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The Glomerular Slit Diaphragm Is a Modified Adherens Junction

JOCHEN REISER^*, WILHELM KRIZ^*, MATTHIAS KRETZLER and PETER MUNDEL

^* Department of Anatomy and Cell Biology, University of Heidelberg, Germany
Department of Medicine, Ludwig-Maximilians-Universität, Munich, Germany
Departments of Medicine and Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York.

Correspondence to Dr. Peter Mundel, Division of Nephrology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Phone: 718-430-3158; Fax: 718-430-8963; E-mail: mundel{at}aecom.yu.edu

	Abstract

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Abstract. The glomerular slit diaphragm between podocyte foot processes shares typical morphologic features with an adherens junction. Differentiated cultured podocytes form cellular structures comparable to filtration slits in vivo. At those sites, zonula occludens-1 (ZO-1) was coexpressed with P-cadherin as well as with -, ß-, and -catenin. In situ, P-cadherin was detected at the slit diaphragm in association with ZO-1 as shown by confocal microscopy and immunogold double labeling electron microscopy. P-cadherin expression in vivo and in vitro was confirmed by reverse transcription-PCR. These findings led to the concept that the slit diaphragm represents an adherens junction composed of P-cadherin, -, ß-, and -catenin, and ZO-1. In contrast to an adherens junction of a similar composition recently described in cultured fibroblasts, the slit diaphragm complex does not contain vinculin, which was found in nearby focal contacts. A P-cadherinbased adherens junction is well-suited to explain the zipper-like structure of the slit diaphragm. The present study should allow new avenues leading to the identification of additional slit diaphragm-associated proteins conferring specificity to this unique cell junction.

	Introduction

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The glomerular capillary wall is highly permeable for water, small solutes, and ions but is tight for plasma proteins and other large molecules, resulting in a virtually protein-free glomerular filtrate. The structural characteristics accounting for this specificity are insufficiently known (1,2,3). Along its route through the filtration barrier, the filtrate has to pass the open pores of the glomerular endothelium, the highly hydrated collagenous network of the glomerular basement membrane (GBM), and the filtration slits. These slits are established by interdigitations of podocyte foot processes and are covered by the slit diaphragm, which is anchored at the sides of the foot processes.

The molecular composition of the slit diaphragm is unknown (3). In 1974, Rodewald and Karnovsky published a model of the substructure of the slit diaphragm, which was based on transmission electron microscopic (TEM) findings of tannic acid-stained material (4). According to their results, the slit diaphragm is made up of rod-like units connected in the center to a linear bar, forming a zipper-like pattern. The intercellular space bridged by the slit diaphragm is 30 to 40 nm wide, and the rectangular pores of the zipper have the approximate size of an albumin molecule (4). Based on the association of the tight junction protein zonula occludens-1 (ZO-1) with the cytoplasmic electron-dense material at the insertion site in foot processes, it was assumed that the slit diaphragm represents a modified tight junction (5,6). However, other characteristic proteins of tight junctions such as symplekin and occludin were not found in podocytes (7,8). Moreover, ZO-1, which was originally thought to be specific for tight junctions, is also present in adherens junctions (9). Recently, expression of P-cadherin in podocyte precursor cells during glomerulogenesis (10) and of catenins in podocytes of the adult kidney (11) was reported. Also, the slit diaphragm shares some typical morphologic features (wide intercellular gap, presence of a central dense line in grazing sections) with an adherens junction (5). We now provide evidence that the glomerular slit diaphragm represents a P-cadherin-based adherens junction.

	Materials and Methods

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Podocyte Cell Culture
Cultivation of conditionally immortalized mouse podocytes was performed, as recently reported (12). In brief, podocytes were maintained in RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 10% fetal calf serum, 100 U/ml penicillin, and 100 mg/ml streptomycin. To propagate podocytes, cells were cultivated at 33°C (permissive conditions), and the culture medium was supplemented with 10 U/ml mouse recombinant -interferon (Sigma, St. Louis, MO) to enhance expression of the T antigen. To induce differentiation, podocytes were maintained on type I collagen at 37°C without -interferon (nonpermissive conditions) for at least 14 d.

Transmission Electron Microscopy
For TEM analysis, podocytes were grown on plastic coverslips (Nunc, Wiesbaden, Germany). Cells were fixed for 2 h with 1.5% glutaraldehyde, washed with phosphate-buffered saline (PBS), and incubated with 1% OsO₄ for 1 h. Then the cells were counterstained with 1% tannic acid, dehydrated in a graded series of ethanol (30, 50, 70, 96, and 100%) at +4°C, and finally embedded in Epon 812 according to standard procedures. Ultrathin sections were observed under a Philips EM 301 electron microscope (Munich, Germany).

Immunofluorescence and Immunoelectron Microscopy
Primary antibodies specific for the following proteins were used: monoclonal anti--, ß-, and -catenin (Transduction Laboratories, Lexington, KY), polyclonal anti-ZO-1 (Zymed Laboratories, South San Francisco, CA), and monoclonal anti-vinculin (Boehringer Mannheim, Mannheim, Germany). For P-cadherin, we used two different antibodies that gave identical results: a mouse monoclonal (Transduction Laboratories) and a rat monoclonal (Zymed Laboratories). Before immunolabeling, the cells were fixed with 2% paraformaldehyde and 4% sucrose at room temperature for 10 min, washed once with PBS, and permeabilized with 0.3% Triton X-100 at room temperature for 10 min. The cells were incubated with blocking solution (2% fetal calf serum, 2% bovine serum albumin, 0.2% fish gelatin) for 30 min at room temperature before further incubation with one of the primary antibodies for 1 h at room temperature. Antigen-antibody complexes were visualized with fluorochrome (Cy2 or Cy3)-conjugated secondary antibodies (Rockland, Gilbertsville, PA). Confocal microscopy and processing of micrographs were performed as reported (12).

For immunogold labeling, ultrathin frozen sections of perfusion-fixed rat kidney were reacted with monoclonal anti-P-cadherin overnight as described previously (13). After rinsing with washing buffer (PBS containing 0.1% bovine serum albumin), rabbit anti-mouse IgG (Zymed Laboratories) was applied at 1:50 dilution for 1 h at room temperature, followed by goat anti-rabbit IgG coupled to 10-nm colloidal gold (BioCell, Cardiff, United Kingdom). After rinsing with washing buffer and PBS, sections were post-fixed with 2% glutaraldehyde and 0.5% tannic acid and counterstained with 2% OsO₄ in PBS. After staining with 2% uranyl acetate for 2 to 5 min, the sections were absorption-stained with 0.003% lead citrate in 2% polyvinyl alcohol (Sigma). After air drying at room temperature, the sections were observed under a Phillips EM 301 electron microscope. For immunogold double labeling, cryosections were reacted with monoclonal anti-P-cadherin and polyclonal anti-ZO-1 followed by 15-nm gold-conjugated anti-mouse IgG and 10-nm gold-conjugated anti-rabbit IgG.

Detection of Cadherin mRNA Expression by Reverse Transcription-PCR
Reverse transcription (RT) and quantitative PCR were performed as described (14), using total RNA from mouse kidney cortex, isolated glomeruli, and differentiated cultured podocytes. For reverse transcriptase negative controls, the enzyme was omitted. For detection of P- and E-cadherin, sequence-specific 20-bp oligonucleotide primers (purchased from Life Technologies) were designed (E-cadherin: sense, 5'-GAT TCT GAT CCT GCT GCT CC-3', antisense, 5'-GGA GCC ACA TCA TTT CGA GT-3', yielding a 204-bp PCR product; P-cadherin: sense, 5'-AAT CGG GAA CTT CAT CAT CG -3', antisense, 5'-TTG AAT CGA CTT CCC CAC TC-3', yielding a 192-bp PCR product. PCR was performed on a serial fivefold dilution series of each cDNA, with assays performed over a 2 to 3 log range. The cDNA reaction mix from RT minus reactions and water controls was consistently negative (data not shown). To assess product abundance, the amplified cDNA was analyzed on a nondenaturing 5% polyacrylamide gel stained with VistraGreen^TM (Amersham, Braunschweig, Germany) and visualized with ImageQuant Software on a Storm FluoroPhosphorImager (Molecular Dynamics, Sunnyvale, CA). PCR product identity was confirmed by sequence-specific restriction analysis.

	Results

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Characterization of Cell-Cell Contacts in Cultured Podocytes
In a novel cell culture system, podocytes can be induced to differentiate into process-bearing cells similar to podocytes in vivo (12). By TEM, the cell-cell contacts of these cultured podocytes share typical morphologic characteristics with the slit diaphragm in vivo (Figure 1). Like in situ (Figure 1a), electron-dense material is located at the cytoplasmic insertion site (Figure 1b). A low-power TEM micrograph shows the longitudinal orientation of the junction between the foot process-like projections of two neighboring podocytes over a longer distance (Figure 1c). The width of the intercellular space bridged by this junction is 25 to 40 nm, which is in the range of the slit diaphragm.

View larger version (108K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Morphologic and molecular analysis of the slit diaphragm complex in vivo and in vitro. (a) Tangential section of the slit diaphragm in situ reveals a zipper-like structure with a central linear bar (asterisk). At the cytoplasmic insertion site, electron-dense material is observed (arrow). (b) A similar junction is observed between processes of cultured podocytes. As with in vivo, the intercellular space (asterisk) is 25- to 40-nm wide, and electron-dense material is found at the cytoplasmic insertion site of this junction (arrow). (c) Low-power micrograph showing the longitudinal orientation of the junction between the foot process-like structures of two adjacent podocytes (arrows). Magnification: x65,000 in a; x70,000 in b; x9,500 in c.

To reveal the molecular composition of this slit diaphragm-like junction, we studied the expression of marker proteins of tight junctions, adherens junctions, and desmosomes by immunocytochemistry in undifferentiated (Figure 2a) and differentiated cultured podocytes (Figure 2b). In undifferentiated cells, ZO-1 was found as a continuous belt outlining the cell borders (Figure 2c). In contrast, in differentiated podocytes ZO-1 was found at process interdigitations of neighboring cells (Figure 2, d and e). In addition, ZO-1 was also found in the nuclei of differentiated podocytes. Because podocytes express neither symplekin (7) nor occludin (an integral membrane protein of tight junctions (reference (8) and this study) (data not shown) and because ZO-1 is also found in adherens junctions (1), we asked whether the cells expressed other markers of adherens junctions or desmosomes. To this end, we analyzed the expression of cadherins and catenins in cultured podocytes. Cadherins constitute a family of glycoproteins involved in homophilic Ca²⁺-dependent cell-cell adhesion and are linked to the actin cytoskeleton via catenins (15). Among several members of the cadherin family, cultured podocytes expressed two forms: E-cadherin and P-cadherin. E-cadherin was observed only in cytoplasmic vesicles (Figure 3, i and j), whereas P-cadherin was found in a pattern comparable to that of ZO-1 at cell-cell contacts in both undifferentiated and differentiated podocytes (Figure 3, a and b). None of the analyzed desmosomal cadherins (desmoglein, desmocollin) was found in cultured podocytes (data not shown).

View larger version (123K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Phase-contrast microscopy and zonula occludens-1 (ZO-1) expression of undifferentiated and differentiated cultured podocytes. (a) Under permissive conditions, the cells form an epithelial monolayer. (b) Under nonpermissive conditions, the cells develop prominent processes, which are only connected at sites of process interdigitations. (c) In undifferentiated cells, ZO-1 is found in a continuous belt along the cell borders. (d) In differentiated cells, ZO-1 is redistributed to sites of cell-cell contacts between interdigitating processes. In addition, ZO-1 is found in the nucleus of differentiated podocytes Magnification: x400 in a and b; x600 in c and d.

View larger version (101K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Expression of adherens junction proteins in cultured podocytes. (a, c, e, g, and i) Undifferentiated cells growing at permissive conditions. (b, d, f, h, and j) Process-bearing differentiated podocytes grown under nonpermissive conditions for 14 d. P-cadherin (a and b), -catenin (c and d), ß-catenin (e and f), and -catenin (g and h) are found along the cell border in undifferentiated cells. Upon differentiation, these proteins are redistributed to sites of interdigitations between neighboring podocytes. Note the additional nuclear staining of P-cadherin, and -, ß-, and -catenins. (i and j) In contrast, E-cadherin is only found in cytoplasmic vesicles, both in undifferentiated (i) and differentiated (j) podocytes. Magnification: x450 in a, c, e, g, i, and j; x800 in b; x650 in d, f, and h.

Next, we studied the expression of -, ß-, and -catenin in cultured podocytes; all of them were expressed in undifferentiated and differentiated cells at sites of intercellular contacts (Figure 3, c through h), corroborating previous in situ results (11). Similar to ZO-1 and P-cadherin, the catenins were redistributed during remodeling of cell-cell junctions. In double-labeling experiments, ZO-1 colocalized with P-cadherin (Figure 4a) and ß-catenin (Figure 4b), as well as with - and -catenin (data not shown) at cell-cell contacts of differentiated podocytes and in the nucleus. Vinculin, another cytoskeleton-associated protein found in P-cadherin-based adherens junctions of nonpolarized fibroblasts (1), did not colocalize with ZO-1; it was detected in nearby focal contacts at the tips of podocyte processes outside the junctional complex (Figure 4c).

View larger version (88K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Double labeling microscopy of differentiated podocytes. (a) Colocalization of ZO-1 (green) and P-cadherin (red) at sites of cell-cell contacts between adjacent podocytes. The overlap results in a yellow staining at the adherens junction and in the nucleus in the merge. (b) Colocalization of ZO-1 (green) and ß-catenin (red). Again, a clear overlap of immunoreactivity results in a yellow staining of cell-cell contacts and in the nucleus in the overlay. (c) In contrast, vinculin (red) does not colocalize with ZO-1 (green) at cell-cell contacts, but is found in nearby focal contacts. Magnification: x800 in a; x650 in b and c.

Characterization of the Glomerular Slit Diaphragm in Vivo
Finally, we studied the expression of P-cadherin in glomeruli of rat kidney. Using a double-labeling approach with P-cadherin and ZO-1, we found overlap of immunofluorescence along the outer surface of the GBM, demonstrating the close proximity of the two proteins (Figure 5). The expression of P-cadherin in the glomerulus was confirmed by RT-PCR, which revealed a PCR product of the expected size identical to that found in cultured podocytes (Figure 6). Interestingly, we also found E-cadherin mRNA (Figure 6), but not protein expression in the glomerulus (data not shown). The absence of E-cadherin expression in podocytes is in line with the findings of Schnabel et al. (5).

View larger version (92K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Immunocytochemical localization of P-cadherin in the glomerulus. Confocal microscopy demonstrating the colocalization of P-cadherin (red) and ZO-1 (green) in a rat kidney glomerulus. The complete overlap of immunoreactivity results in a yellow staining pattern along the glomerular basement membrane (GBM). Magnification, x650.

View larger version (58K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Renal cadherin mRNA expression. Reverse transcription-PCR on serial dilution (1:1 to 1:125) of cDNA obtained from mouse renal cortex (Cortex), isolated glomeruli (Glom), and differentiated cultured podocytes (Podo). Both P-cadherin (P; top lane) and E-cadherin (E; bottom lane) cDNA were amplified from all material examined.

The precise ultrastructural localization of P-cadherin was analyzed by immunogold labeling of ultrathin frozen sections from rat kidney cortex. With this approach, we noted decoration of the slit diaphragm region between podocyte foot processes with gold particles, indicating the association of P-cadherin with the slit diaphragm (Figure 7). The ultrastructural relationship of ZO-1 and P-cadherin was examined by immunogold double labeling. As shown in Figure 8, P-cadherin was found exclusively on the slit diaphragm, whereas ZO-1 was detected on the cytoplasmic face, close to the point of attachment of the slit diaphragm (5,6). Thus, we confirmed the association of both proteins with the junctional complex of the slit diaphragm.

View larger version (123K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. Ultrastructural localization of P-cadherin to the slit diaphragm in situ. (a) Cross section. (b and c) Tangential sections. P-cadherin is found at the level of the slit diaphragm between adjacent podocyte foot processes (arrows). Magnification: x55,000 in a and b; x35,000 in c.

View larger version (114K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 8. Association of P-cadherin and ZO-1 with the slit diaphragm complex. Using immunogold double labeling, P-cadherin was found on the slit diaphragm bridging two adjacent foot processes (large, 15-nm gold particle; arrow). Small (10 nm) gold particles representing ZO-1 were found on the cytoplasmic face of foot processes close to the insertion of the slit diaphragm (arrowheads). Magnification, x65,000.

	Discussion

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The structures and mechanisms responsible for the permselectivity of the glomerular filter are insufficiently understood (3). The filtration barrier is characterized by distinct charge and size selectivities. It is now generally accepted that the charge barrier is constituted mainly of the negative residues of the endothelium and the GBM (3), but only to a lesser extent by the negative moieties on the surface of podocytes. The size selectivity has been attributed to either the GBM or the slit membrane. Recent studies addressing this problem have clearly shown that the slit diaphragm is the most decisive structure for the size selectivity of this filter by restricting the protein passage through the filter, thus being responsible for a largely protein-free filtrate (16,17). On the other hand, the molecular composition of the slit diaphragm represents a doggedly persistent problem in glomerular cell biology (3).

The present study provides considerable progress in this respect. By in vitro and in situ studies, we showed that the slit diaphragm complex—in addition to ZO-1 (6,7)—contains P-cadherin as well as -, ß-, and -catenin (reference 11 and this study). On the basis of these findings, we propose the following molecular organization of the slit membrane complex (Figure 9). P-cadherin represents the core protein with its extracellular domain essentially forming the slit diaphragm. The intracellular domain is connected to ß-catenin and/or plakoglobin (-catenin). The linkage of this complex to the actin cytoskeleton via -actinin is mediated by -catenin, a protein homologous to vinculin (18). Alternatively, the linkage to the actin cytoskeleton can be achieved by interaction of -catenin with ZO-1, which in turn can bind to actin filaments (19). This organization essentially represents an adherens-type junction. A similar P-cadherin-based adherens junction has recently been described in fibroblasts; unlike the slit diaphragm, this junction contains vinculin (1). In podocyte foot processes, vinculin is exclusively expressed in the focal contact connecting the foot processes to the GBM (20). Ultrastructurally, the en face view of the slit diaphragm appearing as a zipper-like structure by TEM is perfectly compatible with the proposed zipper-like model of a cadherin-based adherens junction (2).

View larger version (68K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 9. Cartoon of the molecular organization of the slit diaphragm. According to the present study, the slit diaphragm represents a P-cadherin-based adherens junction. In this model, P-cadherin may serve as the core protein due to homophilic interactions (2). The linkage of the cadherin/catenin complex to the actin cytoskeleton is mediated by ZO-1 (1, 18, 19), which has previously been shown to be associated with the slit diaphragm (5). The relation of the slit diaphragm-associated antigen defined by monoclonal antibody 5-1-6 (21, 22) and nephrin (23, 24) to P-cadherin remains to be established. In contrast to classical adherens junctions (1), vinculin is not part of the junctional complex in podocytes as shown in the present study. The linkage of the foot processes to the GBM is achieved by an 3ß1-integrin complex, which in turn is linked to the actin cytoskeleton via talin, vinculin, and paxillin (reviewed in reference (3). P-C, P-cadherin; , -catenin;, ß, ß-catenin; , -catenin; Z, ZO-1; 3, 3-integrin; ß1, ß1-integrin; V, vinculin; T, talin; P, paxillin.

P-cadherin most likely does not represent the only extracellular protein of the slit diaphragm, since its expression alone may not explain the permselectivity of the glomerular filter. In this context, it will be interesting to reveal the molecular identity of the antigen defined by monoclonal antibody 5-1-6 (21, 22) and its relation to P-cadherin. Interestingly, nephrin, the gene mutated in the congenital nephrotic syndrome (23), appears to be associated with the slit diaphragm as well (24). Thus, it is tempting to speculate that P-cadherin serves as a basic scaffold for the slit diaphragm, whereas the specific properties (e.g., permselectivity) are provided by other proteins (e.g., nephrin, 5-1-6 antigen) associated with the slit diaphragm complex (Figure 9). This idea is supported by the finding that injection of antibody 5-1-6 induces massive proteinuria without detectable structural alterations of the slit membrane itself (21).

Although podocytes do not express classical tight junction proteins such as symplekin (7) and occludin (reference (8) and this study), we cannot rule out a participation of members of a novel class of tight junction proteins termed claudins (25) in slit diaphragm modulation under pathologic conditions. We have begun to address the expression of claudins in podocytes in vivo and in vitro by RT-PCR. In fact, our preliminary data indicate a role of claudins in tight junction formation of podocytes under pathologic conditions, in which podocytes indeed develop tight junctions as shown before (26).

One additional finding of the present study was the prominent expression of all examined junction proteins in the nucleus, both under permissive and nonpermissive conditions (Figures 2,3,4). The nuclear expression of ZO-1 is in line with previous publications showing the translocation of adherens junction proteins to the nucleus during wound healing of Madin-Darby canine kidney cells (27). We have started to address the functional relevance of this nuclear expression in podocytes, which may reflect a state of activation. There is increasing evidence for a dual role of these proteins as structural proteins at cell-cell contacts and as components of transcription complexes in the nucleus (28, 29). The role of these proteins in the podocyte nucleus remains to be established.

In conclusion, the present study provides the basis for the understanding of the molecular composition of the slit diaphragm. Based on these findings, it should be possible to find additional proteins constituting the slit diaphragm complex. It will be of particular interest to see the interactions of P-cadherin with nephrin, the target gene of the Finnish type of congenital nephrotic syndrome, which is also associated with the slit diaphragm (24). For P-cadherin a homophilic interaction has been proven at the molecular level (2); this remains to be established experimentally for nephrin. Furthermore, P-cadherin is already expressed by undifferentiated cultured cells, but is redistributed upon slit diaphragm formation in differentiated podocytes. In contrast, nephrin, which in vivo is found in association with the slit membrane of mature podocytes, is only expressed by differentiated cultured cells (our unpublished data). These data further support the idea that P-cadherin represents the core protein of the slit diaphragm, whereas nephrin may contribute some of the slit diaphragm-specific attributes to this specialized adherens junction. The increasing knowledge of the molecular composition of the slit diaphragm should allow a better understanding of kidney diseases in which the loss of glomerular permselectivity results in the development of proteinuria.

	Acknowledgments

 
This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG) to Drs. Kriz and Mundel. Dr. Mundel was the recipient of a Heisenberg Stipendium of the DFG and is currently supported by the Howard Hughes Medical Research Institute Research Resources for Medical Schools. We thank Dr. Kai Simons (European Molecular Biology Laboratory, Heidelberg, Germany) for critical reading of the manuscript, and Alexandra Zeller and Hiltraud Hosser for expert technical assistance.

	Footnotes

 
This study was presented in part at the 31st Annual Meeting of the American Society of Nephrology, Philadelphia, PA, October 25-28 1998, and has been published in abstract form (J Am Soc Nephrol 9: 507A, 1998).

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