title: Ubiquitination regulates the plasma membrane expression of renal UT-A urea transporters.
abstract: The renal UT-A urea transporters UT-A1, UT-A2, and UT-A3 are known to play an important role in the urinary concentrating mechanism. The control of the cellular localization of UT-A transporters is therefore vital to overall renal function. In the present study, we have investigated the effect of ubiquitination on UT-A plasma membrane expression in Madin-Darby canine kidney (MDCK) cell lines expressing each of the three renal UT-A transporters. Inhibition of the ubiquitin-proteasome pathway caused an increase in basal transepithelial urea flux across MDCK-rat (r)UT-A1 and MDCK-mouse (m)UT-A2 monolayers (P < 0.01, n = 3, ANOVA) and also increased dimethyl urea-sensitive, arginine vasopressin-stimulated urea flux (P < 0.05, n = 3, ANOVA). Inhibition of the ubiquitin-proteasome pathway also increased basolateral urea flux in MDCK-mUT-A3 monolayers (P < 0.01, n = 4, ANOVA) in a concentration-dependent manner. These increases in urea flux corresponded to a significant increase in UT-A transporter expression in the plasma membrane (P < 0.05, n = 3, ANOVA). Further analysis of the MDCK-mUT-A3 cell line confirmed that vasopressin specifically increased UT-A3 expression in the plasma membrane (P < 0.05, n = 3, ANOVA). However, preliminary data suggested that vasopressin produces this effect through an alternative route to that of the ubiquitin-proteasome pathway. In conclusion, our study suggests that ubiquitination regulates the plasma membrane expression of all three major UT-A urea transporters, but that this is not the mechanism primarily used by vasopressin to produce its physiological effects.
authors.affiliation: Faculty of Life Sciences, Core Technology Facility, University of Manchester, Manchester, UK. gavin.s.stewart@manchester.ac.uk
authors.forename: Gavin S
authors.initials: GS
authors.lastname: Stewart
authors.forename: Jennifer H
authors.initials: JH
authors.lastname: O'Brien
authors.forename: Craig P
authors.initials: CP
authors.lastname: Smith
mesh_terms: D000818:Animals; D001127:Arginine Vasopressin / Q000494:pharmacology; D001693:Biological Transport, Active; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D004285:Dogs; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000096:biosynthesis*; D051379:Mice; D054875:Ubiquitination; D019459:Urothelium / Q000166:cytology / Q000378:metabolism; D000096982:Urea Transporters
chemical_list: D001127:Arginine Vasopressin; D026901:Membrane Transport Proteins; D000096982:Urea Transporters