Figure 1.

SB216763, a highly selective small molecule inhibitor of GSK3, reinforces the Nrf2 antioxidant response in podocytes upon doxorubicin injury, attenuates podocyte apoptosis, and preserves actin cytoskeleton integrity. (A) Primary podocytes were prepared from WT mice, pretreated with SB216763 (SB, 10 μmol/L) or vehicle for 30 minutes, and then stimulated with doxorubicin (ADR; 0.25 μg/mL) or an equal volume of vehicle. Cells were harvested at indicated time points, and cell lysates prepared for immunoblot analysis for indicated molecules. GAPDH served as a loading control. (B) Podocytes were pretreated with tBHQ, 20 μmol/L), a standard Nrf2 activator, SB216763 (SB; 10 μmol/L), Trig (30 μmol/L), or an equal volume of vehicle for 30 minutes and then injured with doxorubicin (0.25 μg/mL) for 24 hours. Cells were fixed and subjected to rhodamine phalloidin staining of F-actin (red), fluorescence immunocytochemistry staining for Nrf2 (green), or TUNEL staining (green) and counterstaining with propidium iodide (PI, red) or 4,6-diamidino-2-phenylindole (DAPI, blue). Arrows indicate Nrf2 nuclear accumulation. Arrowheads indicate nuclear fragmentation in cells without Nrf2 nuclear accumulation following doxorubicin exposure, suggestive of cellular apoptosis. Scale bar, 20 μm. (C) Absolute counting of TUNEL-positive podocytes expressed as percentage of the total number of podocyte nuclei per high-power field. The data are expressed as mean±SEM. *P<0.05 versus ADR- or ADR+SB+Trig–treated cells (n=3). (D) Absolute count of Nrf2-positive nuclei expressed as percentage of the total number of podocytes per high-power field. The data are expressed as mean±SEM. *P<0.05 versus ADR- or ADR+SB+Trig-treated cells (n=3). Con, control.