Urea Transporter Proteins Research Articles

Accurate mRNA size determination in northern analysis using individual lane size markers.

BioTechniquesVol. 27, No. 2 BenchmarksOpen AccessAccurate mRNA Size Determination in Northern Analysis Using Individual Lane Size MarkersJohn J. Doran, Jeff M. Sands & Richard T. TimmerJohn J. DoranEmory University School of Medicine, Atlanta, GA, USASearch for more papers by this author, Jeff M. SandsEmory University School of Medicine, Atlanta, GA, USASearch for more papers by this author & Richard T. Timmer*Address correspondence to Dr. Richard T. Timmer, Emory University School of Medicine, Department of Physiology, Rm. 149 Physiology Bldg., Atlanta, GA 30322, USA. Inernet: E-mail Address: timmer@physio2.physio.emory.eduEmory University School of Medicine, Atlanta, GA, USASearch for more papers by this authorPublished Online:22 Aug 2018https://doi.org/10.2144/99272bm14AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail FiguresReferencesRelatedDetailsCited ByGenes and Proteins of Urea Transporters9 October 2014Urea Transport in the Kidney1 April 2011Tissue distribution of UT-A and UT-B mRNA and protein in ratAmerican Journal of Physiology-Regulatory, Integrative and Comparative Physiology, Vol. 290, No. 5Mammalian Urea TransportersAnnual Review of Physiology, Vol. 65, No. 1Molecular Approaches to Urea Transporters1 November 2002 | Journal of the American Society of Nephrology, Vol. 13, No. 11UT-A Urea Transporter Protein in HeartCirculation Research, Vol. 89, No. 2 Vol. 27, No. 2 Follow us on social media for the latest updates Metrics Downloaded 69 times History Published online 22 August 2018 Published in print August 1999 Information© 2018 Author(s)PDF download

Regulation of renal urea transporters.

Urea is important for the conservation of body water due to its role in the production of concentrated urine in the renal inner medulla. Physiologic data demonstrate that urea is transported by facilitated and by active urea transporter proteins. The facilitated urea transporter (UT-A) in the terminal inner medullary collecting duct (IMCD) permits very high rates of transepithelial urea transport and results in the delivery of large amounts of urea into the deepest portions of the inner medulla where it is needed to maintain a high interstitial osmolality for concentrating the urine maximally. Four isoforms of the UT-A urea transporter family have been cloned to date. The facilitated urea transporter (UT-B) in erythrocytes permits these cells to lose urea rapidly as they traverse the ascending vasa recta, thereby preventing loss of urea from the medulla and decreasing urine-concentrating ability by decreasing the efficiency of countercurrent exchange, as occurs in Jk null individuals (who lack Kidd antigen). In addition to these facilitated urea transporters, three sodium-dependent, secondary active urea transport mechanisms have been characterized functionally in IMCD subsegments: (1) active urea reabsorption in the apical membrane of initial IMCD from low-protein fed or hypercalcemic rats; (2) active urea reabsorption in the basolateral membrane of initial IMCD from furosemide-treated rats; and (3) active urea secretion in the apical membrane of terminal IMCD from untreated rats. This review focuses on the physiologic, biophysical, and molecular evidence for facilitated and active urea transporters, and integrative studies of their acute and long-term regulation in rats with reduced urine-concentrating ability.

Molecular characterization of an elasmobranch urea transporter.

Marine elasmobranch fishes retain relatively high levels of urea to balance the osmotic stress of living in seawater. To maintain osmotic balance and reduce the energetic costs of making urea, it is important for these animals to minimize urea excretion to the environment. We have isolated a novel 2.2-kb cDNA from Squalus acanthias (spiny dogfish shark) kidney encoding a 380-amino acid hydrophobic protein (ShUT) with 66% identity to the rat facilitated urea transporter protein UT-A2. Injection of ShUT cRNA into Xenopus oocytes induced a 10-fold increase in 14C-labeled urea uptake, inhibitable by phloretin (0.35 mM). ShUT mRNA is expressed in kidney and brain. Related mRNA species are found in liver, blood, kidney, gill, intestine, muscle, and rectal gland. This is the first facilitated urea transporter to be identified in a marine fish. We propose that the ShUT protein is involved in urea reabsorption by the renal tubules of the dogfish shark, which in turn minimizes urea loss in the urine.

Long-term regulation of renal urea transporter protein expression in rat.

To test the hypothesis that the abundance of the apical urea transporter of the inner medullary collecting duct (IMCD) is regulated in vivo by factors associated with altered water balance, immunoblots of rat inner medullary membrane fractions were probed with rabbit polyclonal antibodies against the renal urea transporter (RUT) gene product. In inner medullas of Brattleboro rats, which manifest severe chronic water diuresis, a 117-kD band was seen, in addition to the previously described 97-kD band. These two bands were detectable by antibodies directed against two different regions of the RUT sequence. When Brattleboro rats were treated with a 5-d infusion of arginine vasopressin (AVP) by osmotic minipump, the 117-kD band was markedly diminished, whereas the 97-kD band was unchanged. Simultaneous infusion of the diuretic agent furosemide prevented the AVP-induced decrease in the 117-kD band. In AVP-infused Sprague Dawley rats, the 117-kD band was barely perceptible. However, when AVP-treated rats were infused with furosemide for 5 d, the 117-kD band was markedly accentuated, whereas the 97-kD band was unchanged. The abundance of the 117-kD RUT protein in the renal papilla was inversely correlated with dietary protein intake. Further immunoblotting studies revealed that the 117-kD protein is heavily expressed in IMCD cells and not in non-collecting duct components of the inner medulla, and is present in low-density microsome fractions from inner medulla. From this study, the following conclusions can be made: (1) The collecting duct urea transporter is present in at least two forms (97 and 117 kD) in the IMCD. (2) The expression level of the 117-kD urea transporter protein is regulated and is inversely correlated with medullary osmolality and urea concentration, but does not correlate with circulating AVP level. (3) Although AVP regulates RUT function on a short-term basis, long-term changes in AVP levels do not increase RUT abundance.

Vasopressin-elicited water and urea permeabilities are altered in IMCD in hypercalcemic rats.

To investigate how hypercalcemia blunts renal concentrating ability, alterations in basal and arginine vasopressin (AVP)-elicited osmotic water (Pf) and urea (Purea) permeabilities were measured in isolated perfused terminal inner medullary collecting ducts (IMCD) from control and chronically hypercalcemic rats after dihydrotachysterol (DHT) (M. Levi, L. Peterson, and T. Berl. Kidney Int. 23: 489-497, 1983) treatment. The IMCD Pf of DHT-treated rats did not increase significantly after AVP and was accompanied by a significant 87 +/- 4% reduction in aquaporin-2 (AQP-2) protein but not mRNA. In contrast, both basal and AVP-elicited IMCD Purea from DHT rats were significantly increased and accompanied by a significant 41 +/- 11% increase in AVP-regulated urea transporter protein content. Immunoblotting with anti-calcium/polyvalent cation-sensing receptor protein (CaR) antiserum revealed specific alterations in CaR bands in endosomes purified from the apical membranes of inner medulla of DHT rats. These data are the first detailed analyses of hypercalcemia-induced alterations in AVP-regulated permeabilities and membrane transporters in IMCD. We conclude that selective alterations in IMCD transport occur in hypercalcemia, permitting the body to dispose of excess calcium without forming calcium-containing renal stones.

Urea production and transport in teleost fishes.

Teleosts appear to have retained the genes for the urea cycle enzymes. A few species express the full complement of enzymes and are ureotelic (e.g., Lake Magadi tilapia) or ammoniotelic (e.g., largemouth bass), whereas most species have low or non-detectable enzyme activities in liver tissue and excrete little urea (e.g., adult rainbow trout). It was surprising, therefore, to find the expression of four urea cycle enzymes during early life stages of rainbow trout. The urea cycle may play a role in ammonia detoxification during a critical time of development. Exposure to alkaline water (pH 9.0-9.5) or NH4Cl (0.2 mmol/l) increased urea excretion by several-fold in trout embryos, free embryos and alevin. Urea transport is either by passive simple diffusion or via carried-mediated transport proteins. Molecular studies have revealed that a specialised urea transport protein is present in kidney tissue of elasmobranchs, similar to the facilitated urea transporter found in the mammalian inner medulla of the kidney.

Urea transporters in kidney and erythrocytes.

Physiological and molecular data demonstrate that urea transport in kidney and erythrocytes is regulated by specific urea transporter proteins. The urea transporter in the terminal inner medullary collecting duct permits very high rates of regulated transepithelial urea transport and results in the delivery of large amounts of urea into the deepest portions of the inner medulla, where it is needed to maintain a high interstitial osmolality for concentrating the urine maximally. The urea transporter in erythrocytes permits these cells to lose urea rapidly as they ascend through the ascending vasa recta, thereby preventing loss of urea from the medulla. Urea lost from the medulla would decrease concentrating ability by decreasing the efficiency of countercurrent exchange, as occurs in individuals who lack the Kidd antigen. The recent cloning of cDNAs for these two urea transporters has begun to yield new insights into the mechanisms underlying acute and long-term regulation of urea transport and should permit exciting new insights in the future. This review focuses on the physiological and biophysical evidence that established the concept of urea transporters, the subsequent cloning of cDNAs for urea transporters, and the recent integrative studies into the regulation of urea transport. We also propose a new systematic nomenclature and a new structural model for urea transporters.

Segmental localization of urea transporter mRNAs in rat kidney.

Renal epithelia express at least two distinct urea transporter mRNAs, termed UT1 and UT2, that are derived from a single UT gene by alternative splicing. Previous immunolocalization studies using a polyclonal antibody that does not distinguish between the protein products of these two transcripts revealed that expression of urea transporter protein is restricted to inner medullary collecting ducts and descending thin limbs of Henle's loop. To identify which transcripts account for protein expression in these two structures, we carried out reverse transcription-polymerase chain reaction studies in microdissected structures using UT1- and UT2-specific primers. UT1 mRNA was detected only in the inner medullary collecting duct, consistent with its identification as the vasopressin-regulated urea transporter. In contrast, UT2-mRNA was detected in the late part of descending thin limbs of short loops of Henle and in the inner medullary part of descending thin limbs of long loops of Henle. This localization is consistent with the predicted role of UT2 in medullary urea recycling. Thus, in conjunction with foregoing physiological studies, our data indicate that these transporters play central roles in the urinary concentrating mechanism.

Ultramicrodetermination of vasopressin-regulated urea transporter protein in microdissected renal tubules.

The vasopressin-regulated urea transporter (VRUT) is a 97-kDa protein (also called "UT-1") responsible for facilitated urea transport across the apical plasma membrane of inner medullary collecting duct (IMCD) cells. To determine the abundance of VRUT protein in collecting duct cells of the rat, we designed a sensitive fluorescence-based enzyme-linked immunosorbent assay capable of detecting <5 fmol of VRUT protein. In collecting duct segments, measurable VRUT was found in microdissected IMCD segments but not in other portions of the collecting duct. In the mid-IMCD, the measured level averaged 5.3 fmol/mm tubule length, corresponding to approximately 5 million copies of VRUT per cell. Thus VRUT is extremely abundant in the IMCD, accounting, in part, for the extremely high urea permeability of this segment. Feeding a low-protein diet (8% protein) markedly decreased urea clearance but did not alter the quantity of VRUT protein in the IMCD. Thus increased urea transport across the collecting duct with dietary protein restriction is not a consequence of increased expression of VRUT. Based on urea fluxes measured in the IMCD and our measurements of the number of copies of VRUT, we estimate a turnover number of > or = 0.3-1 x 10(5) s. In view of the large magnitude of this value and previously reported biophysical properties of urea transport in collecting ducts, we hypothesize that the VRUT may function as a channel rather than a carrier.

Glucocorticoids downregulate the vasopressin-regulated urea transporter in rat terminal inner medullary collecting ducts.

This study tested whether glucocorticoids regulate tubular urea transport. Urea permeability was measured in perfused inner medullary collecting duct (IMCD) subsegments from rats that underwent adrenalectomy, adrenalectomy plus replacement with a physiologic dose of glucocorticoid (dexamethasone), or sham operation. Compared with sham rats, basal urea permeability in terminal IMCD was significantly increased in adrenalectomized rats and reduced in dexamethasone-treated rats. Vasopressin significantly increased urea permeability in all three groups. In contrast, there was no difference in basal or vasopressin-stimulated urea permeability in initial IMCD between the three groups. Next, membrane and vesicle fraction proteins were isolated from inner medullary tip or base and Western analysis was performed by use of an antibody to the rat vasopressin-regulated urea transporter. Vasopressin-regulated urea transporter protein was significantly increased in both membrane and vesicle fractions from the inner medullary tip of adrenalectomized rats. There was no change in vasopressin-regulated urea transporter protein in the inner medullary base, and Northern analysis showed no change in urea transporter mRNA abundance in either inner medullary region. It was concluded that glucocorticoids can downregulate function and expression of the vasopressin-regulated urea transporter in rat terminal IMCD.

Is an intact cytoskeleton required for red cell urea and water transport?

In order to determine the membrane protein(s) responsible for urea and water transport across the human red cell membrane, we planned to reconstitute purified membrane proteins into phosphatidylcholine vesicles. In preparatory experiments, we reconstituted a mixture of all of the red cell integral membrane proteins into phosphatidylcholine vesicles, but found that p-chloromercuribenzenesulfonate (pCMBS), which normally inhibits osmotic water permeability by approximately 90%, has no effect on this preparation. The preparation was also unable to transport urea at the high rates found in red cells, though glucose transport was normal. White ghosts, washed free of hemoglobin and resealed, also did not preserve normal urea and pCMBS-inhibitable water transport. One-step ghosts, prepared in Hepes buffer in a single-step procedure, without washing, retained normal urea and pCMBS-inhibitable water transport. Perturbation of the cytoskeleton in one-step ghosts, by removal of tropomyosin, or by severing the ankyrin link which binds band 3 to spectrin, caused the loss of urea and pCMBS-inhibitable water transport. These experiments suggest that an unperturbed cytoskeleton may be required for normal urea and pCMBS-inhibitable water transport. They also show that the pCMBS inhibition of water transport is dissociable from the water transport process and suggest a linkage between the pCMBS water transport inhibition site and the urea transport protein.

The regulation of urease activity in Aspergillus nidulans.

Aspergillus nidulans can utilize urea as a sole source of nitrogen but not as a carbon source. Urea is degraded by a urease. Mutation at any one of three genes, ureB, ureC, and ureD, may result in deficient urease activity. The ureB gene is closely linked to ureA, the structural gene for the urea transport protein. The heat lability of ureB- revertant strain, intragenic complementation tests, and the linkage of ureB to ureA suggest that ureB is the urease structural gene. The ureD gene is probably involved in the synthesis or incorporation of a nickel cofactor essential for urease activity. The function of the ureC gene is not known. Urease is not induced but is subject to nitrogen regulation. The urease activities of ammonium-derepressed mutants show that the effector of nitrogen regulation is more likely to be glutamine than ammonium. When glutamine is present in the medium, urease appears to be inactivated by some means which does not involve a newly synthesized protease or a direct interaction between glutamine and urease.