View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Kidney International, Vol. 49 (1996), pp.1615—1623 Structure,regulation and physiological roles of urea transporters MATFHIAS A. HEDIGER, ClJG P. SMITH,1 GUOFENG You, WEN-SEN LEE,2 YosHwTsu KANAI,3 and CHAIRAT SHAYAKUL Renal Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, and Department Biological Chemistry & Molecular Phannacology, Harvard Medical School, Boston, Massachusetts, USA Structure, regulation and physiological roles of urea transporters. on land [1]. In aquatic animals, the maintenance of low ammonia Urea is the major constituent of the urine and the principal means for concentrations in the body has not been a problem because of the disposal of nitrogen derived from amino acid metabolism. Specialized phioretin-inhibitable urea transporters are expressed in kidney medulla unlimited water supply. For example, teleost fish maintain their and play a central role in urea excretion and water balance. Thesebody osmolality below that of sea water and dispose waste transporters allow accumulation of urea in the medulla and enable the nitrogen as ammonia with little metabolic expenditure [2—41.By kidney to concentrate urine to an osmolality greater than systemic plasma. Recently, expression cloning with Xenopus oocytes has led to the isolation contrast, land animals have overcome the problem of limited of a novel phioretin-inhibitable urea transporter (UT2) from rabbit, and water supply by synthesizing urea as a mechanism to detoxify subsequently from rat kidney. UT2 from both species has the character- ammonia. istics of the phloretin-sensitive urea transporter previously defined in Certain species also use urea as an important osmolyte [5—71. kidney by in Vitro perfused tubule studies. Based on these advances, Forexample, marine elasmobranch such as sharks, skates and rays Ripoche and colleagues cloned a homologous urea transporter (HUT11) from eiythrocytes. UT2 and HUTI 1 predict 43 kDa polypeptides andpossess large quantities of urea in the blood, body fluids and exhibit 64% amino acid sequence identity. Since regulation of ureatissues [5]. Their strategy for living in a marine environment is to transport in the kidney plays an important role in the orchestration of the maintain their body osmolality isosmotic with sea water, using antidiuretic response, we have studied the regulation of urea transporter in rat kidney at the mRNA level. On Northern blots probed at highurea as an osmolyte. stringency, rat UT2 hybridized to two transcripts of 2.9 kb and 4.0 kb, Given the vital roles urea plays in vertebrates, an important which have spatially distinct distributions within the kidney. Northern question arises as to how urea moves across biological mem- analysis and in situ hybridization of kidneys from rats maintained at different physiologic states revealed that the 2.9 and 4.0 kb transcripts are branes. Urea movement was originally thought to occur by simple regulated by separate mechanisms. The 4 kb transcript was primarily lipid permeation. Subsequent studies, however, revealed that responsive to changes in the dietary protein content, whereas the 2.9 kb several cell types including erythrocytes and epithelial cells of transcript was highly responsive to changes in the hydration state of the certain nephron segments have membrane permeabilities that are animal. We propose that the two UT2 transcripts are regulated by distinct mechanisms to allow optimal fluid balance and urea excretion. considerably higher than can be explained by simple lipid-phase diffusion, suggesting the existence of specialized urea transporters [8]. In the past few years, carrier mediated urea transport has been Urea is the major end product of nitrogen metabolism in mostextensively characterized in kidney inner medulla [9—11] and red mammals. It is synthesized in the liver by the urea cycle andblood cells [12]. In general, these transporters were found to be excreted in the urine (Fig. 1). Although the synthesis of urea frompassive and phloretin-inhibitable, and to exhibit low affinities for ammonia requires the high metabolic expenditure of four ATPurea (Km > 100 mM). The urea permeability of in vitro perfused molecules, urea formation is necessary to eradicate ammonia,kidney tubule segments has been studied by several investigators which is toxic to animals at relatively low concentrations, From an[reviewed in 11]. Moderate permeabilities were detected in thin evolutionary perspective, the development of urea cycle enzymesdescending limb of short loops of Henle [131, and in the inner and the ability of animals to synthesize urea from ammonia has medullaiy portion of long-loop descending and ascending thin become of particular importance with the movement of animalslimbs (Fig. 2) [14—17]. The highest permeability was detected in the terminal inner niedullary collecting duct (IMCD) [18—221. This permeability was found to increase to high values in response Note: In this paper UT2 refers to the urea transporter encoded by the rabbit 3.0 kb or the rat 2.9 kb mRNA, whereas UT refers to the urea to stimulation by vasopressin [18—20, 22]. There was no saturation transporters encoded by the 2.9 and 4 kb mRNAs from rat kidney. of radiolabeled urea transport across terminal IMCD segments in Present address: Department of Physiological Sciences, University of zero trans experiments when the bath urea was varied between Manchester, School of Biological Sciences, Manchester M13 9PT, En- 0 and 800 mM [9]. However, when the urea concentration was gland, United Kingdom. varied in both lumen and bath, the lumen to bath urea flux 2Presentaddress: Cardiovascular Biology Laboratory, Harvard School of Public Health, 677 Huntington Avenue, Boston, MA 02115, USA. approached a limiting value at 400 to 500 m urea. Present address, Department of Pharmacology, Kyorin University Toon and Solomon measured the permeability of ureas and School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181, Japan. amides through the red blood cell membrane and concluded that © 1996 by the International Society of Nephrology permeation is a saturable process with Km values of around 400 1615 1616 Hedigeret al: Mammalian urea transporters Fig. 1. Urea cycle in the liver. Most of the urea excreted by the mammalian kidney is synthesized in the liver. Two ammonium ions are consumed by the urea cycle: One is consumed in the formation of carbamoyl phosphate (the carbamoyl moiety is then transferred to ornithine to form citrulline). The second is consumed in the formation of Urea Kidney aspartate from oxaloacetate (in this reaction, an Arginine cs-amino nitrogen from glutamate is transferred to oxaloacetate by transamination to form aspartate and a-ketoglutarate; this reaction can be considered to consume one ammonium ion NH44-e Urea although it does not directly utilize free ammonium). Citrulline and aspartate then form Liver OrnithinJ Colon argininosuccinate, and subsequent cleavage of argininosuccinate yields arginine and fumarate. Arginine is then hydrolyzed into urea and NR4+CO2 (NH4j ornithine. An equivalent of four ATP molecules are utilized during the formation of each urea 4itl/$1__• molecule. The Figure also highlights the urea Citrulline recycling pathway which exists in some species between the liver and the colon. a Fig. 2. Role of urea transporters in the urinaty 5 concentrating mechanism, and possible sites of V Ot expression of urea transporters in the rat kidney. a, Abbreviations are: DVR, descending vasa recta; 2.9 kb AVR, ascending vasa recta; tDL, thin descending limb; TAL, thick ascending limb; o CD, collecting duct. The 2.9 kb signal is expressed in the inner stripe of the outer medulla (probably in thin descending limbs of short ioops of Henle). The 4.0 kb signal corresponds to the transporter in the IMCD. The Figure shows the thin descending limbs of short ioops of Henle to cross over the long ioops and to the peripheiy of the vascular bundles which include descending and 4kb UT2 ascending vasa recta. This morphological phenomenon was observed in some rodents such as rat and mouse and has implications for the urine concentrating ability. mM [12]. Physiologically, it was thought that facilitated ureaconcentrate urine [25]. Based on their study, Jk(a—b—) patients transport in red cells minimizes volume perturbations when theyhad a maximal urine osmolality of 819 mOsm/kg H20, whereas circulate through the renal medulla. Studies of urea transport inthe maximal urine osmolality in control individuals was —1,000 human erythrocytes of the Kidd blood type Jk(a—b—) revealedmOsm/kg H20. Because of the slightly impaired urine concen- that these cells lack the facilitated urea transporter. Experimen-trating ability in Jk(a—b—) patients, it was proposed that the tally, it was demonstrated that erythrocytes from individuals whoerythrocyte and the kidney urea transporters are encoded by a lack the Kidd antigen exhibit an increased resistance to lysis tosingle gene that is deficient in patients with K.idd blood type. Our aqueous 2 m urea [23]. Froehlich et al also showed that theserecent insights into the molecular identity of urea transporters, erythrocytes have a specific defect in urea transport [24]. Al-however, revealed that there are different urea transporters in though patients with the Jk(a—b—) blood type have no obviouskidney and erythrocytes and that the Kidd blood group is associ- medical problems, recent studies by Sands and colleagues indi-ated with a defect of the urea transporter expressed in erythro- cated that these individuals have a minor defect in the ability tocytes. Hediger et al: Mammalian urea transporters 1617 Role of urea transport in urine concentration suitable for screening cDNA libraries. A powerful expression An important function of the mammalian kidney is to conservecloning technique for studying membrane proteins at the molec- water. Urea transport in the mammalian kidney medulla isular level has subsequently emerged which involves screening of eDNA libraries by functional expression in Xenopus laevis intimately involved in the urinary concentrating mechanism [re- viewed in 11].