Altered expression of junctional adhesion molecule 4 in injured podocytes
http://www.100md.com
《美国生理学杂志》
1Department of Cell Biology, Institute of Nephrology, Niigata University Graduate School of Medical and Dental Sciences, Niigata
2Department of Pediatrics, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
ABSTRACT
Recent investigations have revealed the importance of glomerular podocytes with its diaphragm as the major filtration barrier. Junctional adhesion molecule 4 (JAM4) has been identified as a protein that interacts with membrane-associated guanyl kinase inverted (MAGI)-1 and is reported to be expressed on podocytes. To elucidate the role of JAM4 on podocytes, we examined the expression of JAM4 and MAGI-1 in normal and two different proteinuric rat models: puromycin aminonucleoside (PAN) nephropathy and anti-nephrin antibody-induced (ANA) nephropathy, one model with and one without effacement of podocyte foot processes. JAM4 was detected by immunomicroscopy at the apical membrane of normal podocytes. JAM4 immunostaining was focally increased in the podocytes in PAN nephropathy but not in ANA nephropathy. In proteinuric podocytes, the expression of JAM4 was distinct from that of MAGI-1 or other slit diaphragm molecules such as nephrin and ZO-1. Close colocalization of JAM4 and ezrin was maintained in PAN nephropathy. By immunoelectron microscopy, the signals for JAM4 were detected at the free apical membrane of the podocytes with effaced foot processes. Studies with selective detergent extract revealed that the subcellular localization of JAM4 was altered in PAN nephropathy. Thus the altered expression of JAM4 appears to be associated with morphological changes in podocytes and can be a useful marker of injured podocytes. JAM4 may have a different role at the apical membrane besides the role as a junctional molecule and is likely associated with the unique structure of this epithelium.
podocyte; puromycin aminonucleoside nephropathy; foot process effacement; membrane-associated guanyl kinase inverted-1
THE VISCERAL GLOMERULAR EPITHELIAL cell, also called the podocyte, is a highly specialized, terminally differentiated cell that lines the outer aspect of the glomerular basement membrane (GBM). Podocytes possess interdigitating cell extensions, called foot processes, which are bridged by the slit diaphragm (SD), a podocyte-specific intercellular junction with an electron-dense zipper-like structure (33). The recent discovery of several novel components of the SD (nephrin, Neph1, P-cadherin, etc.) and their physical and functional interactions with intracellular adaptor proteins (ZO-1, CD2AP, podocin, etc.) revealed the region of the SD as the major filtration barrier (1, 32). Another specialized feature of the podocyte is a specific cytoskeletal organization, which plays a critical role in counteracting the high transmural distending force permitting the high-pressure perfusion through glomerular capillaries (21). When podocytes are injured, as in many different pathological settings such as minimal change disease, diabetic nephropathy, lupus nephritis, and several other acquired and genetic kidney diseases, the podocytes react in a particular pattern, retracting their foot processes into their cell bodies to form a flattened, simple epithelium (26, 37). These changes in the podocytes lead to precipitous protein leakage into urine, and if they are not reversed, they ultimately lead to the development of segmental glomerulosclerosis and end-stage renal failure (20, 22, 38). However, the molecular events that cause morphological changes in the podocyte in these etiologically diverse diseases have not been fully clarified.
Junctional adhesion molecule (JAM) is a member of the immunoglobulin superfamily expressed at cell-cell junctions (6). Recently, Hirabayashi et al. (9) have identified JAM4 as a protein that interacts with membrane-associated guanyl kinase inverted (MAGI)-1 in mouse lung tissue. JAM4 has two Ig-like domains, a single transmembrane domain, and a cytoplasmic tail that ends in a PDZ-domain-binding sequence. JAM4 is expressed at podocytes and the apical membranes of renal tubular cells and small intestine epithelial cells (9, 40). JAM4 is speculated to play a role in glomerular permselectivity, because JAM4 reduces paracellular permeability when expressed in Chinese hamster ovary cells (9). However, to date, the functional roles of JAM4 in the glomeruli have not been fully understood.
In an attempt to elucidate the role of JAM4 in maintaining the barrier function of the SD and the interdigitating structure of podocytes, we investigated the expression of JAM4 and MAGI-1 in the developing kidney and in normal and two proteinuric adult rat models. One proteinuric rat model was anti-nephrin antibody-induced (ANA) nephropathy, in which the structural integrity of the SD is largely unaffected despite massive proteinuria. The other was puromycin aminonucleoside (PAN) nephropathy, which is characterized by extensive flattening of the foot processes. We show here that JAM4 is expressed at the apical membrane of podocytes, and its expression is altered in proteinuric glomeruli. JAM4 can be a useful marker for injured podocytes.
MATERIALS AND METHODS
Cloning of Rat JAM4
To clone a rat homolog of JAM4, we employed a PCR with the primers designed from a mouse JAM4 sequence. PCR cloning was performed basically in accordance with the method previously described (17). Total RNA was prepared from normal Wistar rat glomeruli with TRIzol (Invitrogen, Carlsbad, CA) and was utilized to synthesize cDNA with SuperScript II (Invitrogen) according to the manufacturer's protocol. Synthesized cDNA was used as a template. Templates were amplified in DNA with Program Temperature Control System PC-800 (ASTEC, Fukuoka, Japan) for 40–50 cycles at an annealing temperature of 50–58°C. The PCR products that were close to an expected size were cloned into a Topo Vector (Invitrogen), and DNA sequences were determined by an automated DNA sequencer (ABI 310, PerkinElmer Japan, Urayasu, Japan). From a partial clone of rat JAM4, gene-specific primers were designated and PCR was performed again.
Antibodies
To produce an antibody specific to rat JAM4, we chose a peptide of 15 amino acids, YKSDEARAAQIASLP, as the immunogen. This peptide was purchased from GenScript (Scotch Plains, NJ). Three rabbits were immunized with 2.0 mg of the peptide conjugated with the carrier protein KLH. The rabbits were bled 2 wk after the last immunization. The specificity of the anti-JAM4 antibody activity was confirmed by Western blot analysis. Mouse monoclonal antibody to rat nephrin (mAb 5-1-6) and mouse monoclonal antibody to rat Thy-1 antigen (mAb 1-22-3) were prepared in our laboratory (14, 29). Mouse monoclonal anti-ZO-1 antibody and anti-ezrin antibody (3C12) were purchased from Zymed Laboratories (San Francisco, CA). Mouse anti-rat endothelial cell antigen (RECA-1) antibody, goat anti-MAGI-1 antibody, and rabbit anti-laminin antibody were purchased from Serotec (Oxford, UK), Santa Cruz Biotechnology (Santa Cruz, CA), and Sigma (St. Louis, MO), respectively. Mouse anti-podocalyxin antibody (4D5) was donated by Dr. M. Hara (Yoshida Hospital, Niigata, Japan) (8).
Immunofluorescence Microscopy
Immunofluorescence studies were performed in accordance with the method previously described (17). The renal tissue was quickly frozen in n-hexane cooled to –70°C. Three-micrometer-thick frozen sections were cut with a cryostat, fixed with periodate-lysine-paraformaldehyde (PLP) for 1 min, incubated with the primary antibodies described above, and stained with FITC-conjugated anti-rabbit IgG (for anti-JAM4; Dako, Glostrup, Denmark) or FITC-conjugated anti-goat IgG (for anti-MAGI-1; Southern Biotechnology Associates, Birmingham, AL). For dual-labeling immunofluorescence, cryostat sections were incubated with anti-JAM4 antibody, FITC-conjugated donkey anti-rabbit IgG, then anti-Thy-1, anti-RECA-1, anti-podocalyxin, anti-MAGI-1, anti-nephrin, anti-ZO-1, or anti-ezrin antibody. Tetramethyl-rhodamine isothiocyanate (TRITC)-conjugated goat anti-mouse IgG1 (for anti-nephrin, anti-RECA-1, anti-ZO-1 and anti-ezrin; Southern Biotechnology Associates); TRITC-conjugated goat anti-mouse IgG2a (for anti-podocalyxin; Southern Biotechnology Associates); TRITC-conjugated goat anti-mouse IgG3 (for anti-Thy-1; Southern Biotechnology Associates); or TRITC-conjugated donkey anti-goat IgG (for anti-MAGI-1; Santa Cruz Biotechnology) was used as the secondary antibody. The sections were observed with a laser-scanning confocal microscope (MRC-1024, Bio-Rad Laboratories, Hercules, CA) with the appropriate filters for FITC (488-nm excitation, HQ515/30 emission filter) and TRITC (568-nm excitation, 585LP filter). The images were further processed using Adobe Photoshop CS 8.0.1.
Immunoelectron Microscopy
The tissues were fixed with PLP fixative by perfusion and immersion and then embedded in glycol methacrylate. Ultrathin sections of the glycol methacrylate-embedded tissues were collected on nickel grids and incubated with 5% nonfat milk for 1 h and then with the anti-JAM4 antibody for 1 h. The sections were incubated with gold (10 nm)-labeled anti-rabbit IgG (Amersham Life Sciences, Buckinghamshire, UK) for 1 h, washed in PBS several times, and then counterstained with 2% aqueous uranyl acetate and 2% lead citrate for observation by electron microscope (H-600A, Hitachi, Tokyo, Japan).
Western Blot Analysis
Western blot analysis with sequentially solubilized glomerular lysates was performed basically in accordance with the method previously described (17). Normal or proteinuric rat glomeruli were isolated with PBS containing protease inhibitors and solubilized with 1% Triton X-100. After the lysate was centrifuged, the pellet was reextracted with RIPA buffer (consisting of 0.1% SDS, 1% sodium deoxycholate, 1% Triton X-100, 150 mmol/l NaCl, and 10 mmol/l EDTA in 25 mmol/l Tris·HCl, pH 7.2), and the lysate was again centrifuged. The lysate was separated into Triton X-100-soluble (TXS), Triton X-100-insoluble, RIPA-soluble (RIPAS), and RIPA-insoluble fractions (RIPAI). The RIPA-insoluble fraction was solubilized with SDS-PAGE sample buffer (2% SDS, 10% glycerol, 5% 2-mercaptoethanol in 62.5 mM Tris·HCl, pH 6.8). The insoluble material was removed by centrifugation at 15,000 g for 30 min at 4°C. The concentration was measured by the bicinchoninic acid method (Pierce Chemical, Rockford, IL), and equal amounts of these sequentially solubilized fractions were subjected to SDS-PAGE on 7.5% acrylamide gel in accordance with the method of Laemmli and transferred to a PVDF membrane (Pall, Pensacola, FL) by electrophoretic transblotting for 30 min using Trans-Blot SD (Bio-Rad, Hercules, CA). After blocking with bovine skim milk, strips of the membranes were exposed to rabbit anti-actin antibody (Sigma) or rabbit anti-JAM4 antibody. They were washed and then incubated with alkaline phosphatase-conjugated anti-rabbit IgG (Bio Source International, Camarillo, CA). The reaction was developed with an alkaline phosphatase chromogen kit (5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt/nitro blue tetrazolium; Biomedica, Foster City, CA). The density of the positive bands was quantitated by LabWorks (UVP, Upland, CA). This procedure was carried out three times. The percentage of the density of each fraction relative to the total was calculated.
Culture of Podocytes
Cultivation of conditionally immortalized mouse podocytes was conducted as reported previously (25). In brief, podocytes were maintained in RPMI 1640 medium (Nissui Pharmaceutical, Tokyo, Japan), 100 U/ml penicillin (Banyu Pharmaceutical, Tokyo, Japan), and 0.1 mg/ml streptomycin (Meiji Seika Kaisha, Tokyo, Japan). To propagate podocytes, we cultivated cells at 33°C (permissive conditions), and the culture medium was supplemented with 10 U/ml mouse rIFN- (Pepro Tech) to enhance expression of a thermosensitive T antigen. To induce differentiation, we maintained podocytes at 37°C without IFN- (nonpermissive conditions) for at least 1 wk before using in the experiment.
Induction of Proteinuric States
All experiments were performed using specific pathogen-free female Wistar rats (6–8 wk old) purchased from Charles River Japan (Atsugi, Japan). Procedures for the present study were approved by the Animal Committee at Niigata University School of Medicine, and all animals were treated according to the guidelines for animal experimentation of Niigata University.
ANA nephropathy. ANA nephropathy was induced in rats by a single injection of 15 mg of anti-nephrin mAb 5-1-6 into the tail vein. Three rats each were killed just before the injection of mAb 5-1-6 and 24 h and 5 days after the injection. Small pieces of rat kidney tissues were snap-frozen and used for the immunofluorescence study. A portion of the kidney was used for IEM analysis. The glomeruli were isolated from the remaining cortexes using a graded sieving technique and were saved for protein extraction. Twenty-four-hour urine samples were collected, and their protein concentrations were measured by colorimetric assay with a Bio-Rad Protein Assay Reagent (Bio-Rad) using BSA as a standard.
PAN nephropathy. The rats were intravenously injected with 10 mg/100 g body wt of PAN, and three rats each were killed just before the injection and 24 h and 10 days afterward. The cryostat sections for the immunofluorescence study and the glomeruli for the protein extraction were prepared as described above.
RESULTS
Cloning of Rat JAM4 cDNA
The complete nucleotide sequences of the rat homolog of JAM4 and its deduced amino acid sequences are shown in Fig. 1A. The nucleotide sequencing analysis revealed an open reading frame of 1,110 nucleotides encoding a predicted protein of 369 amino acids. The deduced amino acid sequence of rat JAM4 showed 85.9% identity to the amino acid sequence of mouse JAM4 (Fig. 1B). The Expasy search showed that rat JAM4 has a single transmembrane domain and a putative hydrophobic signal peptide at its NH2 terminus. The rat JAM4 has the potential PDZ-binding motif at its COOH terminus.
JAM4 is Expressed at the Apical Surface of the Podocyte
An antibody to the 15-amino acid peptide of rat JAM4 detected a band with a molecular mass of 90–100 kDa in an extract of normal rat glomeruli (Fig. 2A). The size is compatible to the previously reported size (93 kDa) of mouse JAM4 (9). The immunohistochemical study showed that JAM4 was expressed in the glomeruli and also in the proximal tubules (Fig. 2B, a and b). This pattern of JAM4 expression was different from that of MAGI-1, the expression of which was observed in the glomeruli in a podocyte pattern along the capillary wall but not in the proximal or distal tubules. These results were consistent with the previous reports by Patrie et al. (31) and Hirabayashi et al. (9). The dual-labeling immunofluorescence study with cell markers showed that JAM4 was colocalized with podocalyxin in the glomeruli (Fig. 2Cc) but not with Thy-1 (Fig. 2Ca) or RECA-1 (Fig. 2Cb), which indicates that JAM4 is specifically expressed in podocytes in glomeruli.
The precise localization of JAM4 in the podocytes was examined by immunoelectron microscopy (IEM). Immunogold particles for JAM4 were mainly detected above the insertion point of the SD (Fig. 2D, a–c), which indicates that JAM4 is localized at the apical surface of the podocytes. Some gold particles were found in the cytoplasm, where an electron-dense structure was detected. Gold particles labeling JAM4 were also detected at the apical area of the proximal tubules (Fig. 2Dd).
We confirmed that the polyclonal antibody recognizes rat JAM4 expressed on COS cells transfected with a cDNA fragment of rat JAM4 (Fig. 2E).
Expression of JAM4 in Cultured Podocytes
Using the anti-JAM4 antibody, we investigated the expression of JAM4 in cultured podocytes. Weak but clear signals for JAM4 were detected mainly in the cytoplasm of the podocytes (Fig. 2F). The signals were distinguished from those for ZO-1, a marker of a tight junction.
Localization of JAM4 and MAGI-1 in Developing Glomeruli
Next, we studied the expression of JAM4 and MAGI-1 in developing glomeruli of an embryonic rat at embryonic day 18.5. We also performed a dual-labeling immunofluorescence study of JAM4 and MAGI-1 with nephrin. Nephrin is reported to be first expressed at the S-shaped body stage in developing glomeruli (15, 16). JAM4 immunoreactions were already detected at the early vesicle, and the comma-shaped body stages. In these stages, JAM4 staining was broadly detected along the cell surface (Fig. 3Aa). At the S-shaped-body stage, JAM4 staining was detected on the presumptive podocytes and the presumptive proximal tubules (Fig. 3Bb). In contrast to JAM4, MAGI-1 staining was not detected in the renal vesicle or the comma-shaped body. MAGI-1 was first detected at the early capillary loop stage (Fig. 3Ca). Dual-labeling immunofluorescence demonstrated that MAGI-1 and nephrin were in close proximity at the basal margin of the presumptive podocytes (Fig. 3Db).
JAM4 Expression is Altered and Distinct from MAGI-1 in PAN Nephropathy But Not in ANA Nephropathy
To clarify the role of JAM4 in the podocyte, we investigated the expression of JAM4 in ANA and in PAN nephropathy. The average values of urinary protein on days 1 and 5 of ANA nephropathy were 46.6 ± 48.8 and 215.8 ± 47.9 mg/24 h, respectively. Those on days 1, 7, and 10 of PAN nephropathy were 4.5 ± 2.0, 124.2 ± 80.1, and 238.7 ± 96.1 mg/24 h, respectively. We considered day 5 of ANA nephropathy and day 10 of PAN nephropathy as the peak time points of proteinuria and examined the expressions of JAM4 and other molecules at these time points of each model. By electron microscopy, an extensive flattening of the podocyte foot process was observed on day 10 of PAN nephropathy, but not on day 5 of ANA nephropathy, as previously reported (17, 29).
Because JAM4 was first reported to localize at the SD in vivo and to bind to MAGI-1 in vitro (9), we compared the expression of JAM4 with that of MAGI-1 in normal and proteinuric states by confocal dual-labeling immunofluorescence (Fig. 4A).
In normal glomeruli, JAM4 and MAGI-1 showed a close localization along the capillary loop (Fig. 4A, g and j). In ANA nephropathy, the staining pattern of JAM4 did not change significantly on day 1 (data not shown) and not even on day 5, when severe proteinuria was observed (Fig. 4Ab). In PAN nephropathy, increased staining of JAM4 in a dotlike pattern was observed (Fig. 4Ac) on day 10, when severe proteinuria was observed, although no significant change was observed on day 1 (data not shown).
MAGI-1 staining did not change significantly in ANA nephropathy (Fig. 4Ae). In PAN nephropathy, the staining pattern of MAGI-1 was not so uniformly distributed as in normal or in ANA nephropathy, although the overall intensity of the MAGI-1 staining did not change (Fig. 4Af). Some portions of JAM4 staining were clearly distinct from the MAGI-1 staining in PAN nephropathy (Fig. 4A, i and l).
Next, we performed a dual-labeling immunofluorescence study of JAM4 with other SD components, nephrin (34) and ZO-1 (35).
In normal glomeruli, JAM4 staining was closely localized with nephrin (Fig. 4B, g and j) and ZO-1 (Fig. 4C, e and g). In ANA nephropathy, nephrin staining shifted to a discontinuous pattern (Fig. 4Be); however, no similar shift in JAM4 staining was observed. A portion of JAM4 staining was clearly distinct from that of nephrin (Fig. 4B, h and k). Nephrin staining was dramatically lower in PAN nephropathy (Fig. 4Bf) and did not always colocalize with JAM4 staining, which was focally increased in the glomeruli (Fig. 4B, i and l).
The signals for ZO-1 were discontinuously distributed with variable intensity along the capillary wall in PAN nephropathy. This finding is in line with the results reported by Kurihara et al. (23). The localization of JAM4 did not always coincide with that of ZO-1 (Fig. 4C, f and h).
JAM4 and Ezrin Colocalize in the Normal State and PAN Nephropathy
Because signals of JAM4 were detected at the apical membrane of podocytes by IEM, we performed a confocal dual-labeling immunofluorescence study of JAM4 with ezrin, which is known to be expressed along the apical membrane of podocytes. We found that ezrin strikingly overlaps with JAM4 in both glomeruli and apical proximal tubules (Fig. 4D, e and g). In PAN nephropathy, the staining intensity of ezrin was focally increased in the podocytes of most of the glomeruli. The colocalization of ezrin and JAM4 was maintained in PAN nephropathy (Fig. 4D, f and h).
MAGI-1 is Dissociated from Nephrin in PAN Nephropathy
We also compared the localization of MAGI-1 with that of nephrin. In normal glomeruli, MAGI-1 and nephrin were closely localized. The granular staining of nephrin did not always coincide with MAGI-1 staining in ANA nephropathy (Fig. 5, h and k) or in PAN nephropathy (Fig. 5, i and l).
JAM4 Dissociates from the Actin Cytoskeleton in PAN Nephropathy
To analyze whether the amounts of protein of JAM4 are changed in pathological conditions, Western blot analysis was performed using the glomerular lysate solubilized with SDS-PAGE sample buffer. No increase in immunoreactive protein of JAM4 was detected in either ANA or PAN nephropathy (Fig. 6A). Next, we investigated the distribution of JAM4 in normal and pathological conditions with the different detergent extracts. Glomeruli isolated from normal, ANA-, and PAN-treated rats were sequentially solubilized in 1% Triton X-100 and RIPA lysis buffer and separated into TXS, RIPAS, and RIPAI fractions, followed by immunoblotting (Fig. 6B). The ratio of JAM4 in each fraction did not change noticeably in ANA, but the ratio of the amount of JAM4 in the Triton-X-100-soluble fraction to the total amount increased in PAN (Fig. 6C), indicating that JAM4 is dissociated from the actin cytoskeleton in PAN nephropathy.
JAM4 is Expressed on the Apical Membrane in PAN Nephropathy
Results obtained by electron microscopy after immunogold labeling of ultrathin cryosections (Fig. 7) revealed that JAM4 was expressed mainly on the apical plasma membrane of the podocytes with effaced foot processes. The signals were not accumulated at the site of cell-cell contact of podocytes.
DISCUSSION
Although knowledge about the molecular composition of the podocyte has been accumulated, little is known about how these components are organized and how the unique structure of the podocyte foot process is maintained. Here, we have shown that JAM4 is localized at the apical surface of the podocytes and that JAM4 staining is altered and clearly distinct from that of MAGI-1 and other SD-associated molecules in PAN nephropathy.
JAM4 was identified as a molecule interacting with MAGI-1 (9). MAGI-1 is a membrane-associated guanylate kinase (MAGUK) protein located at tight junctions in epithelial cells, and is reported to interact with various molecules, functioning as a scaffold protein at cell junctions (5, 36, 13). JAM4 is a member of the immunoglobulin superfamily and shares a similar molecular structure to the coxsackie and adenovirus receptor (CAR) (2) and endothelial cell-selective adhesion molecule (ESAM) (10): it has two Ig-like domains, a single transmembrane domain, and a cytoplasmic tail that ends in a canonical class-I PDZ domain-binding sequence (6). Hirabayashi et al. (9) have reported that both JAM4 and MAGI-1 are localized at the SD of the podocyte and suggested that they are involved in maintaining the barrier function of the podocyte. However, the precise role of these molecules in the podocyte remains uncertain. To elucidate that, we began this study by investigating the expression of these molecules in normal adult glomeruli, developing glomeruli and glomeruli with podocyte-specific injuries. We cloned a rat homolog of JAM4 and prepared an anti-rat JAM4 antibody in rabbits by immunizing them against the specific peptide of the intracellular site of rat JAM4.
Immunofluorescence study showed that JAM4 was expressed in the glomeruli and also in the proximal tubules but that MAGI-1 staining was restricted to the glomeruli. Developmental analysis in this study showed that the antibody to JAM4 detected signals at all stages of developing glomeruli and the presumptive proximal tubules in the embryonic rat kidney, whereas MAGI-1 was only seen after the capillary loop stage and the expression was restricted to the presumptive podocyte. Tajima et al. (40) also analyzed the expressions of JAM4 and MAGI-1 in several organs and showed that their localization does not always coincide. These findings raise the possibility that JAM4 might interact with other scaffold proteins besides MAGI-1, especially in vivo.
JAM4 is structurally related to other JAMs, and it has many characteristics as a junctional molecule: JAM4 mediates homophilic adhesion, is accumulated at cell-cell contacts in transfected L cells, and reduces permeability of Chinese hamster ovary cell monolayers (9). However, our findings through IEM demonstrated that JAM4 was mainly expressed in the apical area of podocytes, where no junctional structure was seen. Close localization of JAM4 with ezrin, a molecule expressed in the apical plasma membrane of podocytes, also supports the apical localization of JAM4 in podocytes. In proximal tubule cells, signals for JAM4 were detected at the brush border of the proximal tubules by IEM. Tajima et al. (40) have reported that JAM4 expression is mainly observed on the apical surface, not at tight junctions, of the epithelium in the jejunum and ileum. They have also observed that JAM4 is expressed on the apical membrane in transformants of Madin-Darby canine kidney cells. Furthermore, JAM4 is known to be heavily modified with N-glycosylation (9), which is known as an apical sorting signal in epithelial cells (11). All these data suggest that JAM4 can also be expressed at free apical membranes in epithelial cells. The discrepancy between our result and the previous report by Hirabayashi et al. (9) which locates JAM4 at the SD in the podocytes, might be explained by the existence of variants of JAM4. Several cell adhesion molecules are known to have several isoforms with different cytoplasmic domains and different surface localizations in polarized cells (7, 39). In the present study, we used a specific antibody against the rat JAM4 peptides, whereas in the previous study an antibody raised against the fusion protein of intracellular mouse JAM4 was used. It is possible that our antibody reacts to a variant of JAM4 that lacks the signal sequence for the junctional targeting and that the antibody used in the previous study might react more specifically to the junctional variant. In cultured podocytes, JAM4 expression was detected mainly in the cytoplasm and no signal was detected at the cell-cell contact site where ZO-1 expression was found. This observation may also suggest that JAM4 is not always colocalized with junctional proteins or concentrated at the sites of cell-cell contact, although the cytoplasmic localization of JAM4 may be the result of limited differentiation of the cells.
The expression of JAM4 was also analyzed in two proteinuric rat models, in which podocyte injuries are induced by different stimuli. In PAN nephropathy, it has been reported that oxygen radicals, which are produced during the metabolism of PAN, contribute to podocyte damage (3, 28). PAN nephropathy is thought to mimic human idiopathic nephrotic syndrome and has been used to analyze its pathogenesis (4). ANA nephropathy is caused by injecting anti-nephrin antibody, which binds to the extracellular site of nephrin (29, 44). The most remarkable finding by immunofluorescence in these two proteinuric models was that the signals of JAM4 increased focally in the glomeruli of PAN-treated rats. It should be noted that no altered expression of JAM4 was observed in ANA nephropathy, in which the structural integrity of the podocyte foot process is maintained throughout the proteinuric period despite the altered nephrin expression and the resultant massive proteinuria. These findings indicate that the altered expression of JAM4 is likely associated with the rearrangement of the cytoskeleton during foot process effacement of podocytes.
We demonstrated that JAM4 immunostaining was significantly close to ezrin and was distinct from those of the SD components in PAN nephropathy and that the signals for JAM4 by IEM were mainly detected along the free apical plasma membrane of the podocytes with effaced foot processes. Recently, Nagai et al. (27) have reported that CAR, which has molecular structural similarity with JAM4, is expressed at the cell-cell contact site of the podocyte, and that in PAN nephropathy, the expression of CAR is increased and the signals of CAR by IEM are found at the newly formed junctions. These findings indicate that despite their molecular structural similarity, JAM4 and CAR must have different functions in podocytes.
Western blot analysis using sequentially solubilized glomerular materials demonstrated that the change in JAM4 immunostaining in PAN nephropathy was not associated with the increased protein expression. Kerjaschki (18) reported that formation of junctions between foot processes during podocyte injury caused by protamine sulfate is not influenced by cold temperatures (18), which suggests that there is a pool of unassembled proteins in the cytoplasm that is recruited to the junctional areas as a consequence of events. Although JAM4 was not concentrated in cell-cell junctions in PAN nephropathy, the altered immunofluorescent pattern in PAN nephropathy can be explained by redistribution of the unassembled protein. The sporadic gold particles for JAM4 in cytoplasm of the normal podocytes are in accord with this assumption.
It has been shown that podocalyxin, a sialomucin, plays a major role in keeping the urinary space by virtue of the physicochemical properties of its highly negatively charged ectodomain (19, 41) and that Na+/H+ exchanger-regulatory factor-2 (NHERF-2) and ezrin colocalize with podocalyxin along the apical plasma membrane of podocytes, where they form a coimmunoprecipitable complex (24, 30, 43). Takeda et al. (42) also demonstrated that the interaction of the podocalyxin/NHERF-2/ezrin complex with the actin cytoskeleton is disrupted in pathological conditions associated with changes in the foot processes. In this study, we demonstrated that dissociation of JAM4 from the actin cytoskeleton occurred in PAN nephropathy but not in ANA nephropathy. It is possible that JAM4 might be involved in the associated structure with these molecules at the apical membrane, and its interaction with the actin cytoskeleton may be affected by the same signal during the cytoskeletal change in the podocyte. Further study is needed to clarify how JAM4 is coupled to the actin cytoskeleton and whether altered distribution of JAM4 is directly related to the cytoskeletal rearrangement during foot process effacement.
Hugo et al. (12) have reported that ezrin immunostaining intensity is increased in PAN nephropathy, and the staining identified areas of podocytic injury such as adhesion to the parietal glomerular epithelial cells, pseudocyst formation, podocyte vacuolation, and polynucleation of the podocyte. These data and the current results suggest that the altered expression of these apical components sensitively reflects the morphological changes in the podocyte. The expression of these molecules may be a useful marker for detecting injured podocytes in clinical settings.
In conclusion, JAM4 is expressed at the apical surface of podocytes and its altered expression is likely associated with the cytoskeletal changes in injured podocytes.
GRANTS
This work was supported by Grants-Aid for Scientific Research (B13557084 and B14370317 to H. Kawachi and B15390268 to F. Shimizu) from the Ministry of Education, Science, Culture, and Sports of Japan.
ACKNOWLEDGMENTS
Mutsumi Kayaba and Chiharu Nagasawa are gratefully acknowledged for tremendous technical assistance. We also thank Masaaki Nameta for indispensable help in the ultrastructural techniques. We express our gratitude to Dr. Takashi Sekine (Dept. of Pediatrics, The University of Tokyo) for helpful discussions.
FOOTNOTES
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.
REFERENCES
Benzing T. Signaling at the slit diaphragm. J Am Soc Nephrol 15: 1382–1391, 2004.
Cohen CJ, Shieh JT, Pickles RJ, Okegawa T, Hsieh JT, and Bergelson JM. The coxsackie virus and adenovirus receptor is a transmembrane component of the tight junction. Proc Natl Acad Sci USA 98: 15191–15196, 2001.
Diamond JR, Bonventre JV, and Karnovsky MJ. A role for oxygen free radicals in aminonucleoside nephrosis. Kidney Int 29: 478–483, 1986.
Diamond JR and Karnovsky MJ. Focal and segmental glomerulosclerosis following a single intravenous dose of puromycin aminonucleoside. Am J Pathol 122: 481–487, 1986.
Dobrosotskaya I, Guy RK, and James GL. MAGI-1, a membrane-associated guanylate kinase with a unique arrangement of protein-protein interaction domains. J Biol Chem 272: 31589–31597, 1997.
Ebnet K, Suzuki A, Ohno S, and Vestweber D. Junctional adhesion molecules (JAMs): more molecules with dual functions J Cell Sci 117: 19–29, 2004.
El Nemer W, Colin Y, Bauvy C, Codogno P, Fraser RH, Cartron JP, and Le Van Kim C. Isoforms of the Lutheran/basal cell adhesion molecule glycoprotein are differentially delivered in polarized epithelial cells. Mapping of the basolateral sorting signal to a cytoplasmic di-leucine motif. J Biol Chem 274: 31903–31908, 1999.
Han GD, Koike H, Nakatsue T, Suzuki K, Yoneyama H, Narumi S, Kobayashi N, Mundel P, Shimizu F, and Kawachi H. IFN-inducible protein-10 has a differential role in podocyte during Thy-1 glomerulonephritis. J Am Soc Nephrol 14: 3111–3126, 2003.
Hirabayashi S, Tajima M, Yao I, Nishimura W, Mori H, and Hata Y. JAM4, a junctional cell adhesion molecule interacting with a tight junction protein, MAGI-1. Mol Cell Biol 23: 4267–4282, 2003.
Hirata K, Ishida T, Penta K, Razaee M, Yang E, Wohlgemuth J, and Quertermous T. Cloning of an immunoglobulin family adhesion molecule selectively expressed by endothelial cells. J Biol Chem 276: 16223–16231, 2001.
Huet G, Gouyer V, Delacour D, Richet C, Zanetta JP, Delannoy P, and Degand P. Involvement of glycosylation in the intracellular trafficking of glycoproteins in polarized epithelial cells. Biochimie 85: 323–330, 2003.
Hugo C, Nangaku M, Shankland SJ, Pichler R, Gordon K, Amieva MR, Couser WG, Furthmayr H, and Johnson RJ. The plasma membrane-actin linking protein, ezrin, is a glomerular epithelial cell marker in glomerulogenesis, in the adult kidney and in glomerular injury. Kidney Int 54: 1934–1944, 1998.
Ide N, Hata Y, Nishioka H, Hirao K, Yao I, Deguchi M, Mizoguchi A, Nishimori H, Tokino T, Nakamura Y, and Takai Y. Localization of membrane-associated guanylate kinase (MAGI)-1/BAI-associated protein (BAP) 1 at tight junctions of epithelial cells. Oncogene 18: 7810–7815, 1999.
Kawachi H, Orikasa M, Matsui K, Iwanaga T, Toyabe S, Oite T, and Shimizu F. Epitope-specific induction of mesangial lesions with proteinuria by a MoAb against mesangial cell surface antigen. Clin Exp Immunol 88: 399–404, 1992.
Kawachi H, Abrahamson DR, St John PL, Goldstein DJ, Shia MA, Matsui K, Shimizu F, and Salant DJ. Developmental expression of the nephritogenic antigen of monoclonal antibody 5–1-6. Am J Pathol 147: 823–833, 1995.
Kawachi H, Koike H, Kurihara H, Yaoita E, Orikasa M, Shia MA, Sakai T, Yamamoto T, Salant DJ, and Shimizu F. Cloning of rat nephrin: Expression in developing glomeruli and in proteinuric states. Kidney Int 57: 1949–1961, 2000.
Kawachi H, Koike H, Kurihara H, Sakai T, and Shimizu F. Cloning of rat homologue of podocin: expression in proteinuric states and in developing glomeruli. J Am Soc Nephrol 13: 46–56, 2003.
Kerjaschki D. Polycation-induced dislocation of slit diaphragms and formation of cell junctions in rat kidney glomeruli. Lab Invest 39: 430–440, 1978.
Kerjaschki D, Sharkey DJ, and Farquhar MG. Identification and characterization of podocalyxin—the major sialoprotein of the renal glomerular epithelial cell. J Cell Biol 98: 1591–1596, 1984.
Kerjaschki D. Caught flat-footed: podocyte damage and the molecular basis of focal glomerulosclerosis. J Clin Invest 108: 1583–1587, 2001.
Kriz W, Hackenthal E, Nobiling R, Sakai T, and Elger M. A role of podocytes to counteract capillary wall distension. Kidney Int 45: 369–376, 1994.
Kriz W, Gretz N, and Lemley KV. Progression of glomerular disease: is the podocyte the culprit Kidney Int 54: 687–697, 1998.
Kurihara H, Anderson JM, Kerjaschki D, and Farquhar MG. The altered glomerular filtration slits seen in puromycin aminonucleoside nephrosis and protamine sulfate-treated rats contain the tight junction protein ZO-1. Am J Pathol 141: 805–816, 1992.
Li Y, Li J, Straight SW, and Kershaw DB. PDZ domain-mediated interaction of rabbit podocalyxin and Na+/H+ exchange regulatory factor-2. Am J Physiol Renal Physiol 282: F1129–F1139, 2002.
Mundel P and Reiser J, Zuniga Mejia Borja A, Pavenstadt H., Davidson GR, Kriz W, and Zeller R. Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines. Exp Cell Res 236: 248–258, 1997.
Mundel P and Shankland SJ. Podocyte biology and response to injury. J Am Soc Nephrol 13: 3005–3015, 2002.
Nagai M, Yaoita E, Yoshida Y, Kuwano R, Nameta M, Ohshiro K, Isome M, Fujinaka H, Suzuki S, Suzuki J, Suzuki H, and Yamamoto T. Coxsackie virus and adenovirus receptor, a tight junction membrane protein, is expressed in glomerular podocytes in the kidney. Lab Invest 83: 901–911, 2003.
Nosaka K, Takahashi T, Nishi T, Imaki H, Suzuki T, Suzuki K, Kurokawa K, and Endou H. An adenosine deaminase inhibitor prevents puromycin aminonucleoside nephrotoxicity. Free Radic Biol Med 22: 597–605, 1997.
Orikasa M, Matsui K, Oite T, and Shimizu F. Massive proteinuria induced in rats by a single intravenous injection of a monoclonal antibody. J Immunol 141: 807–814, 1988.
Orlando RA, Takeda T, Zak B, Shmieder S, Benoit VM, Mcquistan T, Furthmayr H, and Farquhar MG. The glomerular epithelial cell anti-adhesion podocalyxin associates with the actin cytoskeleton through interactions with ezrin. J Am Soc Nephrol 12: 1589–1598, 2001.
Patrie KM, Drescher AJ, Goyal M, Wiggins RC, and Margolis B. The membrane-associated guanylate kinase protein MAGI-1 binds megalin and is present in glomerular podocytes. J Am Soc Nephrol 12: 667–677, 2001.
Pavenstadt H, Kriz W, and Kretzler M. Cell biology of the glomerular podocyte. Physiol Rev 83: 253–307, 2003.
Rodewald R and Karnovsky MJ. Porous substructure of the glomerular slit diaphragm in the rat and mouse. J Cell Biol 60: 423–433, 1974.
Ruotsalainen V, Ljungberg P, Wartiovaara J, Lenkkeri U, Kestila M, Jalanko H, Holmberg C, and Tryggvason K. Nephrin is specifically located at the slit diaphragm of glomerular podocytes. Proc Natl Acad Sci USA 96: 7962–7967, 1999.
Schnabel E, Anderson JM, and Farquhar MG. The tight junction protein ZO-1 is concentrated along slit diaphragms of the glomerular epithelium. J Cell Biol 111: 1255–1263, 1990.
Shiratsuchi T, Futamura M, Oda K, Nishimori H, Nakamura Y, and Tokino T. Cloning, and characterization of BAI-associated protein 1: a PDZ domain-containing protein that interacts with BAI1. Biochem Biophys Res Commun 247: 597–604, 1998.
Smoyer WE and Mundel P. Regulation of podocyte structure during the development of nephrotic syndrome. J Mol Med 76: 172–183, 1998.
Somlo S and Mundel P. Getting a foothold in nephrotic syndrome. Nat Genet 24: 333–335, 2000.
Sundberg U and Obrink B. CEACAM1 isoforms with different cytoplasmic domains show different localization, organization and adhesive properties in polarized epithelial cells. J Cell Biol 115: 1273–1284, 2002.
Tajima M, Hirabayashi S, Yao I, Shirasawa M, Osuga J, Ishibashi S, Fujita T, and Hata Y. Roles of immunoglobulin-like loops of junctional cell adhesion molecule 4; involvement in the subcellular localization and the cell adhesion. Genes Cells 8: 759–768, 2003.
Takeda T, Go WY, Orlando RA, and Farquhar MG. Expression of podocalyxin inhibits cell-cell adhesion and modifies junctional properties in Madin-Darby canine kidney cells. Mol Biol Cell 11: 3219–3232, 2000.
Takeda T, McQuistan T, Orlando RA, and Farquhar MG. Loss of glomerular foot processes is associated with uncoupling of podocalyxin from the actin cytoskeleton. J Clin Invest 108: 289–301, 2001.
Takeda T. Podocyte cytoskeleton is connected to the integral membrane protein podocalyxin through Na+/H+-exchanger regulatory factor 2 and ezrin. Clin Exp Nephrol 7: 260–269, 2003.
Topham PS, Kawachi H, Haydar SA, Chugh S, Addona TA, Charron KB, Holzman LB, Shia M, Shimizu F, and Salant DJ. Nephritogenic mAb 5–1-6 is directed at the extracellular domain of rat nephrin. J Clin Invest 104: 1559–1566, 1999.(Yutaka Harita, Naoko Miyauchi, Tamaki Ka)
2Department of Pediatrics, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
ABSTRACT
Recent investigations have revealed the importance of glomerular podocytes with its diaphragm as the major filtration barrier. Junctional adhesion molecule 4 (JAM4) has been identified as a protein that interacts with membrane-associated guanyl kinase inverted (MAGI)-1 and is reported to be expressed on podocytes. To elucidate the role of JAM4 on podocytes, we examined the expression of JAM4 and MAGI-1 in normal and two different proteinuric rat models: puromycin aminonucleoside (PAN) nephropathy and anti-nephrin antibody-induced (ANA) nephropathy, one model with and one without effacement of podocyte foot processes. JAM4 was detected by immunomicroscopy at the apical membrane of normal podocytes. JAM4 immunostaining was focally increased in the podocytes in PAN nephropathy but not in ANA nephropathy. In proteinuric podocytes, the expression of JAM4 was distinct from that of MAGI-1 or other slit diaphragm molecules such as nephrin and ZO-1. Close colocalization of JAM4 and ezrin was maintained in PAN nephropathy. By immunoelectron microscopy, the signals for JAM4 were detected at the free apical membrane of the podocytes with effaced foot processes. Studies with selective detergent extract revealed that the subcellular localization of JAM4 was altered in PAN nephropathy. Thus the altered expression of JAM4 appears to be associated with morphological changes in podocytes and can be a useful marker of injured podocytes. JAM4 may have a different role at the apical membrane besides the role as a junctional molecule and is likely associated with the unique structure of this epithelium.
podocyte; puromycin aminonucleoside nephropathy; foot process effacement; membrane-associated guanyl kinase inverted-1
THE VISCERAL GLOMERULAR EPITHELIAL cell, also called the podocyte, is a highly specialized, terminally differentiated cell that lines the outer aspect of the glomerular basement membrane (GBM). Podocytes possess interdigitating cell extensions, called foot processes, which are bridged by the slit diaphragm (SD), a podocyte-specific intercellular junction with an electron-dense zipper-like structure (33). The recent discovery of several novel components of the SD (nephrin, Neph1, P-cadherin, etc.) and their physical and functional interactions with intracellular adaptor proteins (ZO-1, CD2AP, podocin, etc.) revealed the region of the SD as the major filtration barrier (1, 32). Another specialized feature of the podocyte is a specific cytoskeletal organization, which plays a critical role in counteracting the high transmural distending force permitting the high-pressure perfusion through glomerular capillaries (21). When podocytes are injured, as in many different pathological settings such as minimal change disease, diabetic nephropathy, lupus nephritis, and several other acquired and genetic kidney diseases, the podocytes react in a particular pattern, retracting their foot processes into their cell bodies to form a flattened, simple epithelium (26, 37). These changes in the podocytes lead to precipitous protein leakage into urine, and if they are not reversed, they ultimately lead to the development of segmental glomerulosclerosis and end-stage renal failure (20, 22, 38). However, the molecular events that cause morphological changes in the podocyte in these etiologically diverse diseases have not been fully clarified.
Junctional adhesion molecule (JAM) is a member of the immunoglobulin superfamily expressed at cell-cell junctions (6). Recently, Hirabayashi et al. (9) have identified JAM4 as a protein that interacts with membrane-associated guanyl kinase inverted (MAGI)-1 in mouse lung tissue. JAM4 has two Ig-like domains, a single transmembrane domain, and a cytoplasmic tail that ends in a PDZ-domain-binding sequence. JAM4 is expressed at podocytes and the apical membranes of renal tubular cells and small intestine epithelial cells (9, 40). JAM4 is speculated to play a role in glomerular permselectivity, because JAM4 reduces paracellular permeability when expressed in Chinese hamster ovary cells (9). However, to date, the functional roles of JAM4 in the glomeruli have not been fully understood.
In an attempt to elucidate the role of JAM4 in maintaining the barrier function of the SD and the interdigitating structure of podocytes, we investigated the expression of JAM4 and MAGI-1 in the developing kidney and in normal and two proteinuric adult rat models. One proteinuric rat model was anti-nephrin antibody-induced (ANA) nephropathy, in which the structural integrity of the SD is largely unaffected despite massive proteinuria. The other was puromycin aminonucleoside (PAN) nephropathy, which is characterized by extensive flattening of the foot processes. We show here that JAM4 is expressed at the apical membrane of podocytes, and its expression is altered in proteinuric glomeruli. JAM4 can be a useful marker for injured podocytes.
MATERIALS AND METHODS
Cloning of Rat JAM4
To clone a rat homolog of JAM4, we employed a PCR with the primers designed from a mouse JAM4 sequence. PCR cloning was performed basically in accordance with the method previously described (17). Total RNA was prepared from normal Wistar rat glomeruli with TRIzol (Invitrogen, Carlsbad, CA) and was utilized to synthesize cDNA with SuperScript II (Invitrogen) according to the manufacturer's protocol. Synthesized cDNA was used as a template. Templates were amplified in DNA with Program Temperature Control System PC-800 (ASTEC, Fukuoka, Japan) for 40–50 cycles at an annealing temperature of 50–58°C. The PCR products that were close to an expected size were cloned into a Topo Vector (Invitrogen), and DNA sequences were determined by an automated DNA sequencer (ABI 310, PerkinElmer Japan, Urayasu, Japan). From a partial clone of rat JAM4, gene-specific primers were designated and PCR was performed again.
Antibodies
To produce an antibody specific to rat JAM4, we chose a peptide of 15 amino acids, YKSDEARAAQIASLP, as the immunogen. This peptide was purchased from GenScript (Scotch Plains, NJ). Three rabbits were immunized with 2.0 mg of the peptide conjugated with the carrier protein KLH. The rabbits were bled 2 wk after the last immunization. The specificity of the anti-JAM4 antibody activity was confirmed by Western blot analysis. Mouse monoclonal antibody to rat nephrin (mAb 5-1-6) and mouse monoclonal antibody to rat Thy-1 antigen (mAb 1-22-3) were prepared in our laboratory (14, 29). Mouse monoclonal anti-ZO-1 antibody and anti-ezrin antibody (3C12) were purchased from Zymed Laboratories (San Francisco, CA). Mouse anti-rat endothelial cell antigen (RECA-1) antibody, goat anti-MAGI-1 antibody, and rabbit anti-laminin antibody were purchased from Serotec (Oxford, UK), Santa Cruz Biotechnology (Santa Cruz, CA), and Sigma (St. Louis, MO), respectively. Mouse anti-podocalyxin antibody (4D5) was donated by Dr. M. Hara (Yoshida Hospital, Niigata, Japan) (8).
Immunofluorescence Microscopy
Immunofluorescence studies were performed in accordance with the method previously described (17). The renal tissue was quickly frozen in n-hexane cooled to –70°C. Three-micrometer-thick frozen sections were cut with a cryostat, fixed with periodate-lysine-paraformaldehyde (PLP) for 1 min, incubated with the primary antibodies described above, and stained with FITC-conjugated anti-rabbit IgG (for anti-JAM4; Dako, Glostrup, Denmark) or FITC-conjugated anti-goat IgG (for anti-MAGI-1; Southern Biotechnology Associates, Birmingham, AL). For dual-labeling immunofluorescence, cryostat sections were incubated with anti-JAM4 antibody, FITC-conjugated donkey anti-rabbit IgG, then anti-Thy-1, anti-RECA-1, anti-podocalyxin, anti-MAGI-1, anti-nephrin, anti-ZO-1, or anti-ezrin antibody. Tetramethyl-rhodamine isothiocyanate (TRITC)-conjugated goat anti-mouse IgG1 (for anti-nephrin, anti-RECA-1, anti-ZO-1 and anti-ezrin; Southern Biotechnology Associates); TRITC-conjugated goat anti-mouse IgG2a (for anti-podocalyxin; Southern Biotechnology Associates); TRITC-conjugated goat anti-mouse IgG3 (for anti-Thy-1; Southern Biotechnology Associates); or TRITC-conjugated donkey anti-goat IgG (for anti-MAGI-1; Santa Cruz Biotechnology) was used as the secondary antibody. The sections were observed with a laser-scanning confocal microscope (MRC-1024, Bio-Rad Laboratories, Hercules, CA) with the appropriate filters for FITC (488-nm excitation, HQ515/30 emission filter) and TRITC (568-nm excitation, 585LP filter). The images were further processed using Adobe Photoshop CS 8.0.1.
Immunoelectron Microscopy
The tissues were fixed with PLP fixative by perfusion and immersion and then embedded in glycol methacrylate. Ultrathin sections of the glycol methacrylate-embedded tissues were collected on nickel grids and incubated with 5% nonfat milk for 1 h and then with the anti-JAM4 antibody for 1 h. The sections were incubated with gold (10 nm)-labeled anti-rabbit IgG (Amersham Life Sciences, Buckinghamshire, UK) for 1 h, washed in PBS several times, and then counterstained with 2% aqueous uranyl acetate and 2% lead citrate for observation by electron microscope (H-600A, Hitachi, Tokyo, Japan).
Western Blot Analysis
Western blot analysis with sequentially solubilized glomerular lysates was performed basically in accordance with the method previously described (17). Normal or proteinuric rat glomeruli were isolated with PBS containing protease inhibitors and solubilized with 1% Triton X-100. After the lysate was centrifuged, the pellet was reextracted with RIPA buffer (consisting of 0.1% SDS, 1% sodium deoxycholate, 1% Triton X-100, 150 mmol/l NaCl, and 10 mmol/l EDTA in 25 mmol/l Tris·HCl, pH 7.2), and the lysate was again centrifuged. The lysate was separated into Triton X-100-soluble (TXS), Triton X-100-insoluble, RIPA-soluble (RIPAS), and RIPA-insoluble fractions (RIPAI). The RIPA-insoluble fraction was solubilized with SDS-PAGE sample buffer (2% SDS, 10% glycerol, 5% 2-mercaptoethanol in 62.5 mM Tris·HCl, pH 6.8). The insoluble material was removed by centrifugation at 15,000 g for 30 min at 4°C. The concentration was measured by the bicinchoninic acid method (Pierce Chemical, Rockford, IL), and equal amounts of these sequentially solubilized fractions were subjected to SDS-PAGE on 7.5% acrylamide gel in accordance with the method of Laemmli and transferred to a PVDF membrane (Pall, Pensacola, FL) by electrophoretic transblotting for 30 min using Trans-Blot SD (Bio-Rad, Hercules, CA). After blocking with bovine skim milk, strips of the membranes were exposed to rabbit anti-actin antibody (Sigma) or rabbit anti-JAM4 antibody. They were washed and then incubated with alkaline phosphatase-conjugated anti-rabbit IgG (Bio Source International, Camarillo, CA). The reaction was developed with an alkaline phosphatase chromogen kit (5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt/nitro blue tetrazolium; Biomedica, Foster City, CA). The density of the positive bands was quantitated by LabWorks (UVP, Upland, CA). This procedure was carried out three times. The percentage of the density of each fraction relative to the total was calculated.
Culture of Podocytes
Cultivation of conditionally immortalized mouse podocytes was conducted as reported previously (25). In brief, podocytes were maintained in RPMI 1640 medium (Nissui Pharmaceutical, Tokyo, Japan), 100 U/ml penicillin (Banyu Pharmaceutical, Tokyo, Japan), and 0.1 mg/ml streptomycin (Meiji Seika Kaisha, Tokyo, Japan). To propagate podocytes, we cultivated cells at 33°C (permissive conditions), and the culture medium was supplemented with 10 U/ml mouse rIFN- (Pepro Tech) to enhance expression of a thermosensitive T antigen. To induce differentiation, we maintained podocytes at 37°C without IFN- (nonpermissive conditions) for at least 1 wk before using in the experiment.
Induction of Proteinuric States
All experiments were performed using specific pathogen-free female Wistar rats (6–8 wk old) purchased from Charles River Japan (Atsugi, Japan). Procedures for the present study were approved by the Animal Committee at Niigata University School of Medicine, and all animals were treated according to the guidelines for animal experimentation of Niigata University.
ANA nephropathy. ANA nephropathy was induced in rats by a single injection of 15 mg of anti-nephrin mAb 5-1-6 into the tail vein. Three rats each were killed just before the injection of mAb 5-1-6 and 24 h and 5 days after the injection. Small pieces of rat kidney tissues were snap-frozen and used for the immunofluorescence study. A portion of the kidney was used for IEM analysis. The glomeruli were isolated from the remaining cortexes using a graded sieving technique and were saved for protein extraction. Twenty-four-hour urine samples were collected, and their protein concentrations were measured by colorimetric assay with a Bio-Rad Protein Assay Reagent (Bio-Rad) using BSA as a standard.
PAN nephropathy. The rats were intravenously injected with 10 mg/100 g body wt of PAN, and three rats each were killed just before the injection and 24 h and 10 days afterward. The cryostat sections for the immunofluorescence study and the glomeruli for the protein extraction were prepared as described above.
RESULTS
Cloning of Rat JAM4 cDNA
The complete nucleotide sequences of the rat homolog of JAM4 and its deduced amino acid sequences are shown in Fig. 1A. The nucleotide sequencing analysis revealed an open reading frame of 1,110 nucleotides encoding a predicted protein of 369 amino acids. The deduced amino acid sequence of rat JAM4 showed 85.9% identity to the amino acid sequence of mouse JAM4 (Fig. 1B). The Expasy search showed that rat JAM4 has a single transmembrane domain and a putative hydrophobic signal peptide at its NH2 terminus. The rat JAM4 has the potential PDZ-binding motif at its COOH terminus.
JAM4 is Expressed at the Apical Surface of the Podocyte
An antibody to the 15-amino acid peptide of rat JAM4 detected a band with a molecular mass of 90–100 kDa in an extract of normal rat glomeruli (Fig. 2A). The size is compatible to the previously reported size (93 kDa) of mouse JAM4 (9). The immunohistochemical study showed that JAM4 was expressed in the glomeruli and also in the proximal tubules (Fig. 2B, a and b). This pattern of JAM4 expression was different from that of MAGI-1, the expression of which was observed in the glomeruli in a podocyte pattern along the capillary wall but not in the proximal or distal tubules. These results were consistent with the previous reports by Patrie et al. (31) and Hirabayashi et al. (9). The dual-labeling immunofluorescence study with cell markers showed that JAM4 was colocalized with podocalyxin in the glomeruli (Fig. 2Cc) but not with Thy-1 (Fig. 2Ca) or RECA-1 (Fig. 2Cb), which indicates that JAM4 is specifically expressed in podocytes in glomeruli.
The precise localization of JAM4 in the podocytes was examined by immunoelectron microscopy (IEM). Immunogold particles for JAM4 were mainly detected above the insertion point of the SD (Fig. 2D, a–c), which indicates that JAM4 is localized at the apical surface of the podocytes. Some gold particles were found in the cytoplasm, where an electron-dense structure was detected. Gold particles labeling JAM4 were also detected at the apical area of the proximal tubules (Fig. 2Dd).
We confirmed that the polyclonal antibody recognizes rat JAM4 expressed on COS cells transfected with a cDNA fragment of rat JAM4 (Fig. 2E).
Expression of JAM4 in Cultured Podocytes
Using the anti-JAM4 antibody, we investigated the expression of JAM4 in cultured podocytes. Weak but clear signals for JAM4 were detected mainly in the cytoplasm of the podocytes (Fig. 2F). The signals were distinguished from those for ZO-1, a marker of a tight junction.
Localization of JAM4 and MAGI-1 in Developing Glomeruli
Next, we studied the expression of JAM4 and MAGI-1 in developing glomeruli of an embryonic rat at embryonic day 18.5. We also performed a dual-labeling immunofluorescence study of JAM4 and MAGI-1 with nephrin. Nephrin is reported to be first expressed at the S-shaped body stage in developing glomeruli (15, 16). JAM4 immunoreactions were already detected at the early vesicle, and the comma-shaped body stages. In these stages, JAM4 staining was broadly detected along the cell surface (Fig. 3Aa). At the S-shaped-body stage, JAM4 staining was detected on the presumptive podocytes and the presumptive proximal tubules (Fig. 3Bb). In contrast to JAM4, MAGI-1 staining was not detected in the renal vesicle or the comma-shaped body. MAGI-1 was first detected at the early capillary loop stage (Fig. 3Ca). Dual-labeling immunofluorescence demonstrated that MAGI-1 and nephrin were in close proximity at the basal margin of the presumptive podocytes (Fig. 3Db).
JAM4 Expression is Altered and Distinct from MAGI-1 in PAN Nephropathy But Not in ANA Nephropathy
To clarify the role of JAM4 in the podocyte, we investigated the expression of JAM4 in ANA and in PAN nephropathy. The average values of urinary protein on days 1 and 5 of ANA nephropathy were 46.6 ± 48.8 and 215.8 ± 47.9 mg/24 h, respectively. Those on days 1, 7, and 10 of PAN nephropathy were 4.5 ± 2.0, 124.2 ± 80.1, and 238.7 ± 96.1 mg/24 h, respectively. We considered day 5 of ANA nephropathy and day 10 of PAN nephropathy as the peak time points of proteinuria and examined the expressions of JAM4 and other molecules at these time points of each model. By electron microscopy, an extensive flattening of the podocyte foot process was observed on day 10 of PAN nephropathy, but not on day 5 of ANA nephropathy, as previously reported (17, 29).
Because JAM4 was first reported to localize at the SD in vivo and to bind to MAGI-1 in vitro (9), we compared the expression of JAM4 with that of MAGI-1 in normal and proteinuric states by confocal dual-labeling immunofluorescence (Fig. 4A).
In normal glomeruli, JAM4 and MAGI-1 showed a close localization along the capillary loop (Fig. 4A, g and j). In ANA nephropathy, the staining pattern of JAM4 did not change significantly on day 1 (data not shown) and not even on day 5, when severe proteinuria was observed (Fig. 4Ab). In PAN nephropathy, increased staining of JAM4 in a dotlike pattern was observed (Fig. 4Ac) on day 10, when severe proteinuria was observed, although no significant change was observed on day 1 (data not shown).
MAGI-1 staining did not change significantly in ANA nephropathy (Fig. 4Ae). In PAN nephropathy, the staining pattern of MAGI-1 was not so uniformly distributed as in normal or in ANA nephropathy, although the overall intensity of the MAGI-1 staining did not change (Fig. 4Af). Some portions of JAM4 staining were clearly distinct from the MAGI-1 staining in PAN nephropathy (Fig. 4A, i and l).
Next, we performed a dual-labeling immunofluorescence study of JAM4 with other SD components, nephrin (34) and ZO-1 (35).
In normal glomeruli, JAM4 staining was closely localized with nephrin (Fig. 4B, g and j) and ZO-1 (Fig. 4C, e and g). In ANA nephropathy, nephrin staining shifted to a discontinuous pattern (Fig. 4Be); however, no similar shift in JAM4 staining was observed. A portion of JAM4 staining was clearly distinct from that of nephrin (Fig. 4B, h and k). Nephrin staining was dramatically lower in PAN nephropathy (Fig. 4Bf) and did not always colocalize with JAM4 staining, which was focally increased in the glomeruli (Fig. 4B, i and l).
The signals for ZO-1 were discontinuously distributed with variable intensity along the capillary wall in PAN nephropathy. This finding is in line with the results reported by Kurihara et al. (23). The localization of JAM4 did not always coincide with that of ZO-1 (Fig. 4C, f and h).
JAM4 and Ezrin Colocalize in the Normal State and PAN Nephropathy
Because signals of JAM4 were detected at the apical membrane of podocytes by IEM, we performed a confocal dual-labeling immunofluorescence study of JAM4 with ezrin, which is known to be expressed along the apical membrane of podocytes. We found that ezrin strikingly overlaps with JAM4 in both glomeruli and apical proximal tubules (Fig. 4D, e and g). In PAN nephropathy, the staining intensity of ezrin was focally increased in the podocytes of most of the glomeruli. The colocalization of ezrin and JAM4 was maintained in PAN nephropathy (Fig. 4D, f and h).
MAGI-1 is Dissociated from Nephrin in PAN Nephropathy
We also compared the localization of MAGI-1 with that of nephrin. In normal glomeruli, MAGI-1 and nephrin were closely localized. The granular staining of nephrin did not always coincide with MAGI-1 staining in ANA nephropathy (Fig. 5, h and k) or in PAN nephropathy (Fig. 5, i and l).
JAM4 Dissociates from the Actin Cytoskeleton in PAN Nephropathy
To analyze whether the amounts of protein of JAM4 are changed in pathological conditions, Western blot analysis was performed using the glomerular lysate solubilized with SDS-PAGE sample buffer. No increase in immunoreactive protein of JAM4 was detected in either ANA or PAN nephropathy (Fig. 6A). Next, we investigated the distribution of JAM4 in normal and pathological conditions with the different detergent extracts. Glomeruli isolated from normal, ANA-, and PAN-treated rats were sequentially solubilized in 1% Triton X-100 and RIPA lysis buffer and separated into TXS, RIPAS, and RIPAI fractions, followed by immunoblotting (Fig. 6B). The ratio of JAM4 in each fraction did not change noticeably in ANA, but the ratio of the amount of JAM4 in the Triton-X-100-soluble fraction to the total amount increased in PAN (Fig. 6C), indicating that JAM4 is dissociated from the actin cytoskeleton in PAN nephropathy.
JAM4 is Expressed on the Apical Membrane in PAN Nephropathy
Results obtained by electron microscopy after immunogold labeling of ultrathin cryosections (Fig. 7) revealed that JAM4 was expressed mainly on the apical plasma membrane of the podocytes with effaced foot processes. The signals were not accumulated at the site of cell-cell contact of podocytes.
DISCUSSION
Although knowledge about the molecular composition of the podocyte has been accumulated, little is known about how these components are organized and how the unique structure of the podocyte foot process is maintained. Here, we have shown that JAM4 is localized at the apical surface of the podocytes and that JAM4 staining is altered and clearly distinct from that of MAGI-1 and other SD-associated molecules in PAN nephropathy.
JAM4 was identified as a molecule interacting with MAGI-1 (9). MAGI-1 is a membrane-associated guanylate kinase (MAGUK) protein located at tight junctions in epithelial cells, and is reported to interact with various molecules, functioning as a scaffold protein at cell junctions (5, 36, 13). JAM4 is a member of the immunoglobulin superfamily and shares a similar molecular structure to the coxsackie and adenovirus receptor (CAR) (2) and endothelial cell-selective adhesion molecule (ESAM) (10): it has two Ig-like domains, a single transmembrane domain, and a cytoplasmic tail that ends in a canonical class-I PDZ domain-binding sequence (6). Hirabayashi et al. (9) have reported that both JAM4 and MAGI-1 are localized at the SD of the podocyte and suggested that they are involved in maintaining the barrier function of the podocyte. However, the precise role of these molecules in the podocyte remains uncertain. To elucidate that, we began this study by investigating the expression of these molecules in normal adult glomeruli, developing glomeruli and glomeruli with podocyte-specific injuries. We cloned a rat homolog of JAM4 and prepared an anti-rat JAM4 antibody in rabbits by immunizing them against the specific peptide of the intracellular site of rat JAM4.
Immunofluorescence study showed that JAM4 was expressed in the glomeruli and also in the proximal tubules but that MAGI-1 staining was restricted to the glomeruli. Developmental analysis in this study showed that the antibody to JAM4 detected signals at all stages of developing glomeruli and the presumptive proximal tubules in the embryonic rat kidney, whereas MAGI-1 was only seen after the capillary loop stage and the expression was restricted to the presumptive podocyte. Tajima et al. (40) also analyzed the expressions of JAM4 and MAGI-1 in several organs and showed that their localization does not always coincide. These findings raise the possibility that JAM4 might interact with other scaffold proteins besides MAGI-1, especially in vivo.
JAM4 is structurally related to other JAMs, and it has many characteristics as a junctional molecule: JAM4 mediates homophilic adhesion, is accumulated at cell-cell contacts in transfected L cells, and reduces permeability of Chinese hamster ovary cell monolayers (9). However, our findings through IEM demonstrated that JAM4 was mainly expressed in the apical area of podocytes, where no junctional structure was seen. Close localization of JAM4 with ezrin, a molecule expressed in the apical plasma membrane of podocytes, also supports the apical localization of JAM4 in podocytes. In proximal tubule cells, signals for JAM4 were detected at the brush border of the proximal tubules by IEM. Tajima et al. (40) have reported that JAM4 expression is mainly observed on the apical surface, not at tight junctions, of the epithelium in the jejunum and ileum. They have also observed that JAM4 is expressed on the apical membrane in transformants of Madin-Darby canine kidney cells. Furthermore, JAM4 is known to be heavily modified with N-glycosylation (9), which is known as an apical sorting signal in epithelial cells (11). All these data suggest that JAM4 can also be expressed at free apical membranes in epithelial cells. The discrepancy between our result and the previous report by Hirabayashi et al. (9) which locates JAM4 at the SD in the podocytes, might be explained by the existence of variants of JAM4. Several cell adhesion molecules are known to have several isoforms with different cytoplasmic domains and different surface localizations in polarized cells (7, 39). In the present study, we used a specific antibody against the rat JAM4 peptides, whereas in the previous study an antibody raised against the fusion protein of intracellular mouse JAM4 was used. It is possible that our antibody reacts to a variant of JAM4 that lacks the signal sequence for the junctional targeting and that the antibody used in the previous study might react more specifically to the junctional variant. In cultured podocytes, JAM4 expression was detected mainly in the cytoplasm and no signal was detected at the cell-cell contact site where ZO-1 expression was found. This observation may also suggest that JAM4 is not always colocalized with junctional proteins or concentrated at the sites of cell-cell contact, although the cytoplasmic localization of JAM4 may be the result of limited differentiation of the cells.
The expression of JAM4 was also analyzed in two proteinuric rat models, in which podocyte injuries are induced by different stimuli. In PAN nephropathy, it has been reported that oxygen radicals, which are produced during the metabolism of PAN, contribute to podocyte damage (3, 28). PAN nephropathy is thought to mimic human idiopathic nephrotic syndrome and has been used to analyze its pathogenesis (4). ANA nephropathy is caused by injecting anti-nephrin antibody, which binds to the extracellular site of nephrin (29, 44). The most remarkable finding by immunofluorescence in these two proteinuric models was that the signals of JAM4 increased focally in the glomeruli of PAN-treated rats. It should be noted that no altered expression of JAM4 was observed in ANA nephropathy, in which the structural integrity of the podocyte foot process is maintained throughout the proteinuric period despite the altered nephrin expression and the resultant massive proteinuria. These findings indicate that the altered expression of JAM4 is likely associated with the rearrangement of the cytoskeleton during foot process effacement of podocytes.
We demonstrated that JAM4 immunostaining was significantly close to ezrin and was distinct from those of the SD components in PAN nephropathy and that the signals for JAM4 by IEM were mainly detected along the free apical plasma membrane of the podocytes with effaced foot processes. Recently, Nagai et al. (27) have reported that CAR, which has molecular structural similarity with JAM4, is expressed at the cell-cell contact site of the podocyte, and that in PAN nephropathy, the expression of CAR is increased and the signals of CAR by IEM are found at the newly formed junctions. These findings indicate that despite their molecular structural similarity, JAM4 and CAR must have different functions in podocytes.
Western blot analysis using sequentially solubilized glomerular materials demonstrated that the change in JAM4 immunostaining in PAN nephropathy was not associated with the increased protein expression. Kerjaschki (18) reported that formation of junctions between foot processes during podocyte injury caused by protamine sulfate is not influenced by cold temperatures (18), which suggests that there is a pool of unassembled proteins in the cytoplasm that is recruited to the junctional areas as a consequence of events. Although JAM4 was not concentrated in cell-cell junctions in PAN nephropathy, the altered immunofluorescent pattern in PAN nephropathy can be explained by redistribution of the unassembled protein. The sporadic gold particles for JAM4 in cytoplasm of the normal podocytes are in accord with this assumption.
It has been shown that podocalyxin, a sialomucin, plays a major role in keeping the urinary space by virtue of the physicochemical properties of its highly negatively charged ectodomain (19, 41) and that Na+/H+ exchanger-regulatory factor-2 (NHERF-2) and ezrin colocalize with podocalyxin along the apical plasma membrane of podocytes, where they form a coimmunoprecipitable complex (24, 30, 43). Takeda et al. (42) also demonstrated that the interaction of the podocalyxin/NHERF-2/ezrin complex with the actin cytoskeleton is disrupted in pathological conditions associated with changes in the foot processes. In this study, we demonstrated that dissociation of JAM4 from the actin cytoskeleton occurred in PAN nephropathy but not in ANA nephropathy. It is possible that JAM4 might be involved in the associated structure with these molecules at the apical membrane, and its interaction with the actin cytoskeleton may be affected by the same signal during the cytoskeletal change in the podocyte. Further study is needed to clarify how JAM4 is coupled to the actin cytoskeleton and whether altered distribution of JAM4 is directly related to the cytoskeletal rearrangement during foot process effacement.
Hugo et al. (12) have reported that ezrin immunostaining intensity is increased in PAN nephropathy, and the staining identified areas of podocytic injury such as adhesion to the parietal glomerular epithelial cells, pseudocyst formation, podocyte vacuolation, and polynucleation of the podocyte. These data and the current results suggest that the altered expression of these apical components sensitively reflects the morphological changes in the podocyte. The expression of these molecules may be a useful marker for detecting injured podocytes in clinical settings.
In conclusion, JAM4 is expressed at the apical surface of podocytes and its altered expression is likely associated with the cytoskeletal changes in injured podocytes.
GRANTS
This work was supported by Grants-Aid for Scientific Research (B13557084 and B14370317 to H. Kawachi and B15390268 to F. Shimizu) from the Ministry of Education, Science, Culture, and Sports of Japan.
ACKNOWLEDGMENTS
Mutsumi Kayaba and Chiharu Nagasawa are gratefully acknowledged for tremendous technical assistance. We also thank Masaaki Nameta for indispensable help in the ultrastructural techniques. We express our gratitude to Dr. Takashi Sekine (Dept. of Pediatrics, The University of Tokyo) for helpful discussions.
FOOTNOTES
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.
REFERENCES
Benzing T. Signaling at the slit diaphragm. J Am Soc Nephrol 15: 1382–1391, 2004.
Cohen CJ, Shieh JT, Pickles RJ, Okegawa T, Hsieh JT, and Bergelson JM. The coxsackie virus and adenovirus receptor is a transmembrane component of the tight junction. Proc Natl Acad Sci USA 98: 15191–15196, 2001.
Diamond JR, Bonventre JV, and Karnovsky MJ. A role for oxygen free radicals in aminonucleoside nephrosis. Kidney Int 29: 478–483, 1986.
Diamond JR and Karnovsky MJ. Focal and segmental glomerulosclerosis following a single intravenous dose of puromycin aminonucleoside. Am J Pathol 122: 481–487, 1986.
Dobrosotskaya I, Guy RK, and James GL. MAGI-1, a membrane-associated guanylate kinase with a unique arrangement of protein-protein interaction domains. J Biol Chem 272: 31589–31597, 1997.
Ebnet K, Suzuki A, Ohno S, and Vestweber D. Junctional adhesion molecules (JAMs): more molecules with dual functions J Cell Sci 117: 19–29, 2004.
El Nemer W, Colin Y, Bauvy C, Codogno P, Fraser RH, Cartron JP, and Le Van Kim C. Isoforms of the Lutheran/basal cell adhesion molecule glycoprotein are differentially delivered in polarized epithelial cells. Mapping of the basolateral sorting signal to a cytoplasmic di-leucine motif. J Biol Chem 274: 31903–31908, 1999.
Han GD, Koike H, Nakatsue T, Suzuki K, Yoneyama H, Narumi S, Kobayashi N, Mundel P, Shimizu F, and Kawachi H. IFN-inducible protein-10 has a differential role in podocyte during Thy-1 glomerulonephritis. J Am Soc Nephrol 14: 3111–3126, 2003.
Hirabayashi S, Tajima M, Yao I, Nishimura W, Mori H, and Hata Y. JAM4, a junctional cell adhesion molecule interacting with a tight junction protein, MAGI-1. Mol Cell Biol 23: 4267–4282, 2003.
Hirata K, Ishida T, Penta K, Razaee M, Yang E, Wohlgemuth J, and Quertermous T. Cloning of an immunoglobulin family adhesion molecule selectively expressed by endothelial cells. J Biol Chem 276: 16223–16231, 2001.
Huet G, Gouyer V, Delacour D, Richet C, Zanetta JP, Delannoy P, and Degand P. Involvement of glycosylation in the intracellular trafficking of glycoproteins in polarized epithelial cells. Biochimie 85: 323–330, 2003.
Hugo C, Nangaku M, Shankland SJ, Pichler R, Gordon K, Amieva MR, Couser WG, Furthmayr H, and Johnson RJ. The plasma membrane-actin linking protein, ezrin, is a glomerular epithelial cell marker in glomerulogenesis, in the adult kidney and in glomerular injury. Kidney Int 54: 1934–1944, 1998.
Ide N, Hata Y, Nishioka H, Hirao K, Yao I, Deguchi M, Mizoguchi A, Nishimori H, Tokino T, Nakamura Y, and Takai Y. Localization of membrane-associated guanylate kinase (MAGI)-1/BAI-associated protein (BAP) 1 at tight junctions of epithelial cells. Oncogene 18: 7810–7815, 1999.
Kawachi H, Orikasa M, Matsui K, Iwanaga T, Toyabe S, Oite T, and Shimizu F. Epitope-specific induction of mesangial lesions with proteinuria by a MoAb against mesangial cell surface antigen. Clin Exp Immunol 88: 399–404, 1992.
Kawachi H, Abrahamson DR, St John PL, Goldstein DJ, Shia MA, Matsui K, Shimizu F, and Salant DJ. Developmental expression of the nephritogenic antigen of monoclonal antibody 5–1-6. Am J Pathol 147: 823–833, 1995.
Kawachi H, Koike H, Kurihara H, Yaoita E, Orikasa M, Shia MA, Sakai T, Yamamoto T, Salant DJ, and Shimizu F. Cloning of rat nephrin: Expression in developing glomeruli and in proteinuric states. Kidney Int 57: 1949–1961, 2000.
Kawachi H, Koike H, Kurihara H, Sakai T, and Shimizu F. Cloning of rat homologue of podocin: expression in proteinuric states and in developing glomeruli. J Am Soc Nephrol 13: 46–56, 2003.
Kerjaschki D. Polycation-induced dislocation of slit diaphragms and formation of cell junctions in rat kidney glomeruli. Lab Invest 39: 430–440, 1978.
Kerjaschki D, Sharkey DJ, and Farquhar MG. Identification and characterization of podocalyxin—the major sialoprotein of the renal glomerular epithelial cell. J Cell Biol 98: 1591–1596, 1984.
Kerjaschki D. Caught flat-footed: podocyte damage and the molecular basis of focal glomerulosclerosis. J Clin Invest 108: 1583–1587, 2001.
Kriz W, Hackenthal E, Nobiling R, Sakai T, and Elger M. A role of podocytes to counteract capillary wall distension. Kidney Int 45: 369–376, 1994.
Kriz W, Gretz N, and Lemley KV. Progression of glomerular disease: is the podocyte the culprit Kidney Int 54: 687–697, 1998.
Kurihara H, Anderson JM, Kerjaschki D, and Farquhar MG. The altered glomerular filtration slits seen in puromycin aminonucleoside nephrosis and protamine sulfate-treated rats contain the tight junction protein ZO-1. Am J Pathol 141: 805–816, 1992.
Li Y, Li J, Straight SW, and Kershaw DB. PDZ domain-mediated interaction of rabbit podocalyxin and Na+/H+ exchange regulatory factor-2. Am J Physiol Renal Physiol 282: F1129–F1139, 2002.
Mundel P and Reiser J, Zuniga Mejia Borja A, Pavenstadt H., Davidson GR, Kriz W, and Zeller R. Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines. Exp Cell Res 236: 248–258, 1997.
Mundel P and Shankland SJ. Podocyte biology and response to injury. J Am Soc Nephrol 13: 3005–3015, 2002.
Nagai M, Yaoita E, Yoshida Y, Kuwano R, Nameta M, Ohshiro K, Isome M, Fujinaka H, Suzuki S, Suzuki J, Suzuki H, and Yamamoto T. Coxsackie virus and adenovirus receptor, a tight junction membrane protein, is expressed in glomerular podocytes in the kidney. Lab Invest 83: 901–911, 2003.
Nosaka K, Takahashi T, Nishi T, Imaki H, Suzuki T, Suzuki K, Kurokawa K, and Endou H. An adenosine deaminase inhibitor prevents puromycin aminonucleoside nephrotoxicity. Free Radic Biol Med 22: 597–605, 1997.
Orikasa M, Matsui K, Oite T, and Shimizu F. Massive proteinuria induced in rats by a single intravenous injection of a monoclonal antibody. J Immunol 141: 807–814, 1988.
Orlando RA, Takeda T, Zak B, Shmieder S, Benoit VM, Mcquistan T, Furthmayr H, and Farquhar MG. The glomerular epithelial cell anti-adhesion podocalyxin associates with the actin cytoskeleton through interactions with ezrin. J Am Soc Nephrol 12: 1589–1598, 2001.
Patrie KM, Drescher AJ, Goyal M, Wiggins RC, and Margolis B. The membrane-associated guanylate kinase protein MAGI-1 binds megalin and is present in glomerular podocytes. J Am Soc Nephrol 12: 667–677, 2001.
Pavenstadt H, Kriz W, and Kretzler M. Cell biology of the glomerular podocyte. Physiol Rev 83: 253–307, 2003.
Rodewald R and Karnovsky MJ. Porous substructure of the glomerular slit diaphragm in the rat and mouse. J Cell Biol 60: 423–433, 1974.
Ruotsalainen V, Ljungberg P, Wartiovaara J, Lenkkeri U, Kestila M, Jalanko H, Holmberg C, and Tryggvason K. Nephrin is specifically located at the slit diaphragm of glomerular podocytes. Proc Natl Acad Sci USA 96: 7962–7967, 1999.
Schnabel E, Anderson JM, and Farquhar MG. The tight junction protein ZO-1 is concentrated along slit diaphragms of the glomerular epithelium. J Cell Biol 111: 1255–1263, 1990.
Shiratsuchi T, Futamura M, Oda K, Nishimori H, Nakamura Y, and Tokino T. Cloning, and characterization of BAI-associated protein 1: a PDZ domain-containing protein that interacts with BAI1. Biochem Biophys Res Commun 247: 597–604, 1998.
Smoyer WE and Mundel P. Regulation of podocyte structure during the development of nephrotic syndrome. J Mol Med 76: 172–183, 1998.
Somlo S and Mundel P. Getting a foothold in nephrotic syndrome. Nat Genet 24: 333–335, 2000.
Sundberg U and Obrink B. CEACAM1 isoforms with different cytoplasmic domains show different localization, organization and adhesive properties in polarized epithelial cells. J Cell Biol 115: 1273–1284, 2002.
Tajima M, Hirabayashi S, Yao I, Shirasawa M, Osuga J, Ishibashi S, Fujita T, and Hata Y. Roles of immunoglobulin-like loops of junctional cell adhesion molecule 4; involvement in the subcellular localization and the cell adhesion. Genes Cells 8: 759–768, 2003.
Takeda T, Go WY, Orlando RA, and Farquhar MG. Expression of podocalyxin inhibits cell-cell adhesion and modifies junctional properties in Madin-Darby canine kidney cells. Mol Biol Cell 11: 3219–3232, 2000.
Takeda T, McQuistan T, Orlando RA, and Farquhar MG. Loss of glomerular foot processes is associated with uncoupling of podocalyxin from the actin cytoskeleton. J Clin Invest 108: 289–301, 2001.
Takeda T. Podocyte cytoskeleton is connected to the integral membrane protein podocalyxin through Na+/H+-exchanger regulatory factor 2 and ezrin. Clin Exp Nephrol 7: 260–269, 2003.
Topham PS, Kawachi H, Haydar SA, Chugh S, Addona TA, Charron KB, Holzman LB, Shia M, Shimizu F, and Salant DJ. Nephritogenic mAb 5–1-6 is directed at the extracellular domain of rat nephrin. J Clin Invest 104: 1559–1566, 1999.(Yutaka Harita, Naoko Miyauchi, Tamaki Ka)