Figure 10.

Blockade of GSK3β by SB216763 in murine models of nephrotoxic serum nephritis reinforces the Nrf2 antioxidant response in podocytes and protects against podocytopathy and proteinuria. WT FVB/NJ male mice aged 8 weeks were treated with a tail-vein injection of control rabbit IgG or GBM-reactive rabbit anti-mouse glomerular lysate NTS at a dose of 10 µl/g body wt. Mice received a subcutaneous injection of SB216763 (1.5 mg/kg) or vehicle 1 hour before NTS or control IgG injection and afterward every the other day. (A) Quantification of urine albumin levels adjusted by urine creatinine concentrations. The data are expressed as mean±SEM and were subjected to logarithmic transformation and analyzed by repeated-measures ANOVA followed by post hoc Scheffé test. The test for a difference in urine albumin-to-creatinine ratios over time was significant (F[3, 45]=61.061; P<0.01). The test for equality of treatment means over time was also significant (F[2, 15]=45.082; P<0.01). *P<0.05 versus NTS+vehicle (n=6). (B) Kidneys were excised from animals on day 3, and glomeruli were isolated by the magnetic beads–based approach. Glomerular homogenates were prepared for immunoblot analysis for Nrf2, HO-1, pGSK3β, GSK3β, and synaptopodin. GAPDH served as a loading control. (C) Representative micrographs of immunofluorescence staining of kidney specimens procured on day 3 for the GBM-reactive heterologous rabbit IgG in glomeruli. Scale bar, 20 μm. (D) Electron microscopy (EM) of podocyte ultrastructural injury in kidney tissues procured from mice on day 3. Arrowheads indicate lesions of podocyte foot process effacement. Scale bar, 2 μm. (E) Cryosections of kidney specimens were prepared for dual color immunofluorescence staining for Nrf2 (green) and WT-1 (red) to demonstrate Nrf2 nuclear accumulation in glomerular podocytes (indicated by arrowheads). Scale bar, 20 μm. (F) Representative micrographs demonstrate PAS staining of mouse kidneys. Scale bar, 10 μm. (G) Measurement of the width of podocyte foot processes (FP) on electron microscopy in mice 3 days after NTS injury. *P<0.05 versus vehicle-treated mice with NTS nephritis (n=6). (H) Absolute counts of the number of WT-1–positive podocytes in each glomerulus in kidney specimens procured on day 3, as shown by fluorescent immunohistochemistry staining for WT-1 in E. *P<0.05 versus vehicle-treated mice with NTS nephritis (n=6). (I) Morphometric scoring of glomerular damage on PAS staining of kidney tissues procured from mice on day 3. *P<0.05 versus vehicle-treated mice with NTS nephritis (n=6).