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PKC? regulates vasopressin-induced aquaporin-2 trafficking in mouse kidney collecting duct cells in vitro via altering microtubule assembly.

ABSTRACT: AIM: Aquaporin-2 (AQP2) is a vasopressin-regulated water channel located in the collecting tubule and collecting duct cells of mammalian kidney. The aim of this study is to investigate whether PKC? plays a role in vasopressin-induced AQP2 trafficking in mouse inner medullary collecting duct 3 (mIMCD3) cells. METHODS: AQP2-mIMCD3 stable cell line was constructed by transfection of mouse inner medullary collecting duct 3 (mIMCD3) cells with AQP2-GFP construct. Then the cells were transfected with PKC? shRNA, PKC? A/25E, or PKC? scrambled shRNA. The expression levels of PKC?, AQP2, and phospho-S256-AQP2 were analyzed using Western blot. The interaction between AQP2 and PKC? was examined using immunoprecipitation. The distribution of AQP2 and microtubules was studied using immunocytochemistry. The AQP2 trafficking was examined using the biotinylation of surface membranes. RESULTS: Treatment of AQP2-mIMCD3 cells with 100 ?mol/L of 1-desamino-8-D-arginine vasopressin (DdAVP) for 30 min stimulated the translocation of AQP2 from the cytoplasm to plasma membrane through influencing the microtubule assembly. Upregulation of active PKC? by transfection with PKC? A/25E plasmids resulted in de-polymerization of ?-tubulin and redistributed AQP2 in the cytoplasm. Down-regulation of PKC? by PKC? shRNA partially inhibited DdAVP-stimulated AQP2 trafficking without altering ?-tubulin distribution. Although 100 ?mol/L of DdAVP increased AQP2 phosphorylation at serine 256, down-regulation of PKC? by PKC? shRNA did not influence DdAVP-induced AQP2 phosphorylation, suggesting that AQP2 phosphorylation at serine 256 was independent of PKC?. Moreover, PKC? did not physically interact with AQP2 in the presence or absence of DdAVP. CONCLUSION: Our results suggested that PKC? regulates AQP2 trafficking induced by DdAVP via microtubule assembly.


PROVIDER: S-EPMC4010327 | BioStudies | 2012-01-01

REPOSITORIES: biostudies

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