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 antinatriuresis (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 ␣, ␤, and ␥ subunits. ␣ and ␥ blots were further examined for subunit cleavage (a process that activates ENaC). No significant changes were observed in ␣ and ␤ levels throughout the study. However, levels of cleaved ␥ were elevated, suggesting that ENaC was activated. The changes of ␥ persisted at week 10 and were accompanied by additional subunit fragments, indicating potential changes of ␥-cleaving proteases. Enhanced protease activity, and specifically that which could act on the second identified cleavage site in ␥, 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 ␥-ENaC cleavage.Hypercholesterolemia is an established contributor to atherosclerosis and cardiovascular diseases. Increased cholesterol is also known to cause or contribute to renal injury by impairing the filtration permeability barrier and podocyte permeability (1). However, little is known regarding the effects of elevated dietary cholesterol on membrane cholesterol and renal sodium transport. We have recently demonstrated that short term membrane cholesterol enrichment modifies renal sodium transport, leading to increased absorption and antinatriuresis (2). These short term effects were observed within minutes of enrichment and were mediated, in part, by effects on sodium absorption in the loop of Henle. No other reports have examined the effects of membrane cholesterol enrichment on renal Na ϩ transport in vivo and especially so under mild enrichment conditions that precede the vasculature effects of hypercholesterolemia.It is established that membrane cholesterol content can modulate the activity of integral membrane proteins and that experimental depletion or enrichment of cholesterol mod...