NADPH oxidase signaling regulates Epithelial Sodium Channel (ENaC) activity

Abstract

In general, over‐production of superoxide (O2−) in the kidneys leads to serious renal disorders. However, when released in low quantities in a controlled manner, O2− may serve as an important signaling molecule in epithelial cells. Presently, we examine whether sodium transporting epithelial cells can control release of O2− , through regulated NADPH oxidase activity, in order to regulate ENaC in renal epithelia. First, we showed that A6 distal nephron cells express each of the critical NADPH oxidase (NOX) components that catalyze the production of O2− (membrane bound gp91phox and p22phox; cytosolic p67 phox and p47 phox subunits; and small G protein Rac1) using western blot. Since complete activation of NOX is believed to occur following Rac1activation, we then tested whether inhibition or activation of Rac1 influenced ENaC activity. We report that, indeed, 1μM NSC23766 inhibition of Rac1 significantly decreases single channel measurements of ENaC activity from 1.16+.27 to .50+.11 (mean NPo values+SE, n=6, p<0.05). Conversely, 10ng/mL EGF activation of Rac1 significantly increased ENaC NPo values, on average, from .93+.25 to 1.30+.31 in 6 independent observations (p<0.05). HPLC measurements verify that Rac1 inhibition decreases O2− release. Very briefly, we conclude that NADPH‐oxidase induced O2− signaling is a new pathway for ENaC regulation.