UT-A3: localization and characterization of an additional urea transporterisoform in the IMCD

UT-A3 has recently been identified as a splicing variant transcript of the UT-A gene present in the kidney. To study the cellular and subcellular loca lization of UT-A3, we raised a new polyclonal antibody to its COOH-terminal end. Immunoblots identified bands at 44 and 67 kDa predominately in the in ner medulla and showed that the antibody does not recognize UT-A1. Deglycos ylation with PNGase decreased the molecular mass of both forms to 40 kDa. U T-A3 is most abundant in the inner third of the inner medulla and is presen t in membrane fractions. Cell fractionation studies showed that UT-A3 is on ly detectable in inner medullary collecting duct (IMCD) cells. These observ ations were confirmed with immunolocalization studies demonstrating an excl usive labeling of IMCD cells. Double-labeling studies with anti-Na-K-ATPase demonstrated UT-A3 in intracellular membranes and in the apical region but were incompatible with a basolateral site for UT-A3. Although the function of this isoform in the inner medulla is unknown, the large abundance sugge sts that it may be important in the renal handling of urea.