Project Summary One hallmark feature of chronic heart failure (CHF) is sodium and fluid retention. While the cardio-renal syndrome has been recognized, a comprehensive understanding of the precise cellular mechanisms contributing to sodium and water retention in CHF remains elusive. Renal denervation (RDN) has been shown to reduce sodium retention in rats and dogs with CHF. One of the key elements involved in renal sodium retention is activation of epithelial sodium channels (ENaC) of principal cells in the collecting tubule. We have previously shown that ENaC subunit expressions and activity were increased in the kidneys from rats with CHF. We have recent evidence indicating that increased proteases in the tubular fluid may contribute to the enhanced renal ENaC activity, providing a novel mechanistic insight for sodium retention commonly observed in CHF. We have observed that in rats with CHF: 1) the levels of serine proteases were dramatically increased in the urine; 2) protease inhibitor treatment significantly abrogated the enhanced diuretic and natriuretic responses to ENaC inhibitor benzamil in rats with CHF. Furthermore, our preliminary data have shown that RDN decreased the levels of proteases in the urine, reduced protein expressions of ENaC subunits in the kidney of CHF rats, supporting the potential role of sympathetic nerve activation. Based on these data, we will test the hypothesis that RDN reduces renal sodium retention in CHF rats by modulation of the ENaC and protease-ENaC axis. In AIM 1 we will determine the contribution of RDN in the expression/inactivation of ENaC in rats with CHF rats. In AIM 2 we will determine the contribution of tubular proteases in activating ENaC in rats with CHF. These aims will be addressed in rats with CHF using complementary methodologies ranging from cellular to the whole animal level, including physiological measurement of sodium balance, ENaC activity, eletrophysiological recording, protease and ubiquitination of ENaC using molecular biology techniques. The successful completion of the proposed studies will provide significant new information and insight into the contribution of ENaC regulation in altered sodium balance in CHF and the therapeutic benefits of RDN on sodium fluid retention, endemic to CHF.