Kidney Injury Molecule-1 (KIM-1), a Putative Epithelial Cell Adhesion Molecule Containing a Novel Immunoglobulin Domain, Is Up-regulated in Renal Cells after Injury*

Takaharu IchimuraDagger , Joseph V. BonventreDagger §, Véronique Bailly, Henry Wei, Catherine A. Hession, Richard L. Cate, and Michele Sanicola§par

From the Dagger  Renal Unit, Medical Services, Massachusetts General Hospital East and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02129, and  Biogen Incorporated, Cambridge, Massachusetts 02142

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

We report the identification of rat and human cDNAs for a type 1 membrane protein that contains a novel six-cysteine immunoglobulin-like domain and a mucin domain; it is named kidney injury molecule-1 (KIM-1). Structurally, KIM-1 is a member of the immunoglobulin gene superfamily most reminiscent of mucosal addressin cell adhesion molecule 1 (MAdCAM-1). Human KIM-1 exhibits homology to a monkey gene, hepatitis A virus cell receptor 1 (HAVcr-1), which was identified recently as a receptor for the hepatitis A virus. KIM-1 mRNA and protein are expressed at a low level in normal kidney but are increased dramatically in postischemic kidney. In situ hybridization and immunohistochemistry revealed that KIM-1 is expressed in proliferating bromodeoxyuridine-positive and dedifferentiated vimentin-positive epithelial cells in regenerating proximal tubules. Structure and expression data suggest that KIM-1 is an epithelial cell adhesion molecule up-regulated in the cells, which are dedifferentiated and undergoing replication. KIM-1 may play an important role in the restoration of the morphological integrity and function to postischemic kidney.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The dynamic remodeling of tissue architecture which occurs during development must also occur during tissue repair after injury. During development and after injury, cells must decide whether to proliferate, differentiate, or initiate a cell death program based on signals they receive from their extracellular environment. For most cell types, these decisions depend on soluble growth factors and on their attachment to the extracellular matrix or adjacent cells. Thus cells respond both to their growth factor milieu and adhesive environment (for review, see Ref. 1). Cell adhesion molecules/receptors are transmembrane glycoproteins that act at the cell surface to mediate specific binding interactions with other cell adhesion molecules on adjacent cells or with proteins in the extracellular matrix. Cell adhesion molecules (CAMs)1 are classified into families by their structure and include members of the integrin, cadherin, selectin, and immunoglobulin superfamily (IgSF). These molecules can influence growth factor expression, and conversely growth factors can modulate production of cell surface adhesion molecules and the expression of extracellular matrix proteins (for review, see Refs. 1 and 2). The critical importance of adhesion molecules and growth factors for renal development has been demonstrated by severe abnormalities that result from gene disruption of alpha 3 and alpha 8 integrin subunits (3, 4), bone morphogenetic protein-7 (5, 6), and glial-derived neurotrophic factor (7-9).

To study the process of tissue injury and repair, we have chosen an ischemic/reperfusion model of acute renal failure in which the kidney has the capacity for cell renewal (both structurally and functionally) after injury to tubular epithelial cells (10). In this model the nephron is damaged functionally by an ischemic reperfusion injury that results in regional areas of proximal tubule cell death. During the repair process the kidney proximal tubule epithelium undergoes a complex series of events involving 1) cell death and cast formation in the tubule lumen (casts are aggregates of dead, semiviable, and viable cells and other cell debris); 2) proliferation of surviving proximal tubule epithelial cells; 3) formation of a poorly differentiated regenerative epithelium over the denuded basement membrane (this simplified epithelium expresses vimentin, a mesenchymal marker); and 4) differentiation of the regenerative epithelium to form fully functional proximal tubule epithelial cells (10-13). Growth factors such as insulin-like growth factor, epidermal growth factor, and hepatocyte growth factor have been implicated in this repair process as has the endothelial cell adhesion molecule ICAM-1 (for review, see Refs. 10 and 14); however, the mechanisms by which the tubular epithelial cells are restored are still not understood.

To identify molecules involved in processes of injury and repair of the tubular epithelium, we analyzed the difference in the mRNA populations between regenerating and normal kidneys using representational difference analysis (RDA). RDA is a PCR-based method for subtraction originally designed to identify differences at the genomic level (15) and subsequently applied to cDNA analysis (16) to evaluate differences in gene expression. In this report we describe the identification of rat and human cDNAs encoding kidney injury molecule 1 (KIM-1). KIM-1 is a type 1 membrane protein containing a unique Ig-like domain and a mucin domain in its extracellular portion. Structurally, KIM-1 most closely resembles mucosal addressin cell adhesion molecule 1 (MAdCAM-1) (17). These KIM-1 genes show high homology to the monkey gene, hepatitis A virus cell receptor 1 (HAVcr-1), which was identified as a receptor for the hepatitis A virus (18). In situ hybridization and immunohistochemistry demonstrate that KIM-1 mRNA and protein are expressed at high levels in regenerating proximal tubule epithelial cells. Structure and expression data suggest that KIM-1 is a novel epithelial CAM up-regulated on regenerating proximal tubule epithelial cells, which are the cells known to repair and regenerate the damaged region of the nephron in the postischemic kidney.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Animal Model-- Unilateral ischemic rat kidneys were generated as described by Witzgall et al. (12). 190-275-g male Sprague-Dawley rats (Charles River Breeding Laboratories, Inc.) were administered 10 ml of 0.9% saline (37 °C) intraperitoneally. Animals were anesthetized with an intraperitoneal injection of pentobarbital (6.5 mg/100 g body weight). The renal artery and vein were clamped with a microaneurysm clamp (Roboz Surgical Instrument Co.). The incision was closed until 40 min later when the clamp was removed. The incision was then sutured closed, and both kidneys were removed at 24 or 48 h after reperfusion. Both postischemic and contralateral control kidneys were either frozen in liquid nitrogen or fixed for in situ hybridization and/or immunohistochemistry. 40 min of renal vascular occlusion results in a marked reduction in glomerular filtration rate as measured 1-3 h after reperfusion (19) and a marked increase in serum creatinine and blood urea nitrogen if the vascular occlusion is bilateral (20). Because one kidney from the animal is functionally intact, the model of unilateral ischemia used in these studies results in only mild increases in plasma creatinine (from 0.52 ± 0.02 to 0.68 ± 0.08 mg/dl) and blood urea nitrogen (17.5 ± 0.4 to 20.5 ± 1.5 mg/dl) (mean ± S.E.) (n = 4) (19).

Representational Difference Analysis-- Subtraction was performed following the method of Hubank and Schatz (16). Briefly, total RNA from normal and 48 h postischemic kidneys was prepared based on the protocol of Glisin et al. (21). Harvested organs were placed into GNC buffer (4 M guanidine thiocyanate, 0.5% SDS, 25 mM sodium citrate, 0.1% Sigma antifoam) and disrupted on ice with a Polytron. Cell debris was removed with a low speed spin in a clinical centrifuge, and the supernatant was placed on a 5.7 M CsCl, 25 mM sodium acetate, 1 mM EDTA cushion. RNA was pelleted through the cushion in an SW 40 Ti rotor at 85,000 × g for 22.5 h, resuspended, and then precipitated. Poly(A)+ RNA was isolated using an mRNA purification kit (Pharmacia Biotech Inc.). Double-stranded cDNA was synthesized from normal and from 48 h postischemic kidney poly(A)+ RNA using the Life Technologies, Inc. Superscript ChoiceTM.

48 h postischemic kidney cDNA was digested with DpnII and ligated to R-Bgl-12/24 adapter oligonucleotides (R-Bgl-12: GATCTGCGGTGA; R-Bgl-24: AGCACTCTCCAGCCTCTCACCGCA). PCR amplification using R-Bgl-24 oligonucleotides of the adapter-ligated cDNA was used to generate the initial representation designated "tester amplicon." Perkin-Elmer Taq polymerase was used for PCR amplification. The same procedure was used to generate "driver amplicon" from normal rat kidney cDNA. Both amplicons were digested by DpnII; then the tester amplicon was gel purified and ligated to J-Bgl-12/24 (J-Bgl-12: GATCTGTTCATG; J-Bgl-24: ACCGACGTCGACTATCCATGAACA) adapters.

For the first subtractive hybridization, driver and tester amplicons were mixed in the ratio of 100:1. The mixture was phenol extracted, ethanol precipitated, and resuspended in 4 µl of EE×3 buffer (30 mM EPPS, pH 8.0, at 20 °C; 3 mM EDTA). The DNA was denatured for 6 min at 98 °C. The salt concentration was adjusted with 1 µl of 5 M NaCl, annealed for 20 h at 67 °C. Hybridized DNA was diluted with TE containing tRNA. To fill ends of this hybrid DNA, a PCR was set up with diluted samples without adding the primers. The J-Bgl-12 primer was then melted away, ends filled in with Taq DNA polymerase, and J-Bgl-24 primer added to complete amplification. This product was digested with mung bean nuclease for 35 min at 30 °C to remove single-stranded DNA. The digest was heated to 98 °C and used in a PCR with J-Bgl-24 primer. This is designated differential product 1 (DP-1). J adapters on the DP-1 were changed to N-Bgl-12/24 primers (N-Bgl-12: GATCTTCCCTCG; N-Bgl-24: AGGCAACTGTGCTATCCGAGGGAA). A driver:tester ratio of 5,333:1 was used to generate DP-2. N adapters on DP-2 were again changed to J-Bgl-12/24 primers to generate DP-3 with a driver:tester ratio of 4,000:1 and 40,000:1. DP-3 products were gel and Qiaex II purified (Qiagen) for subcloning.

cDNA Cloning-- The DP-3 products were subcloned using the Sureclone kit (Pharmacia) into the SmaI site of pUC18 vector. DP-3 product 1-7 was cloned from DP-3 after 40,000:1 driver:tester ratios, and 3-2 and 3-10 were cloned from DP-3 after 4,000:1.

A cDNA library was generated from 4 µg of poly(A)+ RNA from 48 h postischemic rat kidneys using the Superscript ChoiceTM system for cDNA synthesis and lambda ZapII (Stratagene) cloning kit. 105 clones were screened with the 1-7 RDA and 3-2 RDA products and radiolabeled using the Pharmacia Ready-to-go random priming labeling kit with [32P]dCTP. After tertiary screening, four pure phage clones were isolated for each.

To obtain the human homolog of clone 3-2, a 32P-labeled DNA probe comprising nucleotides 546-969 of the fragment derived from clone 3-2 shown in Fig. 1 was generated and used to screen a human embryonic liver lambda gt10 cDNA library (CLONTECH). 106 clones were screened in duplicate as above at 55 °C. The filters were washed in 2 × SSC at 55 °C. 50 positive phage plaques were identified, plaque purified, and DNA isolated. Phage DNAs were subjected to Southern analysis using the same region 546-969 probe as above and subjected to a final wash with 0.5 × SSC at 55 °C. Two clones were identified as positive.

Northern Analysis-- Poly(A)+ RNA (2.5 µg) from normal adult rat kidney (sham operated), 48 h postischemic adult kidney, and day 18 embryonic kidney was electrophoresed and transferred (22) to a GeneScreen membrane (NEN Life Science Products). For adult rat tissues, a CLONTECH multiple tissue Northern blot was used. Hybridization with the entire coding region of KIM-1 was carried out in plaque screening buffer (PSB, 50 mM Tris, pH 7.5, 1 M NaCl, 0.1% sodium pyrophosphate, 0.2% polyvinylpyrrolidone, 0.2% Ficoll, 0.2% bovine serum albumin, 1% SDS) containing 10% dextran sulfate and 100 µg/ml tRNA at 65 °C.

RNA in Situ Hybridization-- In situ hybridization was carried out according to Finch et al. (23). Briefly, kidneys were perfusion fixed with 4% paraformaldehyde in PBS, further fixed overnight at 4 °C, and processed for paraffin sections. Sections were then dehydrated and hybridized at 55 °C overnight, with 33P-labeled riboprobes generated from 3-2 RDA product subcloned into the BamHI site of pGEM-11Z. After hybridization, sections were washed under high stringency conditions (2 × SSC, 50% formamide at 65 °C). Nonhybridized probe was digested with 20 µg/ml RNase A for 30 min at 37 °C. After further high stringency washing conditions, sections were dehydrated through graded alcohols containing 0.3 M ammonium acetate. NBT-2 emulsion (Eastman Kodak) was used for autoradiography and exposed for at least a week. Silver grains were developed, and sections were counterstained with toluidine blue and photographed with Axioplan microscope (Zeiss).

Affinity-purified Antibodies-- The antigens used to raise antibodies against the rat KIM-1 protein were the peptide R9 HPRAEDNIYIIEDRSRGC cross-linked to keyhole limpet hemocyanin (Pierce) and a fusion protein of glutathione S-transferase (GST) linked to the NH2-terminal Ig domain of the rat KIM-1 protein (amino acids 1-134). 50 µg of the GST-KIM-1 (positions 1-134) or of keyhole limpet hemocyanin-peptide conjugate was used to immunize a rabbit as described (24). Anti-GST-KIM-1 (positions 1-134) antibodies were affinity purified from the antiserum on gel-purified nitrocellulose-bound denatured GST-KIM-1 (positions 1-134). The antibodies raised against the keyhole limpet hemocyanin-peptide conjugates were affinity purified on a Sepharose matrix (SulfoLink coupling gel; Pierce).

Kidney Extracts-- Hemisected kidneys were minced and homogenized in Krebs-Henseleit buffer (20 mM HEPES, pH 7.4, 50 mM beta -glycosphosphate 2 mM EGTA, 250 mM sucrose, 1 mM dithiothreitol, 0.4 mM phenylmethylsulfonyl fluoride, 2.5 µg/ml leupeptin, 2.5 µg/ml aprotinin) with a Polytron. The insoluble material was pelleted by centrifugation at 20,000 × g. It was resuspended once more in Krebs-Henseleit buffer and repelleted by centrifugation. The pellets were weighed and resuspended in 1 g/ml of Krebs-Henseleit buffer containing 1% Triton X-100. Insoluble material was pelleted by centrifugation at 40,000 × g.

Extracts of COS Cells Expressing KIM-1-- A fragment of the full-length KIM-1 cDNA was cloned in the Biogen expression vector pEAG347 for transient expression in COS cells. COS cells were transfected with the control plasmid pEAG347 and with SJR106, the plasmid containing the KIM-1 gene. The cells were grown in 10% fetal bovine serum in Dulbecco's modified Eagle's medium. Cells were harvested 25 or 48 h after transfection, resuspended in 50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM dithiothreitol, containing CompleteTM protease inhibitors (Boehringer Mannheim), and broken by mechanic shearing. The insoluble material was pelleted by centrifugation (5 min at 20,000 × g); the supernatant was collected, and the pellet was resuspended in the breakage buffer containing 1% Triton X-100. Again, insoluble material was pelleted by centrifugation.

Western Blot Analysis-- Aliquots of COS cells and kidney extracts were mixed with Laemmli loading buffer and analyzed by SDS-PAGE on 4-20% precast gels (Daiichi Pure Chemicals Co.). Proteins were transferred onto nitrocellulose membranes in 10 mM CAPS, pH 11, 10% methanol. The blots were blocked in Tris-buffered saline containing 5% non-fat dry milk and 0.05% Tween 20 and probed in the same solution with affinity-purified antibodies raised against peptide R9 or with affinity-purified antibodies raised against GST-KIM-1 (positions 1-134) (0.1 µg/ml). Washes were done with Tris-buffered saline containing 0.05% Tween 20. The secondary probing was done in the blocking solution with horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad) diluted 5,000-fold, and the reacting bands were revealed by chemiluminescence with the supersignal ULTRA chemiluminescent substrate (Pierce).

CNBr Digests-- CNBr digests were done as described (25). Slices (of about 5 × 4 mm) of the polyacrylamide gel containing the putative KIM-1 protein bands were excised with a razor blade using the prestained molecular markers as guide marks. After a 10-min wash in distilled water, they were soaked in 300 µl of 0.1 M HCl, 0.2% beta -mercaptoethanol to which a 50-µl aliquot of fresh CNBr solution in formic acid (700 µg/ml) was added. After 1 h of reaction at room temperature, the slices were washed twice for 5 min with distilled water, once with 0.25 M Tris/HCl, pH 6.8, and then incubated for 10 min at 37 °C in the Laemmli SDS loading buffer. The slices were then inserted into the wells of a 10-20% polyacrylamide gel. After electrophoresis and transfer to nitrocellulose the membrane was exposed to the anti-KIM-1 antibody.

Immunohistochemistry-- An affinity-purified rabbit polyclonal anti-peptide R9 antibody and a Vectastain Elite ABC kit (Vector) (13) were used for immunohistochemistry. Paraformaldehye-fixed paraffin sections were deparaffinized, and endogenous peroxidase activity was ablated by incubation in 2% hydrogen peroxide in methanol for 20 min. The sections were heated with a microwave oven in 0.1 M citrate buffer, pH 6.0, for 10 min (26). The sections were blocked with diluted goat serum (1:67) overnight at 4 °C followed by incubation with affinity-purified or control IgG at a concentration of 5 µg/ml. After 1 h, the sections were washed in PBS and incubated with biotinylated goat anti-mouse IgG for 30 min. After further washes with PBS, the sections were incubated with an avidin-biotinylated horseradish peroxidase complex for 1 h. Sections were washed in PBS and developed by the addition of 50 mM sodium phosphate buffer, pH 7.6, containing 0.12% 3,3-diaminobenzidine tetrahydrochloride (Sigma), 0.0075% nickel chloride, 0.0075% cobalt chloride, and 0.0075% hydrogen peroxide for 2-5 min. The sections were counterstained with 0.01% toluidine blue.

Immunohistochemical Analysis of Bromodeoxyuridine (BrdUrd)-labeled Nuclei and Vimentin in Postischemic Rat Kidney-- Serial sections from rat kidneys fixed in Carnoy's solution after ischemic injury were stained for KIM-1, BrdUrd-labeled nuclei, and regeneration marker vimentin as described (13). Anti-vimentin monoclonal antibody, V-9 (Sigma), was used at a 1:10,000 diluted for the Vectastain Elite ABC kit. For vimentin staining, biotinylated anti-mouse IgG (Vector) was used as the secondary antibody. For KIM-1 immunostaining, rabbit polyclonal affinity-purified anti-peptide R9 antibodies were used (1 µg/ml).

Labeling for BrdUrd was determined by immunostaining of BrdUrd-labeled nuclei in kidney sections prepared from BrdUrd-treated animals as described (13), with modification. Rats were injected intraperitoneally with 60 mg/kg BrdUrd dissolved in dimethyl sulfoxide (60 mg/ml) 2 h before sacrifice. Deparaffinized sections were pretreated with 4 N HCl for 20 min and washed with PBS. Anti-BrdUrd monoclonal antibody (Oncogene Science) was added at a dilution of 1:40 in PBS containing 1.67% normal horse serum. After application of the primary antibody, the Vectastain Elite ABC kit (for mouse IgG) was used. Peroxidase staining was developed for 5 min, as described above, and the sections were counterstained with 0.01% toluidine blue.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Isolation of Rat and Human KIM-1 cDNA Clones-- To identify genes important in the injury/repair process, differences in mRNA populations between regenerating 48 h postischemic adult rat kidneys and normal kidneys were analyzed by RDA. This analysis resulted in the identification of a number of final RDA products, 1-7 RDA (252 base pairs), 3-2 RDA (445 base pairs), and 3-10 RDA (424 base pairs) (see "Experimental Procedures"). Two products, 1-7 RDA and 3-2 RDA, were used to screen a lambda ZapII cDNA library generated from 48 h postischemic rat kidney. The insert of the clone obtained with RDA product 3-2 RDA, clone 3-2 cDNA, was approximately 2.6 kilobases and was subjected to DNA sequencing (Fig. 1A). This sequence contains the sequences of all three RDA products (Fig. 1A). The insert of clone 1-7 cDNA was also sequenced; isolated using the product 1-7 RDA, this insert was a splice variant of 3-2 cDNA which is lacking nucleotides 136-605 (Fig. 1A).


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Fig. 1.   Structure of KIM-1 proteins. Panel A, nucleotide and deduced amino acid sequence of rat KIM-1. The putative signal sequence is underlined. The square boxes indicate cysteines. The junction for the Ig domain and the mucin domain is marked with double bars between Glu130 and Ile131. The rectangular box indicates the putative transmembrane domain. Potential N-glycosylation sites are underscored with broken lines. The fragment that is absent in rat cDNA clone 1-7 is marked with two brackets with stars. RDA fragments originally isolated after subtraction are indicated with two arrows on the both ends with clone numbers. Panel B, alignment of rat and human KIM-1 proteins with monkey HAVcr-1 and mouse EST AA014343. Conserved amino acid residues for four proteins are boxed. Mouse EST was truncated at amino acid Leu146 in the end of Ig domain. Panel C, schematic diagram of rat KIM-1, human KIM-1 clone 85, and human MAdCAM-1 proteins. The branched structure indicates the glycosylation site in the Ig domain.

To confirm the specificity of expression of the 3-2 cDNA in injured kidney, RNA was isolated from sham operated, 48 h postischemic rat kidneys and day 18 embryonic rat kidneys. Northern analysis indicates that a 2.5-kilobase 3-2 cDNA mRNA transcript is expressed at low levels in sham and embryonic kidneys but is dramatically up-regulated in the 48 h postischemic kidney (Fig. 2A).


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Fig. 2.   Northern blot analysis of KIM-1 expression in the normal, 48-h postischemic, and embryonic rat kidney. Northern blots were hybridized with the entire 32P-labeled coding region of the rat KIM-1 cDNA. Hybridization and washing conditions were as described under "Experimental Procedures." Panel A, KIM-1 expression in normal (sham), 48 h postischemic, and day 18 embryonic rat kidneys. 2.5 µg of poly(A)+ RNA was used for each lane. After hybridization with KIM-1 or glyceraldehyde-3-phosphate dehydrogenase probes, the blot was exposed overnight (KIM-1) and 6 h (GAPDH) for autoradiography. Panel B, KIM-1 expression in normal adult rat tissues. 2 µg of poly(A)+ RNA was loaded onto each lane. The blot was then exposed for autoradiography for 4 days.

The expression profile of 3-2 cDNA validated our RDA data, which indicated that the presence of 3-2 cDNA transcripts is different in the normal and injured kidney mRNA populations. Analysis of adult rat tissues indicates that 3-2 mRNA is detectable only at low levels in normal adult kidney, liver, and spleen (Fig. 2B). The presence of different sized mRNAs in liver indicates that the primary transcription product of the 3-2 cDNA undergoes alternative splicing and/or polyadenylation. This variability of mRNA size is consistent with the isolation of two different splice variants from 48 h postischemic kidney.

To clone a human homolog of KIM-1, a probe comprising nucleotides 546-969 of the insert of clone 3-2 (Fig. 1A) was generated and used to screen a human embryonic liver lambda gt10 cDNA library (CLONTECH). The insert of clone 85 was sequenced and encodes a predicted protein of 334 amino acids which is 43.8% identical and 59.1% similar to the predicted rat KIM-1 protein (Fig. 1B).

Structure of KIM-1-- The sequence of rat 3-2 cDNA, which we have named kidney injury molecule-1 (KIM-1), contains an open reading frame of 307 amino acids (Fig. 1A). A signal sequence of 21 amino acids is inferred from von Heijne analysis (27), and a transmembrane region spanning approximately amino acids 235-257 is suggested by hydrophobicity plot analysis (data not shown and Kyte and Doolittle program criteria (28)) indicating that KIM-1 is a type 1 membrane protein. The extracellular domain of KIM-1 is 213 amino acids and contains two distinctive domains. Within the NH2 terminus of the extracellular domain of KIM-1, there is homology to the variable domains of the IgSF seen in many family members such as T-cell receptors and immunoglobulins. Typically, these domains contain two cysteine residues. The KIM-1 Ig domain contains the two sequence motifs, L/I/VXL/IXC (amino acids 33-37) and F/YXCXV/AXH (amino acids 106-112) (Fig. 1B). The former pattern is a signature of the IgSF and is found in IgSF constant regions (29). The latter pattern is a common motif for conserved, disulfide-bonded cysteines in Ig domains (29). Therefore, Cys37 and Cys108 may be able to form an intramolecular disulfide bond. KIM-1 is unique in that in addition to these two cysteine residues, its Ig domain contains four additional cysteines. This six-cysteine Ig domain may define a novel Ig domain structure. The second distinctive domain is a mucin (threonine-, serine-, and proline-rich, T/S/P-rich) domain from amino acid 131 to 201. There are two potential N-linked glycosylation sites between the T/S/P-rich domain and the transmembrane region. The cytoplasmic domain consists of 50 amino acids with no significant peptide motifs observed. The predicted protein sequence of human cDNA clone 85 also contains one Ig, mucin, transmembrane, and cytoplasmic domain each as rat KIM-1. All six cysteines within the Ig domains of both proteins are conserved. Within the Ig domain, the rat KIM-1 and human cDNA clone 85 exhibit 68.3% similarity at the protein level. The mucin domain is longer, and the cytoplasmic domain is shorter in clone 85 than rat KIM-1 (Fig. 1B), with similarity of 49.3 and 34.8%, respectively. We refer to clone 85 as human KIM-1.

A search of NCBI data bases using the BLAST program (30) revealed that rat KIM-1 has homology to HAVcr-1, a monkey gene identified as a receptor for the hepatitis A virus (18) (Fig. 1B). Similarity between rat KIM-1 and HAVcr-1 is 59.8% over their entire amino acid sequences. Similarity between human KIM-1 cDNA and HAVcr-1 is 85.3% over their entire amino acid sequences. Comparison of KIM-1 and HAVcr-1 reveals that both contain a mucin domain and an Ig domain in which all six cysteines align. The combination of an Ig domain and mucin domain suggests that structurally KIM-1 and HAVcr-1 are most closely related to MAdCAM-1, a cell adhesion molecule that has multiple Ig domains and a mucin domain in its extracellular portions (17, 31, 32; shown in Fig. 1C). Mucins are highly repetitive sequences consisting of threonine, serine, and proline and are typically highly O-linked glycosylated sites. Within the mucin domain rat KIM-1 has only two repeats of five amino acids, RPTTT. After this the repetitive sequence pattern starts to degenerate. By contrast, HAVcr-1 has 27 repeats of these six amino acids, indicating a longer mucin domain. The cytoplasmic domain of the human KIM-1 protein is considerably shorter than the HAVcr-1 cytoplasmic domain; within this region they share 65.2% similarity. The BLAST program (30) also revealed that rat KIM-1 has homology to a mouse EST, AA014343 (Fig. 1B).

KIM-1 Protein Is Up-regulated aftere Ischemic Injury-- To determine if the amount of KIM-1 protein is increased after injury, we examined kidney homogenates of contralateral control and postischemic kidneys for the presence of KIM-1 protein. Western blot analysis identifies three proteins (Fig. 3A, arrowheads), detected by two different KIM-1 antibodies after ischemic injury, which are not detectable in homogenates from contralateral control kidneys. The apparent molecular masses of these three proteins are approximately 40, 50, and 70-80 kDa. The three protein species detected by Western blotting could represent glycosylated forms of the same protein given the presence of potential N- and O-linked glycosylation sites in the protein. The fact that each of these proteins reacts with two different sets of polyclonal antibodies supports the interpretation that they are related to KIM-1 and are not cross-reacting bands. Confirmation of this prediction came from the results of partial CNBr cleavage of the three proteins which revealed that they shared common CNBr cleavage fragments (Fig. 3, B and C). Because the cytoplasmic domain of the KIM-1 protein is not predicted to contain any major sites for post-translational modifications, the two smallest products of the digest, 4.7 and 7.4 kDa, detected with antibodies directed against the cytoplasmic domain of KIM-1 (peptide R9), should be the same size for the three different KIM-1 protein bands if they originate from the same protein. We observed that the KIM-1 40-kDa and 70-80-kDa proteins yield fragments migrating at the predicted size (Fig. 3, B and C). Digestion of the 50-kDa protein band also gave the same COOH-terminal signature peptide band confirming that all three KIM-1 species originate from the same polypeptide (data not shown).


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Fig. 3.   KIM-1 protein is up-regulated in the injured kidney. Two sets of equal amounts of postischemic or contralateral kidney extracts were analyzed by SDS-PAGE and Western blot analysis. Panel A, blots were probed with antibodies against peptide R9 (left) or GST-KIM-1 (right). Lanes were loaded with extracts prepared 24 h (lanes 1 and 2) or 48 h (lanes 3 and 4) after reperfusion. Samples in lanes 1 and 3 are from contralateral kidneys, and lanes 2 and 4 are from postischemic kidneys. Arrows indicate the position of the KIM-1-specific bands at approximately 40, 50, and 70-80 kDa seen only in the postischemic kidney lanes. Panel B, the gel slices containing the 40- and 70-80-kDa anti-KIM-1 reactive bands from the 48 h postischemic kidney sample were submitted to a partial digest with CNBr, and the products were analyzed by SDS-PAGE and Western blotting (see "Experimental Procedures"). Lane 1, digest of the 70-80-kDa band; lane 2, digest of the 40-kDa band. The blots were probed with antibodies against peptide R9. Panel C, schematic representation of KIM-1 protein with the leader sequence (LS), the transmembrane domain (TM), the peptide R9 region (R9, indicated with a bar), and the CNBr partial digest products that would react with anti-peptide R9 antibodies (arrows) with their predicted molecular weight as unmodified polypeptides.

The KIM-1 sequence presents two putative sites for N-glycosylation and a mucin domain where O-glycosylation can occur on the polypeptide chain (Fig. 1A). De-N-glycosylation with peptide N-glycosidase (PNGase) F resulted in a shift of all three bands to lower molecular masses, each corresponding to a loss of about 3 kDa, indicating that all three proteins are N-glycosylated (data not shown). Thus, differences in O-glycosylation most probably explain the differences in sizes of these three KIM-1 immunoreactive protein bands.

Expression of the recombinant rat KIM-1 in COS cells has confirmed that the proteins we have detected in the ischemic kidney are KIM-1. COS cells transfected with a full-length rat KIM-1 cDNA produce two major protein bands as detected with KIM-1 antibodies (Fig. 4, A and B), one approximately 40 kDa and the other one, relatively diffuse, at 63 kDa. Partial CNBr digestion of these two bands produces the two COOH-terminal peptides characteristic of the natural KIM-1 protein (Fig. 4C). The absence of a detectable 50-kDa KIM-1 band and the lower molecular mass of the diffuse band can be explained by the fact that glycosylation pathways are known to vary in cells of different origin. In addition, glycosylation pathways may be altered in renal epithelial cells after injury.


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Fig. 4.   Western analysis of KIM-1 recombinant protein expression in COS cells. Panels A and B extracts from the COS cells expressing KIM-1 (lane 1) and control COS cells (lane 2) were analyzed by SDS-PAGE and Western blotting. The blots were probed with antibodies against peptide R9 (panel A) or with antibodies against GST-KIM-1 (panel B). Arrows indicate the position of the KIM-1-specific bands at approximately 40 and 63 kDa. Panel C, gel slices containing the 40- and 63-kDa anti-KIM-1 reactive bands were subjected to partial digestion with CNBr, and the products were analyzed by SDS-PAGE and Western blotting. The blots were probed with antibodies against peptide R9. A digest of the 63-kDa band is loaded on lane 1, and a digest of the 40-kDa band is loaded on lane 2.

KIM-1 Expression Is Localized to the Regenerating Proximal Tubule Epithelial Cells-- To determine the site of KIM-1 up-regulation in the postischemic kidney, RNA in situ hybridization was employed to localize KIM-1 mRNA. KIM-1 mRNA expression was detected in the proximal tubule epithelial cells (Fig. 5, A-D and H), which possess characteristics of injury and regeneration such as loss of brush border, flat structure, and colocalization with exfoliated cells (Fig. 5H) and debris within injured areas of 48 h postischemic kidney. These KIM-1 mRNA-positive cells are localized predominantly in the outer stripe of outer medulla and also in the medullary rays of the cortex (Fig. 5, C and D). The other segments of nephron, including the convoluted part of the proximal tubule, were negative for KIM-1 message at 48 h postischemia. In contrast to the injured kidney, KIM-1 mRNA was almost undetectable in the contralateral kidney (Fig. 5, E and F) or developing kidney after 3 weeks of exposure (data not shown). Hybridization with a KIM-1 sense probe yielded no signal in either postischemic or contralateral kidney control samples.


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Fig. 5.   RNA in situ hybridization analysis of KIM-1 expression in the postischemic kidney. RNA in situ hybridization was performed on tissue sections from 48 h postischemic and contralateral (control) rat kidneys as described under "Experimental Procedures." Panels A, C, E, and G are dark-field, and panels B, D, F, and H are bright-field microphotographs. All photomicrographs were taken with 10 × objectives (scale bar = 50 µm), except for panel H, in which a 40 × objective was used (scale bar = 12.5 µm). KIM-1 mRNA localization is indicated by silver grains in sections. Panels A and B, 48 h postischemic kidney hybridized with an antisense probe showed intense signals in the proximal tubules undergoing regeneration in the outer medulla. The signal is localized in the outer stripe of the outer medulla. Panels C and D, 48 h postischemic kidney hybridized with antisense probe showed intense signals in regenerating proximal tubules in the cortex. Panels E and F, contralateral kidney hybridized with antisense probe. Panel G, 48 h postischemic kidney hybridized with sense probe. Panel H, high magnification of the field in outer stripe of outer medulla in 48 h postischemic kidney. Signal is apparently over the tubular cells (arrowheads) of injured proximal tubule (asterisk) filled with cell debris.

Localization of the KIM-1 protein was established by immunohistochemistry to verify the site of expression because the resolution of RNA in situ data did not allow us to rule out the possibility of KIM-1 expression in the interstitial cell population adjacent to the regenerating tubular cells. KIM-1 immunoreactivity was localized to the regenerating proximal tubule epithelial cells, which possess simplified morphology in the damaged area of 48 h postischemia kidney but not in undamaged parts of nephron in the same section or contralateral kidney (Fig. 6, A-C). High power observation of postischemic kidney sections stained with the antibodies showed that KIM-1 immunoreactivity was visible in cytosol but most apparent in the apical portion of regenerating S3 segment proximal tubule epithelial cells (Fig. 6, E and F). There was an absence of immunoreactivity in the interstitial cell population. There was no significant staining for KIM-1 in contralateral control (Fig. 6C) or developing kidneys (data not shown). To confirm that indeed KIM-1 is expressed on the regenerating epithelial cells, serial sections were immunostained for KIM-1, BrdUrd (to identify proliferating cells), and vimentin (a marker for dedifferentiation) (Fig. 7, A-C) (12, 13). Clear colocalization of KIM-1, BrdUrd, and vimentin in the regenerating tubular epithelial cells in the outer medulla of postischemic kidney after 48 h of reperfusion was observed. This result demonstrates that KIM-1 is expressed in the proliferating BrdUrd-labeled and dedifferentiated vimentin-positive epithelial cells in the damaged area.


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Fig. 6.   Localization of KIM-1 protein expression in the postischemic rat kidney. Affinity-purified antibody against peptide R9 was used for immunostaining of KIM-1 on paraffin sections from 48 h postischemic and contralateral rat kidneys as described under "Experimental Procedures." All photomicrographs were taken by using 10 × objectives (scale bar = 50 µm) except panels E and F, for which a 40 × objective was used (scale bar = 12.5 µm). Panel A, the outer medulla region of 48 h postischemic kidney. Immunoreactivity is indicated with a dark brown stain. The staining in the outer stripe of the outer medulla region of postischemic kidney is absent in controls. Panel B and D, the cortex region of 48 h postischemic kidney. Panel C, contralateral (control) kidney. Panel D, normal rabbit IgG (negative control). Panels E and F, high magnification of KIM-1 staining in the outer medulla (panel E) and the cortex (panel F) of the 48 h postischemic kidney. Relatively intense staining is distributed on the apical portion of cells in injured proximal tubules in the regions of active regeneration (arrowheads). In lumens (asterisks) some cell debris is also visible.


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Fig. 7.   KIM-1 protein is expressed on regenerating epithelial cells. Immunostaining of serial sections from Carnoy's solution-fixed paraffin-embedded 48 h postischemic rat kidney is as described under "Experimental Procedures." KIM-1 immunoreactivity, vimentin (a dedifferentiation marker), colocalized with BrdUrd-labeled nuclei (a marker for cell proliferation) in the same tubule (asterisks and arrowheads) are shown. Photomicrographs are from three serial sections through a damaged region in the outer stripe of the outer medulla. All microphotographs were taken using 40 × objectives (scale bar = 12.5 µm).

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In this report, we describe the isolation and characterization of rat and human cDNA clones for KIM-1. Overall, the human and rat genes are 43.8% identical and exhibit 68.3% similarity within the Ig domain with all six cysteines aligned. Analysis of the KIM-1 predicted protein reveals that it is a type 1 membrane protein containing a unique six-cysteine Ig domain and a mucin domain in its extracellular portion. KIM-1 is expressed at low levels in the normal adult rat kidney, but its expression is dramatically up-regulated in the postischemic kidney. Northern and Western analysis confirmed that the KIM-1 mRNA transcripts and protein are both up-regulated in postischemic kidney in vivo. In situ hybridization and immunohistochemistry demonstrated that KIM-1 is expressed in regenerative proximal tubule epithelial cells in damaged regions, especially in the medulla where the S3 segments of the proximal tubule, which are highly susceptible to insult, are localized. Additionally, immunohistochemical analysis of KIM-1 protein with the dedifferentiation marker vimentin and the proliferation marker BrdUrd demonstrated that KIM-1 is expressed in the regenerating proximal tubule epithelial cells. These data suggest that KIM-1 is a new putative IgSF adhesion molecule that is up-regulated after renal epithelial cell injury and may act at the cell surface of regenerating epithelial cells after postischemic injury.

In the IgSF, one group of molecules, whose extracellular domains contain a combination of Ig domains and a mucin domain, function as membrane receptors/adhesion molecules (32, 33). This group includes MAdCAM-1, CD34, TACTILE (CD96), and HAVcr-1 (18, 32, 33). Among these molecules, HAVcr-1 is probably the monkey homolog of KIM-1 (18). HAVcr-1 was isolated as a virus receptor for hepatitis A virus, but there is no information about its normal function. It is reasonable to predict that HAVcr-1 has an endogenous ligand since other cell surface receptors can serve as virus receptors in addition to binding their legitimate ligands. For example the IgSF member ICAM-1 can function as a rhinovirus receptor, but its native ligands are LFA-1 or Mac-1 (34-36). Structurally, KIM-1 and HAVcr-1 are most reminiscent of MAdCAM-1 in that they contain an atypical Ig domain and a mucin repeat stretch in their extracellular portion. Most IgSF members contain two cysteines within their Ig domains, whereas the MAdCAM-1 NH2-terminal Ig domain contains four cysteines. KIM-1 and HAVcr-1 are unique in that they contain six cysteines in their Ig domain, and this may represent a novel Ig domain structure. Mouse MAdCAM-1 has been shown to act as a bifunctional leukocyte receptor binding both alpha 4beta 7 integrin and L-selectin via its Ig domain and mucin domains, respectively (17, 37, 38). We predict that the KIM-1 Ig domain may play a important role in the interaction of KIM-1 with an as yet unknown specific ligand(s).

Northern blot analysis, RNA in situ studies, and immunohistochemical results all indicate that KIM-1 is expressed at the right time (24 and 48 h postischemia) and in the right place (regenerating proximal tubule epithelial cells) to be implicated in the injury/repair process. One of the cell adhesion molecules examined in this kidney injury model, ICAM-1, has been implicated in renal damage by recruiting neutrophils (39). So far, we have no direct evidence that KIM-1 functions as a positive or negative regulator for the repair process. However, based on its structure, we predict that KIM-1 acts as a CAM/cell surface receptor for proximal tubule epithelial cells during the regeneration process. We believe that KIM-1 is localized apically on the cell, and thus the partner for KIM-1 could be located in the lumen side of injured and regenerating proximal tubule epithelial cells. In the luminal space of 24-48 h postischemic kidney there are a number of different cell populations including infiltrating cells, viable tubule cells that have detached, and dead/dying tubule cells that have detached and will eventually form casts (12, 13). Furthermore, integrins have been shown to be involved in tubular obstruction by forming casts in the postischemic kidney; in particular, reorientation of the alpha 3 integrin subunit from a basal to an apical site has been demonstrated to occur in stressed renal epithelial cells (40). Therefore if KIM-1 is involved in mediating cell interactions, cell surface molecules on these cells located in luminal space are potential targets. It is also possible that KIM-1 is involved in the motogenic response of proximal tubules to cover the area of basement membrane which has become denuded.

The mucin domain of KIM-1 could serve multiple functions. The mucin domain has been proposed to function as a structural domain to present/expose adjacent domains, i.e. it could function to expose the Ig domain well above the plasma membrane as suggested by the "lollipop on a stick" model proposed by Jentoft (41). Mucins also have protective functions for the cell surface of epithelial cells (33). Thus, KIM-1 might play a protective role for regenerative cells by isolating them from an environment filled with dead cells that are releasing their cellular contents. Finally, because the MAdCAM-1 mucin domain can bind L-selectin, the mucin of KIM-1 may be able to interact with a distinct ligand such as the selectins. A recent report by Takada et al. (42) has documented an up-regulation of E-selectin mRNA and an infiltration of periphal mononucleocytes in the postischemic kidneys in uninephrectomized rats. Interestingly, a soluble P-selectin glycoprotein ligand administered 3 h after reperfusion injury resulted in a marked decrease in leukocyte infiltration and prevented an increase in creatinine. This study indicates that selectins are probably involved in the postischemic kidney pathogenesis.

In conclusion, we have identified a putative adhesion molecule that is markedly up-regulated in the S3 segment of proximal tubule cells after ischemic/reperfusion injury. KIM-1 is up-regulated when these cells are undergoing changes that will result in proliferation and regeneration of epithelial cells to reconstruct a functional epithelium. Further characterization of KIM-1 function, particularly identification of a ligand(s), will provide insight as to whether KIM-1 plays a role in kidney injury/repair and whether enhancing or inhibiting KIM-1 function could accelerate the repair process.

    ACKNOWLEDGEMENTS

We thank Blake Pepinsky for design and production of the anti-rat KIM-1 peptide antibodies, Suzanne Robinson for technical assistance, and Kris Bradley for help in preparing the manuscript. We also thank Ellen Garber for pEAG 347 expression vector and Philip Gotwals, Laurie Osborn, Victor Koteliansky, and Werner Meier for helpful discussions.

    FOOTNOTES

* This study was supported in part by National Institutes of Health Grant DK 39773.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF035963.

§ Recipient of National Institutes of Health MERIT Award DK39773.

par To whom correspondence should be addressed: Biogen Inc., 14 Cambridge Center, Cambridge, MA 01242. Tel.: 617-679-3307; Fax: 617-679-2616; E-mail: michele_sanicola{at}biogen.com.

1 The abbreviations used are: CAM(s), cell adhesion molecule(s); IgSF, immunoglobulin superfamily; ICAM-1, intercellular adhesion molecule 1; RDA, representational difference analysis; PCR, polymerase chain reaction; KIM-1, kidney injury molecule-1; MAdCAM-1, mucosal addressin cell adhesion molecule 1; HAVcr-1, hepatitis A virus cell receptor 1; EPPS, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid; DP, differential product; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; CAPS, 3-(cyclohexylamino)propanesulfonic acid; BrdUrd, bromodeoxyuridine.

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Abstract
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Discussion
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