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
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ABSTRACT |
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urea; transporter protein; colon; cAMP
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).
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METHODS |
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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, 1218 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 4872 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) -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.
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RESULTS |
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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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DISCUSSION |
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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 -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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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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