Podocytes cover the outer aspect of the basement membrane of glomerular capillaries as a continuous layer of epithelial cells. The high glomerular filtration rate and—at the same time—the effective retention of plasma proteins is achieved by means of the highly specialized, unique cell-cell contacts of podocytes. The characteristics of podocyte cell-cell contacts are foot process interdigitation and formation of slit diaphragms. Considerable progress has been made in elucidating the molecular composition of the slit diaphragm in recent years1,2,3. The currently known slit diaphragm proteins (SDP) and slit diaphragm-associated proteins (SDAP) include nephrin4,5, FAT6, P-cadherin7, podocin8, CD2AP9, ZO-110, and NEPH111. The dominant role of nephrin among the SDP and SDAP has been confirmed by disruption of the nephrin gene in mice12,13. In these mice, the intercellular spaces between podocyte foot processes are devoid of slit diaphragms, and the mice die soon after birth by nephrotic syndrome. In addition to the discovery of SDP/SDAP, proteins that are preferentially located in podocyte foot processes have been identified (e.g., synaptopodin)14. Despite the increased knowledge of the molecular constituents of the slit diaphragm and foot processes, we do not yet understand the molecular arrangement and the formation of these critically important structures.
To gain further insight into the molecular make-up and the formation of the slit diaphragm and of interdigitating foot processes, suitable cell culture models will be extremely helpful. However, cell-cell contacts of the currently available podocyte cell lines share only limited characteristics with cell-cell contacts of podocytes in situ. The best characterized murine podocyte cell line was generated by Mundel et al15. These cells form multiple processes at low cell density, but they do not organize into a continuous epithelial layer. It was demonstrated that ZO-1, P-cadherin, and catenins localize to areas of cell-cell contact in these cells7. However, these cells express nephrin only at very early passages and in very low abundance16,17, and therefore the nephrin distribution in these cells has never been reported. On the other hand, two recently generated human podocyte cell lines have been reported to express nephrin18,19. In both cell lines, however, nephrin is not located at cell-cell contacts18,19,20.
The aim of our present work was to generate a number of conditionally immortalized murine podocyte cell clones [characterized by Wilm's tumor (WT-1) and podocalyxin expression], and to select those clones that stably express nephrin and that form a continuous epithelial layer in the differentiated state. We reasoned that these selection criteria would yield podocyte cell lines retaining features of podocyte cell-cell contacts in situ. We obtained two podocyte cell lines (PCLs) that fulfilled the selection criteria. These novel PCLs recapitulate several characteristics of cell-cell contacts of podocytes in situ, as demonstrated by expression of SDP/SDAP, by the ultrastructural properties of cell-cell contacts, and by accumulation of nephrin and actin-associated proteins at areas of cell-cell contact.
METHODS
Cell culture and generation of conditionally immortalized mouse PCLs
Glomeruli were isolated from kidneys of ImmortoMouse® mice (Charles River, St. Louis, MO, USA), carrying a temperature-sensitive mutant of the immortalizing SV40 large T antigen under control of the interferon-
(INF-)-inducible H-2Kb promoter15,21. The isolation of glomeruli was done by three-step sieving of renal cortices (250, 100, and 70 
m) as described earlier22. Isolated glomeruli were maintained in RPMI 1640 (Life Technologies, Karlsruhe, Germany) supplemented with 10% fetal bovine serum (FBS) (Boehringer Mannheim, Mannheim, Germany), 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 10 U/mL mouse recombinant INF- (Life Technologies). The outgrowth of podocytes started between days 2 and 3. After 6 days of primary culture, glomeruli were removed and the cells were trypsinized. To propagate podocytes, cells were cultivated at 33°C in the presence of INF- (permissive conditions). Clonal cell lines were generated by limiting dilution of the initial pool of growing cells. To induce differentiation, podocytes were maintained at 38°C without INF- for at least 2 weeks (nonpermissive conditions). A higher temperature to induce differentiation as compared to that described by Mundel et al15 was chosen, because the inactivation of the temperature-sensitive mutant of the SV40 large T antigen is enhanced at 38°C21, and because the higher temperature matches the body temperature of the mouse (38°C to 39°C). Podocyte phenotype of differentiated cell clones was assessed by WT-1 immunofluorescence and podocalyxin reverse transcription-polymerase chain reaction (RT-PCR)23,24. Cell clones being positive for WT-1 and podocalyxin were further analyzed. NIH 3T3 fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies) supplemented with 10% FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin.
Assessment of cell proliferation
PCL between passages 15 and 31 were seeded in 96-well plates for the 3-4,5dimethylthiaozl-2,5-diphenyltetrazolium bromide (MTT) (Sigma, Deisenhofen, Germany) assay or in 6-well plates for counting. Plates were shifted to nonpermissive culture conditions (day 0). During 4 weeks, cell proliferation was assessed in regular time intervals of 2 to 3 days. In the MTT assay, MTT was added at a final concentration of 0.5 mg/mL to the wells, cells were incubated for 5 hours at 38°C, and the purple formazan crystals were solubilized in dimethyl sulfoxide (DMSO). Optical density was measured with a microplate reader (Benchmark) (Bio-Rad, Munich, Germany) at 595 nm against a reference wavelength of 655 nm. Six wells were measured for each time point in each experiment. Data are presented as means 
SEM of three experiments. The linear correlation between cell number and optical density was verified in additional experiments. In counting experiments, cells were trypsinized and counted manually in a hemocytometer. The percentage of viable cells was greater than 95%.
RNA isolation and RT-PCR
Total RNA of cultured podocytes or mouse kidneys was isolated with a mixture of guanidine thiocyanate and phenol (TRI Reagent) (Sigma) according to the manufacturer's protocol. RT was performed at 37°C for 60 minutes with 1 to 2 g RNA. The reaction mixture contained 50 U MuLV reverse transcriptase (Applied Biosystems, Darmstadt, Germany), 10 U RNase inhibitor (Applied Biosystems), 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl2, 10 mmol/L deoxynucleoside triphosphate (dNTP) (Sigma), and 1 mol/L antisense oligonucleotide primer (cf., Table 1). Reaction mixes lacking RT served as negative controls. PCR was performed in the presence of 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl2, 1 mol/L sense oligonucleotide primer (cf. Table 1) and Taq polymerase (Eppendorf). The number of cycles ranged from 25 to 40 with 2 minutes at 94°C, 11/2 minutes at the annealing temperature, and 2 minutes at 72°C. PCR products were separated on 2% ethidium bromide-stained agarose gels, which were recorded with a digitizing video system (Intas, Göttingen, Germany). The sequence specificity of the amplicons was confirmed by size and endonuclease restriction analysis.
Northern blotting
RNA was isolated as described above. Total RNA (30 g per lane) was resolved on 1% agarose-formaldehyde gels and blotted onto Hybond-NX nylon membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK) overnight. Probes were generated by RT-PCR as described above from mouse total kidney RNA using the following primer pairs: mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (GenBank accession # NM_008084), forward 5'-GTG AAG GTC GGT GTG AAC GGA TTT G-3', reverse 5'-ACA TTG GGG GTA GGA ACA CGG AAG G-3', 710 bp product size; mouse nephrin (GenBank accession # AF191090), forward 5'-TGC TGC CCA CGG ATG AGA CA-3', reverse 5'-GGT CAC TGC CCG CAC TTG CT-3', 830 bp product size; mouse CD2AP as indicated in Table 1. Probes were 32P-labeled with the RadPrime DNA Labeling System (Invitrogen, Karlsruhe, Germany). Prehybridization was done in PerfectHyb Plus Hybridization Buffer (Sigma) at 65°C for 2 hours. Membranes were hybridized in 7 mL PerfectHyb Plus Hybridization Buffer containing the 32P-labeled probe at 65°C overnight. Membranes were washed in 1 
standard saline citrate (SSC) containing 0.1% sodium dodecyl citrate (SDS) for 20 minutes at room temperature, and twice in 0.1 SSC containing 0.1% SDS for 20 minutes at 65°C. Blots were exposed to x-ray films (Super RX) (Fuji, Düsseldorf, Germany) for 1 to 4 days at -80°C. Blots were stripped for 30 minutes in 0.1 SSC containing 0.1% SDS, which had been heated to 100°C before, and stripped blots were reprobed for GAPDH expression as a loading control.
Western blotting
Podocytes were lyzed in phosphate-buffered saline (PBS) containing 2% Triton X-100, supplemented with a proteinase inhibitor cocktail (Sigma). Lysates of COS7 cells (kindly provided by Dr. M. Moeller, University of Heidelberg, Germany), which had been transiently transfected with 3xHA-tagged podocin, including or excluding the C-terminus, were employed as positive or negative control, respectively. Protein concentration was determined by the Bradford method. The lysates were heated to 100°C for 3 minutes in SDS gel-loading buffer (50 mmol/L Tris-HCl at pH 6.8, 100 mmol/L dithiothreitol, 2% SDS, 0.1% bromophenol blue, and 10% glycerol) and 30 g protein/lane were separated on a 10% SDS polyacrylamide gel. After blotting on a polyvinylidene difluoride (PVDF) membrane (Millipore, Eschborn, Germany), membranes were blocked overnight at 4°C in blocking buffer (5% nonfat dry milk, 0.9% NaCl, 20 mmol/L Tris-HCl at pH 7.5, and 0.05% Tween). Blots were then incubated with the primary antibody (rabbit antimouse nephrin antibody directed against the 155 C-terminal amino acids4 or rabbit antihuman/antimouse podocin antibody directed against the 17 C-terminal amino acids25) diluted in blocking buffer for 2 hours at room temperature, followed by rinsing with washing buffer (0.9% NaCl, 20 mmol/L Tris-HCl at pH 7.5, and 0.05% Tween). Afterwards the blot was incubated with horseradish peroxidase-labeled secondary antirabbit antibody (Sigma) for 1 hour at room temperature, washed, incubated for 1 minute in chemiluminescence solution (ECL) Amersham Pharmacia Biotech) and finally exposed to x-ray film (Hyperfilm ECL) (Amersham Pharmacia Biotech).
Immunofluorescence and microscopy
For immunofluorescence studies podocytes were cultured on type IV collagen-coated glass coverslips (Biochrom, Berlin, Germany). At room temperature cells were fixed (2% paraformaldehyde, 4% sucrose, PBS) for 8 minutes, permeabilized (0.3% Triton X-100 in PBS) for 10 minutes, and blocked in blocking solution [2% FBS, 2% bovine serum albumin (BSA), 0.2% gelatine, and PBS] for 45 minutes. Primary antibodies were incubated for 60 minutes. The following antibodies were used: rabbit anti-WT-1 (C-19) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit antipodocalyxin (kindly provided by Dr. M.G. Farquhar, University of California, San Diego, CA, USA), mouse antivimentin (clone VIM 13.2) (Sigma), rabbit anti-CD2AP (kindly provided by Dr. A.S. Shaw, Washington University School of Medicine, St. Louis, MO, USA) and H-290 (Santa Cruz Biotechnology), rabbit anticortactin (kindly provided by Dr. X. Zhan, American Red Cross, Rockville, MD, USA), rabbit antipodocin (kindly provided by Dr. C. Antignac, INSERM U423, Hôpital Necker-Enfants Malades, Paris, France), and mouse antisynaptopodin (Progen, Heidelberg, Germany). A polyclonal antipeptide antibody against nephrin was produced in the rabbit as described earlier26. A synthetic peptide covering amino acids 1039 to 1056 of the human nephrin sequence was used as the immunogen (GenBank accession # AF035835). The synthetic peptide in Freund's complete adjuvant was injected into two rabbits. After three booster inmmunizations 4 weeks apart, the respective IgG fractions were immunoaffinity-purified on CNBr-sepharose coupled to the synthetic peptide. The specificity of antisera was tested by indirect immunofluorescence and by Western blotting as well as appropriate preincubations with the immunogen. In addition, a guinea pig antinephrin antibody directed against the fibronectin domain of nephrin was utilized (Progen). Similar results were obtained with both antibodies. Antigen-antibody complexes were visualized with Cy2- or Cy3-conjugated secondary antibodies (Dianova, Hamburg, Germany). F-actin was visualized using fluorochrome-conjugated phalloidins (Molecular Probes, Eugene, OR, USA). Coverslips were washed (PBS), rinsed (H2O), and mounted with 15% Mowiol (Calbiochem, Bad Soden, Germany), 50% glycerol, and PBS. Specimens were viewed with a confocal laser-scanning microscope (TCS-SP) (Leica Microsystems, Mannheim, Germany). Phase contrast images were taken with a Leica IRBE microscope (Leica Microsystems) equipped with a cooled charged coupled device (CCD) camera (Photonic Science, Robertsbridge, UK) and OpenLab software (Improvision, Coventry, UK) for image acquisition.
Transmission electron microscopy
Cells were fixed in 2% glutaraldehyde with 0.2% tannin in PBS, stained in 1% OsO4 for 30 minutes, and contrasted with 1% uranyl acetate for 30 minutes. After dehydration in alcohol, specimens were embedded in Epon 812, and 90 nm sections were cut with an ultramicrotome (Ultracut E) (Leica Microsystems). Sections were placed on Formvar-coated copper grids and examined under a transmission electron microscope (Philips EM 301).
RESULTS
Using limiting dilution we generated about 60 cell clones from the outgrowth of glomeruli which were isolated from H-2Kb-tsA58 transgenic mice carrying a temperature-sensitive variant of the SV40 large T antigen under control of the IFN-–inducible H-2Kb promoter21. Immunofluorescence staining for WT-1 and expression of podocalyxin by RT-PCR was observed in 30 cell clones, indicating that cells were of podocyte and not of parietal epithelial origin24. Transcripts for nephrin were detected in two of the 30 cell clones by RT-PCR. These two nephrin-expressing PCLs were characterized in detail. Since practically identical results were obtained with both PCLs, data will be presented essentially for one PCL.
PCLs could be cultured successfully over 40 passages without noting phenotypic changes. Nephrin was stably expressed in PCLs over all passages as determined by RT-PCR Figure 1a. In addition, stable expression of nephrin in PCLs could be demonstrated by Northern blotting using GAPDH as an internal control Figure 1b. Stable nephrin expression at the protein level was confirmed by Western blotting, yielding bands at about 180 kD Figure 1c. In confluent PCLs, nephrin was located at the perinuclear region and at cell-cell contacts as visualized by immunofluorescence Figure 1d. The pattern of nephrin immunoreactivity at cell-cell contacts varied from discrete dots to a continuous band, reflecting different states of confluence and junction formation (see below).
Figure 1.
Nephrin expression in podocyte cell line (PCL). (A) Ethidium bromide-stained gel of reverse transcription-polymerase chain reaction (RT-PCR) products demonstrates stable nephrin expression in PCL over different passages. (B) Northern blots further demonstrate stable expression of nephrin and CD2AP in PCL over different passages. (C) Western blotting of whole kidney and PCL lysates confirms stable nephrin expression at the protein level over different passages in PCL. (D) In confluent PCL cells, nephrin is located at the perinuclear region (arrow), probably corresponding to the Golgi apparatus, and at cell-cell contacts (arrow head) by immunofluorescence. For immunofluorescence PCL cells above passage 30 were used (scale bar indicates 10 m).
Expression of various podocyte-specific proteins, especially of SDP/SDAP, was examined by RT-PCR and immunofluorescence in PCLs. Transcripts of the podocyte transcription factors WT-1 and Lmx1b23,27 were detected Figure 2. Interestingly, among the 30 cell clones that showed nuclear immunoreactivity for WT-1, the two nephrin-positive cell clones expressed WT-1 at the highest level as observed by RT-PCR. Besides nephrin, transcripts of the SDP/SDAP NEPH1, FAT, P-cadherin, CD2AP, and ZO-1 were present in PCLs Figure 2. Concerning ZO-1, the podocyte-specific 
- splice variant28 was the almost exclusively expressed isoform. Finally, transcripts of the podocyte transmembrane proteins podocalyxin and podoplanin29,30, which are also expressed in other cell types, were detected in PCLs. In kidney RNA, PCR products could be amplified for all transcripts tested Figure 2. Northern blotting showed stable expression of CD2AP in PCLs over different passages Figure 1b. Expression of podocin could be demonstrated at the protein level by Western blotting Figure 3a and by immunofluorescence Figure 3b. In Western blots, bands around 39 to 40 kD were detected for podocin in agreement with previous results25. Podocin assumed a membranous distribution in PCLs accumulating at areas of cell-cell contact (Figure 3b, arrow). Protein expression and localization of WT-1, podocalyxin, and the intermediate filament protein vimentin were visualized in PCLs by immunofluorescence Figure 3c to e. In addition, PCLs stained positively using antibodies against the actin-associated proteins CD2AP, cortactin, and synaptopodin (see Figures 7 and 8), whose cellular distributions are described below in detail.
Figure 2.
Expression of slit diaphragm-associated proteins and further podocyte markers in podocyte cell line (PCL). Transcripts of podocyte-specific transcription factors [Wilm's tumor (WT-1), Lmx1b], slit diaphragm protein (SDP)/slit diaphragm-associated protein (SDAP) (NEPH1, FAT, P-cadherin, and CD2AP, - isoform of ZO-1), and podocyte transmembrane proteins (podocalyxin and podoplanin) were detected by reverse transcription-polymerase chain reaction (RT-PCR) in PCL. Reactions with RNA isolated from PCL were performed in the presence (+RT) and absence of reverse transcriptase (-RT). Total RNA isolated from mouse kidneys and NIH 3T3 fibroblasts served as positive and negative controls, respectively.
Figure 3.
Expression of podocin and subcellular distribution of podocyte markers in podocyte cell line (PCL). (A) Podocin expression was detected in whole kidney and PCL lysates by Western blotting. Lysates of COS7 cells, expressing 3xHA-tagged podocin with or without epitope, served as controls. (B) Podocin immunoreactivity localizes to the membrane and to areas of cell-cell contact in PCL (arrow). (C) PCL show nuclear staining for Wilm's tumor (WT-1). (D) Diffuse membrane staining for podocalyxin. (E) Filamentous staining for vimentin (scale bar indicates 20 m).
Figure 7.
Localization of actin-associated proteins in podocyte cell line (PCL). The actin-associated proteins CD2AP (left column), cortactin (middle column), and synaptopodin (right column) accumulate at areas of cell-cell contact in confluent cells (A to C, arrows). Double staining for F-actin (D to F) demonstrates that CD2AP, cortactin, and synaptopodin colocalize with F-actin along the cell borders [orange to yellow color in merged images (G to I)]. At higher magnification, concentration of synaptopodin in dense F-actin structures at the cell border is visible (I, arrow). In colocalization with F-actin, the staining pattern for CD2AP and cortactin is more continuous, and is not restricted to dense F-actin structures (J and K, arrows) [scale bar indicates 20 m (A to I) and 5 m (J to L), respectively].
Figure 8.
Redistribution of actin-associated proteins upon cell contact in podocyte cell line (PCL). In dispersed cells, CD2AP (left column) and cortactin (middle column) are enriched at some protrusions (A and B, arrows), while large areas of cell margins remain unstained (A and B, arrow heads). Synaptopodin (right column) localizes to stress fibers in dispersed cells (C, arrow). In confluent cells, CD2AP, cortactin, and synaptopodin accumulate at areas of cell-cell contact (D to F, arrows), while accumulation is not observed at cell margins, which line cell-free spaces (D to F, arrow heads) (scale bar indicates 20 m).
When PCL cells were seeded at non-confluent density, cells possessed long and short processes (Figure 4a, arrows), resembling the arborized phenotype of conditionally immortalized podocytes generated by Mundel et al15. At confluence, PCLs formed a monolayer with only a few gaps between cells Figure 4b. When PCL cells were shifted from permissive to nonpermissive culture conditions, proliferation slowed down progressively with a population doubling time of 5.0 0.7 days in the first week and 10.9 1.2 days in the second week (N = 3). Proliferation ceased in the third and fourth week under nonpermissive conditions Figure 4c as measured by the MTT assay. Similar results were obtained in cell counting experiments (data not shown).
Figure 4.
Morphology and proliferation of podocyte cell line (PCL). Phase-contrast images of PCL at nonconfluent (A) and confluent (B) cell density. Nonconfluent PCL cells possess multiple processes (arrows). When PCL are shifted to non-permissive culture conditions (day 0), cell number reaches a plateau after 2 weeks (C). Data are means SEM of three experiments (scale bar indicates 50 m).
In view of the expression of nephrin and all other SDPs/SDAPs in PCLs, we examined the ultrastructure of cell-cell contacts in confluent PCLs by transmission electron microscopy. Electron microscopy of vertical and horizontal cell sections revealed that cell-cell contacts of PCLs share characteristics with those observed in podocytes in situ Figure 5. PCL cells frequently possessed interlacing finger-like processes of about 150 nm width (Figure 5, arrows). These processes formed large areas of contact with a regular spacing of 30 to 40 nm and with some electron-dense material in the intercellular space (Figure 5, arrow heads), being reminiscent of foot process interdigitation in situ31, however, in a very rudimentary and incomplete fashion. Tight junctions were not observed between PCL cells.
Figure 5.
Ultrastructure of cell-cell contacts in podocyte cell ine (PCL). Areas of cell-cell contact were examined in horizontal (A and B) and vertical (C and D) sections through confluent PCL cells by electron microscopy. Cell borders are characterized by interlacing finger-like processes (arrows). Cell-cell contacts frequently show a regular spacing of 30 to 40 nm with electron-dense material in-between (arrow heads) (scale bars indicate 200 nm).
Full figure and legend (275K)In differentiated PCL cells, the nephrin distribution was altered upon the presence of cell-cell contacts Figure 6a to d. Dispersed cells, which possessed no or very few cell-cell contacts, showed prominent intracellular nephrin staining Figure 6a. Nephrin localized to the perinuclear region and was distributed in a pattern reminiscent of focal contacts (Figure 6a, arrow), as described by Saleem et al19 in a human podocyte cell line. Subconfluent PCL cells with an increased cell-cell contact area still showed the focal adhesion-like pattern of nephrin staining (Figure 6b, arrow), but also demonstrated accumulation of nephrin in areas of cell-cell contact (Figure 6b, arrow heads). In confluent PCL cells Figure 6c and d, nephrin was present predominantly along cell-cell contacts in a punctate pattern (Figure 6c, arrow head). A fraction of confluent PCL cells displayed even stronger immunoreactivity for nephrin at cell-cell contacts, staining as continuous bands Figure 6d. Intracellular staining for nephrin was weak in confluent cells. Nephrin completely colocalized with F-actin at areas of cell-cell contact in confluent PCL cells Figure 6e to g.
Figure 6.
Redistribution of nephrin upon cell contact in podocyte cell line (PCL). In dispersed cells with rare cell-cell contacts (A) and also in subconfluent cells (B), nephrin is concentrated in the perinuclear region and at focal adhesion-like sites (arrows). If cells possess larger areas of cell-cell contact (B) and form a continuous monolayer (C and D), nephrin is redistributed to areas of cell-cell contact (arrow heads). In most confluent cells, nephrin is concentrated in dots at cell-cell contacts (B and C) and, in some cells, continuous bands of nephrin staining are visible (D). Nephrin (E) colocalizes with F-actin (F) at areas of cell-cell contact in confluent cells (orange to yellow color in the merged image (G) (scale bar indicates 20 m).
Podocytes in situ are characterized by cytoskeletal compartmentalization (i.e., F-actin and several actin-associated proteins are concentrated in foot processes32). Among the actin-associated proteins with preferential localization in foot processes are synaptopodin14, CD2AP16,33, and cortactin2[abstract; Besse-Eschmann V, et al, J Am Soc Nephrol, 13: 530A, 2002]. Immunofluorescence staining revealed that CD2AP, cortactin, and synaptopodin were concentrated at areas of cell-cell contact in confluent PCL cells (Figure 7a to c, arrows). At areas of cell-cell contact, CD2AP, cortactin, and synaptopodin colocalized with F-actin Figure 7g to l. While CD2AP and cortactin colocalized with F-actin in a rather continuous, ruffle-like band along the cell border Figure 7j and k, synaptopodin was predominantly located at sites enriched with F-actin Figure 7l. In dispersed PCL cells, CD2AP, cortactin, and synaptopodin were largely absent from cell margins Figure 8a to c. CD2AP and cortactin staining in dispersed cells was restricted to the edge of a few processes (Figure 8a and b, arrows); the remaining areas of the cell margin were devoid of both proteins (Figure 8a and b, arrow heads). Synaptopodin was essentially confined to stress fibers in nonconfluent cells (Figure 8c, arrows). A further indication, that redistribution of CD2AP, cortactin, and synaptopodin to areas of cell-cell contact occurred in a contact-dependent fashion, was obtained in confluent PCL cells where cell-free spaces were encountered occasionally. CD2AP, cortactin, and synaptopodin were enriched at areas of cell-cell contact (Figure 8d to f, arrows), whereas cell margins without neighbors demonstrated only weak staining for the three proteins (Figure 8d to f, arrow heads).
DISCUSSION
We have generated PCLs in which we could demonstrate expression of 14 podocyte proteins (WT-1, Lmx1b, nephrin, NEPH1, FAT, P-cadherin, podocin, CD2AP, ZO-1, podocalyxin, podoplanin, vimentin, cortactin, and synaptopodin). The novel murine PCLs represent a major advance in podocyte culture because of two unique features. First, the novel PCLs are the only murine podocyte cell lines with stable nephrin expression that have been generated so far. Second, the novel PCLs is the only podocyte culture system in which nephrin localizes to cell-cell contacts. Though nephrin expression has been reported in two other cell lines of podocyte origin to date, nephrin does not localize to cell-cell contacts in these human cell lines18,19,20. In these human cell lines, nephrin shows either diffuse membrane staining18 or localizes to focal adhesion-like sites19,20. This distribution of nephrin to sites reminiscent of focal adhesions can also be observed in our novel PCL cells, but only in a nonconfluent state (cf. Figure 6). Obviously, expression of nephrin per se is not sufficient to result in localization of nephrin at cell-cell contacts. Possibly, the nephrin-expressing human cell lines lack some nephrin-interacting proteins needed to direct and/or to stabilize nephrin at cell-cell contacts. Given the critical importance of nephrin for the formation of slit diaphragms12,13,34, localization of nephrin at cell-cell contacts in our novel PCLs suggests that at least some questions concerning the molecular biology of podocyte cell-cell contacts can now be addressed in vitro.
In situ, the actin-associated proteins CD2AP, cortactin, and synaptopodin are highly concentrated in podocyte foot processes2,14,16,33[abstract; Besse-Eschmann V, et al, J Am Soc Nephrol, 13: 530A, 2002]. Similar to the redistribution of nephrin, we documented an enrichment of CD2AP, cortactin, and synaptopodin at areas of cell-cell contact in PCLs in a cell contact-dependent manner. CD2AP was discovered due to its ability to cluster CD2 at the immunologic synapse35. Later, CD2AP was shown to interact also with nephrin9. Two domains of CD2AP have been described to mediate nephrin-binding, a novel C-terminal domain as demonstrated in transfected HeLa cells16, and the third SH3 domain as determined in the M1 collecting duct cell line36. Thus, CD2AP could accumulate at cell-cell contacts in PCLs because of its ability to cluster nephrin. On the other hand, CD2AP is not only observed in podocyte foot processes in vicinity to the slit diaphragm16,33, but also in other cytosolic areas33. The cytosolic localization may point to another possible function of CD2AP, being the regulation of actin nucleation. In this respect, we have localized CD2AP as well as cortactin, which is known to be able to activate actin nucleation37, to small actin spots in cultured podocytes33. These small actin spots are likely to represent sites of highly dynamic actin turnover38,39. Localization of CD2AP to one end of actin tails in ARF6 overexpressing cells provides further evidence that CD2AP might be involved in dynamic actin processes40. Thus accumulation of CD2AP and cortactin at areas of cell-cell contacts in PCLs could indicate contact-dependent activation of actin nucleation with recruitment of actin nucleating proteins or, alternatively, immobilization of actin nucleating proteins in cell protrusions upon contact. Interestingly, besides activating actin nucleation, cortactin has been shown to stabilize newly formed F-actin branches41. Whether areas of cell-cell contact in PCLs and in podocytes in situ are characterized by stable actin filaments or by dynamic actin turnover will be important to determine, since pathologic alterations of podocyte cell-cell contacts depend on the reorganization of the actin cytoskeleton42.
Synaptopodin is an actin-bundling protein that is mainly located in stress fibers in cultured podocytes, in contrast to podocytes in situ where synaptopodin is almost exclusively found in foot processes14. In the PCLs presented, synaptopodin is redistributed from stress fibers to dense F-actin structures at areas of cell-cell contact. The mechanism by which synaptopodin is recruited to areas of cell-cell contact is unknown. Recently, it has been shown that synaptopodin binds to the membrane-associated guanylate kinase MAGI-143. Since MAGI-1 associates with megalin in podocytes44, interaction of synaptopodin with MAGI-1 and other proteins might be responsible for synaptopodin targeting to areas of cell-cell contact. Whether synaptopodin, in addition to its actin-bundling activity, stimulates actin nucleation and polymerization as reported for the homologues protein fesselin45 remains to be determined.
CONCLUSION
We present novel PCLs recapitulating characteristics of podocyte cell-cell contacts in situ. These cell lines will be of great use to study aspects of the molecular arrangement and formation of podocyte cell-cell contacts. Furthermore, our results suggest that formation of cell-cell contacts and accumulation of nephrin and actin-associated proteins in foot processes are interconnected events.
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Acknowledgments
We thank Claudia Kocksch for skilled and committed technical assistance, Hiltraud Hosser for expert electron microscopy imaging, Ingrid Ertl for photography, and Rolf Nonnenmacher for excellent graphic work. This study was supported by grants of the Medical Faculty of the University of Heidelberg (grant 313/2000) and the German Research Foundation (DFG, grant En 329/7–1) to K.E.
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