Skip to main content
. 2022 Mar 17;36(7-9):550–566. doi: 10.1089/ars.2021.0047

FIG. 6.

FIG. 6.

ENaC is regulated through aldosterone activation of NOXA1-dependent NOX in M1 renal epithelial cells. (A) Real-time PCR analysis of Nox1, Noxa1, Ncf1/2 (p47phox/p67phox), and Nox4 expression levels in M1 renal epithelial cells treated with vehicle or aldosterone for 2 h. Data are mean ± SEM of mRNA expression adjusted to 18s RNA levels. (B) DHE fluorescence was measured in M1 cells treated with vehicle or aldosterone for 30 min, or cells pretreated with 10 μM DPI, 50 μM oxypurinol, 10 μM L-NAME, or 20 mM NAC and then treated with aldosterone. Data are mean ± SEM of DHE fluorescence as percentage of control vehicle-treated cells. (C) DHE fluorescence was determined in control- or PEG-SOD treated M1 cells, or cells transduced with Noxa1, Nox1, or scrambled siRNA, and then treated with vehicle or aldosterone for 30 min. Data are mean ± SEM of DHE fluorescence as percentage of control-untreated cells. (D) Real-time PCR analysis of Nox1 mRNA expression in control and scrambled siRNA or Nox1 siRNA-transduced cells treated with aldosterone for 2 h. Data are mean ± SEM of mRNA expression fold change relative to control adjusted for 18s RNA levels. (E) Real-time PCR analysis of Noxa1 mRNA expression in control and scrambled siRNA or Noxa1 siRNA-transduced cells treated with aldosterone for 2 h. Data are mean ± SEM of mRNA expression fold change relative to control adjusted to 18s RNA. (F) Real-time PCR analysis of Scnn1a (αENaC) gene expression in control, PEG-SOD treated cells, or cells transduced with Noxa1, Nox1, or scrambled siRNA and treated with aldosterone for 2 h. Data are mean ± SEM of mRNA expression adjusted to 18s RNA. (G) Representative confocal microscopy z-stack images of polarized M1 renal epithelial cells treated with vehicle or aldosterone for 30 min, and stained for γENaC (green) and OCLN (red). Fluorescence quantification of apical and basolateral localization of γENaC in control and Noxa1 siRNA- or scrambled siRNA-transduced M1 cells treated with vehicle or aldosterone. DPI, diphenyliodinium; NAC, N-acetyl cysteine; L-NAME, L-nitro arginine methyl ester OCLN, occluding; PEG-SOD, polyethylene glycol-conjugated superoxide dismutase; siRNA, small-interfering RNA.