ENaC inhibition stimulates Cl− secretion in the mouse cortical collecting duct through an NKCC1-dependent mechanism

In cortical collecting ducts (CCDs) perfused in vitro, inhibiting the epithelial Na+ channel (ENaC) reduces Cl− absorption. Since ENaC does not transport Cl−, the purpose of this study was to determine how ENaC modulates Cl− absorption. Thus, Cl− absorption was measured in CCDs perfused in vitro that were taken from mice given aldosterone for 7 days. In wild-type mice, we observed no effect of luminal hydrochlorothiazide on either Cl− absorption or transepithelial voltage ( V T). However, application of an ENaC inhibitor [benzamil (3 μM)] to the luminal fluid or application of a Na+-K+-ATPase inhibitor to the bath reduced Cl− absorption by ∼66–75% and nearly obliterated lumen-negative V T. In contrast, ENaC inhibition had no effect in CCDs from collecting duct-specific ENaC-null mice (Hoxb7:CRE, Scnn1aloxlox). Whereas benzamil-sensitive Cl− absorption did not depend on CFTR, application of a Na+-K+-2Cl− cotransport inhibitor (bumetanide) to the bath or ablation of the gene encoding Na+-K+-2Cl− cotransporter 1 (NKCC1) blunted benzamil-sensitive Cl− absorption, although the benzamil-sensitive component of V T was unaffected. In conclusion, first, in CCDs from aldosterone-treated mice, most Cl− absorption is benzamil sensitive, whereas thiazide-sensitive Cl− absorption is undetectable. Second, benzamil-sensitive Cl− absorption occurs by inhibition of ENaC, possibly due to elimination of lumen-negative V T. Finally, benzamil-sensitive Cl− flux occurs, at least in part, through transcellular transport through a pathway that depends on NKCC1.