Figure 3.

Inhibition of GSK3β is necessary and sufficient for the induced Nrf2 antioxidant response and protection against actin cytoskeleton disorganization in podocytes upon doxorubicin injury. Primary podocytes were prepared from WT mice and transiently transfected with vectors encoding the empty vector (EV), the HA-conjugated WT, dominant negative KD, or constitutively active (S9A) GSK3β. (A) Representative micrographs of fluorescent immunocytochemistry staining for HA (green). Cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI; blue). Scale bar, 20 μm. Transfection efficiency in the primarily cultured podocytes typically ranged from 30% to 60% and varied depending on the condition of the cultures. The batch of cultures with transfection efficiency >50% was used for subsequent experiments. (B) After transfection, cells were treated with 0.25 µg/ml of doxorubicin for 24 hours in the presence or absence of SB216763 (SB; 10 µmol/L). Cell lysates were collected and subjected to immunoblot analysis for Nrf2, HO-1, GSK3, HA, cleaved caspase 3, and GAPDH. (C) Cells, treated as stated in B, were fixed and subjected to immunofluorescence staining for Nrf2 (green) and rhodamine phalloidin labeling of F-actin (red) and counterstained with propidium iodide (PI, red) or DAPI (blue). Arrowheads indicate nuclear fragmentation in cells without Nrf2 nuclear accumulation after doxorubicin exposure, suggestive of cellular apoptosis. Arrows indicate Nrf2 nuclear accumulation. Scale bar, 20 μm. (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 all other groups; ^#P<0.05 versus S9A (n=3).