The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 9 September 2013
Chronic epithelial kidney injury molecule-1
expression causes murine kidney fibrosis
Benjamin D. Humphreys,1,2 Fengfeng Xu,1 Venkata Sabbisetti,1 Ivica Grgic,1,3
Said Movahedi Naini,1 Ningning Wang,1,4 Guochun Chen,1,5 Sheng Xiao,6 Dhruti Patel,1
Joel M. Henderson,7 Takaharu Ichimura,1 Shan Mou,1,8 Savuth Soeung,1
Andrew P. McMahon,2,9,10,11 Vijay K. Kuchroo,6 and Joseph V. Bonventre1,2
1Renal Division, Brigham and Women’s Hospital, Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA.
2Harvard Stem Cell Institute, Cambridge, Massachusetts, USA. 3Department of Internal Medicine and Nephrology, Philipps University, Marburg, Germany.
4Department of Internal Medicine and Nephrology, The First Affiliated Hospital with Nanjing Medical University, Nanjing, People’s Republic of China.
5Division of Nephrology, Second Xiangya Hospital, Central South University, Changsha, Hunan, People’s Republic of China. 6Center for Neurologic Disease,
Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA. 7Department of Pathology and Laboratory Medicine,
Boston University School of Medicine, Boston, Massachusetts, USA. 8Renal Division, Renji Hospital, Shanghai JiaoTong University School of Medicine,
Shanghai, People’s Republic of China. 9Department of Stem Cell and Regenerative Biology, Harvard University,
Cambridge, Massachusetts, USA. 10Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA.
11Department of Stem Cell Biology and Regenerative Medicine, Keck School of Medicine of the University of Southern California, Los Angeles, California, USA.
Acute kidney injury predisposes patients to the development of both chronic kidney disease and end-stage
renal failure, but the molecular details underlying this important clinical association remain obscure. We
report that kidney injury molecule-1 (KIM-1), an epithelial phosphatidylserine receptor expressed transiently
after acute injury and chronically in fibrotic renal disease, promotes kidney fibrosis. Conditional expression
of KIM-1 in renal epithelial cells (Kim1RECtg) in the absence of an injury stimulus resulted in focal epithelial
vacuolization at birth, but otherwise normal tubule histology and kidney function. By 4 weeks of age, Kim1RECtg
mice developed spontaneous and progressive interstitial kidney inflammation with fibrosis, leading to renal
failure with anemia, proteinuria, hyperphosphatemia, hypertension, cardiac hypertrophy, and death, analo-
gous to progressive kidney disease in humans. Kim1RECtg kidneys had elevated expression of proinflamma-
tory monocyte chemotactic protein-1 (MCP-1) at early time points. Heterologous expression of KIM-1 in an
immortalized proximal tubule cell line triggered MCP-1 secretion and increased MCP-1–dependent macro-
phage chemotaxis. In mice expressing a mutant, truncated KIM-1 polypeptide, experimental kidney fibrosis
was ameliorated with reduced levels of MCP-1, consistent with a profibrotic role for native KIM-1. Thus, sus-
tained KIM-1 expression promotes kidney fibrosis and provides a link between acute and recurrent injury with
progressive chronic kidney disease.
Acute kidney injury (AKI) is characterized by a rapid decline in kid-
ney function, often triggered by an ischemic or toxic insult. This clin-
ical syndrome is associated with substantial short-term morbidity,
mortality, and cost, but it had previously been assumed that patients
surviving the episode made a full renal recovery (1). However, AKI
is now appreciated to be markedly associated with increased risk of
future chronic kidney disease (CKD), end-stage renal disease (ESRD)
(2, 3), and long-term mortality (4). The population rate of AKI is
increasing at greater than 7% per year (5, 6), and some estimates indi-
cate that the incidence of AKI-related ESRD is equal to the incidence
of ESRD from diabetes (7). The mechanisms that might explain the
link between AKI and future CKD/ESRD are poorly understood,
but peritubular capillary loss, a known consequence of AKI (8), is
proposed to lead to chronic hypoxia and later development of tubu-
lointerstitial fibrosis and CKD (9, 10). How chronic ischemia might
trigger parenchymal loss at a molecular level is unresolved.
Kidney injury molecule-1 (KIM-1), originally identified as hepa-
titis A virus receptor (HAVCR1, also known as Tim-1), is a type 1
transmembrane protein strongly induced by ischemic and toxic
insults to kidney. It also plays diverse roles in T and B cell biology
(11). In healthy kidney, KIM-1 is undetectable, but after injury,
it is induced more than any other protein, in which case it local-
izes to the apical surface of surviving proximal tubule epithelial
cells (12). The extracellular KIM-1 Ig variable domain binds and
internalizes oxidized lipid as well as phosphatidylserine exposed
on the outer leaflet of luminal apoptotic cells (13, 14), thereby
aiding in nephron repair and tissue remodeling through phago-
cytosis of cells and debris (15). KIM-1 is expressed in CKD (16–20)
where it colocalizes with areas of fibrosis and inflammation (21),
and its expression correlates directly with interstitial fibrosis in
human allografts (22). Increased urinary KIM-1 is an indepen-
dent predictor of long-term renal graft loss and is also elevated in
human nondiabetic, proteinuric CKD (23, 24). The expression of
KIM-1 in chronic and progressive kidney disease, settings with-
out significant numbers of apoptotic cells in the tubule lumen,
the epidemiologic association of AKI with future CKD (25), and
the temporal and spatial association of KIM-1 with inflammation
and fibrosis suggest that it might play a pathogenic role in link-
ing AKI to CKD and renal fibrosis.
In this study, we examined the functional consequences of
chronic KIM-1 expression in renal epithelial cells. To dissociate
the effects of KIM-1 expression from the pleiotropic effects of
Conflict of interest: Joseph V. Bonventre is an inventor on KIM-1 patents, which
have been licensed by Partners Healthcare to Johnson & Johnson, Genzyme, Biogen
Idec, and other companies.
Citation for this article: J Clin Invest. 2013;123(9):4023–4035. doi:10.1172/JCI45361.
4024 The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 9 September 2013
ischemic kidney injury used to induce KIM-1, we created a genetic
model in which KIM-1 is expressed chronically in the absence of
any injury stimulus. Using this model, we demonstrate here that
chronic KIM-1 expression leads to inflammation, tubulointersti-
tial fibrosis characterized by elevated monocyte chemotactic pro-
tein-1 (MCP-1) levels and a murine CKD phenotype. In contrast,
mice with mutant endogenous KIM-1 were protected from fibro-
sis in a mouse model of CKD and had a reduced level of MCP-1.
Together, these results indicate that persistent KIM-1 expression
after AKI promotes interstitial fibrosis and correlates with MCP-1
expression and further suggest that KIM-1 may represent a novel
therapeutic target in CKD (26). The mouse model we have devel-
oped also recapitulates the renal and extrarenal manifestations
of CKD seen in humans. These studies provide insight into how
recurrent tubular injury, as reflected by persistent KIM-1 expres-
sion, might facilitate progressive CKD and lead to ESRD.
To determine the kinetics of KIM-1 induction during fibrotic
disease, we examined the time course for KIM-1 expression in a
rodent model of renal fibrosis, unilateral ureteral obstruction
(UUO). KIM-1 protein was strongly upregulated 2 days after ure-
teral obstruction and fell thereafter, but remained significantly
elevated at day 14 (Figure 1A). KIM-1 was expressed on the api-
cal aspect of proximal tubule epithelia, in tubules surrounded
by expanded interstitium with abundant interstitial smooth
muscle actin–positive myofibroblasts (Figure 1B). To distin-
guish between KIM-1 expression as a cause or consequence of
epithelial injury and fibrosis in vivo, we created a conditional
Z/Kim1-AP transgene enabling Cre recombinase-dependent activa-
tion of KIM-1 and alkaline phosphatase (AP) expression (Figure 2,
A–E). Crossing the Z/Kim1-AP mouse with Six2-GFPCre mice
(hereafter referred to as Six2-GC) (27), generated bigenic Kim1RECtg
(Kim1 renal epithelial cell transgenic) mice with KIM-1 and AP
expression in metanephric mesenchyme-derived kidney epithelia.
Kim1RECtg kidneys expressed the Z/Kim1-AP transgene primarily in
cortical and outer medullary epithelia, with rare transgene expres-
sion in inner medulla (Figure 2E and Supplemental Figure 1; sup-
plemental material available online with this article; doi:10.1172/
JCI45361DS1). Mosaic transgene activity was observed with
10%–20% of renal tubules positive for AP activity, with a similar
fraction of positive podocytes. Note, however, that despite the AP
expression pattern, KIM-1 protein was only seen in tubules and
never in podocytes (Figure 3C and data not shown). A comparison
of the distribution of endogenous KIM-1 after UUO versus AP
expression in Kim1RECtg kidneys is presented in Table 1.
Kim1RECtg mice were born at expected Mendelian ratios and
expressed Kim1 mRNA at birth (Figure 3A). KIM-1 protein was
properly sorted to the apical membrane of cortical proximal
tubule epithelia (Figure 3, B and C). There was no difference in the
birth weights of transgenic versus littermate control mice (n = 3
Kim1RECtg or 7 littermate controls), but Kim1RECtg mice did not gain
weight as quickly as littermate controls (Supplemental Figure 2).
At birth, kidneys from Kim1RECtg mice were 23% smaller by weight,
however, than those of littermate controls (Figure 3, D and E,
P < 0.05, n = 5 Kim1RECtg or 13 control kidneys). This was associated
with 43% fewer nephrons in Kim1RECtg kidneys without significant
differences in glomerular diameter at P14 (Figure 3, F and G). Kid-
ney histology at P1 showed a mild reduction in cortical thickness,
with occasional microcysts that appeared to be glomerular (about
10% of total glomeruli; Figure 3H) and rare large cysts (fewer than
1 per section). A detailed histologic analysis at P15 revealed nor-
mal glomeruli including foot processes, however (Figure 3, I and
J), as well as normal interstitium and vasculature. Focal coarse
vacuolization and focal epithelial degeneration were noted only
in Kim1RECtg mouse kidneys (n = 3 Kim1RECtg and 3 control kidneys;
Tables 2 and 3). These coarse vacuoles, suggestive of local inju-
ry, were found in about 1% of tubules (Figure 3, K and L). There
were no histologic differences in other organs of Kim1RECtg mice
when compared with organs from littermate controls (data not
shown). Thus, P15 kidneys from Kim1RECtg mice were characterized
by reduced nephron endowment and rare tubular epithelial vacu-
olization, but kidney histology was otherwise normal.
At 5 weeks, kidneys from Kim1RECtg mice developed a patchy
mononuclear interstitial infiltrate with occasional hyaline casts and
focal tubular damage. KIM-1 continued to be expressed in a subset
of tubular epithelial cells along the apical membrane (Supplemen-
tal Figure 3). By 12 weeks, interstitial inflammation was extensive,
together with tubular dedifferentiation, microcystic tubular dila-
tion, hyaline casts, and fibrosis. This inflammatory, tubular injury,
and fibrotic phenotype was observed in all Kim1RECtg mice older
than 6 weeks that were examined (Figure 4A and Supplemental
Figure 4). In the oldest mice, prominent periarterial inflammation
was present resembling ectopic lymph nodes. Tubular injury scores
confirmed these histologic observations (Figure 4B). Serum creati-
nine was equal between transgenic and control mice at P14, but
KIM-1 is highly induced in fibrotic kidney injury adjacent to interstitial myofibroblasts. (A) KIM-1 protein is highly induced by 2 days after ureteral
ligation, with persistent expression at days 7 and 14 as assessed by Western blot. (B) Tubular KIM-1 expression in the UUO renal fibrosis model.
After fibrotic injury, KIM-1–positive epithelia are adjacent to SMA-positive interstitial myofibroblasts. Scale bar: 10 μm.
4034 The Journal of Clinical Investigation http://www.jci.org Volume 123 Number 9 September 2013
lected with a metabolic cage and processed in a similar fashion. MCP-1,
IL-6, and TGF-β microbead-based assays were developed and validated
in the lab. Approximately 6000 beads/50 μl were incubated with 30 μl
of sample or recombinant proteins (R&D Systems) for 1 hour, washed 3
times with PBST, and incubated in corresponding biotinylated antibod-
ies (R&D Systems) for 45 minutes on an orbital shaker at 300 rpm. Beads
were washed again with PBST and incubated for 15 minutes with strepta-
vidin-PE solution (Invitrogen). The signal from the fluorochrome, which
is directly proportional to the amount of antigen bound at the micro-bead
surface, ws captured using the Bio-Plex 200 system (Bio-Rad). Data were
generated and interpreted using parametric logistic regression analysis.
Boyden chamber assay. Cell chemotaxis assay was performed in a modi-
fied Boyden chamber using 24-well flat-bottom tissue plates with 5-μm
polyethylenterephtylan membrane inserts (BD). The lower compartment
of each chamber was filled with 500 μl of the conditioned medium. Mem-
brane inserts were filled with 300 μl of cell suspension and placed in the pre-
filled lower compartments. The chambers were then incubated for 3 hours
in 37°C, 5% CO2. After incubation, nonmigrated cells in the upper wells
were removed by scraping and the migrated cells were stained with eosin on
the membrane. Adherent cells on the lower surface of the membrane were
counted from 5 high power fields by light microscopy (×40). Data are pre-
sented as cells per high-power field. Neutralizing antibody against MCP-1
was from Sigma-Aldrich.
Statistics. All results are reported as mean ± SEM. All error bars on graphs
represent SEM. Statistical tests are 2-tailed, unpaired t tests except for sur-
vival analysis (Figure 4E), which used the log rank test, and Figures 7B and
Supplemental Figure 2, which used a repeated measures t test.
Study approval. All animal studies were approved by the Harvard Institu-
tional Animal Care and Use Committee.
We thank Corrine Lobe for the Z/AP plasmid and Jordan Kriedberg
for the Podocin-Cre transgenic mice. This work was supported by NIH
grants DK73628, DK84316, DK088923, funds from the Harvard
Stem Cell Institute, and an Established Investigator Award from the
American Heart Association to B.D. Humphreys and DK39773 and
DK72381 to J.V. Bonventre. Work in A.P. McMahon’s laboratory is
supported by the National Institute of Diabetes and Digestive and
Kidney Diseases (NIDDK). N. Wang was supported by the China
Scholarship Council–Harvard University Exchange. S. Xiao was sup-
ported by NIDDK K01 DK090105. I. Grgic was supported by a fel-
lowship of the Deutsche Forschungsgemeinschaft.
Received for publication July 25, 2012, and accepted in revised
form June 17, 2013.
Address correspondence to: Benjamin D. Humphreys, Brigham and
Women’s Hospital, Harvard Institutes of Medicine, Rm 550, 4 Black-
fan Circle, Boston, Massachusetts 02115, USA. Phone: 617.525.5971;
Fax: 617.525.5965; E-mail: firstname.lastname@example.org.
forward: CTGGGATTCACCTCAAGAACATC, reverse: CAGGGT-
CAAGGCAAGCCTC; CXCL2 forward: CCAACCACCAGGCTACAGG,
reverse: GCGTCACACTCAAGCTCTG; CXCL10 forward: CCAAGT-
GCTGCCGTCATTTTC, reverse: GGCTCGCAGGGATGATTTCAA;
GAPDH forward: CATGTTCCAGTATGACTCCACTC, R: GGCCT-
CACCCCATTTGATGT; IL-1β forward: CCTTCCAGGATGAGGA-
CATGA, reverse: AACGTCACACACCAGCAGGTT; IL-6 forward:
TAGTCCTTCCTACCCCAATTTCC, reverse: TTGGTCCTTAGC-
CACTCCTTC; MCP-1 forward: TGCATCTGCCCTAAGGTCTTC, reverse:
AAGTGCTTGAGGTGGTTGTGG; TGF-β forward: GCAACAATTCCTG-
GCGTTACC, reverse: CGAAAGCCCTGTATTCCGTCT; TNF-α forward:
CCCTCACACTCAGATCATCTTCT, reverse: GCTACGACGTGGGCTA-
CAG; αSMA forward: CTGACAGAGGCACCACTGAA, reverse: CATCTC-
CAGAGTCCAGCACA; fibronectin forward: ATGTGGACCCCTCCT-
GATAGT, R: GCCCAGTGATTTCAGCAAAGG.
Physiologic measurements. Serum creatinine was measured using a Beckman
Creatinine Analyzer 2 by the Jaffe rate method. Hematocrit was calculated
after centrifugation of a hematocrit capillary tube. Proteinuria was assessed
by the micro pyrogallol red method (total protein kit; Sigma-Aldrich) or by
separation of 1 μl of urine on a 10% SDS-PAGE gel followed by coomassie
stain. Blood pressure was assessed by tail-cuff analyzer (Visitech Systems
Inc.; Apex). After training conscious mice for 3 days, systolic blood pressure
and heart rate were collected for 3 consecutive days between 9 am and 11 am,
with an average of 10 reads each day. KIM-1 protein in urine was measured
by microsphere-based Luminex technology. NAG in urine was measured by
colorimetric assayed using a commercial kit (Roche). For serum electrolytes,
BUN, and creatinine, mice were anesthetized and the blood samples were
collected from carotid artery. Analyses were assessed by a core laboratory
(Children’s Hospital Boston, Boston, Massachusetts, USA).
Cell culture. Cos7 cells were transfected using Lipofectamine 2000
(Invitrogen). Porcine proximal tubular epithelial cells (LLC-PK1) cells were
grown in DMEM supplemented with 10% FBS and maintained at 37°C in 5%
CO2. To create stable cell lines expressing full-length KIM-1 (KIM-1–LLC),
LLC-PK1 cells were transfected with pcDNA3–KIM-1 or pcDNA3–KIM-1–
Y350F plasmid and the stable population was selected using G418 treatment
(400 μg/ml). Control cell lines (pcDNA-LLC) stably expressing pcDNA3-neo
were generated the same way. To generate mBMDM, femurs and tibias were
removed from 20- to 25-g BALB/c mice. BM was isolated from these by stan-
dard sterile techniques and matured for 7 days in uncoated Petri dishes using
DMEM/F12 medium with 10% FCS, penicillin (100 U/ml), and streptomycin
(100 mg/ml) and conditioned with M-CSF from L929 cells. The U937 cell
line, a human monomyelocytic cell line, was cultured in RPMI 1640 medium
supplemented with 10% FCS, 1% penicillin/streptomycin, and 2 mM l-gluta-
mine. Cells were subcultured 3 times a week and maintained at a concentra-
tion of 0.5–1.0 × 106 cells/ml. Monocytic differentiation of U937 cells was
achieved by adding 10 nM PMA for 48 hours. PMA-differentiated U937 was
washed 3 times by sterile PBS before experiments.
Cytokine measurement. Both KIM-1–LLC and pcDNA-LLC cells were
grown to confluence, and supernatant was collected, centrifuged, and
stored at –80°C until further analysis. Alternatively, mouse urine was col-
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