Regulation of human colonic UT-B urea transporters (360G-Wellcome-092524_Z_10_Z)

This project will investigate the regulation of UT-B urea transporters within the human colon. Tissue samples will be surgically obtained from the four different subsections of the colon (i.e. ascending, transverse, descending and sigmoid). Using RT-PCR analysis, all UT-B urea transporters present will be cloned and sequenced. UT-B urea transporter protein expression will be investigated using two previously characterised antibodies, with additional isoform-specific antibodies being produced as required. Protein investigations will involve western blotting and immunolocalisation using frozen cryo-sections of colonic tissue. To detail urea transport regulation, several stable MDCK cell lines will be produced - each one expressing a single UT-B isoform (e.g. MDCK-hUT-B1). In addition, the Caco-2 intestinal cell model will also be utilised. After cellular localisation has been confirmed, the relevant 14C-labelled urea flux experiments will be performed to investigate urea transport functi on (e.g. phloretin and dimethylurea inhibition). Further studies will investigate the regulation of UT-B-mediated transport both by intracellular pathways (e.g. cAMP) and by extracellular factors (e.g. short chain fatty acids). Finally, further cell lines expressing UT-B isoforms with mutated glycosylation sites will also be produced and investigated. Through these pioneering studies, we will determine the precise functional regulation of human colonic UT-B urea transporters.