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1 Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom
* To whom correspondence should be addressed. E-mail: gavin.s.stewart{at}manchester.ac.uk.
Renal facilitative urea transporters play a vital role in the urinary concentrating mechanism. UT-A3 is a phloretin-sensitive urea transporter that in mouse is expressed on the basolateral membrane of renal inner medullary collecting duct (IMCD) cells. In this study, we engineered a MDCK I cell line that stably expresses mouse UT-A3 (MDCK-mUT-A3). Immunoblotting using the UT-A antibody ML446 detected a ~40 kDa signal in MDCK-mUT-A3 protein that corresponds to mUT-A3. Using cultured epithelial monolayers, radioactive 14C-urea flux experiments determined that basolateral urea transport was no different between MDCK-mUT-A3 and control MDCK-FLZ cells under basal conditions (NS, ANOVA). However, exposure to arginine vasopressin (AVP) significantly stimulated basolateral urea flux in MDCK-mUT-A3 monolayers (P<0.05, ANOVA), while it had no effect in control MDCK-FLZ monolayers (NS, ANOVA). The AVP-stimulated basolateral urea transport in MDCK-mUT-A3 was inhibited by 1,3 dimethyl urea (P<0.05, ANOVA) or phloretin (P<0.05, ANOVA), both known inhibitors of facilitative urea transporters. MDCK-mUT-A3 basolateral urea flux was also stimulated by increasing intracellular levels of cAMP, via forskolin (P<0.05, ANOVA), or intracellular calcium, via ATP (P<0.05, ANOVA). Finally, one hour pre-incubation with a specific protein kinase A inhibitor, H89, significantly inhibited the increase in urea transport produced by AVP (P<0.05, ANOVA). In conclusion, we have produced the first renal cell line to stably express the mUT-A3 urea transporter. Our results indicate that mUT-A3 is acutely regulated by AVP, via a PKA-dependent pathway. These findings have important implications for the regulation of urea transport in the renal IMCD and the urinary concentrating mechanism.
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M. A. Blount, J. D. Klein, C. F. Martin, D. Tchapyjnikov, and J. M. Sands Forskolin stimulates phosphorylation and membrane accumulation of UT-A3 Am J Physiol Renal Physiol, October 1, 2007; 293(4): F1308 - F1313. [Abstract] [Full Text] [PDF]
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