Evaluation of glomerular sirtuin-1 and claudin-1 in the pathophysiology of nondiabetic focal segmental glomerulosclerosis

Figures

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  Figure 1. In focal segmental glomerulosclerosis, the glomerular loss of sirtuin-1 was accompanied by claudin-1 upregulation and podocyte injury. (a) Representative periodic acid-Schiff (PAS) staining of kidney sections (4 µm thick) from CTL or FSGS patients. The black arrows indicate positive staining for segmental glomerulosclerosis. (b) Representative immunohistochemistry micrographs (4 µm thick) and graphic presentation (c) show that nuclear SIRT1 was significantly reduced in FSGS patients. The black arrows indicate positive staining for nuclear SIRT1. (d) Representative immunohistochemistry micrographs (4 µm thick) and graphic presentation (e) show the upregulation of glomerular claudin-1 in FSGS patients. The black arrows indicate positive staining for glomerular claudin-1. (f) Representative immunohistochemistry micrographs (4 µm thick) and graphic presentation (g) show that glomerular podocin staining was greatly reduced in FSGS patients. The black arrows indicate positive staining for glomerular podocin. Statistical analyses were performed by parametric unpaired t test with Welch’s correction. Values are expressed as the mean ± SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001. Scale…

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  Figure 2. Chronic SRT1720 administration alleviates albuminuria and glomerulosclerosis in mice with adriamycin-induced nephropathy. (a) The 10% SDS-PAGE analysis and graphic presentation (b) show the abundance of urinary albumin, which was markedly reduced by SRT1720 chronic administration. Statistical analysis was performed by two-way ANOVA. A significant interaction between ADR/SRT1720 was observed by two-way ANOVA (F = 5.76; DF = 1; P = 0.043; Table 1). A bovine serum albumin standard (BSA, 1 mg/ml) was loaded on the adjacent lane (left). The cropped gel is displayed, and the full-length gel is included in the supplementary files. (c) Graphic presentation of the urinary albumin-to-creatinine ratio (mg/g). Statistical analysis was performed by two-way ANOVA. A significant interaction between ADR/SRT1720 was observed by two-way ANOVA (F = 12.8; DF = 1; P = 0.0072; Table 1). (d) Representative PAS micrographs (4 µm thick) and graphic presentation (e) show that chronic SRT1720 administration attenuated ADR + vehicle-induced glomerular sclerotic lesions, as well as protein cast deposition inside tubular structures. The yellow arrow shows protein casts inside tubules, and black arrows…

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  Figure 3. Chronic SRT1720 administration fails to attenuate glomerular claudin-1 in mice with adriamycin-induced nephropathy, although WT1 levels are maintained. (a) Representative micrographs (4 µm thick) and graphic presentation (b) confirming the upregulation of SIRT1 (red) by chronic SRT1720 administration. The white arrows indicate positive staining for nuclear SIRT1. Statistical analysis was performed by two-way ANOVA. (c) Representative micrographs (4 µm thick) and graphic presentation (d) show that chronic SRT1720 administration increases glomerular claudin-1 expression in ADR + vehicle-treated mice. The white arrows indicate positive staining for glomerular claudin-1. Statistical analysis was performed by two-way ANOVA. A significant interaction between ADR/SRT1720 was observed by two-way ANOVA (F = 4.37; DF = 1; P = 0.046; Table 1). (e) Representative micrographs (4 µm thick) and graphic presentation (f) show that chronic SRT1720 administration attenuates WT1 depletion in ADR + SRT1720-treated mice. The white arrows indicate positive staining for WT1. Statistical analysis was performed by two-way ANOVA. A significant interaction between ADR/SRT1720 was observed by two-way ANOVA…

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  Figure 4. Prior stimulation of sirtuin-1 with SRT1720 does not attenuate podocyte adriamycin-induced apoptosis in vitro. (a) Representative flow cytometry analysis and graphic presentation (b) show that ADR increases podocyte apoptosis in a dose-dependent manner. Cells were treated with various concentrations of ADR for 24 h, as indicated. Q1, cells in necrosis; Q2, cells in late apoptosis; Q3, cells in early apoptosis; and Q4, healthy cells. Total apoptosis means the sum of early and late apoptosis. Statistical analysis was performed by one-way ANOVA. (c) Representative immunoblots (10% SDS-PAGE) and graphic presentation (d) show SIRT1 protein abundance in SRT1720-treated cells, as indicated. The cropped blot is displayed, and the full-length gel is included in the supplementary files. (e) Representative flow cytometry analysis and graphic presentation (f) show that SRT1720 did not attenuate ADR-induced podocyte apoptosis. ADR cells were treated with 1 µM ADR for 24 h. ADR + SRT1720 cells were pretreated with SRT1720 [5 µM] for 24 h and then treated with ADR [1 µM] for 24 h. Total apoptosis means the sum of early and late apoptosis. Statistical analysis was performed by one-way ANOVA.…

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  Figure 5. Adriamycin-induced podocyte apoptosis is preceded by a rapid increase in claudin-1 expression, which is counteracted by sirtuin-1 acute stimulation with SRT1720. (a) Representative immunoblots (15% SDS-PAGE) and graphic presentation (b) show claudin-1 protein abundance in ADR [1 µM]-treated cells at various time points, as indicated. The cropped blot is displayed, and the full-length gel is included in the supplementary files. Statistical analysis was performed by one-way ANOVA. (c) Representative immunoblots (15% SDS-PAGE) and graphic presentation (d) show claudin-1 protein abundance in SRT1720 [5 µM, 24 h] + ADR [1 µM, 30 min] treated cells, as indicated. The cropped blot is displayed, and the full-length gel is included in the supplementary files. Statistical analysis was performed by two-way ANOVA. Values are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. CTL control cells (graph circles), ADR adriamycin-treated cells (graph squares), SIRT1 sirtuin-1, SRT1720 SIRT1-specific agonist, SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis, ANOVA analysis of variance.

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  Figure 6. Adriamycin treatment leads to podocyte sirtuin-1 translocation from the nucleus to the cytoplasm, as well claudin-1 upregulation. (a) Representative micrographs and graphic presentation (b) show the nuclear output of SIRT1 (green) in ADR [1 µM, 30 min]-treated cells. White arrows indicate cytoplasmic SIRT1. (c) Representative micrographs and graphic presentation (d) show increased claudin-1 staining in ADR [1 µM, 30 min]-treated cells. White arrows indicate concentrated claudin-1 (green) spots on cell membranes, as well as the perinuclear region. Values are expressed as the mean ± SEM. **P < 0.01, ****P < 0.0001. Statistical analyses were performed by unpaired t test with Welch’s correction. CTL control cells (graph circles), ADR adriamycin-treated cells (graph squares), SIRT1 sirtuin-1.