Kidney collecting duct cells are continuously exposed to the changes of extracellular pH (pHe). We aimed to study the effects of altered pHe on desmopressin (dDAVP)-induced phosphorylation (Ser256, Ser261, Ser264, and Ser269) and apical targeting of aquaporin-2 (AQP2) in rat kidney inner medullary collecting duct (IMCD) cells. When freshly prepared IMCD tubule suspensions exposed to HEPES buffer with pH 5.4, 6.4, 7.4, or 8.4 for 1 h were stimulated with dDAVP (10-10 M, 3 min), AQP2 phosphorylation at Ser256, Ser264, and Ser269 was significantly attenuated under acidic conditions. Next, IMCD cells primary cultured in transwell chambers were exposed to a transepithelial pH gradient for 1 h (apical pH 6.4, 7.4, or 8.4 vs. basolateral pH 7.4 and vice versa). Immunocytochemistry and cell surface biotinylation assay revealed that exposure to either apical pH 6.4 or basolateral pH 6.4 for 1 h was associated with decreased dDAVP (10-9 M, 15 min, basolateral)-induced apical targeting of AQP2 and surface expression of AQP2. Fluorescence resonance energy transfer analysis revealed that the dDAVP (10-9 M)-induced increase of PKA activity was significantly attenuated when LLC-PK1 cells were exposed to pHe 6.4 compared with pHe 7.4 and 8.4. In contrast, forskolin (10-7 M)-induced PKA activation and dDAVP (10-9 M)-induced increases of intracellular Ca2+ were not affected. Taken together, dDAVPinduced phosphorylation and apical targeting of AQP2 are attenuated in IMCD cells under acidic pHe, likely via an inhibition of vasopressin V2 receptor-G protein-cAMP-PKA actions.
- Collecting duct
- Fluorescence resonance energy transfer
- Vasopressin V2 receptor
ASJC Scopus subject areas