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The UT-A1 urea transporter is a glycoprotein with two different glycosylated forms of 97 and 117 kDa. In this study, we found the 117-kDa UT-A1 preferentially resides in lipid rafts, suggesting that the glycosylation status may interfere with UT-A1 lipid raft trafficking. This was confirmed by a site-directed mutagenesis study in MDCK cells. The nonglycosylated UT-A1 showed reduced localization in lipid rafts. By using sugar-specific binding lectins, we further found that the UT-A1 in nonlipid rafts contained a high amount of mannose, as detected by concanavalin A, while the UT-A1 in lipid rafts was the mature P6acetylglucosamine-containing form, as detected by wheat germ agglutinin. In the inner medulla (IM) of diabetic rats, the more abundant 117-kDa UT-A1 in lipid rafts was the mature glycosylation form, with high amounts of N-acetylglucosamine and sialic acid. In contrast, in the IM of normal rats, the predominant 97-kDa UT-A1 was the form enriched in mannose. Functionally, inhibition of glycosylation by tunicamycin or elimination of the glycosylation sites by mutation significantly reduced UT-A1 activity in oocytes. Taken together, our study reveals a new role of N-linked glycosylation in regulating UT-A1 activity by promoting UT-A1 trafficking into membrane lipid raft subdomains. [ABSTRACT FROM AUTHOR]

Of the three major protein variants produced by the UT-A gene (UT-A1, UT-A2, and UT-A3) UT-A1 is the largest. It contains UT-A3 as its NH2-terminal half and UT-A2 as its COOH-terminal half. When being part of UT-A1, UT-A3 and UT-A2 are joined by a segment, Lp, whose central pan, Lc, is not part of UT-A3 or UT-A2 but is present only in UT-A1. Lc contains the phosphorylation sites S486 and S499 that are involved in protein kinase A-dependent activation, as well as the binding site for snapin, a protein involved in soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE)-mediated vesicle trafficking and fusion to the plasma membrane. We attached Lc to UT-A2 and UT-A3 to test how these pbosphorylation sites influenced their urea transport activity. Adding Lc to UT-A2 conferred stimulation by cAMP to the cAMP-unresponsive UT-A2, and adding Lc to UT-A3 did not further enhance its already existing cAMP response. These findings suggest that the responsiveness to vasopressin that is observed with UT-A1 can be introduced into the unresponsive UT-A2 variant through the Lc segment that is unique to UT-A1. In UT-A3, however, the Lc segment plays no significant role in its activation by cAMP. In addition, the Lc segment also gave UT-A2 the ability to bind snapin and, in Xenopus oocytes, to be stimulated in its urea transport activity by snapin and syntaxins 3 and 4, in the same way as UT-A1. [ABSTRACT FROM AUTHOR]

Osmotic reabsorption of water through aquaporin-2 (AQP2) in the inner medulla is largely dependent on the urea concentration gradients generated by urea transporter (UT) isoforms. Vasopressin (AVP) increases expression of both AQP2 and UT-A isoforms. Activation of the P2Y2 receptor (P2Y2-R) in the medullary collecting duct inhibits AVP-induced water flow. To gain further insights into the overarching effect of purinergic signaling on urinary concentration, we compared the protein abundances of AQP2 and UT-A isoforms between P2Y2-R knockout (KO) and wild-type (WT) mice under basal conditions and following AVP administration. Under basal conditions (a gel diet for 10 days), KO mice concentrated urine to a significantly higher degree, with 1.8-, 1.66-, and 1.29-fold higher protein abundances of AQP2, UT-A1, and UT-A2, respectively, compared with WT, despite comparable circulating AVP levels in both groups. Infusion of 1-desamino-8-D-arginine vasopressin (dDAVP; desmopressin; 1 ng/h sc) for 5 days resulted in 2.14-, 2.6-, and 2.22-fold higher protein abundances of AQP2, AQP3, and UT-A1, respectively, in the inner medullas of KO mice compared with WT mice. In response to acute (45 min) stimulation by AVP (0.2 unit/mouse sc), UT-A1 protein increased by 1.39- and 1.54-fold in WT and KO mice, respectively. These data suggest that genetic deletion of P2Y2-R results in increased abundances of key proteins involved in urinary concentration in the inner medulla, both under basal conditions and following AVP administration. Thus purinergic regulation may play a potential overarching role in balancing the effect of AVP on the urinary concentration mechanism. [ABSTRACT FROM AUTHOR]

Volume depletion due to persistent glucosuria-induced osmotic diuresis is a significant problem in uncontrolled diabetes mellitus (DM). Angiotensin II receptor blockers (ARBs), such as candesartan, slow the progression of chronic kidney disease in patients with DM. However, mice with genetic knockout of components of the renin-angiotensin system have urine concentrating defects, suggesting that ARBs may exacerbate the volume depletion. Therefore, the effect of candesartan on UT-A1, UT-A3, NKCC2, and aquaporin-2 (AQP2) protein abundances was determined in control and 3-wk DM rats. Aldosterone levels in control rats (0.36 ± 0.06 nM) and candesartan-treated rats (0.34 ± 0.14 nM) were the same. DM rats had higher aldosterone levels (1.48 ± 0.37 nM) that were decreased by candesartan (0.97 ± 0.26 nM). Western analysis showed that UT-A1 expression was increased in DM rats compared with controls in inner medullary (IM) tip (158 ± 13%) and base (120 ± 25%). UT-A3 abundance was increased in IM tip (123 ± 11%) and base (146 ± 17%) of DM rats vs. controls. UT-A3 was unchanged in candesartan-treated control rats. In candesartan-treated DM rats, UT-A3 increased in IM tip (160 ± 14%) and base (210 ± 19%). Candesartan-treated DM rats had slightly higher AQP2 in IM (46%, P < 0.05) vs. control rats. NKCC2/BSC1 was increased 145 ± 10% in outer medulla of DM vs. control rats. We conclude that candesartan augments compensatory changes in medullary transport proteins, reducing the losses of solute and water during uncontrolled DM. These changes may represent a previously unrecognized beneficial effect of type 1 ARBs in DM. [ABSTRACT FROM AUTHOR]

To identify novel gene tar- gets of vasopressin regulation in the renal medulla, we performed a cDNA microarray study on the inner medullary tissue of mice following a 48-h water restriction protocol. In this study, 4,625 genes of the possible ~12,000 genes on the array were included in the analysis, and of these 157 transcripts were increased and 63 transcripts were decreased by 1.5-fold or more. Quantitative, real-time PCR measurements confirmed the increases seen for 12 selected transcripts, and the decreases were confirmed for 7 transcripts. In addition, we measured transcript abundance for many renal collecting duct proteins that were not represented on the array; aquaporin-2 (AQP2), AQP3, Pax-8, and α- and β-Na-K-ATPase subunits were all significantly increased in abundance; the β- and γ-subunits of ENaC and the vasopressin type 1A receptor were significantly decreased. To correlate changes in mRNA expression with changes in protein expression, we carried Out quantitative immunoblotting. For most of the genes examined, changes in mRNA abundances were not associated with concomitant protein abundance changes; however, AQP2 transcript abundance and protein abundance did correlate. Surprisingly, aldolase B transcript abundance was increased but protein abundance was decreased following 48 h of water restriction. Several transcripts identified by microarray were novel with respect to their expression in mouse renal medullary tissues. The steroid hormone enzyme 3β-hydroxysteroid dehydrogenase 4 (3βHSD4) was identified as a novel target of vasopressin regulation, and via dual labeling immunofluorescence we colocalized the expression of this protein to AQP2-expressing collecting ducts of the kidney. These studies have identified several transcripts whose abundances are regulated in mouse inner medulla in response to an increase in endogenous vasopressin levels and could play roles in the regulation of salt and water excretion. [ABSTRACT FROM AUTHOR]

The article presents a scientific research on the tissue distribution of urea transporter-A (UT-A) and urea transporter-B (UT-B) messenger RNA and protein in rats. Researchers were able to identify UT-A and or UT-B messenger RNA and or protein forms in several organs not typically associated with the metabolism of urea.

We have identified a urea transporter from the mucosa of the human colon that has characteristics consistent with a Kid antigen/UT-B urea transporter. This intestinal urea transporter encodes a 389-amino acid peptide with a sequence identical to that previously reported for the UT-B urea transporter in erythrocytes. Expression of a UT-B 2-kb mRNA transcript and of ∼50- and ∼98-kDa UT-B proteins is detected in human colonic mucosa by Northern and Western blot analysis. The UT-B protein is localized in the cell membrane and cytoplasm of the superficial intestinal epithelium and in the epithelial cells in the crypts. A 2-kb UT-B mRNA transcript and the UT-B protein were also identified in the intestinal cell line Caco-2. The transepithelial flux of 14C urea was examined in Caco-2 cells growing on porous membrane support and was significantly inhibited by phloretin, 1,3-dimethylurea, and thiourea, suggesting that the transfer of urea across the Caco-2 monolayer could be mediated, at least in part, by the UT-B urea transporter. We conclude that the Kidd antigen/UT-B urea transporter is physiologically expressed in the human colon epithelium, where it could participate in the transport of urea across the colon mucosa. [ABSTRACT FROM AUTHOR]

Progress in understanding the cell biology of urea transporter proteins has been hampered by the lack of an appropriate cell culture system. The goal of this study was to create a polarized epithelial cell line that stably expresses the largest of the rat renal urea transporter UT-A isoforms, UT-A1. The gene for UT-A1 was cloned into pcDNA5/FRT and transfected into Madin-Darby canine kidney (MDCK) cells with an integrated Flp recombination target site. The cells from a single clone were grown to confluence on collagen-coated membranes until the resistance was >1,500 Ω·cm². Transepithelial [14C]urea fluxes were measured at 37°C in a HCO3-/ CO2 buffer, pH 7.4, with 5 mM urea. The baseline fluxes were not different between unstimulated UT-Al-transfected MDCK cells and nontransfected or sham-transfected MDCK cells. However, only in the UT-Al-transfected cells was UT-Al protein expressed (as measured by Western blot analysis) and urea transport stimulated by forskolin or arginine vasopressin. Forskolin and arginine vasopressin also increased the phosphorylation of UT-Al. Thionicotinamide, dimethylurea, and phloretin inhibited the forskolin-stimulated [14C]urea fluxes in the UT-Al-transfected MDCK cells. These characteristics mimic those seen in rat terminal inner medullary collecting ducts. This new polarized epithelial cell line stably expresses UT-Al and reproduces several of the physiological responses observed in rat terminal inner medullary collecting ducts. [ABSTRACT FROM AUTHOR]

Localization of the urea transporter UT-B protein in human and rat erythrocytes and tissues. Am J Physiol Cell Physiol 281: C1318-C1325, 2001.—A new polyclonal antibody to the human erythrocyte urea transporter UT-B detects a broad band between 45 and 65 kDa in human erythrocytes and between 37 and 51 kDa in rat erythrocytes. In human erythrocytes, the UT-B protein is the Kidd (Jk) antigen, and Jk(a+b+) erythrocytes express the 45- to 65-kDa band. However, in Jk null erythrocytes [Jk(a b-)], only a faint band at 55 kDa is detected. In kidney medulla, a broad band between 41 and 54 kDa, as well as a larger band at 98 kDa, is detected. Human and rat kidney show UT-B staining in nonfenestrated endothelial cells in descending vasa recta. UT-B protein and mRNA are detected in rat brain, colon, heart, liver, lung, and testis. When kidney medulla or liver proteins are analyzed with the use of a native gel, only a single protein band is detected. UT-B protein is detected in cultured bovine endothelial cells. We conclude that UT-B protein is expressed in more rat tissues than previously reported, as well as in erythrocytes. [ABSTRACT FROM AUTHOR]

Presents information on a study which investigated the structural basis of UT-A1 protein forms. Role of the protein forms in urinary concentrating mechanism; Effect of chronic vasopressin treatment on electrophoretic mobility of UT-A1; Preparation of polyclonal antibodies to multiple UT-A1 epitopes; Stabilization of UT-A1 protein complexes by chemical cross-linking.

Trafficking of the urea transporter UT-A1 to the plasma membrane is key in vasopressin-regulated urea transport, but the mechanisms remain unclear. Our experiments show UT-A1 involvement with several SNARE-mediated accessory proteins involved in orientation of proteins in the plasma membrane. One group of proteins involved in membrane orientation is the syntaxin family. We recently reported that snapin is important in the interaction of UT-A1 with syntaxin-4 and SNAP23 (t-SNARE). UT-A1-MDCK cells with adenoviral-mediated over-expression of both snapin and syntaxin-4, show that the association of UT-A1 with syntaxin-4 is enhanced snapin. Over-expression of syntaxin-4 alone showed minimal UT-A1/syntaxin-4 binding. Confocal microscopy of UT-A1 and snapin show colocalization in both cytoplasm and plasma membrane. Co-injection of UT-A1 with snapin cRNA (2 ug) in Xenopus oocytes increased urea influx. Co-injection of UT-A1 with t-SNARE without snapin, decreased urea influx. These results suggest that snapin promotes the initial docking of UT-A1 at the plasma membrane to form a SNARE complex. When we treated UT-A1-MDCK cells with vasopressin or forskolin, UT-A1 and snapin were redistributed to the plasma membrane. Collectively, our data support a role for a snapin syntaxin-4/SNAP23 mediated UT-A1 docking complex in vasopressin-regulated trafficking of UT-A1 in UT-A1-MDCK cells. In addition, we verified that the components of the snapin/t-SNARE/UT-A1 complex are present in rat inner medulla, suggesting that this mechanism may be physiologically important. [ABSTRACT FROM AUTHOR]

The 14-3-3 proteins are a group of highly conserved 30-kD regulatory proteins that mainly bind to phosphorylated residues in target proteins. 14-3-3 proteins interact with numerous cellular proteins and influence a large number of cellular processes. UT-A1 urea transporter contains a potential 14-3-3 recognition motif in the large cytoplasmic loop, suggesting a potential role of 14-3-3 in the regulation of UT-A1. To test this model, we examined the expression pattern of seven 14-3-3 protein isoforms in kidney cortex, outer medulla (OM) and inner medulla (IM). Zeta, beta and tau are highly and widely expressed in kidney cortex, OM and IM, as well as in brain, heart and liver. Gamma, eta, and epsilon are found in cortex, OM, and IM, but mainly expressed in IM. There was no detectable sigma in kidney tissue. We then prepared seven 14-3-3 isoform-specific GST fusion proteins for 14-3-3/UT-Al interaction studies. GST pull-down assays with UT-A1-MDCK cell lysate demonstrated that UT-A1 is in a protein complex with selected 14-3-3 isoforms: gamma eta and zeta. R18 peptide, a high-affinity competitive inhibitor of 14-3-3 interactions, prevented UT-A1 from binding to 14-3-3. To examine whether phosphorylation regulates 14-3-3 and UT-A1 binding, UT-A1-MDCK cells were treated with cAMP/adenylyl cyclase stimulator forskolin (10 µM) for 5, 15, and 30 min. Cell lysates were prepared and used for GST 14-3-3 gamma, eta and zeta pull-down assay. Phosphorylation significantly induced UT-A1 binding to 14-3-3. Our study identified 14-3-3 as a novel and potentially important regulator of urea transport activity. [ABSTRACT FROM AUTHOR]

The UT-A1 urea transporter mediates rapid transepithelial urea transport across the inner medullary collecting duct and plays a major role in the urinary concentrating mechanism. To transport urea, UT-A1 must be present in the plasma membrane. The purpose of this study was to screen for UT-A1-interacting proteins and to study the interactions of one of the identified potential binding partners with UT-A1. Using a yeast two-hybrid screen of a human kidney cDNA library with the UT-A1 intracellular loop (residues 409-594) as bait, we identified snapin, a ubiquitously expressed SNARE-associated protein, as a novel UT-Al binding partner. Deletion analysis indicated that the C-terminal coiled-coil domain (H2) of snapin is required for UT-A1 interaction. Snapin binds to the intracellular loop of UT-A1 but not to the N- or C-terminal fragments. Glutathione S-transferase pulidown experiments and co-immunoprecipitation studies verified that snapin interacts with native UT-A1, SNAP23, and syntaxin-4 (t-SNARE partners), indicating that UT-A1 participates with the SNARE machinery in rat kidney inner medulla. Confocal microscopic analysis of immunofluorescent UT-A1 and snapin showed co-localization in both the cytoplasm and in the plasma membrane. When we co-injected UT-A1 with snapin cRNA in Xenopus oocytes, urea influx was significantly increased. In the absence of snapin, the influx was decreased when UT-A1 was combined with t-SNARE components syntaxin-4 and SNAP23. We conclude that UT-A1 may be linked to the SNARE machinery via snapin and that this interaction may be functionally and physiologically important for urea transport. [ABSTRACT FROM AUTHOR]