Regulation of Epithelial Na+ Transport by Soluble Adenylyl Cyclase in Kidney Collecting Duct Cells

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 Na⁺ 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 (mpkCCD_c14) cells. These cells exhibit aldosterone-stimulated transepithelial Na⁺ currents that depend on both the apical epithelial Na⁺ channel (ENaC) and basolateral Na⁺,K⁺-ATPase. RNA interference-mediated 60-70% knockdown of sAC expression comparably inhibited basal transepithelial short circuit currents (I_sc) in mpkCCD_c14 cells. Moreover, the sAC inhibitors KH7 and 2-hydroxyestradiol reduced I_sc 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 I_sc over 4 h, an effect that was abolished in the presence of KH7. The sAC contribution to I_sc 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 mpkCCD_c14 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 Na⁺ reabsorption in the kidney collecting duct, acting to enhance Na⁺,K⁺-ATPase activity.Maintenance of intracellular pH depends in part on the extracellular to intracellular Na⁺ gradient, and elevation of intracellular Na⁺] can lead to acidification of the cytoplasm. It has been shown that acidification of the cytoplasm of cells from frog skin and toad bladder by increased partial pressure of CO₂ reduces Na⁺ transport and permeability (1, 2). Conversely, the rise in plasma bicarbonate caused by metabolic alkalosis with chronic diuretic use has been shown to increase net renal Na⁺ reabsorption independently of volume status, electrolyte depletion, and/or increased aldosterone secretion (3, 4). However, the underlying mechanisms involved in these phenomena remain unclear.The soluble adenylyl cyclase (sAC)² is a chemosensor that mediates the elevation of cAMP in intracellular microdomains (5-7). Unlike transmembrane adenylyl cyclases (tmACs), sAC is insensitive to regulation by forskolin or heterotrimeric G proteins (8) and is directly activated by elevations of intracellular calcium (9, 10) and/or bicarbonate ions (11). Thus, sAC mediates localized intracellular increases in cAMP in response to variations in bicarbonate levels or its closely related parameters, partial pressure of CO₂ and pH. Mammalian sAC is more similar to bicarbonate-regulated cyanobacterial adenylyl cyclases than to other mammalian nucleotidyl cyclases, which may indicate that there is a unifying mechanism for the regulation of cAMP signaling by bicarbonate across biological systems. Although sAC appears to be encoded by a single gene, there is significant isoform diversity for this ubiquitously expressed enzyme (11, 12) generated by alternative splicing (reviewed in Ref. 13). sAC has been shown to regulate the subcellular localization and/or activity of membrane transport proteins such as the vacuolar H⁺-ATPase (V-ATPase) and cystic fibrosis transmembrane conductance regulator in epithelial cells (14, 15). Functional activity of sAC has been reported in the kidney (16), and sAC has been localized to epithelial cells in the distal nephron (14, 17).Given that natriuresis is decreased during metabolic alkalosis, when bicarbonate is elevated, and Na⁺ reabsorption is impaired by high partial pressure of CO₂, we hypothesized that bicarbonate-regulated sAC may play a key role in the regulation of transepithelial Na⁺ transport in the distal nephron. Reabsorption of Na⁺ in the kidney and other epithelial tissues is mediated by the parallel operation of apical ENaC and basolateral Na⁺,K⁺-ATPase, and both transport proteins can be stimulated by cAMP via the cAMP-dependent protein kinase (PKA) (18, 53). The aims of this study were to investigate the role of sAC in the regulation of transepithelial Na⁺ transport in the kidney through the use of specific sAC inhibitors and electrophysiological measurements. We found that sAC inhibition blocks transepithelial Na⁺ reabsorption in polarized mpkCCD_c14 cells under both basal and hormone-stimulated conditions. Selective membrane permeabilization studies revealed that although ENaC activity appears to be unaffected by sAC inhibition, flux through the Na⁺,K⁺-ATPase is sensitive to sAC modulation. Inhibiting sAC decreases ATPase activity without affecting plasma membrane expression of the pump; thus, tonic sAC activity appears to be required for Na⁺ reabsorption in kidney collecting duct.