Vasopressin‐Regulated Phosphorylation of Urea Transporter Proteins, UT‐A1 and UT‐A3, in Rat Inner Medullary Collecting Duct (IMCD)

Abstract

Vasopressin‐regulated urea transport in the renal IMCD is mediated by two transporters, UT‐1A and UT‐A3, derived from the same gene by alternative splicing. The initial 459 amino acids are the same in both. To study UT‐A1/3 phosphorylation, we made phospho‐specific antibodies to UT‐A sequences targeting phospho‐serines at positions 84 and 486. Both antibodies proved specific, recognizing only the phosphorylated forms of UT‐A1 and ‐A3. Immunoblotting of rat IMCD suspensions or inner medullas from intact rats showed that the V2R‐selective vasopressin analog dDAVP increases phosphorylation at both S84 and S486 by ~8‐fold. The increase in S84 phosphorylation was seen in both UT‐A1 and ‐A3. Time‐course studies in rat IMCD suspensions showed phosphorylation within 1 minute of dDAVP exposure, consistent with the time course of urea transport increases in isolated perfused tubules. Confocal immunofluorescence immunocytochemistry in rat medullary tissue showed labeling limited to the IMCD, which increased markedly in response to dDAVP. These studies demonstrate regulated phosphorylation of both UT‐A1 and UT‐A3 in response to vasopressin, potentially contributing to the regulation of urea transport in the IMCD.