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 bicarbonatedependent 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 sACdependent 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.

Soluble Adenylyl Cyclase in Vascular Endothelium

“…Two sAC isoforms (≈55 and ≈80 kDa), identified by Hallows et al 20 in cell lysates of mpkCCDc14 kidney cells, were confirmed in IHKE cells, whereas we detected an additional isoform of ≈65 kDa predominantly in ECs. We confirmed the identity of all 3 detected protein bands by an anti-sAC siRNA approach.…”

“…In the kidney, sAC has been reported to be involved in blood pressure homeostasis, whereas specific inhibition of sAC by its inhibitor KH7 resulted in abrogation of basic and agonist-stimulated Na + reabsorption. 20 ENaC, Na + /K + -ATPase, and MR expression may involve binding of phosphorylated CREB (CREB-p) to cAMP response elements (CRE) at the promoter level. [21][22][23] In…”

Noncanonical Control of Vasopressin Receptor Type 2 Signaling by Retromer and Arrestin

“…In principal, the cells of the collecting duct, the immediate consequence of cAMP generation by V2R, is a PKA-dependent increase in the permeability of the apical membrane to water and sodium (44,45). Sodium transport involves ENaC, an ion channel that is regulated in part by its expression level at the apical plasma membrane.…”

Regulation of nephron water and electrolyte transport by adenylyl cyclases

“…In particular, Pastor-Soler et al (54) reported colocalization between sAC and the vacuolar ATPase (V-ATPase), but not aquaporin-2 (AQP2), in rat kidney, suggesting intercalated cells selectively express sAC within the kidney. In contrast, two other studies using a different antibody reported that sAC immunostaining was present in both rat principal and intercalated cells (33,55). Paunescu et al (55) noted that sAC and the V-ATPase colocalized apically and subapically in type A intercalated cells, while they colocalized basolaterally in type B intercalated cells.…”

“…Immunostaining for AC3 was present in the mouse connecting segment and CCD (60). Soluble AC protein appears to be abundantly present in the CCD (33,54,55). The outer medullary CD (OMCD) has a similar pattern of AC isoform expression as the CCD (7,12,36,79).…”