesis of Ngal protein is markedly upregulated within hours
of ischemia-reperfusion injury (44). However, if Ngal is present-
ed before, or in the early stages of, cell damage, there is rescue of
N-cadherin (a pathway not previously described in renal disease),
enhanced expression of HO-1 (a protective enzyme), and the sup-
pression of endogenous Ngal (a marker of epithelial damage) and
blunting of cell death. The preservation of renal function was due
not to Ngal itself, but to a siderophore bound in the Ngal calyx.
While this cofactor is a bacterial product obtained from cloning
of the protein in bacteria, surely a serendipitous finding, we sug-
gest that mouse urine contains an equivalent factor. Hence Ngal
is a siderophore delivery protein for the proximal tubule, and this
study is one of the first to use a bacterial siderophore in vivo (62).
It is currently unknown how the proximal tubule captures Ngal.
Indeed, an unambiguous identification of receptors for most lipo-
calins is still lacking. Perhaps megalin, which is necessary for rec-
lamation of RBP, is also the Ngal receptor (63). In fact, knockout
of megalin leads to the appearance of Ngal in the urine (E.I. Chris-
tensen and T. Willnow, personal communication; as shown in Sup-
plemental Figure; supplemental material available online with this
article; doi:10.1172/JCI200523056DS1), but these animals were
also, unexpectedly, found to have much higher levels of Ngal mes-
sage in the kidney (64), which suggests that urinary Ngal might
have derived from local synthesis rather than a failure to capture
the filtered load. Despite this ambiguity, we believe that Ngal is
similar to other lipocalins, such as RBP and the α-2u-globulin
lipocalin (65), that enter the cell by a megalin pathway and traffic
to lysosomes (dextran+, LAMP1+) for degradation. These data con-
trast with the trafficking of Ngal in cell lines that do not express
megalin (such as embryonic metanephric mesenchymal cells) and
where the protein escapes degradation (39). Similarly, transferrin
is degraded by a megalin-cubilin–based pathway in the proximal
tubule (66), whereas the protein recycles after endocytosis in cell
lines. Hence it is reasonable to propose that after filtration, Ngal
is captured by megalin, is degraded by the proximal tubule, and is
not recycled. This hypothesis is supported by the observation that
full-length Ngal does not reappear in the blood at delayed time
points after injection.
Members of the lipocalin superfamily are transport proteins
for low–molecular weight hydrophobic chemicals. Well-known
ligands include pheromones (67) and fatty acids, which bind
α-2u-globulins and the major urinary proteins, and retinoids,
which bind RBP (68). Less-known ligands include iron and its
cofactors, but nitrophorins provide a precedent. Insect nitropho-
rins, for example, contain iron-loaded heme groups that trans-
port NO to the site of a bite (69). α1-Microglobulin (70) binds
heme and produces the yellow-brown pigment in the urine. A
wide range of siderophores were recently demonstrated to bind
tear lipocalin (71). Ngal’s ligand is a bacterial siderophore (entero-
chelin) that can transfer iron to cells or chelate iron from cells. We
showed this in vitro by monitoring changes in the level of expres-
sion of genes carrying iron-responsive elements (IREs), such as
ferritin and transferrin receptor 1 (39), and more sensitively by
monitoring changes in the level of expression of reporter con-
structs that contain IREs coupled to fluorescent proteins (43).
In contrast to these responses, we were unable to show activa-
tion or suppression of a variety of signaling pathways measured
with 18 phospho-antibodies (including those for Raf, MEK, ERK,
p38, JNK, and PKC isoforms) in developing kidneys treated with
Ngal for 20 minutes or for 1 hour. Hence Ngal is a siderophore:Fe
transport and delivery protein, and we suggest that this is its pro-
tective mechanism. Indeed, most of the Ngal protein (125I-Ngal
and fluorescent) and its bound 55Fe were delivered to lysosomes
in the S1 and S2 segments of the proximal tubule, but neither
free iron (54) nor free siderophore (which binds serum albumin;
ref. 72) could be delivered. It seems likely that siderophore:Fe is
transferred to the cytoplasm from the complex, since the protein
is degraded in the proximal tubule.
We previously showed that induction of epithelia from mesen-
chyme was enhanced by loading of Ngal with enterochelin and
with iron (43). In the current work we again show that the Ngal:
siderophore complex, rather than apo-Ngal, is the active factor,
implicating the ligands of Ngal, rather than the carrier protein
itself. We argue here that siderophore:Fe delivery rather than iron
chelation is the mechanism of action of Ngal. First, iron-free Ngal:
siderophore probably obtains iron during transit from the peri-
toneum to the proximal tubule, because enterochelin is so avid
for iron (Kd = 10–49 M) (42). Indeed, iron-free enterochelin can
strip iron from transferrin (55), and iron-free Ngal:siderophore
redirects 55Fe from the spleen and the liver (54) and produces a
threefold increase in kidney and urinary 55Fe contents after injec-
tion of iron-free Ngal:siderophore and 55Fe in vivo. Because Ngal
is cleared by glomerular filtration, the shift in 55Fe to the kidney
and to the urine indicates that iron-free Ngal became iron loaded
during transit. Hence iron-loaded and iron-unloaded Ngal:sidero-
phore delivers iron to the proximal tubule.
We also argue that iron chelation is not like the mechanism of
protection by Ngal:siderophore:Fe, because in order to act as an
iron chelator, iron would first have to dissociate from enterochelin,
which, at least in solution, occurs too slowly to be relevant (Figure
8) and, in any case, would not produce a net loss of iron. More-
over, if enterochelin were liberated from Ngal after endocytosis, it
would degrade both spontaneously and by the action of esterases
(73), and the decay products probably cannot cause a net loss of
iron (74). Third, while Ngal:siderophore:Fe was protective, the gal-
lium homologue was inactive, despite the fact that enterochelin:
gallium derivatives (75) and Ngal:enterochelin:gallium traffic to
the kidney and despite the fact that gallium might even exchange
for iron in the siderophore complex (76). Gallium DFO can even
be an effective scavenger of catalytic iron (77) and reduce reactive
oxygen species. Hence Ngal:enterochelin:gallium is ineffective not
because it can never chelate iron, but because it does not deliver
the siderophore:Fe complex. Fourth, the pharmacology of the iron
chelators differs from the actions of Ngal:siderophore. For exam-
618 The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 3 March 2005
ple, in most studies, desferri-DFO (16) is continuously infused and
single doses, unlike Ngal, are not protective. In fact, while DFO
is mostly excreted in the urine, Ngal is recovered by the proximal
tubule, which indicates that the Ngal:siderophore is more likely to
transfer iron to proximal tubule cells rather than remove iron from
these cells. Similarly, iron chelation by apo-transferrin reduced
azotemia in vivo, but unlike Ngal, it did not mitigate apoptosis
(18). Desferri-exochelin (20), another type of apo-siderophore, pro-
tected the heart from ischemic damage in an in vitro model of cor-
onary artery ligation (20), but this has not been shown by systemic
infusion, where the siderophore would be exposed to many sourc-
es of iron. Hence while many studies unequivocally demonstrate
that iron chelation ameliorates ischemia-reperfusion damage, and
that the lipophilicity of siderophores allows tissue penetration,
and perhaps access to membrane-associated iron that generates
hydroxyl radicals (6, 20), siderophore:Fe donation by Ngal, rather
than iron chelation, explains the data.
The delivery of iron could be protective and enhance cell repair
for a number of reasons. Low doses of iron or heme, or other iron
donors (78, 79), induce HO-1 (56, 80). This enzyme is both nec-
essary and sufficient in a dose-dependent fashion to protect epi-
thelia (refs. 56, 81, 82; reviewed in 83) from ischemia (57, 58) and
nephrotoxicity (84). In fact, the enzyme is specifically induced in
the proximal tubule (85) by a large number of insults (56), and it
prevents iron overload in both mouse (86) and human (85) proxi-
mal tubules. HO-1 is thought to protect cells by limiting uptake
and enhancing the release of iron and thus to reduce the cell con-
tent of non–ferritin-bound iron (31). In addition, it synthesizes
antioxidant biliverdin and CO and can induce p21 (reviewed in
refs. 79, 87, 88). Treatment with Ngal:siderophore resulted in
low levels of expression of HO-1 in normal proximal tubules, but
markedly enhanced expression of HO-1 in the ischemic tissues.
We speculate that this induction is the critical pathway by which
Ngal protects the proximal tubule, an idea supported by loss of
Ngal activity after introduction of the HO inhibitor. However, in
addition to HO-1 there are many other iron regulatory pathways
(ferritin) and non-iron pathways (cell replication) that Ngal may
stimulate by delivering iron. For example, iron is necessary for the
R2 subunit of ribonuclease reductase, the enzyme that synthesizes
DNA (89); it enhances the expression of many cyclin genes (90)
and stress-related proteins (91); and it inhibits apoptosis medi-
ated by p38 MAPK phosphorylation (92) and NIP3 (93). Last, it is
even possible that enterochelin:Fe or its degradation products may
scavenge free radicals.
A model of the postischemic kidney might focus on the idea of
“cell shift,” rather than on the pathologic consequences of cata-
lytic iron alone. Transport of iron into viable proximal tubule cells
could supply iron for cell proliferation as well as activate the dis-
posal of excess iron by HO-1 (31), whereas loss of iron from the cell
is proapoptotic. Chelation of extracellular iron, on the other hand,
would reduce free radical formation and hence provide protection
from ischemic damage by a different mechanism at a later point in
the disease process. To test these hypotheses it will be necessary to
detect the effect of the Ngal complex on iron levels in the proximal
tubular cells in vivo (available cell lines have little uptake of Ngal)
and to distinguish between damaged and viable cells. Perhaps
expression of our IRE–yellow fluorescent protein construct (43)
in the proximal tubules of animals could provide real-time moni-
toring of iron in the first hours after Ngal is captured by these cells
and over the course of ischemic disease.
Bacterial siderophore–mediated rescue of the proximal tubule
raises the fundamental question of whether mammalian-expressed
Ngal has the same type of ligand as the recombinant protein. Early
studies from the laboratories of J.A. Fernandez-Pol (59) and A.
Cerami (60) suggested that low–molecular weight iron carriers
were expressed by mammalian cells, but their identification was
not established, nor were carrier proteins like Ngal implicated in
their traffic. However, we found a factor in the mouse urine that
was competitive with the bacterial siderophore. Hence if such a fac-
tor was available in the ischemic kidney, it would suggest that Ngal
mediates autocrine or paracrine iron trafficking. These hypotheses
might be validated, first, by protection of the kidney with Ngal
loaded with the mammalian factor, rather than the bacterial sid-
erophore, and, second, by demonstration that ischemic damage is
prolonged in mice or humans with defective expression of Ngal or
its putative cofactor. Creation of Ngal knockout mice, and knock-
outs specific to kidney Ngal, would be a first step.
Lastly, because Ngal has been suggested to regulate leukocytes
by inducing apoptosis after receptor-mediated signaling (94), it
remains possible that Ngal is a bifunctional molecule and has
actions other than siderophore transport. Perhaps endogenously
expressed Ngal, which peaks hours after ischemia-reperfusion
injury, might reduce the population of invading leukocytes and,
conversely, Ngal-deleted animals have a greater burden of these
cells. However, rather than a proapoptotic action, we could only
document that Ngal reduced, rather than increased, TUNEL stain-
ing in the ischemic kidney. Most importantly, it is improbable that
exogenous Ngal regulates populations of leukocytes, because it
was effective before or in the first hours of ischemia-reperfusion,
and its actions depend on its siderophore:Fe ligand.
In sum, ischemia-reperfusion has been shown to change the
activity of a few components of iron metabolism. We demonstrate
that a siderophore-binding protein is highly overexpressed in
these diseases and that a single systemic administration of holo-
protein, the iron-transporting form of Ngal, mitigates injury.
While liver cells may also accumulate iron in the initial phases
of ischemia reoxygenation by upregulating the transferrin recep-
tor, the proximal tubule may have special mechanisms to acquire
iron, because these cells do not express transferrin receptors (66).
In addition to the previously described apical megalin-transferrin
(66), the current data demonstrate an apical Ngal-iron pathway.
Our data are applicable to the human, where we found Ngal to be
highly expressed in renal diseases.
Patients. We analyzed healthy volunteers and patients diagnosed with either
acute or chronic renal failure. Acute renal failure was diagnosed by a doubling
of the serum creatinine in less than 5 days. The definition of chronic renal fail-
ure was a serum creatinine greater than 2 mg/dl, but unchanged during the
prior 2 months. The presumed etiology of acute renal failure included sepsis,
which was defined by the presence of the following criteria: (a) positive blood
cultures or evidence of local infection in the lung, skin, or urinary tract, and
(b) fever or an elevated white blood cell count. Some of these patients required
blood pressure support. Other etiologies of ATN included hypotension due
to bleeding or heart failure, nephrotoxins, and post-transplantation isch-
emia. The presumed etiologies of chronic renal failure included obstructive
uropathy, chronic interstitial nephritis, and diabetes. Samples of blood and
urine were collected from patients evaluated at Columbia University Medical
Center and at Kyoto University Hospital with approval of both Institutional
Review Boards and then analyzed in a blinded fashion.
The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 3 March 2005
Measurement of Ngal. An anti–mouse Ngal polyclonal antibody was
raised in rabbits, purified on a column of Sepharose 4 Fast Flow beads
(Amersham Biosciences) coupled to recombinant mouse Ngal (see below),
and then eluted at pH 2.5. Monoclonal anti–human Ngal (AntibodyShop
A/S; 1:1,000) was also used to detect Ngal. Human Ngal was better recog-
nized by the mAb, while mouse Ngal was recognized only by the affinity-
purified polyclonal antibody.
Human blood samples were initially collected in citrate, EDTA, or heparin,
but all of these preparations showed similar Ngal immunoreactivity, and
we chose to study human serum and mouse plasma. The samples were
centrifuged through a 100-kDa cutoff filter (Amicon YM-100; Millipore
Corp.), and the flow-through was used for immunoblot. In patients
undergoing hemodialysis, samples were taken immediately before dialysis.
Fresh urine samples were centrifuged at low speed to remove debris and
then used without further concentration.
Pathologic specimens. Pathologic specimens included ischemic ATN (10
cases), toxic ATN (11 cases: 5 caused by antibiotics, 2 by zoledronate [ref.
95], 1 by carboplatinum, 2 by nonsteroidal anti-inflammatory agents, and
1 by hemoglobinuria), and glomerulopathies (10 cases, including dia-
betic nephropathy, anti–glomerular basement membrane disease, pauci-
immune crescentic glomerulonephritis, IgA nephropathy, minimal-change
disease, and focal segmental glomerulosclerosis), and also normal kidneys
(3 cases). Formalin-fixed, paraffin-embedded tissues were sectioned (5 μm)
and subjected to antigen retrieval using microwave in a citrate buffer (pH
6.0) for 30 minutes. Endogenous peroxidase was blocked with 5% H2O2 for
30 minutes, followed by blocking in 10% goat serum/1% BSA. Affinity-puri-
fied anti–mouse Ngal (0.4 μg/ml) was applied overnight at 4°C, followed
by biotinylated goat anti-rabbit IgG (1:100; Vector Laboratories Inc.) and
avidin-HRP, each for 30 minutes. Slides were developed with DAB/0.3%
H2O2 for 2.5 minutes and counterstained with hematoxylin. Nonimmune
rabbit IgG (0.4 μg/ml; Vector Laboratories Inc.) was used as a control.
Recombinant Ngal. Recombinant human and mouse glutathione-S-
transferase–Ngal was expressed in BL21 or XL1-Blue strains of E. coli (Strat-
agene) (39, 41, 96) with additional ferric sulfate (50 μM; Sigma-Aldrich).
Ngal was isolated using Glutathione Sepharose 4B beads (Amersham
Biosciences), eluted by thrombin cleavage (Sigma-Aldrich), and then fur-
ther purified by gel filtration (Superdex 75, SMART system; Amersham
Biosciences) and examined by Coomassie gels (Bio-Rad Laboratories Inc.).
BL21-derived Ngal was loaded with iron-free or iron-saturated enteroche-
lin (0.7 kDa; EMC Microcollections GmbH) using a 5-fold molar excess.
Unbound siderophore was removed by washing in a Microcon YM-10
centrifugal filter (Millipore Corp.) with PBS. To produce 55Fe- or gallium-
loaded Ngal, we incubated the iron-free Ngal:enterochelin complex with
equimolar 55Fe or gallium in 150 mM NaCl/20 mM HEPES (pH 7.4), and
the complex was washed 3 times on a 10-kDa filter. Iodobeads (Pierce) were
used to label Ngal with 125I, and unincorporated 125I was removed by gel
filtration (PD-10 column, Amersham Biosciences) followed by extensive
dialysis (7-kDa cutoff membrane; Pierce) against PBS. Alexa 568 and FITC
(Molecular Probes) were coupled to Ngal, according to the manufactur-
er’s instructions, and then extensively dialyzed. Protein was measured by
Coomassie gels in comparison with BSA standard.
Ngal trafficking. To detect Ngal delivery to the kidney, recombinant Ngal
(10 or 100 μg), Alexa 568–Ngal (100 μg), 125I-Ngal (10 μg, 2 × 106 cpm), or
Ngal:enterochelin:55Fe (10 μg, 1 × 106 cpm) was injected into the peritone-
um, and the blood, urine, kidney, liver, and spleen were obtained. Ngal was
detected by immunoblot. Alexa 568–Ngal was detected by confocal micros-
copy (LSM META detector, Carl Zeiss), and Ngal-mediated iron trafficking
was detected by scintillation counter and by light microscopic radioautog-
raphy of Epon-embedded kidneys. Slides were exposed to emulsion (Poly-
sciences Inc.) for 1 week and then developed with MICRODOL (Eastman
Kodak Co.) and counterstained with toluidine blue. To detect lysosomes in
the proximal tubule, mice were injected with fluorescein-dextran (43 kDa,
0.5 mg; Sigma-Aldrich) 24 hours before Alexa 568–Ngal (100 μg) was intro-
duced. LAMP1 (Santa Cruz Biotechnology Inc.) was detected in cryostat
sections of kidneys fixed in 4% paraformaldehyde.
Mouse ATN. Use of mice was approved by the Institutional Animal Care
and Use Committee of Columbia University. Male C57BL/6 mice (20–25 g;
Charles River Laboratories Inc.) were anesthetized with i.p. pentobarbital
(50 mg/kg) and placed on a heating pad under a warming light to maintain
37°C core body temperature. Kidneys were exposed through an abdominal
section, and the right kidney was removed or its vascular pedicle and ureter
ligated. The vascular pedicle of the left kidney was clamped by a micro-
aneurysm clip (Kent Scientific Corp.) for 30 minutes after right nephrec-
tomy. This period of ischemia generated reproducible renal injury, but it
minimized mortality (97). In our series, we had 11% mortality in untreated
animals, but Ngal:siderophore or Ngal:siderophore:Fe treatment reduced
mortality to 2%. During surgery, PBS (0.5 ml) was used to dampen the
peritoneum, and the animal was then closed with 5-0 nylon. Ngal, reti-
nol-loaded RBP (a kind gift of W.S. Blaner, Columbia University) (98),
enterochelin, or DFO mesylate (Sigma-Aldrich) was injected into the peri-
toneum or s.c. 15 minutes before ischemia or 1–2 hours after reperfusion.
These substances were diluted in 0.05 ml of PBS, whereas control animals
received 0.05 ml of PBS alone. The HO inhibitor zinc protoporphyrin IX
(Sigma-Aldrich) (99) and vehicle (both 10 μmol/kg dissolved in 2 mM
NaOH, neutralized with equimolar HCl, and then diluted to 0.05 ml with
PBS) were introduced into the peritoneum 20 minutes before ischemia,
and also 4 hours after reperfusion.
After 6 or 24 hours of reperfusion, heparinized plasma, urine, and
kidney were obtained to measure Ngal (polyclonal, 1:500), HO-1 (Stress-
Gen Biotechnologies Corp.; 1:2,000), E-cadherin (BD Transduction
Laboratories; BD Biosciences — Pharmingen; 1:2,000), N-cadherin (BD
Transduction Laboratories; BD Biosciences — Pharmingen; 1:3,000), and
GAPDH (Chemicon International Inc.; 1:3,000) using immunoblots.
Plasma was also used for creatinine and blood urea nitrogen colorimetric
assays (Sigma-Aldrich) (100). Sagittal sections of the kidneys were fixed in
4% formalin or were snap-frozen for mRNA and protein analysis. Paraf-
fin-embedded sections (5 μm) were stained with H&E or by an in situ kit
(fluorescein-TUNEL; Roche Applied Science) for apoptotic nuclei or total
nuclei (TO-PRO-l; Molecular Probes). For cell proliferation, BrdU (50 mg/
kg) was injected into the peritoneum 1 hour before sacrifice, and cryostat
sections were stained with anti-BrdU (Roche Applied Science) according to
the manufacturer’s instructions.
Real-time RT-PCR. Total RNA was extracted from mouse kidneys using
RNeasy Mini Kit (QIAGEN Inc.) with on-column DNase digestion accord-
ing to the manufacturer’s instructions. The cDNA template was synthesized
using Omniscript Reverse Transcriptase and oligo-dT primer (QIAGEN
Inc.). The PCR reaction was carried out using iQ SYBR Green Supermix and
MyiQ single-color real-time PCR detection system (Bio-Rad Laboratories
Inc.) with incubation times of 2 minutes at 95°C, followed by 40 cycles of
30 seconds at 95°C and 30 seconds at 60°C. Specificity of the amplification
was checked by melting curve analysis and by agarose gel electrophoresis.
Primer sequences for mouse Ngal mRNA (GenBank NM_008491) were
CTCAGAACTTGATCCCTGCC (forward primer, positions 93–112) and
TCCTTGAGGCCCAGAGACTT (reverse, 576–557). Sequences for mouse
β-actin mRNA (GenBank X03672) were CTAAGGCCAACCGTGAAAAG
(forward, 415–434) and TCTCAGCTGTGGTGGTGAAG (reverse, 696–677).
Each plate included a dilution series of standard samples, which were used
to quantify Ngal mRNA. Values were normalized for β-actin mRNA.
Iron-binding cofactor. Cofactor-dependent iron binding to Ngal was
measured in 150 mM NaCl/20 mM Tris (pH 7.4) buffer (100 μl) with apo-
620 The Journal of Clinical Investigation http://www.jci.org Volume 115 Number 3 March 2005
Ngal (10 μM), 55Fe (1 μM), and a low–molecular weight fraction (<3,000
Da) of mouse urine (0–30 μl). The urine fraction was obtained by passing
of fresh urine sequentially through 10-kDa and 3-kDa membranes (Ami-
con YM-10 and YM-3; Millipore Corp.). After 60 minutes at room tem-
perature, the mixture was then washed 3 times on a 10-kDa membrane
(Amicon YM-10; Millipore Corp.). Ngal loaded with iron-free enterochelin
(rather than apo-Ngal) served as a positive control for iron capture. Ferric
citrate (1 mM) or iron-loaded enterochelin (siderophore:Fe, 50 μM) was
used as a competitor of 55Fe binding.
Statistics. The data were expressed as means ± SEM and analyzed by
1-way ANOVA with Bonferroni’s post hoc test for comparison across
groups. Ngal levels in humans were log-transformed for statistical analysis.
The Jablonski score of kidney damage was analyzed by the Kruskal-Wallis
test with Dunn’s post hoc test.
The authors would like to thank G. Bittenham, R. Strong, and
Q. Al-Awqati for advice. We are very grateful to E.I. Christensen
and T. Willnow for discussion of the megalin knockout mice. This
work was supported by the NIH (DK55388 and DK58872) and by
a March of Dimes Research Grant.
Received for publication August 17, 2004, and accepted in revised
form December 20, 2004.
Address correspondence to: Jonathan Barasch, College of Phy-
sicians and Surgeons of Columbia University, 630 West 168th
Street, New York, New York 10032, USA. Phone: (212) 305-1890;
Fax: (212) 305-3475; E-mail: firstname.lastname@example.org.
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