PKCα regulates vasopressin-induced aquaporin-2 trafficking in mouse kidney collecting duct cells in vitro via altering microtubule assembly.
OBJECTIVE
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 PKCα 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 PKCα shRNA, PKCα A/25E, or PKCα scrambled shRNA. The expression levels of PKCα, AQP2, and phospho-S256-AQP2 were analyzed using Western blot. The interaction between AQP2 and PKCα 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 μmol/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 PKCα by transfection with PKCα A/25E plasmids resulted in de-polymerization of α-tubulin and redistributed AQP2 in the cytoplasm. Down-regulation of PKCα by PKCα shRNA partially inhibited DdAVP-stimulated AQP2 trafficking without altering α-tubulin distribution. Although 100 μmol/L of DdAVP increased AQP2 phosphorylation at serine 256, down-regulation of PKCα by PKCα shRNA did not influence DdAVP-induced AQP2 phosphorylation, suggesting that AQP2 phosphorylation at serine 256 was independent of PKCα. Moreover, PKCα did not physically interact with AQP2 in the presence or absence of DdAVP.


CONCLUSIONS
Our results suggested that PKCα regulates AQP2 trafficking induced by DdAVP via microtubule assembly.
Department of Trauma Surgery, Tongji Hospital, Huazhong University of Science & Technology, Wuhan, China.
Hong
H
Zhao
Xi
X
Yao
Tao-Xia
TX
Wang
Wen-Min
WM
Jin
Qian-Qian
QQ
Ji
Xiao
X
Yang
Qiu-Hong
QH
Duan
Li-Jun
LJ
Yao
D000818:Animals; D050034:Antidiuretic Agents / Q000494:pharmacology*; D051399:Aquaporin 2 / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003894:Deamino Arginine Vasopressin / Q000494:pharmacology*; D007668:Kidney / Q000166:cytology*; D051379:Mice; D010766:Phosphorylation / Q000187:drug effects; D051571:Protein Kinase C-alpha / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D034741:RNA, Small Interfering / Q000235:genetics; D014404:Tubulin / Q000378:metabolism*; D015854:Up-Regulation
D050034:Antidiuretic Agents; D051399:Aquaporin 2; D034741:RNA, Small Interfering; D014404:Tubulin; D051571:Protein Kinase C-alpha; D003894:Deamino Arginine Vasopressin