Aldosterone Induces Oxidative Stress via NADPH Oxidase and Downregulates the Endothelial NO Synthase in Human Endothelial Cells

Aldosterone (Aldo) is traditionally viewed as a hormone regulating electrolyte homeostasis and blood pressure. Recent studies suggest that Aldo can cause microvascular damage, oxidative stress and endothelial dysfunction. However, its exact cellular mechanism in vascular complications remains unclearly defined. This study was conducted to determine the mechanisms responsible for Aldo-induced oxidative stress and the possible involvment of Aldo in the expressionnal regulation of the endothelial NO synthase (eNOS) in human umbilical artery endothelial cells (HUAECs). HUAECs were incubated for 24 h with Aldo (100 nmol/L) in the presence and absence of Spiro (1 μmol/L). NADPH oxidase protein expression and its activity were estimated. Moreover, eNOS protein level and 3-nitrotyrosine (3-NT) contents were also determined. Results showed that stimulation of HUAECs with Aldo (100 nmol/L) resulted in a significant upregulation in NADPH oxidase subunits (Nox2, p47phox and p22phox protein levels), and these alterations were significantly abolished on pre-incubation with the MR antagonist, Spiro. However, Aldo did not exhibit any significant change in Nox4 protein level. Using MR-reactive antibodies, we investigated the MR protein expression in HUAECs in response to Aldo. Of note, MR protein expression is upregulated by Aldo and downregulated by Spiro. Functionally, these effects were reflected on increased oxidative stress markers as evident by increased NADPH oxidase activity and 3- NT. Moreover, Aldo significantly inhibited eNOS protein expression and pretreatment with Spiro restored eNOS to the normal level. Taken together, the present results demonstrate that Aldo stimulates NADPH oxidase-mediated oxidative stress thereby reducing eNOS expression and Nox2 appears to be the most relevant isoform in this setting. Moreover, the MR antagonist, Spiro efficiently inhibited these changes.