Figure 4.

Inhibition of GSK3β by TDZD-8 reduces the oxidative stress, improves mitochondrial dysfunction and prevents GSK3β overactivity in adriamycin-injured podocytes. (A) Differentiated podocytes in culture were administered adriamycin (0.25 μg·mL⁻¹) or vehicle (Ctrl) 1 h following pretreatment with TDZD-8 (TDZD or T, 5 μM), vehicle, or NAC (2 mM), a quintessential antioxidant and ROS scavenger. Before termination of the treatments at the indicated time points, podocytes were incubated with 10 μM DCF-DA for 30 min and the amount of reactive oxygen species was measured by fluorometric analysis of the fluorescence intensity of DCF in cell suspensions and expressed as fold induction over the value at time 0. *P < 0.05 versus other groups (n = 6). (B) Differentiated podocytes in culture were treated as described in (A) for 24 h. Cell viability was estimated by the MTT assay. *P < 0.05 versus Ctrl; ^†P < 0.05 versus ADR; (n = 6). (C) Representative fluorescent micrographs demonstrating TUNEL staining of cells that were treated as described in (A) for 24 h; (D) TUNEL-positive cells were counted and expressed as % of 100 cells that were labelled with DAPI. **P < 0.01 versus Ctrl; ^††P < 0.01 versus ADR; (n = 6). (E) Cell lysates were prepared from cells that were differently treated for 24 h as described in (A). Cell lysates were subjected to immunoblot analysis for cleaved of caspase-3 and GAPDH. Arbitrary units of cleaved caspase-3/GAPDH ratios are expressed as immunoblot densitometric ratios of the molecules as fold of Ctrl. **P < 0.01 versus Ctrl; ^†P < 0.05 versus ADR; (n = 4). (F) Cell lysates were prepared from cells that were differently treated for 24 h as described in (A). Cell lysates were subjected to immunoblot analysis for inhibitory phosphorylation of GSK3β (p-GSK3β) and total GSK3β. Arbitrary units of p-GSK3β/GSK3β ratios are expressed as immunoblot densitometric ratios of the molecules as fold of Ctrl. Both TDZD-8 and NAC effectively prevented the inhibitory effect of adriamycin on the phosphorylation of GSK3β. **P < 0.01 versus Ctrl; ^†,&P < 0.05 versus ADR; (n = 6). (G) Mitochondria were isolated from cells that were differently treated for 24 h as described in (A). Mitochondrial dysfunction, reflected by the propensity of mitochondria to swell when incubated with 20 mM CaCl₂ for 20 min, was spectrophotometrically monitored at 540 nm. **P < 0.01 versus Ctrl; ^†P < 0.05 versus adriamycin; (n = 6). (H) Cell lysates were prepared from cells that were differently treated for 24 h as described in (A). These lysates were then subjected to immunoprecipitation for Cyp-F and immunoprecipitates were processed for immunoblot analysis for phosphorylated serine (p-Ser).