Vasopressin regulation of the renal UT-A3 urea transporter

Abstract

Facilitative urea transporters in the mammalian kidney play a vital role in the urinary concentrating mechanism. The urea transporters located in the renal inner medullary collecting duct, namely UT-A1 and UT-A3, are acutely regulated by the antidiuretic hormone vasopressin. In this study, we investigated the vasopressin regulation of the basolateral urea transporter UT-A3 using an MDCK-mUT-A3 cell line. Within 10 min, vasopressin stimulates urea flux through UT-A3 transporters already present at the plasma membrane, via a PKA-dependent process. Within 1 h, vasopressin significantly increases UT-A3 localization at the basolateral membrane, causing a further increase in urea transport. While the basic trafficking of UT-A3 to basolateral membranes involves both protein kinase C and calmodulin, its regulation by vasopressin specifically occurs through a casein kinase II-dependent pathway. In conclusion, this study details the effects of vasopressin on UT-A3 urea transporter function and hence its role in regulating urea permeability within the renal inner medullary collecting duct. arginine vasopressin; urea transport; membrane localization; casein kinase II IN MAMMALS, THE PASSIVE MOVEMENT of urea across cell membranes is facilitated by phloretin-sensitive urea transporters derived from two distinct genes, UT-A (Slc14a2) and UT-B (Slc14a1) (20). In the kidney, there are four major isoforms of these facilitative urea transporters, three are derived from alternative splicing of the UT-A gene (namely UT-A1, UT-A2, and UT-A3), plus one UT-B isoform (UT-B1) (22). Renal facilitative urea transporters are vital to the urinary concentrating mechanism and mouse knockout models have shown that UT-A1 and UT-A3 (8), UT-A2 (27), and UT-B1 (1, 29) all play a significant role in producing concentrated urine.UT-A1 and UT-A3 are both located in the inner medullary collecting duct (IMCD) (7,21,24). Indeed, it has now been shown that the UT-A urea transporter promoter, UT-A alpha, is responsible for this specific targeting to principal cells of the renal IMCD (10). At a subcellular level, UT-A1 is located to the apical membrane of IMCD cells (18). In contrast, the UT-A3 isoform, originally cloned from rat kidney (15), has been localized to the basolateral membrane in both mouse (24) and rat (17). In combination, UT-A1 and UT-A3 are responsible for urea reabsorption from the IMCD lumen to the inner medullary interstitium, and the absence of these transporters causes a urea-dependent osmotic diuresis (9).In perfused isolated IMCD, vasopressin stimulates phloretin-sensitive trans-epithelial urea transport (23,28). Recent studies of rat UT-A1 (11) and mouse UT-A3 (25) expressed in Madin-Darby canine kidney (MDCK) type I cell lines showed that both isoforms can be acutely regulated by arginine vasopressin (AVP), via a cAMP-dependent pathway. Interestingly, the protein kinase A (PKA) inhibitor H89 reduced the vasopressin activation of both UT-A1 (12) and UT-A3 (25). Vasopressin is known to rapidly increase phosphorylation of UT-A1 via PKA...

Novel bUT-B2 urea transporter isoform is constitutively activated

“…AVP is known to regulate the transport function of renal UT-A transporters (7,18,29). In this study, experiments revealed that preincubation for 1 h in 10 Ϫ6 M AVP did not increase DMU-sensitive urea flux in MDCK-bUT-B2 monolayers (NS, n ϭ 3) (see Fig.…”

“…Acute exposure to vasopressin causes a sustained functional increase in both UT-A1 (7) and UT-A3 (27,29), while having a more transient effect on UT-A2 (19). In contrast, for bUT-B2-mediated urea flux, vasopressin had no significant effect (see Fig.…”

Structure and permeation mechanism of a mammalian urea transporter

“…One notable difference between the UT-B and UT-A orthologs is that the latter is upregulated by the antidiuretic hormone vasopressin via phosphorylation of multiple sites on its long cytoplasmic N-terminus (16). Some of this increase in activity can be accounted for by increased localization of UT-A in the plasma membrane (38), but there is also evidence for an increase in urea transport activity that occurs on a more rapid time scale than the rate of accumulation of UT-A in the plasma membrane, possibly due to modulation of UT activity through phosphorylation of proteins already present at the cell surface (39). Given the high conservation of pore-lining residues between UT-B and UT-A, it is tempting to consider the possibility that the S m site barrier is also present in UT-A, but with phosphorylation rather than osmotic stress as the trigger for modulation.…”

Functional characterization of the central hydrophilic linker region of the urea transporter UT-A1: cAMP activation and snapin binding

“…Thus, while there appear to be two mechanisms by which LcUT-A3 could be activated, these two actions are not additive in their effects. The intrinsic activation of UT-A3 might be mechanistically related to the mechanism proposed for activation in UT-A3-expressing MDCK cells that involves casein kinase II, protein kinase C, and calmodulin (23). Nevertheless, the consideration that UT-A1 possesses all amino acid residues of UT-A3 (with the exception of its very COOH-terminal residue) and that UT-A1 activation by cAMP is completely abolished by mutating the Lc-residing S486 and S499 to alanines, it would appear that the mechanism(s) that are responsible for activating UT-A3 are of lesser importance for UT-A1 activation.…”

“…When heterologously expressed in Madin-Darby canine kidney (MDCK) cells, UT-A1 mediated a urea permeability that was stimulated up to 20-fold over background by vasopressin and forskolin, an activator of adenylate cyclase (6,7). In UT-A3-expressing MDCK cells, basolateral urea influx was enhanced as well (22,23). In cRNA-injected oocytes, UT-A1 and UT-A3 also mediated cAMP-stimulated urea influx (8,21).…”