Acute Cholesterol-induced Anti-natriuretic Effects ROLE OF EPITHELIAL Na+ CHANNEL ACTIVITY, PROTEIN LEVELS, AND PROCESSING

The epithelial Na+ channel (ENaC) is modulated by membrane lipid composition. However, the effect of an in vivo change of membrane composition is unknown. We examined the effect of a 70-day enhanced cholesterol diet (ECD) on ENaC and renal Na+ handling. Rats were fed a standard chow or one supplemented with 1% cholesterol and 0.5% cholic acid (ECD). ECD animals exhibited marked anti-diuresis and anti-natriuresis (40 and 47%), which peaked at 1-3 weeks. Secondary compensation returned urine output and urinary Na+ excretion to control levels by week 10. During these initial changes, there were no accompanying effects on systolic blood pressure, serum creatinine, or urinary creatinine excretion, indicating that the these effects of ECD preceded those which modify renal filtration and blood pressure. The effects of ECD on ENaC were evaluated by measuring the relative protein content of alpha, beta, and gamma subunits. alpha and gamma blots were further examined for subunit cleavage (a process that activates ENaC). No significant changes were observed in alpha and beta levels throughout the study. However, levels of cleaved gamma were elevated, suggesting that ENaC was activated. The changes of gamma persisted at week 10 and were accompanied by additional subunit fragments, indicating potential changes of gamma-cleaving proteases. Enhanced protease activity, and specifically that which could act on the second identified cleavage site in gamma, was verified in a newly developed urinary protease assay. These results predict enhanced ENaC activity, an effect that was confirmed in patch clamp experiments of principal cells of split open collecting ducts, where ENaC open probability was increased by 40% in the ECD group. These data demonstrate a complex series of events and a new regulatory paradigm that is initiated by ECD prior to the onset of elevated blood pressure. These events lead to changes of renal Na+ handling, which occur in part by effects on extracellular gamma-ENaC cleavage.