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1 School of Biological Sciences, University of Manchester, Manchester, M13 9PT, United Kingdom
2 Laboratory of Kidney and Electrolyte Metabolism, NHLBI, NIH, Bethesda, Maryland 20892, USA
3 The Water and Salt Research Center, University of Aarhus, Aarhus, DK-8000, Denmark
4 School of Biomedical Sciences, University of Leeds, Leeds, LS2 9JT, United Kingdom
5 Department of Biomedical Sciences, University of Sheffield, Sheffield, S10 2TN, United Kingdom
* To whom correspondence should be addressed. E-mail: craig.smith{at}man.ac.uk.
Facilitative UT-A urea transporters play a central role in the urinary concentrating mechanism. There are three major UT-A isoforms found in the mouse kidney - mUT-A1, mUT-A2 and mUT-A3. The major aim of this study was to identify the location and function of mUT-A3. UT-A proteins were investigated using three novel mouse UT-A targeted antibodies - ML446, MQ2 and ML194. ML446 detected mUT-A1 and mUT-A3. ML194 detected mUT-A1 and mUT-A2. Importantly, MQ2 was found to be selective for mUT-A3. MQ2 detected a 45-65 kDa signal in the mouse kidney inner medulla, which was deglycosylated to a 40 kDa protein band. Immunolocalization studies showed that mUT-A3 was strongly detected in the papillary tip, mainly in the basolateral regions of inner medulla collecting duct cells. Immunoblotting of sub-cellular fractions of inner medulla protein suggested that in mouse kidney mUT-A3 was present in plasma membranes. Consistent with this, immunoelectron microscopy demonstrated that mUT-A3 was predominantly localized at the basal plasma membrane domains of the inner medullary collecting duct cells in mouse kidney. Heterologous expression of mUT-A3-EGFP in MDCK cells showed that the protein localized to the basolateral membrane. In conclusion, our study indicates that mUT-A3 is a basolateral membrane transporter expressed in IMCD cells.
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