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

作　　者：Hong ZHAO Xi YAO Tao-xia WANG Wen-min JIN Qian-qian JI Xiao YANG Qiu-hong DUAN Li-jun YAO 

机构地区：[1]Department of Trauma Surgery,Tongji Hospital,Huazhong University of Science&Technology,Wuhan 430030,China [2]Departmentsof Nephrology,Union Hospital,Huazhong University of Science&Technology,Wuhan 430030,China [3]Department of Biochemistryand Molecular Biology,Tongji Medical College,Huazhong University of Science&Technology,Wuhan 430022,China

出　　处：《Acta Pharmacologica Sinica》2012年第2期230-236,共7页中国药理学报（英文版）

摘　　要：Aim： Aquaporin-2 （AQP2） is a vasopressin-regulated water channel located in the collecting tubule and collecting duct cells of mamma- lian kidney. The aim of this study is to investigate whether PKC（x plays a role in vasopressin-induced AQP2 trafficking in mouse inner medullary collecting duct 3 （mlMCD3） cells.Methods： AQP2-mlMCD3 stable cell line was constructed by transfection of mouse inner medullary collecting duct 3 （mlMCD3） cells with AQP2-GFP construct. Then the cells were transfected with PKCa shRNA, PKC（x A/25E, or PKCa scrambled shRNA. The expres- sion levels of PKCa, 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-mlMCD3 cells with 100 pmol/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 PKCa by transfection with PKCa A/25E plasmids resulted in de-polymerization of a-tubulin and redistributed AQP2 in the cytoplasm. Down-regulation of PKCa by PKCa shRNA partially inhibited DdAVP-stimulated AQP2 trafficking without altering a-tubulin distribution. Although 100 pmol/L of DdAVP increased AQP2 phosphorylation at serine 256, down-regulation of PKCa by PKCa shRNA did not influ- ence DdAVP-induced AQP2 phosphorylation, suggesting that AQP2 phosphorylation at serine 256 was independent of PKCa. Moreover PKCa did not physically interact with AQP2 in the presence or absence of DdAVP.Conclusion： Our results suggested that PKCa regulates AQP2 trafficking induced by DdAVP via microtubule assembly.Aim： Aquaporin-2 （AQP2） is a vasopressin-regulated water channel located in the collecting tubule and collecting duct cells of mamma- lian kidney. The aim of this study is to investigate whether PKC（x plays a role in vasopressin-induced AQP2 trafficking in mouse inner medullary collecting duct 3 （mlMCD3） cells.Methods： AQP2-mlMCD3 stable cell line was constructed by transfection of mouse inner medullary collecting duct 3 （mlMCD3） cells with AQP2-GFP construct. Then the cells were transfected with PKCa shRNA, PKC（x A/25E, or PKCa scrambled shRNA. The expres- sion levels of PKCa, 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-mlMCD3 cells with 100 pmol/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 PKCa by transfection with PKCa A/25E plasmids resulted in de-polymerization of a-tubulin and redistributed AQP2 in the cytoplasm. Down-regulation of PKCa by PKCa shRNA partially inhibited DdAVP-stimulated AQP2 trafficking without altering a-tubulin distribution. Although 100 pmol/L of DdAVP increased AQP2 phosphorylation at serine 256, down-regulation of PKCa by PKCa shRNA did not influ- ence DdAVP-induced AQP2 phosphorylation, suggesting that AQP2 phosphorylation at serine 256 was independent of PKCa. Moreover PKCa did not physically interact with AQP2 in the presence or absence of DdAVP.Conclusion： Our results suggested that PKCa regulates AQP2 trafficking induced by DdAVP via microtubule assembly.

关 键 词：PKCa 1-desamino-8-D-arginine vasopressin （DdAVP） AQUAPORIN-2 MICROTUBULE kidney medullary collecting duct 

分 类 号：Q575.32[生物学—生物化学] Q51