Epithelial Sodium Channel in Aldosterone-Induced Endothelium Stiffness and Aortic Dysfunction

Abstract

Enhanced activation of the endothelial mineralocorticoid receptor contributes to the development of arterial stiffness, which is an independent predictor of cardiovascular disease. Previously, we showed that enhanced endothelium mineralocorticoid receptor signaling in female mice prompts expression and translocation of the α-subunit of the epithelial sodium channel to the endothelial cell (EC) surface (EnNaC) inducing vascular fibrosis and stiffness. Further, amiloride, an epithelial sodium channel antagonist, inhibits vascular fibrosis, remodeling, and stiffness induced by feeding a Western diet high in saturated fat and refined carbohydrates. However, how this occurs remains unknown. Thereby, we hypothesized that endothelial cell-specific EnNaC activation is necessary for aldosterone-mediated endothelium stiffness. To address this notion, EnNaC α-subunit knockout (EnNaC^-/-) and wild-type littermate female mice were administrated aldosterone (250 µg/kg per day) via osmotic minipumps for 3 weeks beginning at 25 to 28 weeks of age. In isolated mouse endothelial cells, inward sodium currents were significantly reduced in amiloride controls, as well as in EnNaC^-/-. Likewise, aldosterone-induced endothelium stiffness was increased and endothelium-dependent relaxation less in EnNaC^-/- versus wild-type. Further, EnNaC^-/- mice exhibited attenuated responses to aldosterone infusion, including aortic endoplasmic reticulum stress, endothelium nitric oxide synthase activation, endothelium permeability, expression of proinflammatory cytokines, oxidative stress, and aortic collagen 1 deposition, supporting the notion that αEnNaC subunit activation contributes to these vascular responses.

Keywords: aldosterone; cardiovascular disease; endothelium; epithelial sodium channel; inflammation.

Fig 1

Effect of Aldo and ENaC on inward Na⁺ currents in ECs. (A) Na⁺ current tracings in ECs with or without amiloride stimulation. (B) Amiloride repressed inward Na⁺ currents in cultured ECs, n=4–5 cells. Na⁺ current tracings (C), group I–V curves of Na⁺ currents (D), and comparison of peak inward Na+ currents at −80 mV (E) in EnNaC WT and EnNaC^−/− mice treated with Aldo, n=16 cells. *p<0.05 compared with control or EnNaC WT group in multiple comparison analysis.

Fig 2

EnNaC^−/− mice prevented Aldo infusion-induced endothelium stiffness and aortic relaxation dysfunction. (A) The ex vivo measurement of endothelium stiffness by using atomic force microscopy, n=4–5. (B) Aortic pulse wave velocity measured in vivo, n=3–6. (C) Vasodilator responses of isolated aortic rings to the endothelium-dependent dilators, acetylcholine (C) and to the endothelium-independent vasodilator, sodium nitroprusside (D), n=4. *p<0.05 compared with EnNaC WT group; #p<0.05 compared with Aldo in EnNaC WT group in multiple comparison analysis.

Fig 3

EnNaC^−/− mice prevented Aldo infusion-induced endoplasmic reticulum stress and reduction of eNOS activity. (A) The protein abundance of GRP78 and eNOS in aortic tissues were performed with immunoblotting. Quantitative analysis of protein abundance in GRP78 (B) and p-eNOS (C). *p<0.05 compared with EnNaC WT group; #p<0.05 compared with Aldo in EnNaC WT group in multiple comparison analysis.

Fig 4

EnNaC^−/− mice prevented Aldo-induced expression of endothelium adhesion molecules, permeability, and pro-inflammatory cytokines. (A) The protein abundance of ICAM-1 and VCAM-1 in aortic tissues. (B) ex vivo aortic endothelium permeability assay, n=5. mRNA levels of CD68 (C), IL1 (D) and IL6 (E) in aortic tissues, n=4. *p<0.05 compared with EnNaC WT group; #p<0.05 compared with Aldo in EnNaC WT in multiple comparison analysis.

Fig 5

EnNaC^−/− mice prevented Aldo infusion-induced aortic oxidative stress and remodeling. (A) Representative images of aortic sections stained for 3-NT, a marker of oxidant stress from accumulation of oxidant peroxynitrite (ONOO⁻). (B) Representative image immunostaining for NOX2 with corresponding measures of average gray scale intensities (C–D). Representative image immunostaining for collagen 1 (E) with corresponding measures of average gray scale intensities (F). Scale bar = 50 μm. n=5–6 per group. *p<0.05 compared with EnNaC WT group; #p<0.05 compared with Aldo in EnNaC WT in multiple comparison analysis.

Fig 6

EnNaC mediates Aldo-induced endothelium stiffness and aortic dysfunction. Aldo increases EnNaC membrane abundance in ECs which promotes Na⁺ entry into the endothelium, resulting in increases in GRP78, inflammation, 3-NT and NOX2 and a reduction of eNOS activity and NO production and a subsequent increase in vascular stiffness.