Characterization of a human colonic cDNA encoding a structurally novel urea transporter, hUT-A6

Craig P. Smith,1 Elizabeth A. Potter,1 Robert A. Fenton,2 and Gavin S. Stewart1

1School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom; and 2Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1603

Submitted 27 August 2003 ; accepted in final form 5 June 2004


    ABSTRACT
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
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Two closely related genes, UT-A (Slc14a2) and UT-B (Slc14a1), encode specialized transporter proteins that modulate the movement of urea across cell membranes. In this article, we report the characterization of a cDNA isolated from human colonic mucosa encoding a novel UT-A urea transporter, hUT-A6. The encoded protein is 235 amino acids (aa) in length, making it the smallest UT-A member characterized. On the basis of previous structural predictions, hUT-A6 is structurally unique in that it consists of a single hydrophobic core flanked by hydrophilic NH2- and COOH-terminal domains. The transcript encoding hUT-A6 contains a novel 129-bp exon, exon 5a, which, as a result of alternative splicing, introduces a unique 19-aa segment and a stop codon. Functionally, the protein transports urea, and this activity is inhibited by phloretin. Interestingly, despite the lack of a protein kinase A (PKA) consensus site {[RK](2)-X-[ST]}, transport of urea by hUT-A6 is stimulated by PKA agonists. Deletion of the two PKA consensus sites from murine UT-A3 (mUT-A3) did not affect the stimulatory response of PKA agonists, which, together with the lack of PKA consensus sites in hUT-A6, indicates that regulation of hUT-A6 and mUT-A3 is not mediated through a classic PKA phosphorylation consensus.

urea; transporter protein; colon; cAMP


UREA TRANSPORTERS DERIVED from the UT-A (Slc14a2) and UT-B (Slc14a1) genes belong to a family of specialized membrane proteins that regulate urea movement across plasma membranes. Members of both subfamilies play integral roles in the urinary concentrating mechanism (5, 18, 23). The UT-B gene encodes one isoform, whereas in comparison, five UT-A isoforms have been characterized to date (1, 6, 15, 16).

The major isoforms expressed at a protein level in the kidney are UT-A1, UT-A2, and UT-A3. In mouse and rat kidney, UT-A1 and UT-A3 are localized to the inner medullary collecting ducts, and the transport activities of these proteins are acutely sensitive to cAMP (2, 8, 13, 21, 28, 30). These proteins are integral to the urinary concentrating mechanism because animals lacking these transporters have a concentrating defect (5). UT-A2 is expressed in descending thin limbs of the loop of Henle and is insensitive to acute stimulation by cAMP (8, 20, 30). Together, these proteins function to establish and maintain the hypertonic medullary compartment that is central to the urinary concentrating mechanism.

Another renal isoform, UT-A4, has been detected in the kidney by PCR, but a protein representing this isoform has yet to be characterized; therefore, its precise role is still unclear (13). In contrast, UT-A5 is expressed in testis and has been suggested to play a role in spermatogenesis (7).

Urea is a major source of nitrogen for the commensal bacteria that inhabit the colon. It passes from the circulation of the host into the digestive tract, possibly by carrier-mediated transport, and is broken down by the resident bacterial enzyme urease into ammonia and carbon dioxide. The ammonia is then used to synthesize amino acids (aa) and nucleotides (nt) required for bacterial growth (10). Previously, investigators at our laboratory (25, 26) as well as other researchers (12, 29) reported the presence of UT-A and UT-B mRNA and proteins in the monogastric colon. In this report, we describe a novel UT-A cDNA isolated from human colonic mucosa. The encoded protein is the smallest functional UT-A described to date and has a unique structure that challenges current structural tenets of the facilitated urea transporter superfamily (19).


    METHODS
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 METHODS
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 DISCUSSION
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Clone isolation. A 1,789-bp cDNA was isolated from human colonic mucosa and subcloned into pME18S-FL3 at the DraIII restriction site (work performed by the New Energy and Industrial Technology Development Organization's full-length human cDNA sequencing project, Institute of Medical Science, University of Tokyo, Tokyo, Japan). This clone (GenBank accession no. AK074236) was provided to us, and confirmatory sequencing was performed (Lark Technologies, Essex, UK). Protein analysis was performed using the ScanProsite tool on the Expert Protein Analysis System web site of the Swiss Institute of Bioinformatics (http://ca.expasy.org/).

Xenopus oocyte expression experiments. Xenopus oocyte expression experiments were performed as previously described (22). Briefly, the clone was digested using EcoRI and NotI, blunt ended using PfuTurbo DNA polymerase (Stratagene), and ligated into an EcoRV-digested, dephosphorylated pT7TS transcription vector. The vector was linearized using Xba1, and complementary RNA (cRNA) was prepared using the T7 mMessage mMachine (Ambion). Defolliculated oocytes were injected with 0.3 ng of cRNA or deionized H2O and incubated for 3 days at 18°C. [14C]urea uptake was measured in the presence or absence of 0.5 mM phloretin. Treatment with phloretin consisted of 15-min preincubation followed by 90-s [14C]urea uptake with phloretin added to the uptake solution. The effect of protein kinase A (PKA) agonists was tested by preincubation of oocytes for 10 or 60 min in 500 µM 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), 500 µM 3-isobutyl-1-methylxanthine (IBMX), and 50 µM forskolin.

Site-directed mutagenesis. Mutational deletion of PKA consensus sites [RK](2)-X-[ST] from murine UT-A3 (mUT-A3) [GenBank accession no. AF258602 (7)] was performed using the QuickChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. The following mutagenic primers (MWG Biotech) were obtained in each case to substitute the terminal serine in the consensus sequence for alanine. For S85A substitution, the sense primer 5'-AATAAAAGGAGAGAAGCTGAGGTGTCCCGCCGG-3' and the antisense primer 5'-CCGGCGGGACACCTCAGCTTCTCTCCTTTTATT-3' were used. For S92A substitution, the sense primer 5'-GTGTCCCGCCGGGCTGCTGCGGGCCGGGGAGGC-3' and the antisense primer 5'-GCCCCTCCGGCCCGCAGCAGCCCGGCGGGACAC-3' were used. Mutations were incorporated via PCR [cycling parameters: 95°C for 30 s, 12–18 cycles: 95°C for 30 s, 55°C for 1 min, 68°C for 2 min per kb plasmid length] and used to transform TOP 10 One Shot Escherichia coli (Invitrogen). Colonies were screened by analytical digest, visualized by agarose gel electrophoresis, and then sequenced. Cassettes containing the mutations were then subcloned into appropriately digested wild-type mUT-A3.

Northern blot analysis. A commercially available human mRNA blot (no. 7782-1; BD Biosciences) containing esophagus, stomach, duodenum, ileocecum, ileum, jejunum, cecum, colon, rectum, and liver (~1 µg/lane) was probed at medium stringency using a 32P-labeled, full-length human urea transporter A6 (hUT-A6) cDNA probe as described previously (7). Hybridization was performed for 16 h at 42°C (50% formamide), and washing was conducted at 48°C in 0.1x SSC-0.1% SDS for 60 min. Autoradiography was performed at –80°C for 48–72 h.

RT-PCR of colon, testis, and kidney mRNA. Total RNA (1 µg) from human colon and testis (BD Biosciences) was reverse transcribed using oligo(dT) and Superscript II reverse transcriptase (Invitrogen) according to the manufacturer's recommended protocol. Human kidney inner medullary, outer medullary, and cortical cDNA were a kind gift from Dr. Boye L. Jensen (Dept. of Physiology and Pharmacology, University of Southern Denmark, Odense, Denmark). PCR amplification was performed using 0.2 µl of cDNA, 10 pmol forward and reverse primers, and HotStarTaq (Qiagen). The primers used were 1) UT-A6 specific (primers situated in exon 5a), designed to amplify a 128-bp UT-A6 product (5'-GATGGAGACGGATTTTAACTGGAGTA and 5'-GCATGTTCATGGATATCACTCTAATCT); 2) UT-A1/3/A6 specific (forward primer situated in exon 5, reverse primer located in exon 10), designed to amplify a 682-bp UT-A1/3 product or a 712-bp UT-A6 product (5'-GTGGCTTCTGTTTCCTGTGACCTT and 5'-CCTCGGGGTAGGTGACTTTGCTGAGT); and 3) {beta}-actin specific (5'-GTGCTGTCTGGCGGCACCACCAT and 5'-CCTGTAACAACGCATCTCATAT). After an initial denaturation step at 95°C for 15 min, PCR cycling conditions were 35 cycles of denaturation at 94°C for 30 s, annealing at 63°C for 30 s, extension at 72°C for 60 s, and a final extension at 72°C for 8 min. All PCR products were sequenced for verification.

UT-A gene assembly. The BLAT alignment tool (14) (available at: http://genome.ucsc.edu/cgi-bin/hgBlat) and the finished human genome assembly (freeze date July 2003) were used to determine the exon-intron boundaries of the hUT-A6 gene. The sequence assembly at chromosome 18 between nt 43065681 and nt 43168141 was used for further analysis.

Statistics. Quantitative data were compared using one-way ANOVA. The Student-Newman-Keuls post hoc test was used to detect significant differences between groups. Values are means ± SE, and statistical significance was assumed at the 5% level.


    RESULTS
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Characterization of cDNA AK074236. Analysis of the 1,789-bp cDNA nt sequence revealed a putative open reading frame from nt 101 to nt 806 preceded by an in-frame stop codon at bp 61 and a polyadenylation signal (AGUAAA) 21 bp 5' to a poly(A) tail. The longest complete coding sequence results in a 235-aa protein, and in accordance with the nomenclature proposed by Sands et al. (19), we have named the protein hUT-A6 (human urea transporter A6). The 5'-UTR of hUT-A6 consists of 100 bp; however, it is possible that the initial exon is truncated at the 5'-end and in reality is the same as the one in hUT-A1 (see below). The 3'-UTR contains 921 bp followed by the poly(A) tail.

hUT-A6 is the smallest member of the UT-A family yet characterized. It shares 99% identity with hUT-A1 (GenBank accession no. AAL08485) from aa 1 to aa 216 (Fig. 1). There is a single substitution at aa 195, which in hUT-A1 is alanine, whereas in hUT-A6 this residue is valine. Following aa 216 is an anomalous region, unique among the UT-A family, consisting of 19 hydrophilic aa. This region is the result of a novel, 127-bp, alternatively spliced exon that encodes both the 19 residues and a stop codon. Also contained within this exon is a single putative N-glycosylation site (N-I/N-T-W/G) at NH2-terminal 223. The protein encoded by hUT-A6 contains three potential PKC ([ST]-X-[RK]) sites at S15, S71, and S197 and no putative PKA {[RK](2)-X-[ST]} sites (Fig. 2). The Kyte-Doolittle plot of hUT-A6 suggests that this protein shares essentially the same structural configuration as the NH2-terminal 216 aa of hUT-A1 (1), with a hydrophobic core flanked by hydrophilic NH2 and COOH termini.



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Fig. 1. A: human UT-A1 (hUT-A1) amino acid (aa) sequence (GenBank accession no. AAL08485) and hUT-A6 aa sequence (GenBank accession no. AK074236). Asterisks represent conserved aa. Period represents stop codon. B: Kyte-Doolittle mean hydrophobicity profile of hUT-A6 using a window setting of 9. The NH2 and COOH termini are composed of hydrophilic residues.

 


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Fig. 2. Scaled schematic representation of a hUT-A1 protein. Solid rectangles represent hydrophobic domains. Asterisks denote protein kinase C (PKC) consensus sites [ST]-X-[RK]. Squares denote protein kinase A consensus sites [RK](2)-X-[ST]. N-glycosylation sites are represented as diamonds. Numbers correspond to UT-A1 aa. Solid lines show aa composition of hUT-A2 and hUT-A6 relative to UT-A1. hUT-A6 truncates in the predicted extracellular loop. The shaded line denotes 19 aa encoded in exon 5a.

 
Genomic structure of hUT-A gene. The human Slc14a2 gene covers a total of 68,293 bp, from position 43082758 to 43151050 on the direct strand of chromosome 18 and encodes three known transcripts: hUT-A1 (1), hUT-A2 (17), and hUT-A6. The genomic organization of the gene was previously described in detail (1); however, we have incorporated the novel exon into the gene structure and present the revised version in Fig. 3.



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Fig. 3. Schematic representation of organization and splicing of the human urea transporter UT-A gene (Slc14a2) revised to incorporate novel exon 5a, unique to hUT-A6. The gene covers a total of 68,293 bp, from position 43082758 to position 43151050 on chromosome 18 (top). The 22 exons of the UT-A gene are shown; exon width is representative of actual size, and intronic distance is scaled. The cassette exon (exon 5a) of hUT-A6 is shown as a hatched box, with the unique sequence and flanking intronic region highlighted. Lines indicate the splicing patterns for UT-A1, UT-A2, and UT-A6.

 
Analysis of the genomic structure of the gene reveals that hUT-A1 and hUT-A6 (two different transcripts encoding two different protein isoforms) result from the presence or absence of a cassette exon (exon 5a) and the use of alternative splicing sites within common exons. The 1,729-bp hUT-A6 cDNA covers 28,930 bp and contains 11 UT-A exons. All exon-intron boundaries of hUT-A6 conform to the consensus (GT-AG) rule (Table 1). The initial intron of the hUT-A6 pre-mRNA is 9,084 bp in length (compared with 9,048 bp in hUT-A1) and results from the use of an alternative splice donor site within the hUT-A gene. hUT-A6 also contains a unique exon, termed exon 5a, that is the result of alternative splice donor sites within intron 5 (2,343 bp). Furthermore, a cryptic splice site (TTGCAG) in exon 6 results in a truncation of this exon in hUT-A6 compared with hUT-A1 (95 rather than 193 bp). Thereafter, both hUT-A6 and hUT-A1 share common exons and splice sites.


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Table 1. Summary of hUT-A6 gene structure

 
Xenopus oocyte expression. Oocytes injected with hUT-A6 cRNA showed a fourfold increase in urea uptake (Fig. 4A). Inclusion of 0.5 mM phloretin in the uptake solution did not significantly affect urea uptake in H2O-injected oocytes. However, consistent with previously characterized UT-A transporters (7, 13, 21, 22), phloretin reduced urea influx into oocytes injected with hUT-A6 cRNA to the level of H2O-injected control oocytes. Exposure of oocytes expressing either mUT-A3 (positive control) or hUT-A6 to 500 µM 8-BrcAMP, 500 µM IBMX, and 50 µM forskolin resulted in a significant increase in urea transport compared with non-cAMP-treated controls. (Fig. 4, B and C). These findings demonstrate that urea transport by hUT-A6 is sensitive to cAMP. In contrast to hUT-A6, mUT-A3 contains two putative PKA consensus sites. To test whether mUT-A3 is regulated via these sites and therefore is different from hUT-A6, S85 or S92 was mutated to A, alone or together. Despite the absence of S85, S92, or S85 and S92, all mutant proteins responded to stimulation by the PKA agonist 8-BrcAMP (Fig. 4, B and C), indicating that these sites are not required in UT-A proteins for PKA agonist sensitivity.



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Fig. 4. Functional characteristics of hUT-A6 expressed in oocytes. A: [14C]urea accumulated in 90-s period by H2O-injected control oocytes or oocytes expressing hUT-A6. Solid bars represent oocytes preincubated (15 min) with 500 µM phloretin. Phloretin reduced urea uptake in hUT-A6-expressing oocytes to that of H2O-injected control oocytes. *Significant (P < 0.05) difference in urea uptake compared with the H2O-injected control group. +Phloretin inhibition caused a difference (P < 0.05) in [14C]urea uptake compared with the paired control group. B: effect of PKA agonists on [14C]urea uptake by oocytes expressing human UT-A6, S85A, or S92A PKA consensus sequence mutants of murine UT-A3 (mUT-A3). Open bars represent urea uptake under control conditions. Solid bars show urea uptake in the presence of 500 µM 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), 500 µM 3-isobutyl-1-methylxanthine, and 50 µM forskolin. C: effect of deletion of both PKA sites S85 and S92 from mUT-A3. Treatment of mUT-A3 mutant at S82A and S92A had no effect on PKA agonist stimulation of urea uptake. All values are means ± SE; n = 8. **Significant (P < 0.01) difference in urea uptake of cAMP-stimulated groups compared with the unstimulated controls. ***Significant (P < 0.001) difference in urea uptake of cAMP-stimulated groups compared with the unstimulated controls. All three graphs represent separate experiments with oocytes from different toads; therefore, only qualitative comparisons are possible between experiments.

 
Northern blot analysis and tissue distribution. Medium-stringency Northern blot analysis of poly(A)+ mRNA from human gastrointestine, using a full-length hUT-A6 probe, resulted in strong signals of 2.2 and 5.6 kb in descending and ascending colon. Weaker signals of the same size were present in cecum, jejunum, ileum, and ileocecum (Fig. 5). After prolonged exposure, signals were evident in duodenum and stomach (not shown); however, no detectable UT-A mRNA species were observed in esophagus, liver, rectum, or transverse colon.



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Fig. 5. Northern blot analysis of human gastrointestinal tract poly(A)+ mRNA (1 µg/lane) probed with full-length 32P-labeled hUT-A6 cDNA at medium stringency. Strong hybridization to mRNA species is evident at 2.2 and 5.6 kb in descending and ascending colon. Weaker signals of the same size were present in cecum, jejunum, ileum, and ileocecum.

 
RT-PCR of colon, testis, and kidney mRNA. To determine whether UT-A6 mRNA was present in kidney, we performed RT-PCR analysis using primers designed to differentiate UT-A1/3 from UT-A6 (Fig. 6). A primer set specific for exon 5a containing cDNA produced a 128-bp product in human colon only. Primer set B was designed to amplify a 712-bp product if UT-A6 was present or a 682-bp product if UT-A1/3 was present. In human colon, only a 712-bp band was detected, corresponding to the UT-A6 amplicon. In kidney inner medulla, the only amplification product detected was 682 bp, corresponding to UT-A1/3 (unfortunately, we were unable to distinguish UT-A1 from UT-A3 using this method). Therefore, from this analysis, we conclude that UT-A6 mRNA is present in colon, but not in kidney, and that UT-A1/3 mRNA is present in inner medulla, but not in the remainder of the kidney.



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Fig. 6. RT-PCR determination of UT-A3, UT-A6, and {beta}-actin mRNA expression in human tissues. A: isoform-specific primers detect expression of UT-A6 in colon alone. No product is observed in absence of reverse transcriptase (RT). B: isoform-selective primers detect UT-A6 expression alone in colon and only UT-A1/3 expression in kidney inner medulla. C: {beta}-actin is expressed in all tissues tested. IM, inner medulla; OM, outer medulla; CTX, kidney cortex.

 

    DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In this study, we have characterized a cDNA isolated from human colonic mucosa encoding a member of the UT-A urea transporter family. Although on first acquaintance the encoded protein resembled UT-A3, further examination revealed that the cDNA encoded a novel isoform, hUT-A6. In terms of genomic organization, structure, and mode of regulation, as well as from a physiological standpoint, hUT-A6 has important implications in the urea transporter field.

At 235 aa, hUT-A6 is the smallest member of the UT-A subfamily to be characterized. It is composed of the NH2-terminal 216 aa of hUT-A1; therefore, it represents the first of the four hydrophobic domains that are contained within hUT-A1 (see Fig. 2). According to the model proposed by Sands et al. (19), hUT-A6 completely spans the membrane three times and has a fold (putative {beta}-turn) that partially spans the membrane and terminates within the hydrophilic extracellular loop. It is therefore likely that hUT-A6 has an NH2 terminus that projects into the cytoplasm and a COOH terminus that extends into the extracellular space. In favor of this suggestion is the presence of the consensus for an N-glycosylation site in the 19 COOH-terminal hydrophilic aa encoded by the novel exon 5a.

Using the Xenopus oocyte expression system, we have shown that hUT-A6 functions as a urea transporter and, like all other characterized UT-A family members, is inhibited by phloretin (1, 8, 24, 32, 33). Even more intriguing is the discovery that, despite its not having distinct PKA consensi, hUT-A6 is acutely activated by cAMP. If activation of UT-A6 results from direct phosphorylation of the protein, as Zhang et al. (34) suggested for UT-A1, then the site of phosphorylation must lie within the primary sequence of hUT-A6. However, the absence of a distinct PKA site suggests that cAMP is acting either via a cryptic PKA site or perhaps indirectly via a kinase other than PKA (4). In contrast to hUT-A6, both rat and mouse UT-A3 have two potential PKA sites in the NH2 hydrophobic domain and are also sensitive to cAMP (8, 13, 21). The increase in urea permeability after cAMP stimulation suggested that in UT-A3, one of these sites may be responsible for mediating the cAMP response and that hUT-A6 may have a different mode of regulation. To test this hypothesis, we mutated the two sites (S to A) in mUT-A3 either individually or together and tested the sensitivity of the mutants to cAMP. Regardless of whether both sites were deleted or a single site was mutated, murine UT-A3 remained sensitive to cAMP stimulation. Therefore, we conclude that cAMP does not modulate hUT-A6 or mUT-A3 by individual PKA consensus sites with the classic motif [RK](2)-X-[ST].

Comparative genomic analysis of the revised gene structure incorporating exon 5a revealed that this exon is unique to the hUT-A gene and is not evident in other species from which cDNA have been isolated (6, 16). Interestingly, taxonomy database searches (Protein families database of alignments and hidden Markov models, Wellcome Trust Sanger Institute, Hinxton, UK; available at: http://www.sanger.ac.uk/Software/Pfam/) revealed that ancestors of hUT-A6 were limited to the vertebrata whereas ancestors of UT-A1 are present in the bacteria and throughout eukaryota, indicating that hUT-A6 is a recent addition to the UT-A family. Because UT-A mRNA and protein have been detected in species in which UT-A6 is not present, the prospective role of hUT-A6 may be fulfilled by other isoforms.

Using Northern blot analysis, we determined the distribution of UT-A mRNA in the human gastrointestinal system. Strong signals were detected for 2.2- and 5.6-kb transcripts in descending and ascending colon, and weaker signals were observed in cecum, jejunum, ileum, and ileocecum. If all of the initial exon of the UT-A gene (exon 1) were incorporated into hUT-A6, the predicted size of the full-length transcript would be ~2.2 kb. We therefore suggest that the cDNA AK074236 [GenBank] represents a truncated hUT-A6 transcript and that the 2.2-kb signal we detected is the full-length hUT-A6 mRNA. Interestingly, Bagnasco et al. (1) reported transcripts of 2.2, 4.4, and 6.5 kb in human kidney inner medulla, suggesting that hUT-A6 may also be present in kidney. Alternatively, because a cDNA encoding the human ortholog of UT-A3 has not been isolated, we could not rule out that the observed 2.2-kb transcript in kidney might encode the human homolog of UT-A3. Analysis of mRNA from colon, testis, and kidney cortex and outer and inner medullas clearly showed that UT-A6 was present in colon but was not detectable in testis or kidney. Furthermore, UT-A1/3 was detected only in kidney inner medulla and not in colon or testis. It is therefore likely that the 2.2-kb signal revealed by Northern blot analysis in kidney medulla by Bagnasco et al. (1) represents UT-A3 and not UT-A6. We did not detect the 4.4-kb transcript corresponding to hUT-A1 in any of the tissues we tested, indicating that hUT-A1 is unlikely to be expressed in the gastrointestinal tract. The molecular identity of the larger 5.6-kb transcript is currently not known, but it is likely to represent another as yet uncharacterized UT-A isoform.

Evidence for the presence of colonic facilitative urea transporters has been suggested in several species, including rabbits (33), rats (3), humans (12), and mice (26), but why are urea transporters expressed in the colon? For many years, it has been known that in ruminant animals (e.g., cattle, sheep) that consume a diet low in protein, urea is a valuable reservoir of nitrogen that can be salvaged from the gastrointestinal tract (11). This process of urea salvaging can be summarized as follows. Urea passes from the circulation of the mammalian host into the rumen, where it is broken down by the bacterial enzyme urease into ammonia and carbon dioxide. Ammonia is then used by the bacteria to synthesize amino acids required for growth. Finally, the mammalian host can reabsorb some of these amino acids, or indeed the ammonia, and hence salvage the nitrogen from the urea molecule (10).

Because monogastric animals do not possess a rumen, it was originally thought that they were unable to use this process. However, significant urea hydrolysis by the intestinal bacteria in humans was observed as early as the 1950s (31). Furthermore, supplementary evidence has emerged suggesting that monogastrics can indeed salvage urea nitrogen under certain circumstances, such as during pregnancy (9) or at times of low dietary protein intake (27). The predominant tissue involved in these processes is the colon. The discovery of a functional facilitative urea transporter in human colon argues that, indeed, urea passes into the colon in a regulated manner and that this may represent a key aspect of the commensal interaction between gut microflora and their mammalian hosts.

In conclusion, we have characterized a cDNA isolated from human colonic mucosa encoding a novel UT-A urea transporter isoform, and despite its being the smallest member of the UT-A family, hUT-A6 functions as a facilitative urea transporter whose activity is sensitive to both phloretin and cAMP.


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This work was supported by grants from the Biotechnology and Biological Sciences Research Council and the Royal Society (to C. P. Smith).


    ACKNOWLEDGMENTS
 
We thank James Holroyd for helpful comments.


    FOOTNOTES
 

Address for reprint requests and other correspondence: C. P. Smith, School of Biological Sciences, Univ. of Manchester, G.38, Stopford Bldg., Oxford Road, Manchester M13 9PT, United Kingdom (E-mail: cpsmith{at}man.ac.uk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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