Molecular Mechanisms of Urea Transport

Physiologic data provided evidence for specific urea transporter proteins in red blood cells and kidney inner medulla. During the past decade, molecular approaches resulted in the cloning of several urea transporter cDNA isoforms derived from two gene families: UT-A and UT-B. Polyclonal antibodies were generated to the cloned urea transporter proteins, and their use in integrative animal studies resulted in several novel findings, including: (1) UT-B is the Kidd blood group antigen; (2) UT-B is also expressed in many non-renal tissues and endothelial cells; (3) vasopressin increases UT-A1 phosphorylation in rat inner medullary collecting duct; (4) the surprising finding that UT-A1 protein abundance and urea transport are increased in the inner medulla during conditions in which urine concentrating ability is reduced; and (5) UT-A protein abundance is increased in uremia in both liver and heart. This review will summarize the knowledge gained from studying molecular mechanisms of urea transport and from integrative studies into urea transporter protein regulation.     

Ultrastructural localization of UT-A and UT-B in rat kidneys with different hydration status

Urea transport in the kidney is mediated by a family of transporter proteins, including renal urea transporters (UT-A) and erythrocyte urea transporters (UT-B). We aimed to determine whether hydration status affects the subcellular distribution of urea transporters. Male Sprague-Dawley rats were divided into three groups: dehydrated rats (WD) given minimum water, hydrated rats (WL) given 3% sucrose in water for 3 days before death, and control rats given free access to water. We labeled kidney sections with antibodies against UT-A1 and UT-A2 (L194), UT-A3 (Q2), and UT-B using preembedding immunoperoxidase and immunogold methods. In control animals, UT-A1 and UT-A3 immunoreactivities were observed throughout the cytoplasm in inner medullary collecting duct (IMCD) cells, and weak labeling was observed on the basolateral plasma membrane. UT-A2 immunoreactivity in the descending thin limbs (DTL) was observed mainly on the apical and basolateral membranes of type I epithelium, and very faint labeling was observed in the long-loop DTL at the border between the outer and inner medulla. UT-A1 immunoreactivity intensity was markedly lower, and UT-A3 immunoreactivity was higher in IMCD of WD vs. controls. UT-A2 immunoreactivity intensities in the plasma membrane and cytoplasm of type I, II, and III epithelia of DTL were greater in WD vs. controls. In contrast, UT-A1 expression was greater and UT-A2 and UT-A3 expressions were lower in WL vs. controls. The subcellular distribution of UT-A in DTL or IMCD did not differ between control and experimental animals. UT-B was expressed in the plasma membrane of the descending vasa recta of both control and experimental animals. UT-B intensity was higher in WD and lower in WL vs. controls. These data indicate that changes in hydration status over 3 days affected urea transporter protein expression without changing its subcellular distribution.     

The Erythrocyte Urea Transporter UT-B

During the past decade significant progress has been made in our understanding of the role played by urea transporters in the production of concentrated urine by the kidney. Urea transporters have been cloned and characterized in a wide range of species. The genomic organization of the two major families of mammalian urea transporters, UT-A and UT-B, has been defined, providing new insight into the mechanisms that regulate their expression and function in physiological and pathological conditions. Beside the kidney, the presence of urea transporters has been documented in a variety of tissues, where their role is not fully known. Recently, mice with targeted deletion of the major urea transporters have been generated, which have shown variable impairment of urine concentrating ability, and have helped to clarify the physiological contribution of individual transporters to this process. This review focuses on the erythrocyte urea transporter UT-B.     

Molecular and functional characterization of an amphibian urea transporter.

We report the characterization of a frog (Rana esculenta) urea transporter (fUT). The cloned cDNA is 1.4 kb long and contains a putative open reading frame of 1203 bp. In frog urinary bladder, the gene is expressed as two mRNAs of 4.3 and 1.6 kb. The fUT protein is 63.1 and 56.3% identical to rat UT-A2 and UT-B1, respectively. The internal duplication of UT-A2 and UT-B, as well as the double LP box urea transporter signature sequence were found in this amphibian urea transporter. When expressed in Xenopus oocytes, fUT induced a 10-fold increase in urea permeability, which was blocked by both phloretin and mercurial reagents. The fUT protein did not transport thiourea, but the fUT-mediated urea transport was strongly inhibited by this compound. Thus, this amphibian urea transporter displays transport characteristics in between those of UT-A2 and UT-B.     

Urea transporter expression in aging kidney and brain during dehydration

Aging is commonly associated with defective urine-concentrating ability. The present study examined how the kidney and the brain of senescent (30-mo-old) female WAG/Rij rats respond to dehydration induced by 2 days of water deprivation in terms of urea transporter (UT) regulation. In euhydrated situation, senescent rats exhibited similar vasopressin plasma level but lower urine osmolality and papillary urea concentration and markedly reduced kidney UT-A1, UT-A3, and UT-B1 abundances compared with adult (10-mo-old) rats. Senescent rats responded to dehydration similarly to adult rats by a sixfold increase in vasopressin plasma level. Their papillary urea concentration was doubled, without, however, attaining that of dehydrated adult rats. Such an enhanced papillary urea sequestration occurred with a great fall of both UT-A1 and UT-A3 abundances in the tip of inner medulla and an increased UT-A1 abundance in the base of inner medulla. UT-A2 and UT-B1 were unchanged. These data suggest that the inability of control and thirsted senescent rats to concentrate urine as much as their younger counterparts derives from lower papillary urea concentration. In aging brain, UT-B1 abundance was increased twofold together with a fourfold increase in aquaporin-4 abundance. Dehydration did not alter the abundance of these transporters.     

Structure-activity analysis of thiourea analogs as inhibitors of UT-A and UT-B urea transporters

Small-molecule inhibitors of urea transporter (UT) proteins in kidney have potential application as novel salt-sparing diuretics. The urea analog dimethylthiourea (DMTU) was recently found to inhibit the UT isoforms UT-A1 (expressed in kidney tubule epithelium) and UT-B (expressed in kidney vasa recta endothelium) with IC₅₀ of 2-3 mM, and was shown to have diuretic action when administered to rats. Here, we measured UT-A1 and UT-B inhibition activity of 36 thiourea analogs, with the goal of identifying more potent and isoform-selective inhibitors, and establishing structure-activity relationships. The analog set systematically explored modifications of substituents on the thiourea including alkyl, heterocycles and phenyl rings, with different steric and electronic features. The analogs had a wide range of inhibition activities and selectivities. The most potent inhibitor, 3-nitrophenyl-thiourea, had an IC₅₀ of ~ 0.2 mM for inhibition of both UT-A1 and UT-B. Some analogs such as 4-nitrophenyl-thiourea were relatively UT-A1 selective (IC₅₀ 1.3 vs. 10 mM), and others such as thioisonicotinamide were UT-B selective (IC₅₀ > 15 vs. 2.8 mM).     

UT-B is expressed in bovine rumen: potential role in ruminal urea transport

The UT-A (SLC14a2) and UT-B (SLC14a1) genes encode a family of specialized urea transporter proteins that regulate urea movement across plasma membranes. In this report, we describe the structure of the bovine UT-B (bUT-B) gene and characterize UT-B expression in bovine rumen. Northern analysis using a full-length bUT-B probe detected a 3.7-kb UT-B signal in rumen. RT-PCR of bovine mRNA revealed the presence of two UT-B splice variants, bUT-B1 and bUT-B2, with bUT-B2 the predominant variant in rumen. Immunoblotting studies of bovine rumen tissue, using an antibody targeted to the NH2-terminus of mouse UT-B, confirmed the presence of 43- to 54-kDa UT-B proteins. Immunolocalization studies showed that UT-B was mainly located on cell plasma membranes in epithelial layers of the bovine rumen. Ussing chamber measurements of ruminal transepithelial transport of (14)C-labeled urea indicated that urea flux was characteristically inhibited by phloretin. We conclude that bUT-B is expressed in the bovine rumen and may function to transport urea into the rumen as part of the ruminant urea nitrogen salvaging process.     

Molecular cloning of urea transporters from the kidneys of baleen and toothed whales

Urea transport in the kidney is important for the production of concentrated urine. This process is mediated by urea transporters (UTs) encoded by two genes, UT-A (Slc14a2) and UT-B (Slc14a1). Our previous study demonstrated that cetaceans produce highly concentrated urine than terrestrial mammals, and that baleen whales showed higher concentrations of urinary urea than sperm whales. Therefore, we hypothesized that cetaceans have unique actions of UTs to maintain fluid homeostasis in marine habitat. Kidney samples of common minke (Balaenoptera acutorostrata), sei (B. borealis), Bryde's (B. brydei) and sperm whales (Physeter macrocephalus) were obtained to determine the nucleotide sequences of mRNAs encoding UT. The sequences of 2.5-kb cDNAs encode 397-amino acid proteins, which are 90-94% identical to the mammalian UT-A2s. Two putative glycosylation sites are conserved between the whales and the terrestrial mammals, whereas consensus sites for protein kinases are not completely conserved; only a single protein kinase A consensus site was identified in the whale UT-A2s. Two protein kinase C consensus sites are present in the baleen whale UT-A2s, however, a single protein kinase C consensus site was identified in the sperm whale UT-A2. These different phosphorylation sites of whale UT-A2s may result in the high concentrations of urinary urea in whales, by reflecting their urea permeability.     

Ubiquitination regulates the plasma membrane expression of renal UT-A urea transporters

The renal UT-A urea transporters UT-A1, UT-A2, and UT-A3 are known to play an important role in the urinary concentrating mechanism. The control of the cellular localization of UT-A transporters is therefore vital to overall renal function. In the present study, we have investigated the effect of ubiquitination on UT-A plasma membrane expression in Madin-Darby canine kidney (MDCK) cell lines expressing each of the three renal UT-A transporters. Inhibition of the ubiquitin-proteasome pathway caused an increase in basal transepithelial urea flux across MDCK-rat (r)UT-A1 and MDCK-mouse (m)UT-A2 monolayers (P < 0.01, n = 3, ANOVA) and also increased dimethyl urea-sensitive, arginine vasopressin-stimulated urea flux (P < 0.05, n = 3, ANOVA). Inhibition of the ubiquitin-proteasome pathway also increased basolateral urea flux in MDCK-mUT-A3 monolayers (P < 0.01, n = 4, ANOVA) in a concentration-dependent manner. These increases in urea flux corresponded to a significant increase in UT-A transporter expression in the plasma membrane (P < 0.05, n = 3, ANOVA). Further analysis of the MDCK-mUT-A3 cell line confirmed that vasopressin specifically increased UT-A3 expression in the plasma membrane (P < 0.05, n = 3, ANOVA). However, preliminary data suggested that vasopressin produces this effect through an alternative route to that of the ubiquitin-proteasome pathway. In conclusion, our study suggests that ubiquitination regulates the plasma membrane expression of all three major UT-A urea transporters, but that this is not the mechanism primarily used by vasopressin to produce its physiological effects. ubiquitin-proteasome pathway; urea transport; membrane localization     

Glycoforms of UT-A3 urea transporter with poly-N-acetyllactosamine glycosylation have enhanced transport activity

Urea transporters UT-A1 and UT-A3 are both expressed in the kidney inner medulla. However, the function of UT-A3 remains unclear. Here, we found that UT-A3, which comprises only the NH(2)-terminal half of UT-A1, has a higher urea transport activity than UT-A1 in the oocyte and that this difference was associated with differences in N-glycosylation. Heterologously expressed UT-A3 is fully glycosylated with two glycoforms of 65 and 45 kDa. By contrast, UT-A1 expressed in HEK293 cells and oocytes exhibits only a 97-kDa glycosylation form. We further found that N-glycans of UT-A3 contain a large amount of poly-N-acetyllactosamine. This highly glycosylated UT-A3 is more stable and is enriched in lipid raft domains on the cell membrane. Kifunensine, an inhibitor of α-mannosidase that inhibits N-glycan processing beyond high-mannose-type N-glycans, significantly reduced UT-A3 urea transport activity. We then examined the native UT-A1 and UT-A3 glycosylation states from kidney inner medulla and found the ratio of 65 to 45 kDa in UT-A3 is higher than that of 117 to 97 kDa in UT-A1. The highly stable expression of highly glycosylated UT-A3 on the cell membrane in kidney inner medulla suggests that UT-A3 may have an important function in urea reabsorption.     

UT-B1 Mediates Transepithelial Urea Flux in the Rat Gastrointestinal Tract

The process of urea nitrogen salvaging plays a vital role in the symbiotic relationship between mammals and their intestinal bacteria. The first step in this process requires the movement of urea from the mammalian bloodstream into the gastrointestinal tract lumen via specialized proteins known as facilitative urea transporters. In this study, we examined both transepithelial urea fluxes and urea transporter protein abundance along the length of the rat gastrointestinal tract. Urea flux experiments that used rat gastrointestinal tissues showed significantly higher transepithelial urea transport was present in caecum and proximal colon (P < 0.01, n = 8, analysis of variance [ANOVA]). This large urea flux was significantly inhibited by 1,3,dimethylurea (P < 0.001, n = 8, ANOVA) and thiourea (P < 0.05, n = 6, unpaired t-test), both known blockers of facilitative urea transporters. Immunoblotting analysis failed to detect any UT-A protein within rat gastrointestinal tissue protein samples. In contrast, a 30-kDa UT-B1 protein was strongly detected in both caecum and proximal colon samples at significantly higher levels compared to the rest of the gastrointestinal tract (P < 0.01, n = 4, ANOVA). We therefore concluded that UT-B1 mediates the transepithelial movement of urea that occurs in specific distal regions of the rat gastrointestinal tract.     

Hyperosmotic NaCl and urea synergistically regulate the expression of the UT-A2 urea transporter in vitro and in vivo

The UT-A2 urea transporter is involved in the recycling of urea through the kidney, a process required to maintain high osmotic gradients. Dehydration increases UT-A2 expression in vivo. The tissue distribution of UT-A2 suggested that hyperosmolarity, and not vasopressin, might mediate this effect. We have analyzed the regulation of UT-A2 expression by ambiant osmolarity both in vitro (mIMCD3 cell line) and in vivo (rat kidney medulla). The UT-A2 mRNA was found to be synergistically up-regulated by a combination of NaCl and urea. Curiously, the UT-A2 protein was undetectable in this hypertonic culture condition, or after transfection of the UT-A2 cDNA, whereas it could be detected in HEK-293 transfected cells. Treating rats with furosemide, a diuretic which decreases the kidney interstitium osmolarity without affecting vasopressin levels, led to decreased levels of the UT-A2 protein. Our results show that the UT-A2 urea transporter is regulated by hyperosmolarity both in vitro and in vivo.     

Molecular characterization of a novel UT-A urea transporter isoform (UT-A5) in testis

Urea movement across plasmamembranes is modulated by specialized transporter proteins that areproducts of two genes, termed UT-A and UT-B. These proteins play keyroles in the urinary concentrating mechanism and fluid homeostasis. Wehave isolated and characterized a 1.4-kb cDNA from testes encoding anew isoform (UT-A5) belonging to the UT-A transporter family. Forcomparison, we also isolated a 2.0-kb cDNA from mouse kidneyinner medulla encoding the mouse UT-A3 homologue. The UT-A5 cDNAhas a putative open reading frame encoding a 323-amino acidprotein, making UT-A5 the smallest UT-A family member in terms ofmolecular size. Its putative topology is of particular interest,because it calls into question earlier models of UT-A transporterstructure. Expression of UT-A5 cRNA in Xenopus oocytesmediates phloretin-inhibitable urea uptake and does not translocatewater. The distribution of UT-A5 mRNA is restricted to the peritubularmyoid cells forming the outermost layer of the seminiferous tubuleswithin the testes and is not detected in kidney. UT-A5 mRNA levels arecoordinated with the stage of testes development and increase 15 dayspostpartum, commensurate with the start of seminiferous tubule fluid movement.