REVIEW J Am Soc Nephrol 13: 2795–2806, 2002 Molecular Approaches to Urea Transporters JEFF M. SANDS Renal Division, Department of Medicine, and Department of Physiology, Emory University School of Medicine, Atlanta, Georgia. Abstract. Urea plays a critical role in the urine-concentrating abundance and urea transport are increased in the inner me- mechanism in the inner medulla. Physiologic data provided dulla during conditions in which urine concentrating ability is evidence that urea transport in red blood cells and kidney inner reduced; (2) vasopressin increases UT-A1 phosphorylation in medulla was mediated by specific urea transporter proteins. rat inner medullary collecting duct; (3) UT-A protein abun- Molecular approaches during the past decade resulted in the dance is upregulated in uremia in both liver and heart; and (4) cloning of two gene families for facilitated urea transporters, UT-B is expressed in many nonrenal tissues and endothelial UT-A and UT-B, encoding several urea transporter cDNA cells. This review will summarize the knowledge gained from isoforms in humans, rodents, and several nonmammalian spe- using molecular approaches to perform integrative studies into cies. Polyclonal antibodies have been generated to the cloned urea transporter protein regulation, both in normal animals and urea transporter proteins, and the use of these antibodies in in animal models of human diseases, including studies of integrative animal studies has resulted in several novel find- uremic rats in which urea transporter protein is upregulated in ings, including: (1) the surprising finding that UT-A1 protein liver and heart. Urea is a small (molecular weight, 60 Da), highly polar mol- across cell membranes slowly and achieve equilibrium in the Ϫ ϭ Ϫ ecule ([NH2] [C O] [NH2]) that has low lipid solubility steady state. However, the transit time for tubule fluid through through artificial lipid bilayers (4 ϫ 10Ϫ6 cm/s (1)). Thus, urea the collecting duct or for red blood cells through the vasa recta should have a low permeability across cell membranes that is too fast to allow urea concentrations to reach equilibrium lack a transport protein to facilitate its transfer. The high urea solely by passive diffusion. Furthermore, the finding that urea permeability across red blood cells and the kidney terminal transporters are expressed in several nonrenal tissues suggests inner medullary collecting duct (IMCD) was the initial evi- that urea was mistakenly considered freely permeable because dence suggesting the presence of specific urea transporter of a lack of knowledge regarding the wide-spread distribution proteins (2,3). Transport studies of red blood cells and isolated of urea transporters. perfused tubules, performed in the 1970s and 1980s, provided the physiologic evidence that established the concept of urea The Urinary-Concentrating Mechanism: transporters (reviewed in references 4 and 5). In the last 10 yr, Role of Urea Transporters two genes and several cDNA isoforms for urea transporters The original quest for a urea transporter resulted from urea’s were cloned (Table 1). Through the use of these cDNAs and key role in the urinary-concentrating mechanism. Urea’s im- polycolonal antibodies to the urea transporter proteins, urea portance to the generation of concentrated urine has been transporters have now been found in liver, heart, testis, and appreciated since Gamble et al. (9) described “an economy of brain, and in some of these tissues, their abundance is altered water in renal function referable to urea” in 1934. Protein- by uremia (6–8). This review will focus on the knowledge deprived animals and humans have an impaired ability to gained from molecular approaches to the study of urea trans- concentrate their urine, but this is corrected by an infusion of porters in kidney and other tissues. urea (9–16). Thus, any hypothesis regarding the mechanism by Given that most textbooks state that urea is freely permeable which the kidney concentrates urine needs to take into account across cell membranes, the reader may be puzzled at the some effect derived from urea. existence of urea transporters in red blood cells, kidney, and The passive mechanism hypothesis for urinary concentration other organs. Although urea’s permeability across artificial was proposed in 1972 by Kokko and Rector (17) and Stephen- lipid bilayers is very low, it is not zero, and urea will diffuse son (18) (Figure 1). This hypothesis requires that the inner medullary interstitial urea concentration exceed the urea con- centration in the lumen of the thin ascending limb. The in- Correspondence to Dr. Jeff M. Sands, Emory University School of Medicine, Renal Division, WMRB Room 338, 1639 Pierce Drive, NE, Atlanta, GA equality of urea concentration permits the interstitial NaCl 30322. Phone: 404-727-2525; Fax: 404-727-3425; E-mail: [email protected] concentration to be less than the NaCl concentration in the 1046-6673/1311-2795 lumen of the thin ascending limb, thereby establishing a gra- Journal of the American Society of Nephrology dient for passive NaCl absorption in the absence of an osmotic Copyright © 2002 by the American Society of Nephrology gradient. If an inadequate amount of urea is delivered to the DOI: 10.1097/01.ASN.0000035084.94743.7C deep inner medullary interstitium, then the chemical gradients 2796 Journal of the American Society of Nephrology J Am Soc Nephrol 13: 2795–2806, 2002 Table 1. Facilitated urea transporter genes and isoformsa Gene Isoform RNA (kb) Protein (kD) AVP Location References Slc14a2 UT-A1 (UT1) 4.0 97, 117 Yes IMCD (20,23,39) UT-A1b 3.5 Medulla* (36,43) UT-A2 (UT2) 2.9 55 No tDL, liver (21,24,27,29,99,124) UT-A2b 2.5 Medulla*, heart (7,31,36) UT-A3 2.1 44, 67 Yes* IMCD (31,33,34,34,35) UT-A3b 3.7 Medulla* (36) UT-A4 2.5 43 Yes Medulla*, liver? (31) UT-A5c 1.4 Testis (33) Slc14a1 UT-B1 (UT3) 3.8 43 DVR, RBCb (94,96,105,106) UT-B2 (UT11) 4.4 45 DVR, RBCb (24,95,106) a Isoform names are based on the urea transporter nomenclature proposed in reference 4, with alternative names in parenthesis; AVP, urea flux is stimulated by vasopressin in Xenopus oocytes or HEK-293 cells (Yes*, urea flux is stimulated in two studies but not in a third); IMCD, inner medullary collecting duct; tDL, thin descending limb; Medulla* (exact tubular location unknown); DVR, descending vasa recta; RBC, red blood cells. b Also expressed in several other tissues and endothelial cells. c Cloned from mouse only. necessary for passive NaCl absorption from the thin ascending testis but not in kidney (33), and the effect of vasopressin on limb cannot be established, and urine-concentrating ability is UT-A5 has not been tested. UT-A5 is the shortest member of reduced. Urea absorption from the terminal IMCD, mediated the UT-A family; its deduced amino acid sequence begins at by a facilitated urea transporter, is the primary mechanism for methionine 139 of mouse UT-A3, after which it shares 100% delivering urea to the deep inner medullary interstitium (2,19). homology and a common C-terminal amino acid (3' cDNA) On the basis of functional measurements in isolated perfused end with UT-A3 (33). IMCD and red blood cells that defined the properties of puta- Three UT-A cDNA transcripts that have alternative 3'-un- tive facilitated urea transporters, two genes encoding several translated regions have been cloned and named UT-A1b, UT- cDNA isoforms have been cloned (Table 1). A2b, and UT-A3b, respectively (36). UT-A1b and UT-A2b transcripts are approximately 0.4 kb shorter than the original The UT-A Urea Transporter Family cDNAs, whereas the UT-A3b transcript is approximately 1.5 UT-A1 is the largest UT-A protein (Figure 2) and is ex- kb longer than the original cDNA (36). Northern analysis pressed in the apical membrane of the IMCD in humans (20) shows that UT-A1b, UT-A2b, and UT-A3b mRNAs are ex- and rats (21,22). Human and rodent UT-A1 are stimulated by pressed in rat inner medulla (36). cyclic AMP (cAMP) when expressed in Xenopus oocytes Polyclonal antibodies have been made to three regions of (20,23–26). UT-A2, which was the first urea transporter to be UT-A1: the C-terminus (21,37); the N-terminus (28); and the cloned (27), is expressed in thin descending limbs (21,22,28) intracellular loop region of UT-A1 (38). Due to the high degree and is not stimulated by cAMP analogs when expressed in of homology between the kidney UT-A cDNA isoforms, the either Xenopus oocytes or human embryonic kidney (HEK) C-terminus antibody should detect UT-A1 (97 and 117 kD), 293 cells (23–25,27,29–31). UT-A1 and UT-A2 share identical UT-A2 (55 kD), and UT-A4 (43 kD), the N-terminus antibody C-terminal amino acid and 3' cDNA sequences, but they differ should detect UT-A1, UT-A3 (44 and 67 kD), and UT-A4, and at their N-terminal (5') ends (20,23,32). Thus, UT-A2 is basi- the loop region antibody should detect only UT-A1 cally the C-terminal (3') half of UT-A1. UT-A3 has the same (5,31,35,38). Western analyses of inner medullary tip proteins N-terminal amino acid and 5' cDNA sequence as UT-A1 but show bands at both 117 and 97 kD using any of the anti-UT-A1 has a unique C-terminal (3') end and is basically the N-terminal antibodies (21,37,38); both protein bands represent glycosy- (5') half of UT-A1 (31,33,34). UT-A3 is also expressed in the lated versions of a non-glycosylated 88-kD UT-A1 protein apical membrane of the IMCD (35) and is stimulated by cAMP (39).