FIG. 5.

Aldosterone-induced activation of Nrf2 in LLC-PK1 cells is mediated by an increase of reactive oxygen and nitrogen species. (A) Kinetics of oxidant production caused by aldosterone in LLC-PK1 cells. ROS production was measured using the probe H₂DCF-DA. LLC-PK1 cells were treated for the indicated time periods with 10 or 100 nM aldosterone. Oxidation of H₂DCF-DA was detected by monitoring the increase in fluorescence (λexc: 485 nm; λem: 535 nm). (B) LLC-PK1 cells were incubated in the absence (C) and presence of 10 and 100 nM aldosterone for 4 and 24 h. The GSH/GSSG ratio was measured at 405 nm. Results are expressed as GSH/GSSG ratio. (C) LLC-PK1 cells were incubated in the absence (C) and the presence of 100 nM aldosterone (Ald) for 30 min. Aldosterone-treated cells were simultaneously incubated with 0.5 mM LA (LA), 0.5 mM NAC (NAC), or 50 μM tempol (Tem). Nuclear fractions were isolated and analyzed by EMSA. The intensity of the bands corresponding to the Nrf2-DNA complexes was measured. (A, B, E) Shown are mean values±SEM, *p≤0.05 versus time point 0, ^#p≤0.05 versus the aldosterone-treated group, tested by ANOVA with subsequent post-hoc comparisons by Scheffé. (C, D) Shown is the median±the standard error of the median, *p≤0.05 versus the control group, evaluated by Mann–Whitney test. H₂DCF-DA, 5-(and-6)-carboxy-2′7′-dichlorodihydro-fluorescein diacetate; LA, (±)-α-lipoic acid; NAC, N-acetyl cysteine; Tem, tempol.