Nephrin Is Critical for the Action of Insulin on Human Glomerular Podocytes
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Figures
- FIG. 1.
Nephrin expression in NM mutant podocytes. A: IF of WT and NM podocytes. Both were thermoswitched for 14 days with the same antibody concentrations and incubation time. WT shows peripheral staining (arrowed). In NM, nephrin is located within the cytoplasm. B: NM quantitatively shows nephrin expression as demonstrated by Western blot. Single band corresponds with positive control from human glomeruli. Western blot and IF were performed with mouse monoclonal antibody 50A9.
- FIG. 2.
Insulin-stimulated 2-DOG uptake in human nephrin mutant podocyte cell lines. 2-DOG uptake in WT and NM and FM immortalized podocytes. Basal wells (−) were compared with 15 min of stimulation with 100 nmol/l insulin (+). Seven to 14 independent experiments were performed for each condition (SEM shown). Significant difference between groups, P < 0.001 (ANOVA). Post hoc Bonferroni reveals a significant increase in WT insulin response (*) compared with either nephrin mutant cell lines (FM or NM); P < 0.01.
- FIG. 3.
Nephrin knockdown and glucose uptake in WT human podocytes. A: Densitometry of three independent experiments showing the nephrin/actin ratio between vehicle, nephrin, and scramble siRNA. Nephrin knockdown results in a 62% reduction in nephrin/actin signal (SEM shown, analyzed using ANOVA). Significant difference between groups, P < 0.05. Post hoc Bonferroni analysis; *P < 0.05 vs. vehicle. B: 2-DOG uptake in WT podocytes with nephrin-specific siRNA (nephrin siRNA), scrambled siRNA (scram siRNA), and vehicle-only treated podocytes (veh); five to seven independent experiments for each condition. SEM shown. ANOVA, P = 0.01 for all groups. Post hoc Bonferroni analysis with significant decrease in nephrin siRNA-treated cells (*). P < 0.05 in comparison to WT podocytes. No significant difference in scramble siRNA-treated cells. In this set of experiments, vehicle alone resulted in an insulin-stimulated increased glucose uptake of 59% compared with 105% seen in previous work (12).
- FIG. 4.
A: Stable nephrin transfection in FM podocytes. Nephrin IF using monoclonal antibody 48E11 (i). Peripheral nephrin staining (indicated by arrow) shown in FMnt. Mouse monoclonal isotype control and FM show minimal staining for this antibody. Western blot for nephrin (ii). Monoclonal 50A9 antibody used signal in human glomerular positive control and FMnt lanes. FM lane negative for nephrin. Equal amounts of protein were loaded for each (90 μg). B: 2-DOG uptake in nephrin-transfected FM podocytes. 2-DOG uptake after 15 min of 100 nmol/l insulin (+) stimulation or in basal state (−). Comparison of nephrin-transfected FMnt cells and nontransfected FM cells. Significant increase in glucose uptake in response to insulin in FMnt cells. **P < 0.001 using a paired two-tailed Student's t test; n ≥6 experiments for each condition studied.
- FIG. 5.
A: Nephrin enables GLUT1 and GLUT4 to translocate and fuse with the plasma membrane of the podocyte. GLUT1 bis-glucose demonstrated that in FMnt cells, GLUT1 was present at the plasma membrane of the podocyte and was also increased with a 15-min treatment of 100 nmol/l insulin. FM demonstrated no GLUT1 signal, despite more protein being precipitated with streptavadin in the FM preparation (288 μg) compared with FMnt cells (166.5 μg). B: Nephrin enables GLUT1 and GLUT4 to translocate and fuse with the plasma membrane of the podocyte. IF for GLUT4 using the 1F8 antibody in insulin-treated cells (100 nmol/l for 15 min) showed that in the nephrin mutant cells (NM and FM), vesicular GLUT4 vesicles were present in a subplasma membrane location (arrowed) after insulin stimulation, but in WT and FMnt cells, GLUT4 signal appeared to be fused with the plasma membrane (*).
- FIG. 6.
A: Glutathionine precipitation of the COOH of nephrin with VAMP2. Glutathione precipitation of pEGFP-VAMP2 by pcDNA-GST-COOH nephrin (NPHN) (lane 1) and pEGFP-VAMP2 by pcDNA-GST (lane 3) using glutathione-Sepharose beads (Amersham/Pharmacia). Precipitated samples were subjected to SDS-PAGE and then immunoblotted with GFP antibody (Vector Labs). No band was detected in lane 3, which indicates that no precipitation occurred between pcDNA-GST alone and pEGFP-VAMP2. Conversely, a band of 46 kDa is detected by the GFP antibody in lane 1. This verifies the presence and therefore successful precipitation of pEGFP-VAMP2 by nephrin. Lane 2 demonstrates 10% input. B: Glutathione precipitation of pEGFP-VAMP2 by pcDNA-GST-NPHN (1057–1241) (lane 1) and pEGFP-VAMP2 by pcDNA-GST (empty) (lane 3) subsequently immunoblotted with GST antibody (Clontech; BD Biosciences) to confirm presence of GST-tagged reagents. The detection of bands of 26 kDa, corresponding to the size of pcDNA-GST (empty) (lane 3), and of 42kDa, corresponding to the size of pcDNA-GST-NPHN (lane 1), verifies the presence of pcDNA-GST alone and pcDNA-GST-NPHN within the precipitation. Lane 2 shows 10% input.
- FIG. 7.
Nephrin–VAMP2 coimmunoprecipitation in human glomeruli. A: VAMP2 immunoprecipitation with monoclonal mouse anti-VAMP2 antibody and probing of Western blot with anti-nephrin rabbit antibody. A negative control of normal mouse antibody (Sigma) was used for pull-down in lane 1. Positive glomerular whole cell lysate in lane 4. Lane 2 is insulin-stimulated glomeruli, which gave increased signal compared with basal glomeruli (lane 3). Equal amounts of protein were immunoprecipitated in lanes 1–3 (750 μg) B: Nephrin pull-down of human glomeruli followed by VAMP2 Western blot. The Profound system was used for this (research design and methods). Whole glomerular lysate positive control (lane 2) and normal rabbit immunoglobulin negative control (lane 1) are shown. Equal amounts of antibody were used for immunoprecipitation (100 μg) and equal amounts of glomeruli were immunoprecipitated (700 μg). Pull-down of nephrin–VAMP2 complex is shown by probing the blot with anti-VAMP2 antibody (bottom panel). Then blot was stripped and reprobed for nephrin (top panel). Nephrin IP results in pull-down of VAMP2 (lane 3).
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