Aquaporin-2 Levels in vitro and in vivo are Regulated by VACM-1, a Cul 5 GeneLe I.P.a,d · Schultz S.a,d · Andresen B.T.a,d · Dewey G.L.a · Zhao P.b · Listenberger L.a · Deen P.M.c · Buchwalter A.a · Barney C.C.a · Burnatowska-Hledin M.A.a
a Departments of Biology and Chemistry, Schaap Science Center, Hope College, Holland, MI 49422-9000; b Van Andel Research Institute, 333 Bostwick NE, Grand Rapids, MI 49503; c Department of Physiology, UMC Nijmegen Nijmegen, The Netherlands; d Contributed equally to the work reported in this manuscript
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Background: In the renal collecting duct, vasopressin regulates water permeability by a process that involves stimulation of adenylyl cyclase activity, cAMP production and subsequent translocation of water channel aquaporin-2 (AQP2) into the apical plasma membrane. We have previously shown that in cos 1 cells in vitro, both adenylyl cyclase activity and cAMP production can be regulated by VACM-1, a cul 5 gene that forms complexes involved in protein ubiquitination and subsequent degradation. Methods: To extend these observations further, the effects of changes in hydration state on the expression of VACM-1 at the mRNA and the protein level were examined in rats deprived of water (WD) for 24 hrs. Results: In the kidney of WD rats Western blot analyses of kidney tissue showed that the decrease in VACM-1 protein concentration was correlated with the increase in the AQP2 protein level. The immunostaining data suggested that VACM-1/cul5 may be decreased in renal collecting duct but increases in the vasculature of the inner medullary region in response to WD. To determine the possible consequences of the WD dependent decrease in VACM-1/cul5, we next examined the effects of VACM-1 expression on AQP2 protein in vitro. Immunocytochemistry and Western blot analyses data indicate that VACM-1/cul5 expression in MDCK line stably expressing AQP2 gene and in cos 1 cells co-transfected with the AQP2 and VACM-1/cul5 cDNAs decreased AQP2 protein concentration when compared to the vector transfected control groups. Conclusion: In summary, our data demonstrate that VACM-1 is involved in the regulation of AQP2 protein concentration and may play a role in regulating water balance.
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