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First published online June 28, 2004
doi: 10.1242/10.1242/jcs.01163

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Enhanced podocalyxin expression alters the structure of podocyte basal surface

¹ Institute of Biology, National Center for Scientific Research `Demokritos', Agia Paraskevi, 15310 Athens, Greece
² Laboratory of Histology and Embryology, Medical School, University of Athens, 11527 Goudi, Athens, Greece
³ Department of Pediatrics, University of Michigan, 1500 E. Medical Center Drive, Ann Arbor, MI 48109, USA
⁴ Department of Clinical Pathology, University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria

View larger version (28K): [in a new window]   Fig. 1. Western blot analysis of PC expression in HGEC. Total protein was extracted from cells cultured either on plastic (control) or on increasing concentrations of collagen IV (A,D), laminin (B,E), or GBM (C,F) substrate. Equal amounts of total protein per substrate concentration (40 µg for A, 50 µg for B,C) were analyzed on SDS-PAGE and immunoblotted with 3D3 anti-PC antibody diluted 1:20 in TBS-5% milk. Blots were stripped and re-probed with anti-tubulin antibody. Lanes 1-5 (A-C), represent cell culture on substrates (0-20 µg/cm2). Quantification of the protein content of PC was performed by densitometric analysis (D-F). The bars represent the mean±s.d. from three independent experiments after normalizing to the density of ß-tubulin. Results were analyzed using single factor ANOVA and Newman-Keuls test (SNK) (*P<0.05 by ANOVA and SNK test; comparison of each different substrate concentration with zero concentration).

View larger version (27K): [in a new window]   Fig. 2. Northern blot analysis of PC mRNA in HGEC. (A) Total RNA was isolated from cells cultured on dishes either uncoated (control, lane 1) or coated with 20 µg/cm2 of each collagen IV (lane 2), laminin (lane 3), or GBM (lane 4). 10 µg of each sample were probed with [32P]dCTP-labeled human PC-cDNA. Filters were re-hybridized with [32P]dCTP-labeled cDNA probe for GAPDH. (B) Densitometric analysis of the mRNA level probed in panel A. The bars represent the mean±s.d. from three independent experiments after normalizing to the density of GAPDH. Results were analyzed using single factor ANOVA and SNK test (*P<0.05 by ANOVA and SNK test; comparison of each different substrate with plastic).

View larger version (18K): [in a new window]   Fig. 3. FACS analysis of PC cell-surface expression. Cells were cultured on dishes either uncoated (control-shaded histograms), or coated with 20 µg/cm2 of each collagen IV (panel a, unshaded histogram), laminin (panel b, unshaded histogram), or GBM (panel c, unshaded histogram). First peaks (interrupted lines) represent cells incubated only with fluorescein-conjugated anti-mouse IgG (negative control). Cells were incubated with 3D3 anti-PC antibody diluted 1:50 in FACS buffer and in sequence with fluorescein-conjugated anti-mouse IgG as a secondary antibody. Mean of fluorescence and percentage of positive cells were calculated for the histogram section indicated as M1.

View larger version (100K): [in a new window]   Fig. 4. Confocal microscopic analysis of PC distribution in HGEC. Cells were cultured on coverslips either uncoated (control) or coated with 20 µg/cm2 of each laminin or GBM substrate. HGEC were fixed in formaldehyde and stained with 3D3 anti-PC antibody diluted 1:50 in PBS and fluorescein-conjugated anti-mouse IgG as a secondary antibody. Panels a-c correspond to serial X-Y sections of the basal surface generated with a motor step of 0.5 µm. Panels d correspond to the lateral surface, and panels e to the apical surface. Scale bar, 15 µm.

View larger version (60K): [in a new window]   Fig. 5. Effects of laminin and GBM substrates on the expression of nephrin in HGEC. Western blotting (A) and confocal microscopy (B) were used to determine nephrin expression. (A) Total protein was isolated from cells cultured either on plastic (lane 1) or dishes coated with 20 µg/cm2 of each GBM (lane 2), or laminin (lane 3). 50 µg of total protein were analyzed on SDS-PAGE and immunoblotted with anti-nephrin antibody at a concentration of 2.5 µg/ml in TBS-5% milk. Blots were stripped and reprobed with anti-tubulin antibody (upper panel). Quantification of the protein content of nephrin was performed by densitometric analysis (lower panel). The bars represent the mean±s.d. from three independent experiments after normalizing to the density of ß-tubulin. Results were analyzed using single factor ANOVA and SNK test (*P<0.05 by ANOVA and SNK test; comparison of each different substrate with plastic). (B) For confocal microscopy, cells were cultured on coverslips, either uncoated (control) or coated with substrates, fixed and stained with anti-nephrin antibody at a concentration of 1 µg/ml in PBS and fluorescein-conjugated anti-mouse IgG as a secondary antibody. Panels a-c correspond to X-Y sections of the basal, lateral and apical surface respectively. Scale bar, 15 µm.

View larger version (62K): [in a new window]   Fig. 6. Confocal microscopic analysis of the distribution of ZO-1 in HGEC cultured on GBM. Cells were cultured on coverslips coated with 20 µg/cm2 of GBM substrate. HGEC were fixed in formaldehyde, permeabilized with Triton-X100 and stained with anti-ZO-1 antibody at a concentration of 5 µg/ml in PBS, 5% FCS and FITC-conjugated anti-mouse IgG as a secondary antibody. Scale bar, 10 µm.

View larger version (56K): [in a new window]   Fig. 7. Effects of high glucose concentration on the expression of PC. (A) Western blotting of PC expression in HGEC cultured in the presence of 5 mM or 25 mM glucose. Equal amounts of total protein (50 µg) were analyzed and blotted with 3D3 anti-PC antibody. Blots were stripped and reprobed with anti-tubulin antibody to verify equal loading. (B) Northern blot analysis of PC mRNA in HGEC. Total RNA was isolated from cells grown in 5 mM or 25 mM glucose. 10 µg of each sample were probed with [32P]dCTP-labeled human PC-cDNA. Filters were rehybridized with [32P]dCTP-labeled cDNA probe for GAPDH to verify equal loading. (C) FACS analysis of PC surface expression in HGEC cultured in 5 mM (unshaded histogram) or 25 mM glucose (shaded histogram). First peaks (interrupted lines) represent cells (cultured in 5 mM or 25 mM glucose) incubated only with FITC-conjugated IgG. Mean of fluorescence and percentage of positive cells was calculated in the histogram section indicated as M1. (D) Western blotting of PC expression in HGEC cultured on dishes either uncoated (lane 2) or coated with 20 µg/cm2 of each laminin (lane 3), or GBM (lane 4), in the presence of 25 mM glucose. Control cells were cultured on plastic in the presence of 5 mM glucose (lane 1). (E) PC expression in streptozotocin-treated diabetic rats. Micrographs represent PC staining in control (upper panel) or diabetic (lower panel) glomerulus. Scale bar, 50 µm.

View larger version (85K): [in a new window]   Fig. 8. Scanning electron micrographs. HGEC were cultured on coverslips either uncoated as a control (a) or coated with 20 µg/cm2 of laminin (b), or GBM (c). Arrow points to thin elongated end process, and arrowheads, spike-like processes. Scale bars, 10 µm in A; 2 µm in B.

View larger version (46K): [in a new window]   Fig. 9. Transmission electron micrographs HGEC were cultured on dishes either uncoated (control) or coated with 20 µg/cm2 of laminin, or GBM substrate. Arrows show laminin (b) and GBM (c); asterisks point to raised areas appearing on the basal surface in places of cell-cell contact; arrowhead indicates locally lifted areas at the basal cell surface. Scale bar, 1.5 µm.

View larger version (143K): [in a new window]   Fig. 10. Scanning electron micrographs of HGEC. Cells were cultured on coverslips either uncoated (b,e) or coated with 20 µg/cm2 of laminin (c,f), or GBM (d,g) in the presence of 25 mM glucose. Control cells were cultured on uncoated coverslips in the presence of 5 mM glucose (a). Scale bars, 15 µm in panels a,b; 10 µm in panels c,d; 2 µm in panels e-g.

View larger version (21K): [in a new window]   Fig. 11. HGEC adhesion to collagen IV or laminin in the presence of anti-PC antibody. [35S] methionine labeled HGEC grown in either 5 mM (A) or 25 mM (B) glucose, were allowed to adhere to collagen IV or laminin substrates in the presence of serial dilutions of 3D3 anti-PC antibody (1:50-1:2500). Control cells adhered in the absence of antibodies. Adherent cells were lysed and lysates were counted in a scintillation counter. Each value was plotted relative to the extent of cell adhesion achieved in the absence of antibodies, and represents the mean±s.d. of three independent experiments. Control values (adhesion in the absence of antibodies) were expressed as 100% adhesion. Single factor ANOVA and additionally SNK test were used independently in each experiment to determine whether the effect of monoclonal antibodies on cell adhesion was significant (*P<0.05).

View larger version (16K): [in a new window]   Fig. 12. Cell adhesion to collagen IV in the simultaneous presence of anti-ß1 integrin and anti-PC monoclonal antibodies. [35S] methionine labeled HGEC cultured in either 5 mM (A) or 25 mM (B) glucose, were allowed to adhere to collagen IV in the presence of anti-ß1 integrin monoclonal antibody which was used at 1:400 and 1:1500 dilution. The effects of anti-ß1 antibody on HGEC adhesion were examined in the presence or absence of serial dilutions of 3D3 anti-PC monoclonal antibody. Control cells adhered in the absence of mAbs. Adherent cells were lysed and lysates were counted in a scintillation counter. Each value, was plotted relative to the extent of cell adhesion achieved in the absence of antibodies, and represents the mean±s.d. of three independent experiments. Control values were expressed as 100% adhesion. Single-factor ANOVA and additionally Newman-Keuls test were used independently in each experiment to determine whether the effect of anti-PC monoclonal antibody on inhibition of cell adhesion, caused by anti-ß1 integrin mAb, was significant (*P<0.05).

View larger version (51K): [in a new window]   Fig. 13. HGEC spreading on collagen IV or laminin in the presence of monoclonal antibodies. Cells grown in either 5 mM (A) or 25 mM (B) glucose concentration, were spread on substrates as described, in the absence (panels a) or presence of 3D3 anti-PC antibody (panels b) or anti-HLA antibody. Each value represents the mean ± s.d. of three independent experiments and shows the percentage of cells in each size range. Single-factor ANOVA and SNK test was used to determine whether the presence of monoclonal antibody affected cell spreading in each size range (*P<0.05). Scale bar, 20 µm.

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