Facilitative UT-A and UT-B urea transporters regulate the movement of urea across biological membranes and play a central role in the urinary concentrating mechanism (Smith & Rousselet, 2001). We have previously reported that at least three different UT-A isoforms (mUT-A1, mUT-A2 and mUT-A3) are present in the mouse kidney (Fenton et al. 2002a). We have also shown that mUT-A3 mRNA levels are increased during thirsting (Fenton et al. 2002b). The aim of this study was to identify the specific renal location and function of mUT-A3.
MQ2, a novel antibody targeted to the C-terminal of mUT-A3, was raised in rabbits. Tissue for Western analysis was obtained from humanely killed male adult NMR1 mice. For immunolocalisation studies, mice were anaesthetized with Inactin (100 mg kg-1 I.P.) before perfusion-fixation of kidneys with 4 % paraformaldehyde. Western analysis of dissected mouse kidney protein showed that MQ2 only detected mUT-A3 (a 45-55 kDa protein). This isoform specificity is in contrast to our previously reported antibodies, ML446 and ML194 (Stewart et al. 2002). Immunolocalisation studies using MQ2 showed that mUT-A3 was only present in inner medullary collecting duct (IMCD) segments IMCD2 and IMCD3, and was located on the basolateral membranes. Finally, using 14C-labelled urea, Xenopus oocyte flux experiments confirmed that mUT-A3 was stimulated by short-term exposure to PKA stimulants (P < 0.01, ANOVA, n = 6).
These results indicate renal mUT-A3 is a basolateral transporter, regulated in the short-term by PKA. We therefore suggest that mUT-A3 plays an important role in transcellular urea transport across IMCD epithelia. Our findings also agree with reports that vasopressin increases urea permeability in both apical and basolateral membranes.
This work was funded by the BBSRC and The Royal Society.