Enhanced levels of proteases in tubular fluid activate ENaC in chronic heart failure

Abstract

One of the key mechanisms involved in renal sodium retention in chronic heart failure (CHF) is activation of epithelial sodium channels (ENaC) in collecting tubules. Proteolytic cleavage and ubiquitylation/deubiquitylation have important roles in activating ENaC. We hypothesized that enhanced levels of proteases in tubular fluid activate ENaC resulting in renal sodium retention in rats with CHF. CHF was produced by left coronary artery ligation in rats. By immunoblotting, we found that several urinary serine proteases were significantly increased in CHF compared to sham rats (increased fold: furin 6.7, prostasin 23.6, plasminogen 2.06 and plasmin 3.57 vs. sham, P<0.05). Whole‐cell patch‐clamp was conducted in cultured renal collecting duct M‐1 cells to record Na+ currents. Protease‐rich urine (from rats with CHF vs. sham rats) significantly increased the Na+ inward current in M‐1 cells (7.2 folds). Ubiquitin assay demonstrated reduced ubiquitylated γ‐ENaC in the kidneys of CHF rats. We also found that ubiquitin‐protein Nedd4 was decreased (79%) and deubiquitylation enzyme Usp2–45 was increased (75%) in the isolated pure populations of tubular fragments from CHF rats. These findings demonstrate for the first time that increased proteases in the tubular fluid contribute to the enhanced ENaC activity, providing a novel mechanistic insight for sodium retention commonly seen in CHF.