Regulation of epithelial Na+ transport by soluble adenylyl cyclase in kidney collecting duct cells

Abstract

Alkalosis impairs the natriuretic response to diuretics, but the underlying mechanisms are unclear. The soluble adenylyl cyclase (sAC) is a chemosensor that mediates bicarbonate-dependent elevation of cAMP in intracellular microdomains. We hypothesized that sAC may be an important regulator of Naa transport in the kidney. Confocal images of rat kidney revealed specific immunolocalization of sAC in collecting duct cells, and immunoblots confirmed sAC expression in mouse cortical collecting duct (mpkCCDc14) cells. These cells exhibit aldosterone-stimulated transepithelial Naa currents that depend on both the apical epithelial Naa channel (ENaC) and basolateral Na+,K+-ATPase. RNA interference-mediated 60-70% knockdown of sAC expression comparably inhibited basal transepithelial short circuit currents (Isc) in mpkCCDc14 cells. Moreover, the sAC inhibitors KH7 and 2-hydroxyestradiol reduced Isc in these cells by 50-60% within 30 min. 8-Bromoadenosine-3′,5′-cyclic-monophosphate substantially rescued the KH7 inhibition of transepithelial Na+ current. Aldosterone doubled ENaC-dependent Isc over 4 h, an effect that was abolished in the presence of KH7. The sAC contribution to Isc was unaffected with apical membrane nystatin-mediated permeabilization, whereas the sAC-dependent Na+ current was fully inhibited by basolateral ouabain treatment, suggesting that the Na+,K+-ATPase, rather than ENaC, is the relevant transporter target of sAC. Indeed, neither overexpression of sAC nor treatment with KH7 modulated ENaC currents in Xenopus oocytes. ATPase and biotinylation assays in mpkCCDc14 cells demonstrated that sAC inhibition decreases catalytic activity rather than surface expression of the Na%+,K+-ATPase. In summary, these results suggest that sAC regulates both basal and agonist-stimulated Naa reabsorption in the kidney collecting duct, acting to enhance Na+,K+-ATPase activity. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.