REVIEW J Am Soc Nephrol 13: 2795–2806, 2002

Molecular Approaches to 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 inner reduced; (2) increases UT-A1 phosphorylation in medulla was mediated by specific . rat inner medullary collecting duct; (3) UT-A 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 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 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). In addition, UT-A1 exists as a 206-kD protein complex in analogs when expressed in HEK-293 cells or Xenopus oocytes native inner medullary membranes (39). UT-A1 protein is most in two studies (26,31) but not in a third study (34). Although abundant in the inner medullary tip, present in the inner med- UT-A4 has the same N- and C-terminal amino acid (5' and 3' ullary base, but only as a 97-kD protein, and is not present in cDNA) sequence as UT-A1, it is smaller than UT-A1 and outer medulla or cortex (21,40,41). basically consists of the N-terminal (5') quarter of UT-A1 Even though the C-terminus of UT-A3 differs from UT-A1 spliced to the C-terminal (3') quarter of UT-A1 (31). Although by only a single amino acid, Terris et al. (35) succeeded in its exact tubular location is unknown, UT-A4 mRNA is ex- making a UT-A3–specific antibody that detects bands at both pressed in kidney medulla and is stimulated by cAMP analogs 67 and 44 kD; both bands represent glycosylated versions of a when expressed in HEK-293 cells (31). UT-A5 is expressed in non-glycosylated 40-kD UT-A3 protein. Both UT-A3 glyco- J Am Soc Nephrol 13: 2795–2806, 2002 Molecular Approaches to Urea Transporters 2797

Figure 1. Diagram showing the location of the major medullary transport proteins involved in the urine concentrating mechanism. Shown are a loop of Henle (left) and collecting duct (right). UT, urea transporter; AQP, aquaporin; NKCC/BSC, Na-K-2Cl ; ROMK, renal outer medullary K channel. proteins are most abundant in the inner medullary tip, weakly spliced to exons 14 to 23. UT-A3 is encoded by exons 1 to 12. detected in the inner medullary base and outer medulla, and not UT-A4 is encoded by exons 1 to 7 spliced to exons 18 to 23. detected in cortex (35). These three UT-A isoforms share a common transcription start Although the apical membrane is the rate-limiting barrier for site in exon 1 and translation start site in exon 4. UT-A2 is vasopressin-stimulated urea transport, functional studies show unique because it is the only isoform that uses exon 13. It is that -inhibitable urea transport is present in both the encoded by exons 13 to 23 with a translation start site in exon apical and basolateral membranes of rat terminal IMCD (42). 16. Exon 24 contains the alternative 3' untranslated region used Both UT-A1 and UT-A3 immunostaining is detected in the by UT-A1b and UT-A2b. apical plasma membrane and intracellular cytoplasmic vesicles The rat UT-A isoforms originate from a single gene of terminal IMCD, but not in the basolateral plasma membrane (Slc14a2) that has two promoter elements: promoter I, which is (21,35). Thus, the molecular identity of the IMCD basolateral upstream of exon 1 and drives transcription of UT-A1, UT- membrane urea transporter remains undetermined. A1b, UT-A3, UT-A3b, and UT-A4; and promoter II, which is located within intron 12 and drives transcription of UT-A2 and The UT-A Gene UT-A2b (36,43). The initial 1.3 kb of UT-A promoter I con- The UT-A gene Slc14a2 was initially cloned from rat (43), tains 3 CCAAT elements but does not contain a TATA box but it has now been cloned from both human and mouse (44). However, its expression in a luciferase reporter gene (20,32). The rat UT-A gene contains 24 exons and extends for construct and transfection into MDCK, mIMCD3, or LLC-PK1 approximately 300 kb (43). UT-A1 is encoded by exons 1 to 12 cells results in promoter activity (44,45). Hyperosmolality in- 2798 Journal of the American Society of Nephrology J Am Soc Nephrol 13: 2795–2806, 2002

Figure 2. Diagram showing relationship between the four rat kidney UT-A proteins. There is a high degree of homology between these protein isoforms. There is no difference in the coding region of UT-A1, UT-A2, and UT-A3, and their respective b variants; therefore, the latter are not shown separately in this diagram. UT-A1, UT-A3, and UT-A4 share common amino-termini (N versus N'). UT-A1, UT-A2, and UT-A4 share common carboxy-termini (C versus C'). Consensus sites for phosphorylation and glycosylation are indicated. S, serine; T, threonine. creases promoter I activity, consistent with the presence of a The mouse UT-A gene has a similar structure (26,32). Like tonicity enhancer (TonE) element (44). Dexamethasone re- the rat (43), it contains 24 exons, is over 300 kb in length, has duces promoter I activity and the mRNA abundance of UT-A1, two promoter elements, and promoter I contains a TonE ele- UT-A3, and UT-A3b in the inner medulla of rats given a stress ment, the activity of which is increased by hypertonicity (32). dose of dexamethasone (45). Although a consensus glucocor- In contrast to rat, mouse UT-A promoter I activity is increased ticoid response element (GRE) is present in promoter I, the by cAMP, even though no consensus CRE element is present repressive effect of dexamethasone does not occur via this (32). Although all mouse UT-A isoforms, including UT-A5, GRE (45). originate from a single mouse UT-A gene, the transcription In contrast to the other UT-A isoforms, the transcription start start site of UT-A5 has not been determined and could be site for UT-A2 is located in exon 13, almost 200 kb down- located downstream from mouse UT-A1 and UT-A3 (32). stream from exon 1 (36,43). This distance suggested the hy- The human UT-A gene is located on 18, con- pothesis that there may be a second, internal promoter within tains 20 exons, and extends for approximately 67.5 kb. The intron 12. Cloning and sequencing of intron 12 shows that it human gene is substantially shorter than the rodent gene: (1) does contain a TATA box 40 bp upstream of the UT-A2 the 5'-untranslated region is almost entirely located in exon 1, transcription start site in exon 13 and a cAMP response ele- whereas in rat and mouse it spans the first three widely spaced ment (CRE) 300 bp upstream of the UT-A2 transcription start exons; and (2) the 3'-untranslated region does not contain an site (36,43). Transfection of a luciferase reporter gene from this exon analogous to rat exon 24 (20,32,43). region into mIMCD3 cells shows evidence of promoter activity Although the mechanism is not known, single nucleotide when the cells are stimulated with cAMP, but not under basal polymorphisms in human UT-A2 are associated with variation conditions (36,43). in BP in men but not in women (46). Facilitated urea trans- J Am Soc Nephrol 13: 2795–2806, 2002 Molecular Approaches to Urea Transporters 2799 porter cDNAs that are most homologous to UT-A2 have been UT-A4’s mRNA abundance in the renal medulla is too low to cloned from frog, elasmobranch, eel, gulf toadfish, Lake detect by Northern analysis (31) and UT-A5 is not expressed in Migadi tilapia, and pilot whale (47–53). A detailed discussion kidney (33). Thus, there may be multiple mechanisms by of these urea transporters is beyond the scope of this review, which vasopressin regulates the different UT-A protein and and the reader is referred to the original citations for more mRNA isoforms. information about them. UT-A Proteins during Development Long-Term Regulation of UT-A UT-A immunostaining is not detected in the fetal kidney but Vasopressin appears in 1-d-old rats, both in the IMCD (UT-A1) and the thin Increasing plasma vasopressin (also called antidiuretic hor- descending limb (UT-A2), and increases progressively in both mone [ADH]) by administering it exogenously, or by water segments until adult levels are achieved at 21 d of age (22). restriction, decreases the abundance of both the 117- and Thus, the time course for the development of urine-concentrat- 97-kD UT-A1 proteins in rat inner medulla (38) and decreases ing ability in rats coincides with the increase in UT-A1 and basal urea permeability in the perfused terminal IMCD (54). UT-A2 immunostaining. Thus, these studies led to the surprising finding that urea transport and UT-A1 protein abundance are decreased when Impaired Urine-Concentrating Ability and UT-A1 vasopressin levels are increased. This decrease in UT-A1 pro- The long-term regulation of UT-A1 protein abundance has tein abundance and basal urea permeability is not a result of a been studied in several conditions associated with reduced decrease in UT-A1 or UT-A1b mRNA, because Northern urine-concentrating ability: water diuresis; low-protein diet; analysis shows no change in either mRNA abundance in re- hypercalcemia; furosemide diuresis; adrenalectomy; and lith- sponse to either water loading or restriction in most studies ium administration (30,37,38,41,54,60–64). Surprisingly, in (24,25,29,32,36,55), although one study does report that every condition except lithium administration (which is dis- UT-A1 is decreased in water-restricted or vasopressin-treated cussed in more detail below), both UT-A1 protein abundance Brattleboro rats (which have central diabetes insipidus) (56). and basal facilitated urea permeability are increased in the Feeding rats a low (10%) or high (40%) protein diet for 1 deepest portion of the IMCD during conditions with reduced wk, compared with a control diet containing 20% protein, also urine-concentrating ability. The increase in UT-A1 protein has no effect on UT-A1 mRNA abundance in any portion of abundance and urea absorption could be a mechanism for the the kidney medulla (30,57). However, in Brattleboro rats, rapid increase in urine-concentrating ability that occurs within UT-A1 mRNA is decreased in the inner medullary tip of 5 to 10 min after urea is infused into malnourished or low- low-protein–fed rats (57), suggesting that there may be an protein-fed people or rats (9,10,12,65): UT-A1 protein abun- interaction between dietary protein and vasopressin on UT-A1 dance is increased when urine-concentrating ability is im- mRNA abundance. Overall, transcriptional regulation does not paired, and this response “prepares” the IMCD to restore inner appear to be the mechanism for changes in UT-A1 protein medullary urea rapidly once urea (or protein) intake rises. abundance in response to changes in hydration and/or vaso- pressin level. Glucocorticoids In contrast, UT-A2, UT-A2b, UT-A3, and UT-A3b mRNA Adrenalectomy causes a urinary-concentrating defect in hu- abundances fall in the inner medulla of water-loaded rats and mans and rats (66–68). Administering dexamethasone to adre- rise in rodents with increased vasopressin levels, due either to nalectomized rats decreases UT-A1 protein abundance and vasopressin administration or water restriction (24,29,32, facilitated urea permeability in the rat terminal IMCD (37). 36,55,56,58). UT-A2 mRNA abundance is increased in the Administering dexamethasone to normal rats decreases UT- base of the inner medulla from rats fed an 8% (low) protein diet A1, UT-A3, and UT-A3b mRNA abundances, but not UT-A2 for 1 wk (30), but not in Sprague-Dawley or Brattleboro rats mRNA abundance, in the tip of the inner medulla (45). This fed a 10% protein diet (57). UT-A2 protein abundance is repressive effect is likely to be transcriptionally regulated increased by administering dDAVP (Desmopressin, a V2-se- because dexamethasone decreases the activity of promoter I lective vasopressin receptor agonist) to Brattleboro rats (28) (which controls transcription of UT-A1 and UT-A3) but has no and decreased by treating rats with furosemide (59). Thus, effect on promoter II (which controls transcription of UT-A2) vasopressin may regulate UT-A2 by a transcriptional mecha- (45). nism, consistent with promoter II containing a CRE element and promoter activity being increased by cAMP (44). UT-A2 Volume Expansion expression can also be induced in mIMCD3 cells grown in Rats (and people) become volume-expanded when given hypertonic culture media in which osmolality is increased by aldosterone and a high-NaCl diet, but do not become volume adding equiosmolar NaCl and urea; mIMCD3 cells grown in expanded when given aldosterone and a NaCl-free diet (69). isotonic culture media do not express UT-A2 (or any other Volume-expanded rats have decreased levels of UT-A1 and UT-A isoform) (43,59). UT-A3 protein also increases in water- UT-A3 protein abundances in the inner medulla, whereas deprived rats (35), which could be transcriptionally mediated UT-A2 protein is unchanged (69). After volume expansion, the by the TonE element in promoter I (44). The long-term regu- decrease in UT-A1 protein parallels the decrease in serum urea lation of UT-A4 and UT-A5 have not been studied because concentration while the decrease in UT-A3 is delayed (69). 2800 Journal of the American Society of Nephrology J Am Soc Nephrol 13: 2795–2806, 2002

Inhibition of AT1-receptors (for 2 d) also decreases UT-A1 and Rapid Regulation of UT-A in the IMCD UT-A3 protein abundances, suggesting that the suppression of Studies of perfused IMCD have been the primary method the renin-angiotensin system that accompanies aldosterone- for investigating the rapid regulation of urea transport. This induced volume expansion may mediate the reduction in these method provides physiologically relevant, functional data, two urea transporters (69). but it cannot determine which urea transporter isoform is responsible for a functional effect because the terminal UT-A1 Protein in Animal Models of IMCD expresses both UT-A1 and UT-A3. Thus, the func- Human Diseases tional studies reviewed below may be due to urea transport Diabetes Mellitus mediated by UT-A1, UT-A3, or both. In the past few years, In rats, uncontrolled diabetes mellitus (induced by strepto- new findings have been published for two regulators of urea zotocin) increases corticosterone production and urea excretion transport: vasopressin and angiotensin II, and these studies (70). UT-A1 protein abundance is downregulated in the inner will be reviewed below. The reader is referred to older medullary tip of rats at 3 d after streptozotocin-injection (62). reviews (4,5,79) that discuss other agents that rapidly reg- However, UT-A1 protein abundance does not decrease in ulate urea transport. adrenalectomized rats injected with streptozotocin, suggesting that the diabetes-induced increase in glucocorticoids is the Vasopressin mechanism for reducing UT-A1 protein in rats with uncon- Adding vasopressin to the bath of a perfused rat terminal trolled diabetes for 3 d (62). In contrast, at 21 d post-strepto- IMCD results in binding to V2-receptors, stimulating adenylyl zotocin, UT-A1 mRNA and protein are increased in the inner cyclase, generating cAMP, and ultimately increasing facilitated medulla (71). However, UT-A1 protein is decreased in 6-mo- urea transport (2,80–82). One possible mechanism for rapid old, obese Zucker rats, a model of type II diabetes (72). Thus, regulation is that vasopressin alters the phosphorylation of UT-A1 abundance may vary with time from streptozotocin UT-A1 and/or UT-A3. The deduced amino acid sequences for treatment, and hence with the duration of diabetes, and/or the UT-A1 and UT-A3 contain several consensus sites for phos- type of diabetes. phorylation by protein kinase A (PKA), as well as PKC and tyrosine kinase (Figure 2) (31). Vasopressin increases the Renal Failure phosphorylation of both the 117- and 97-kD UT-A1 proteins Administering adriamycin to rats for 3 wk results in protein- within 2 min in rat IMCD suspensions (83); it is not known uria and decreased UT-A1 protein abundance in the inner whether vasopressin also alters UT-A3 phosphorylation. The medulla (73). Administering cisplatin to rats for 5 d results in time course and dose response for vasopressin-stimulated acute renal failure, accompanied by an increase in urine vol- phosphorylation of UT-A1 is consistent with the time course ume and a decrease in urine osmolality, but no change in and dose response for vasopressin-stimulated urea transport in UT-A1, UT-A2, or UT-A4 protein abundances in the outer or the perfused rat terminal IMCD (80,82–84). Both dDAVP and inner medulla (74). Inducing uremia in rats by 5/6 nephrec- cAMP also increase UT-A1 phosphorylation, and PKA inhib- tomy results in an increase in urine output and a decrease in itors block the phosphorylation of UT-A1 by vasopressin (85). urine osmolality at 5 wk post-nephrectomy accompanied by These findings strongly support the hypothesis that vasopressin undetectable levels of UT-A1 mRNA and protein and reduced rapidly increases urea transport in the rat terminal IMCD by levels of UT-A2 mRNA and protein (8). increasing UT-A1 phosphorylation. Another possible mechanism by which vasopressin could Lithium rapidly increase urea transport is regulated trafficking of UT-A1 and/or UT-A3. However, regulated trafficking of Lithium is widely used to treat patients with manic-depres- UT-A1 does not occur in the rat IMCD (86). Whether UT-A3 sive (bipolar) disorders but can cause nephrogenic diabetes undergoes regulated trafficking has not been studied. insipidus and an inability to concentrate urine (reviewed in reference 75). Although the mechanisms by which lithium causes nephrogenic diabetes insipidus are not entirely under- Angiotensin II stood, lithium-treated rats do have a marked reduction in AQP2 Both RT-PCR and in situ hybridization studies show that protein (64,76,77) and inner medullary interstitial osmolality mRNA for the type 1 angiotensin II (AT1) receptor is present (78). in rat IMCD (87–89), and radioligand binding studies show

Lithium-fed rats have a marked reduction in UT-A1 protein that AT1 receptors are present (90). Angiotensin II has no abundance in both the inner medullary tip and base (64). In effect on basal urea permeability, but it increases vasopressin- addition, vasopressin does not increase UT-A1 phosphoryla- stimulated facilitated urea permeability in rat terminal IMCD tion in IMCD suspensions from lithium-fed rats, in contrast to and 32P incorporation into both the 117- and 97-kD UT-A1 vasopressin’s effect on IMCD suspensions from normal rats proteins via a PKC-mediated effect (91). Mice that lack tissue (64). Thus, lithium differs from the other conditions associated angiotensin converting enzyme (ACE.2 mice) have a histolog- with reduced urine-concentrating ability (discussed above) be- ically normal medulla and a urine-concentrating defect (92). In cause it reduces UT-A1 protein abundance. The reason for this the inner medulla of these mice, UT-A1 protein is decreased to difference will require future studies. 25% of the level in wild-type mice (93). Neither the urine- J Am Soc Nephrol 13: 2795–2806, 2002 Molecular Approaches to Urea Transporters 2801 concentrating defect nor the reduction in UT-A1 protein is blood cells (97,106). In kidney outer or inner medulla, a broad corrected by administering angiotensin II to ACE.2 mice (93). band between 41 to 54 kD is detected; deglycosylation con- Thus, angiotensin II may play a physiologic role in the urinary- verts the broad band seen by Western analysis of either red concentrating mechanism by augmenting the maximal urea blood cells or kidney medulla to a sharp 32 kD band (106,110). permeability response to vasopressin. In addition, a 98-kD band is detected in kidney (106). How- ever, the molecular explanation for this 98 kD band is uncer- UT-B Urea Transporter tain (106). The facilitated urea transporter, UT-B, was Human and rodent kidney show UT-B immunostaining in originally cloned from a human erythropoietic cell line (94), nonfenestrated endothelial cells that are characteristic of de- but it has also been cloned from rodents (4,95–97). The human scending vasa recta (8,97,106,109,110). UT-B protein is also Slc14a1 (UT-B) gene arises from a single locus located on present in rodent testis, brain, colon, heart, liver, lung, aorta, chromosome 18q12.1-q21.1, which is close to, but distinct bladder, spinotrapezius muscle, and mesenteric artery from, the human Slc14a2 (UT-A) gene (98–100). The mouse (8,106,110,111) and in several cultured endothelial cell lines Scl14a1 (UT-B) and Scl14a2 (UT-A) genes also occur in (106,111). UT-B promotes urea entry into cultured endothelial tandem on (101). A minor blood group anti- cells, thereby increasing intracellular urea and inhibiting L- gen, the Kidd (or Jk) antigen, is also located in the same region arginine transport (111). If a similar mechanism is present in of human chromosome 18 as are the two urea transporter genes patients with chronic kidney disease, then the inhibition of (100). In humans, the Kidd antigen is the UT-B protein (98– arginine transport, a precursor of nitric oxide, could be another 100). Several mutations of the Kidd antigen/UT-B (Scl14a1) mechanism contributing to hypertension in these patients gene have been reported (100,102). Red blood cells from (111). individuals that lack the Kidd antigen (Jk(a-b-) or Jk null) do UT-B immunostaining is only weakly detected in rat kidney not have phloretin-sensitive facilitated urea transport (103). at fetal day 20, but it increases progressively after birth in the The human UT-B gene includes 11 exons, with the coding descending vasa recta, both in terms of the intensity of staining region beginning in exon 4 and extending through exon 11, and and the number of endothelial cells that stain for UT-B, until is approximately 30 kb in length (100). Reticulocytes express adult levels are achieved at 21 d of age (22). Thus, the time two mRNA transcripts, 4.4 and 2.0 kb, due to alternative course for the development of urine concentrating ability in polyadenylation signals; both mRNA transcripts encode a sin- rats coincides with the increase in UT-B staining in the de- gle 45-kD protein (100). Although two rat cDNA sequences scending vasa recta. have been reported (UT-B1, UT-B2), they differ by only a few nucleotides at their 3' end (95,96), and it is uncertain whether UT-B1 and UT-B2 truly represent different rat UT-B isoforms, Role of UT-B in Urine-Concentrating Ability a polymorphism, or a sequencing artifact. At present, most Kidd antigen null individuals are unable to concentrate their investigators favor the hypothesis that rat UT-B1 and UT-B2 urine above 800 mOsm/kg H2O, even following overnight are not distinct isoforms because humans have only a single water deprivation and exogenous vasopressin administration isoform, but this hypothesis has not been tested. UT-B1/UT-B2 (112). A UT-B knockout mouse has a similar impairment in mRNA is widely expressed and has been detected in kidney urine-concentrating ability, achieving a maximal urine osmo- and several other organs, including brain, testis, bone marrow, lality of 2400 mOsm/kg H2O compared with 3400 in a wild- spleen, prostate, bladder, thymus, heart, skeletal muscle, lung, type mouse (97). These findings support the hypothesis that liver, colon, small intestine, and pancreas (8,24,94–97,99,104– facilitated urea transport in red blood cells or descending vasa 106). However, Northern analysis has not detected UT-B1/ recta is necessary to preserve the efficiency of countercurrent UT-B2 mRNA in placenta, salivary glands, ovary, leukocytes, exchange (113). UT-B protein and phloretin-inhibitable urea monocytes, or B lymphocytes (24,94,96,104). transport are present in both red blood cells and perfused rat Three studies have addressed the question of whether UT-B descending vasa recta (8,106,109,114–116), suggesting that transports urea only, or water and urea, by injecting UT-B1/ urea transport in red blood cells and descending vasa recta UT-B2 cRNA into Xenopus oocytes. Two studies report that occurs via UT-B protein. UT-B can function as a water channel when expressed in Mathematical models of microcirculatory exchange between oocytes (97,107). However, a third study reports that UT-B is the ascending and descending vasa recta predict that urea specific for urea transport if a physiologic expression level is transporters (UT-B) are necessary to counterbalance the effect achieved in oocytes, but that higher levels of UT-B expression of aquaporin-1 water channels in the descending vasa recta, result in an increase in water permeability (108). i.e., the efficiency of small solute trapping within the renal Antibodies have been made to the N- or C-terminus of UT-B medulla will be decreased in the absence of UT-B, thereby (8,106,109,110) that should detect both UT-B1 and UT-B2 decreasing the efficiency of countercurrent exchange and proteins, if indeed there are two rat isoforms. Thus in this urine-concentrating ability (117,118). Consistent with this hy- review, I will refer to the rat protein(s) detected by the anti- pothesis, urea recycling is impaired in the UT-B knockout UT-B antibodies as UT-B protein. UT-B protein appears on mouse (97). Thus, the production of maximally concentrated Western analysis as a broad band between 45 to 65 kD in urine appears to require UT-B protein expression in red blood human red blood cells and 37 to 51 kD in rat or mouse red cells and/or descending vasa recta (119). 2802 Journal of the American Society of Nephrology J Am Soc Nephrol 13: 2795–2806, 2002

Long-Term Regulation of UT-B in Kidney analysis shows that rat heart expresses 3 UT-A proteins: 56, 51, The long-term regulation of UT-B has not been studied as and 39 kD (7). Uremia increases the abundance of the 56-kD extensively as UT-A. In Brattleboro rats, administering vaso- UT-A glycoprotein (7). The abundance of the 56-kD UT-A pressin or dDAVP for 6 h reduces UT-B mRNA abundance in protein also increases in hypertrophic hearts from non-uremic both the inner and outer medulla (55). However, administering DOCA/salt hypertensive rats, and in short-term hypertension vasopressin or dDAVP for 5 d increases UT-B mRNA abun- induced bya3dinfusion of angiotensin II (7). Rat heart dance in the inner stripe of the outer medulla and the inner expresses only a single 2.7-kb UT-A mRNA transcript (7,31), medullary base, but it decreases it in the inner medullary tip and cDNA sequencing shows that this mRNA is UT-A2b (7). (55). In normal rats, dDAVP administration for 7 d decreases Human heart expresses four UT-A proteins: 97, 56, 51, and UT-B protein abundance in the inner medulla, but furosemide 39 kD (7). The abundance of the 56- and 51-kD UT-A proteins administration also results in a more modest decrease in UT-B increases in terminally failing (NYHA class IV) human hearts protein (110). Varying dietary protein between 10 and 40% had (7). Thus, UT-A proteins are expressed in human and rat heart, no effect on UT-B mRNA abundance in any portion of the and the abundance the 56-kD UT-A protein increases in con- medulla in either Brattleboro or normal rats (57). Inducing ditions such as uremia and hypertension that predispose to left uremia in rats by 5/6 nephrectomy results in a reduction of ventricular hypertrophy. UT-B mRNA and protein after 5 wk (8). Lastly, lithium-fed rats have a marked reduction in UT-B protein abundance in the Testis inner medullary base (64). Seminiferous tubules have phloretin-inhibitable urea trans- port and express four UT-A mRNA transcripts (approximately Effect of Uremia on Urea Transporters in 4.0, 3.3, 2.8, and 1.7 kb), some of which have not been detected in other rodent tissues (31,33,124,126); the 1.7-kb transcript is Nonrenal Tissues UT-A5 (33). UT-B protein and mRNA are also expressed in Liver seminiferous tubules (8,95,96,106,126). Uremic does not The liver performs ureagenesis and has phloretin-inhibitable change UT-B mRNA abundance in rat testis (8). urea transport, suggesting that liver expresses a urea trans- porter, possibly to accelerate urea efflux after ureagenesis Brain (6,120–122). HepG2 cells are a cultured human hepatoblas- Both UT-A and UT-B mRNA and protein are expressed in toma cell line that has a high rate of urea influx that is inhibited brain (8,95,96,105,106,123,124). UT-B mRNA is unchanged by two urea transport inhibitors: phloretin and thionicoti- at 1 wk post-5/6 nephrectomy, but it is reduced to about 30% namide (123). Western blot analysis of HepG2 cells and rat of control levels after 5 wk (8). liver reveals two protein bands: a 49-kD UT-A protein in the plasma membrane and a 36-kD UT-A protein in the cytoplasm Acknowledgments (123). Rat liver expresses a 2.6-kb UT-A mRNA (124). This This work was supported by National Institutes of Health grants size is consistent with either UT-A2b or UT-A4 (Table 1), and R01-DK41707, R01-DK63657, and P01-DK50268. sequencing of the liver UT-A mRNA will be required to identify the isoform. References The abundance of the 49-kD UT-A protein in liver varies 1. Galluci E, Micelli S, Lippe C: Non-electrolyte permeability with uremia and/or acidosis in rats (123,125); it increases in across thin lipid membranes. 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