ATP is an important paracrine regulator of renal tubular water and urea transport. The activity of P2Y2, the predominant P2Y receptor of the medullary collecting duct, is mediated by ATP, and modulates urinary concentration. To investigate the role of purinergic signaling in the absence of urea transport in the collecting duct, we studied wild-type (WT) and UT-A1/A3 null (UT-A1/A3 KO) mice in metabolic cages to monitor urine output, and collected tissue samples for analysis. We confirmed that UT-A1/A3 KO mice are polyuric, and concurrently observed lower levels of urinary cAMP as compared to WT, despite elevated serum vasopressin (AVP) levels. Because P2Y2 inhibits AVP-stimulated transport by dampening cAMP synthesis, we suspected that, similar to other models of AVP-resistant polyuria, purinergic signaling is increased in UT-A1/A3 KO mice. In fact, we observed that both urinary ATP and purinergic-mediated prostanoid (PGE2) levels were elevated. Collectively, our data suggest that the reduction of medullary osmolality due to the lack of UT-A1 and UT-A3 induces an AVP-resistant polyuria that is possibly exacerbated by, or at least correlated with, enhanced purinergic signaling.

Two urea transporters, UT-A1 and UT-A3, are expressed in the kidney terminal inner medullary collecting duct (IMCD) and are important for the production of concentrated urine. UT-A1, as the largest isoform of all UT-A urea transporters, has gained much attention and been extensively studied; however, the role and the regulation of UT-A3 are less explored. In this study, we investigated UT-A3 regulation by glycosylation modification. A site-directed mutagenesis verified a single glycosylation site in UT-A3 at Asn279. Loss of the glycosylation reduced forskolin-stimulated UT-A3 cell membrane expression and urea transport activity. UT-A3 has two glycosylation forms, 45 and 65 kDa. Using sugar-specific binding lectins, the UT-A3 glycosylation profile was examined. The 45-kDa form was pulled down by lectin concanavalin A (Con A) and Galant husnivalis lectin (GNL), indicating an immature glycan with a high amount of mannose (Man), whereas the 65-kDa form is a mature glycan composed of acetylglucosamine (GlcNAc) and poly-N-acetyllactosame (poly-LacNAc) that was pulled down by wheat germ agglutinin (WGA) and tomato lectin, respectively. Interestingly, the mature form of UT-A3 glycan contains significant amounts of sialic acid. We explored the enzymes responsible for directing UT-A3 sialylation. Sialyltransferase ST6GalI, but not ST3GalIV, catabolizes UT-A3 α2,6-sialylation. Activation of protein kinase C (PKC) by PDB treatment promoted UT-A3 glycan sialylation and membrane surface expression. The PKC inhibitor chelerythrine blocks ST6GalI-induced UT-A3 sialylation. Increased sialylation by ST6GalI increased UT-A3 protein stability and urea transport activity. Collectively, our study reveals a novel mechanism of UT-A3 regulation by ST6GalI-mediated sialylation modification that may play an important role in kidney urea reabsorption and the urinary concentrating mechanism.