INTRODUCTION

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UREA SERVES DIFFERENT PURPOSES in different organisms. Urea represents a source of nitrogen for microorganisms like yeasts and bacteria. Urea is present in high concentrations in the tissues of marine elasmobranchs, where it serves as an osmolyte to balance the high salinity of seawater. Excretion of urea provides a good vehicle for eliminating waste products from nitrogen metabolism in mammals. Although urea can diffuse slowly across cell membranes along concentration gradients, in many cases a more efficient and rapid movement of this solute is necessary. This can be achieved by use of specific carriers, resulting in facilitated diffusion or active transport. Urea transporters have now been cloned and characterized in bacteria, yeasts, amphibians, marine organisms, and mammalians (Table 1). The urea transporter genes described in Saccharomyces cerevisiae (DUR3) (10), in Helicobacter pylori (UreI ) (50), and the ABC-type permeases in cyanobacteria (46) do not have homologous counterparts in higher organisms, and only minimal identity (20-25%) exists between the urea transporter genes of Yersinia pseudotuberculosis (Yut), and Brucella melitensis (8) and those in mammals and lower vertebrates. However, substantial homology is emerging in the structure of urea transporters described in mammals, amphibians, and elasmobranch and teleost fishes. Two similar but distinct urea transporters have been identified in rodents and humans: the UT-A urea transporter, encoded by the Slc14A2 gene, and the UT-B urea transporter, encoded by the Slc14A1 gene. The organization of the Slc14A2 gene has now been elucidated in rats (25), humans (2), and mice (12). The genomic structure of the UT-B urea transporter, encoded by the Slc14A1 gene, is also known (23). These advances have made possible the identification of some important regulatory mechanisms involved in the long-term expression of these transporters (3, 25, 26, 31) and will facilitate the study of how urea transporter expression is regulated in other species.

	THE UT-A TRANSPORTER AND Slc14A2 GENE

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Most information on the UT-A transporter has been generated in studies on urea transport in rat kidney, where three major transcripts are detected by Northern hybridization in the medullary region of the kidney: UT-A1 (4.0 kb), UT-A2 (2.9 kb), and UT-A3 (2.1 kb). The mRNA of UT-A4 (2,584 bp) is present in very low abundance, and it is only detectable by PCR. Additional mRNA isoforms are evident by Northern hybridization: UT-A1b (3.5 kb), UT-A2b (2.5 kb), and UT-A3b (3.7 kb), and they differ from the major UT-A transcripts by expressing alternative 3'-untranslated sequences (UTR) (3). All of these transcripts are localized in the medullary region of the kidney. UT-A2 is expressed in the thin descending limb of Henle's loop (tDL), and UT-A1 and UT-A3 are localized in the inner medullary collecting duct (IMCD) (36, 43). There is also evidence that two other UT-A isoforms are expressed in rat testis (11, 19), one comprising 3.3 kb and the other 1.7 kb, the latter probably representing the rat equivalent of mouse UT-A5. On the basis of Western blot analysis, it has been proposed that the UT-A transporter may be present in liver (21) and heart (9); however, the specific UT-A mRNA species expressed in those organs have yet to be characterized.

The UT-A transporter is encoded by the Slc14A2 gene, which was first cloned in rats (25). The rat gene is large, extends for ~300 kb, and encodes all the known rat transcript sequences with 24 exons (Fig. 1). The Slc14A2 gene includes at least two promoters. A promoter in the 5'-flanking region controls transcription of UT-A1, UT-A3, and their 3'-UTR variants, as well as UT-A4. Another promoter in intron 12 controls transcription of UT-A2 and UT-A2b. Exon 13 includes the transcription start of UT-A2 and is transcribed only in UT-A2 in rats and mice. It is likely that additional promoters may be present in the gene, which may regulate transcription of the testis UT-A isoforms, but they have not yet been described. The murine Slc14A2 gene has also been characterized (12) and appears very similar to the rat gene, with two promoters, two large introns separating exons 2, 3, and 4, which encode the 5'-UTR sequence of UT-A1, UT-A3, and UT-A4, respectively, and an additional exon in intron 5 from which the 5'-end of UT-A5 originates (the transcription start site of mUT-A5 has not been described). In humans, only UT-A1 and UT-A2 have been cloned, although Northern blot analysis shows a 2.2-kb mRNA consistent with UT-A3 expression in the kidney (2). The human Slc14A2 gene is smaller than the rat and mouse genes, and most of the 5'-UTR of UT-A1 is encoded by a single initial exon (Fig. 2). On the basis of the cloned cDNA sequence, there is no evidence that the 5'-end of human UT-A2 is encoded by a unique exon as in rodents or that a distinct promoter controls its transcription. However, this possibility cannot be ruled out until a specific transcription start site for human UT-A2 is identified and the structure of its 5'-flanking genomic region is known. The Slc14A2 gene is adjacent to the Slc14A1 gene, which encodes the UT-B transporter, on chromosome 18 in humans and mice (13, 29).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   A: organization of the rat Slc14A2 gene (scale: 10 kb). Vertical lines indicate the position of the exons encoding urea transporter UT-A transcripts (B). PI and PII indicate the 5' and 3' promoters, respectively. PI controls transcription of UT-A1, UT-A1b, UT-A3, UT-A3b, and UT-A4. PII controls transcription of UT-A2 and UT-A2b. B: splicing pattern of the Slc14A2 gene generates different UT-A transcripts, shown as boxed segments of rectangles (scale: 1 kb). A similar filling pattern among transcripts indicates regions of homology. Nos. underneath each segment indicate the first and last exon spliced together to encode a particular segment of each transcript. The UT-A isoforms designated b represent variants with a 3'-untranslated region (UTR) different from that of the parent transcript. The 3'-UTRs of UT-A1b and UT-A2b are provided by alternative use of exon 24, skipping part of exon 23. The UT-A3b isoform has a longer 3'-UTR resulting from extended transcription from exon 12.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Human Slc14A2 gene (A) and human Slc14A1 gene (B; scale: 10 kb). Horizontal brackets indicate the exons encoding individual transcripts. Illustration of the splicing pattern of the Slc14A1 gene for the UT-B transcripts is based on the original description of this gene by Lucien et al. (23).

	THE UT-B TRANSPORTER AND Slc14A1 GENE

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The urea transporter UT-B (HUT11), which is expressed in erythrocytes, was cloned in 1994 from human bone marrow cells (30), encodes a protein that is 71% identical to the UT-A2 protein, and corresponds to the Kidd blood group antigen (29). By Northern hybridization, two mRNA species of 2.5 and 4.7 kb are present in the human kidney (30), and transcripts of similar size (2.0 and 3.8 kb) have been found in mouse brain, spleen, kidney, ureter, and urinary bladder (52) and in rat testis (3.8 kb) (11). In the kidney, UT-B is expressed in descending vasa recta (44, 51). The Kidd/UT-B transporter is encoded by the Slc14A1 gene. The Slc14A1 gene has been characterized in humans (23). It is ~30 kb long, includes 11 exons, and encodes two transcripts, arising from usage of different polyadenylation signals, separated by ~2 kb in exon 11 (Fig. 2). The Slc14A1 gene on chromosome 18, according to current maps of the human genome (http://genome.ucsc.edu), could be separated by <100 kb from the human Slc14A2 gene. The close position of these two genes and the considerable number of highly homologous splice isoforms that they encode suggest a possible origin by duplication of an ancestral gene.

Interestingly, individuals lacking the Kidd erythrocyte antigen show a mild deficit (20%) in the ability to concentrate urine after water deprivation (34). The cause of this deficit in Kidd null subjects was recently linked to mutations in the Slc14A1 gene, resulting in a truncated UT-B protein unable to mediate urea uptake when expressed in Xenopus laevis oocytes (23). A relatively mild defect in urinary concentration has been found in a recently created UT-B knockout mouse (52), which lacks expression of the UT-B protein in vasa recta. UT-B null mice show a selective impairment in the capacity to concentrate urea in the urine and a 35% decrease in maximum urinary concentration after water deprivation. Overall, these data suggest that lack of UT-B expression in the kidney of Kidd null humans and UT-B knockout mice does not result in profound impairment of the urinary concentrating mechanism. It is not known whether in these conditions upregulation of the UT-A transporters may occur, but this possibility needs to be tested. Whether disruption of the UT-A transporter system would have more dramatic consequences is not clear. No specific condition has been identified so far that may be associated with defective function of the UT-A transporter and/or with mutation of the Slc14A2 gene in humans. Although instances of familial hyperazotemia with normal glomerular filtration rate have been described (16), a genetic analysis of those affected by this syndrome is not available. Generation of UT-A knockout/transgenic mice could clarify the role and importance of the UT-A transporter in renal function.

	REGULATION OF UT-A AND UT-B TRANSPORTER EXPRESSION IN THE KIDNEY

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UT-A

Several conditions affecting the level of mRNA and/or protein abundance of UT-A and UT-B in the kidney have been reported in different studies, which are listed in Table 2.

Hydration is a very important physiological determinant of urinary concentration and urea transport, and its effects on urea transporter expression have been examined in several articles. Water restriction is associated with production of highly concentrated urine, increased content of sodium chloride and urea in the renal medullary interstitium, and increased vasopressin levels (reviewed in Ref. 4). Increased interstitial urea content is due to stimulation of the facilitated transport of this solute by the terminal segment of IMCD and urea recycling by influx from the ascending vasa recta into descending vasa recta and into the tDL to prevent escape into the renal venous circulation. It is therefore not surprising that UT-A3 and UT-A2 mRNA levels are increased in the renal medulla during water deprivation (UT-A1 does not change significantly) (3). This pattern is similar to the increased expression of the inner medullary osmolyte sodium-myo-inositol cotransporter (SMIT) and betaine-GABA cotransporter BGT-1 during thirsting, which occurs in response to the increased extracellular tonicity in the medullary interstitium (5, 15). Upregulation of renal tonicity-responsive genes is usually mediated by transcriptional activation via the binding of a transactivating factor [tonicity enhancer binding protein (TonEBP)/ NF-AT5] to a tonicity enhancer DNA sequence (TonE/osmotic response element) (24, 41). Transcription of UT-A1 and UT-A3 is controlled by the 5'-promoter of the UT-A gene (promoter I), which includes a TonE sequence within 400 bp of the transcription start site common to UT-A1 and UT-A3 (26). We showed that the activity of UT-A promoter I is stimulated by hypertonicity, through binding of TonEBP to TonE, similar to the medullary genes involved in osmoregulation (26). Thus during water deprivation, increased extracellular tonicity activates transcription of UT-A1 and UT-A3 (and their 3'-UTR variants) by stimulation of UT-A promoter I. The regulation of UT-A1 in response to hydration is obviously more difficult to study in humans than in rodents, but it is likely to be similar. Within the initial 2-kb 5'-flanking sequence of the human gene are two closely spaced TonE motifs at ~1.3 kb from the beginning of exon 1, suggesting that hypertonicity may also stimulate expression of human UT-A1.

UT-A2 mRNA consistently increases in the inner medulla of dehydrated animals. Transcription of UT-A2 is controlled by UT-A promoter II in intron 12, which, unlike promoter I, has several cAMP response elements (25). The activity of UT-A promoter II is stimulated by cAMP and forskolin but not by hypertonicity (25). Vasopressin induces generation of cAMP by activation of the vasopressin V₂ receptor. During water deprivation, higher levels of vasopressin leading to increased intracellular cAMP could stimulate transcription of UT-A2 by activating UT-A promoter II. The importance of vasopressin for UT-A2 expression has been confirmed by analysis of protein abundance (47) and is underscored by studies in Brattleboro rats, which lack this hormone and in which UT-A2 mRNA is usually undetectable even after thirsting, unless exogenous DDAVP, a V₂-receptor agonist, is given (37). Because the V₂ receptor is not present in the tDL, the exact mechanism linking vasopressin to UT-A2 expression remains obscure and will require further study. Similar responses to hypertonicity and cAMP have been reported for the two promoters of the mouse UT-A gene (12).

During water diuresis in rats, UT-A3 decreases in the inner medulla compared with controls (UT-A1 does not change significantly), whereas UT-A2 is significantly decreased in the outer medulla but does not change in the inner medulla (3). It is likely that dilution of the inner medullary osmotic gradient and absence of vasopressin stimulation may dampen the activity of the two promoters, reducing transcription.

Transcription also seems to play a substantial role in the downregulation exerted by glucocorticoids on urea transport. Previous studies showed that urea fractional excretion increases in adrenalectomized rats treated with dexamethasone for 3 days (22) and that urea permeability of perfused IMCD segments and UT-A1 protein abundance in the inner medullary tip decrease in adrenalectomized rats treated with dexamethasone for 7 days (27). Our laboratory recently demonstrated that rats treated with stress doses of glucocorticoids for 3 days show an ~70% decrease in UT-A1 and UT-A3 mRNA in the renal inner medulla (31). In the same study, we show that this effect correlates with 70% inhibition of UT-A promoter I activity by dexamethasone, whereas the abundance of UT-A2 mRNA and the activity of UT-A promoter II are not affected. This downregulation is not mediated by glucocorticoid response elements, and the specific sequences in promoter I involved in this response have not yet been identified. Thus the decrease in UT-A1 protein induced by glucocorticoids can be explained by reduced transcription of UT-A1. The role of mRNA stability in modulating the abundance of UT-A transcripts in these conditions has not been established.

The relative importance of transcriptional control vs. posttranscriptional mechanisms in regulating UT-A abundance is not known, but the latter may be predominant in certain instances of impaired urinary concentrating ability. An increase in the 117-kDa form of the UT-A1 protein has been reported in rats when their ability to concentrate urine is impaired after furosemide and water diuresis (42). This is somewhat surprising in light of unchanged or decreased UT-A1 mRNA reported in this condition and suggests that increased translation efficiency and/or reduced protein degradation may intervene to sustain the level of UT-A1 protein, in an attempt to maximize urea transport and to preserve the medullary osmotic gradient in the face of diuresis.

UT-B

	REGULATION OF UT-A AND UT-B TRANSPORTER EXPRESSION DURING DEVELOPMENT OF THE KIDNEY AND OTHER ORGANS

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The expression of UT-A and UT-B and of other transporters has been examined in the developing rat kidney (20). UT-A begins to appear in IMCD and tDL after birth, and its intensity increases afterward. UT-B is apparent in descending vasa recta of 20-day-old fetal kidneys, and its intensity also increases after birth. These observations suggest that increased abundance of urea transporters may be an important component for the maturation of urinary concentrating ability by the developing kidney.

UT-A and UT-B transcripts are expressed in testis. However, not much is known about the factors influencing testicular expression of the UT-A and UT-B transporters. UT-A5 mRNA is detected in testis ~15 days postpartum and reaches high expression ~25 days after birth (14). The mechanisms regulating its expression, as well as the promoter sequences controlling transcription of UT-A5, are still unknown.

UT-B has been detected in brain astrocytes (45), and UT-B mRNA abundance has been found to be decreased in the brain of rats with chronic renal failure induced by nephrectomy (17). These animals had high plasma urea levels, and whether urea by itself has a depressive effect on UT-B remains to be determined.

	UREA TRANSPORTERS IN LOWER VERTEBRATES

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Fascinating progress is occurring in our understanding of the physiological role of urea transport in elasmobranch and teleost fishes and amphibians, and elucidation of the molecular structure of urea transporters in lower organisms may greatly contribute to trace the evolution of urea transporter systems.

Table 3 summarizes the degree of protein homology of urea transporters cloned in lower vertebrates and mammalian transporters (the homology of these transporters with the urea transporter proteins in Y. pseudotuberculosis and B. melitensis is only 25-27%, and there is no similarity with those of other microorganisms). Figure 3 shows the hydrophilicity plots of the urea transporters listed in Table 3 and illustrates the remarkable similarity in the predicted structural configuration of these transporters in different species. All transporters include two conserved amino acid sequences, WDLPVFTLPFN and PVGXGQVXGCDNPW (X indicates conservative substitutions), which are found in similar location within the peptide of different transporters (Fig. 4), including UT-A3, UT-A4, UT-A5, and UT-A1. However, only UT-B shows an ALE domain (residues 205-207 in rat UT-B, residues 219-221 in human UT-B), which may be considered a signature sequence for the UT-B transporter. On the basis of structural analysis, it seems that nonmammalian transporters show a slightly higher homology with UT-A, more specifically with UT-A2, than with UT-B, and a phylogenetic relationship between the elasmobranch urea transporter ShUT and UT-A2 has been previously suggested by Smith and Wright (40). Similar considerations apply to the urea transporter of another elasmobranch, the Atlantic stingray, leading to the hypothesis that the urea transporters in elasmobranch and teleost fish and the mammalian UT-A2 transporters may all derive from a common ancestral form and that, among the mammalian urea transporters, UT-A2 may be the most representative of the common ancestral form (18).

View larger version (36K): [in this window] [in a new window]   Fig. 3.   Hydrophilicity plots of urea transporter peptides in lower vertebrates and mammalians (Kyte-Doolittle). The protein sequences, predicted from the cDNAs listed in Table 1, are aligned according to their respective size, indicated as no. of amino acids (top).

View larger version (51K): [in this window] [in a new window]   Fig. 4.   Putative peptide sequences of lower vertebrates and mammalian transporters aligned to show amino acid motifs (boxed areas) WDLPVFTLPFN and PVGV/IGQV/IYGCDNPW, which are conserved among different species, and the ALE motif, which appears to be present only in the UT-B transporters. Nos. on the left, no. of amino acids in each sequence.

The cDNAs, but not the genes encoding the transporters listed in Table 3, have been cloned. However, high-stringency Northern blot analysis suggests that additional transcripts may exist for some of the urea transporters described. In the bladder of Rana esculenta, 1.6- and 4.3-kb mRNA species have been detected (6). Two mRNA species, 10 and 2.2 kb, have been reported in the kidney and brain of Squalus achantias (40); two mRNA species of 1.8 and 3.5 kb are described in the gills of the gulf toadfish (49). In addition to whUT-A2 (2.7 kb, 91% identical to hUT-A2), a 4.0-kb transcript is expressed in the kidney of the short-finned whale, possibly representing the cetacean counterpart of UT-A1 (18). UT-A1 is the largest urea transporter cloned so far, although there is evidence that a UT-B large transcript may exist, and it will be very interesting to see whether any of the elasmobranch and teleost large transcripts shows structural analogies with UT-A1.

Not much is known about the regulation of urea transport and transporter expression in lower vertebrates in different physiological states and in response to environmental changes. In the gills of the gulf toadfish, urea excretion occurs through pulses that last from 0.5 to 3 h. This process apparently does not require variation in the abundance of tUT mRNA in the gills, suggesting that the main regulatory events are not acting at the mRNA level (49). Some elasmobranch species can survive and reproduce in waters of different salinities and adapt to these environmental changes by modifying renal function and urea reabsorption (18). It is possible that these changes in habitat may require a relatively long-term modulation of the abundance of urea transporter, but it is hard to predict how similar the mechanisms regulating these responses may be to those operating in the mammalian kidney. This may become clearer once the urea transporter genes in marine organisms are cloned and characterized.