Cloning and characterization of the human urea transporter UT-A1 and mapping of the human Slc14a2 gene

Serena M. Bagnasco1, Tao Peng1, Michael G. Janech2, Alexander Karakashian1, and Jeff M. Sands3

1 Department of Pathology, 3 Division of Nephrology, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322; and 2 Marine Biomedicine and Environmental Sciences, Medical University of South Carolina, Charleston, South Carolina 29425


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have isolated and characterized the human homolog of the rat largest urea transporter of the UT-A family (hUT-A1). The 4.2-kb hUT-A1 cDNA encodes a 920-amino acid peptide, which is 89% identical to the rat UT-A1 protein. By Northern hybridization, hUT-A1 expression is detected in the human inner medulla as a ~4.4-kb mRNA transcript. By Western analysis, hUT-A1 is identified as a ~100-kDa protein in the human inner medulla. By immunohistochemistry, hUT-A1 expression is localized to the inner medullary collecting duct (IMCD). When transfected into HEK-293 cells hUT-A1 cDNA is translated into a ~98-kDa protein. Expression of hUT-A1 in Xenopus oocytes results in phloretin-inhibitable uptake of 14C-urea, which shows only modest stimulation by cAMP, suggesting that in the human IMCD vasopressin may have a limited role in the short-term regulation of hUT-A1-mediated urea transport. We determined the organization of the human Slc14a2 gene and identified 20 exons distributed over ~67.5 kb on chromosome 18, from which hUT-A1 and the other human urea transporter, hUT-A2, are transcribed.

urea transport; urine transporter; vasopressin; kidney


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

FACILITATED TRANSPORT OF UREA in the renal inner medulla is important to maintain a sufficiently high level of urea in the medullary interstitium. Urea and NaCl are the major solutes involved in generating an osmotic gradient along the cortico-medullary axis in the mammalian kidney, which is crucial for the urine concentration process.

Two major types of mammalian urea transporters are known: the renal tubular urea transporter (UT-A), and the erythrocyte urea transporter (UT-B). Four UT-A isoforms have been identified in the rat kidney, rUT-A1 (10), rUT-A2 (11), rUT-A3 (2), and rUT-A4 (2). The activity of rat UT-A1, UT-A3, and UT-A4 expressed in vitro appears to be sensitive to cAMP stimulation, suggesting that the function of these transporters may be regulated by vasopressin in vivo. UT-A1 and UT-A3 are expressed in the inner medullary collecting ducts (IMCD) (9, 12), UT-A2 is expressed in the thin descending limb of Henle's loop (TDL) (9), and the renal tubular localization of UT-A4 is not known. The Slc14a2 gene encoding the four rUT-A renal transporters has been recently cloned and characterized in the rat (4). Only UT-A2 has been identified in the human kidney (7). We report the cloning and characterization of the human urea transporter hUT-A1 and describe the organization of the human Slc14a2 gene, from which hUT-A1 and hUT-A2 are transcribed.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

RT-PCR. Normal human kidney tissue was obtained from surgical nephrectomy specimens or kidneys that could not be used for transplantation. No human tissue was obtained specifically for its use in this study. Portions of kidney cortex and medulla were dissected and frozen until use. Total RNA was extracted with TRI pure isolation reagent (Boehringer-Mannheim, Indianapolis, IN). RT-PCR from 1 µg of total RNA and amplification of the first strand cDNA were performed with the Titan RT-PCR System (Boehringer-Mannheim) after the protocol suggested by the manufacturer. A 1.2-kb amplification product was initially obtained using sense primer 5'-GTTTGTCAACAACCCCCTTAGTGG-3' and antisense primer 5'-CAAAGATGGAAATAGAGTCTCG-3', which spans the region between nucleotides 790-2005 of rat UT-A1 cDNA. The amplicon was subcloned into the pGEMT vector (Promega, Madison, WI) and sequenced to verify homology with the rat UT-A1 cDNA.

RACE. The 5'- and 3'- ends of the human UT-A1 transcript were identified by rapid amplification of cDNA ends (RACE), with the reagents provided in the 5'/3'-RACE kit (Boehringer-Mannheim), as described previously (2). For 5'-RACE, first strand cDNA synthesis was performed with antisense primer 5'-AAGCCACCAGTAGTAGTCTAACTTCTCC-3', followed by PCR amplification with antisense primer 5'-AGAGCTGTTAAGGTCGAGACCACTG-3'. The PCR product was gel purified and sequenced. 3'-RACE was performed by using nested sense primers 5'-TGGAAGTTCCATCGATAGAAGAGTGG-3', 5'-GTTCTACGTCATCACCTGGCAGAC-3', and 5'-GAGAAACAGAAGGGCATCAATCATAAC-3'. The PCR products were gel purified and sequenced with an automatic DNA sequencing system (ABI PRISM Genetic Analyzer, PerkinElmer, Foster City, CA) to verify overlapping of the RACE products and the RT-PCR segment. Analysis of DNA sequences was performed by using the Wisconsin Sequence Analysis Package and Lasergene softwares. To determine the exon-intron organization of the Slc14a2 gene, the human genome databases of National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) and Celera (Celera Genomics Group, Rockville, MD) were searched to find matches with the sequence of our hUT-A1 clone (GenBank accession no. AF349446) and with the sequence of a previously identified human UT-A2 cDNA (GenBank accession no. X96969).

Primer extension. Primer extension was performed as previously described (1), using the primer 5'-GTGGCACACACTTGTAGTCCTA-3' from the first 150 bp of the 5'-urine transporter (UTR) of hUT-A1 cDNA.

Northern hybridization. Total RNA (10 µg) from human renal cortex and medulla was separated on a 1.3% agarose-2.2 M formaldehyde gel, immobilized onto a nylon membrane (Hybond-N+, Amersham, Arlington Heights, IL), and hybridized with 32P-labeled DNA probe spanning 454 bp (nt 980-1434) of hUT-A1 cDNA, under high-stringency conditions, as described previously (2).

Transcription and translation of hUT-A1 cDNA in cultured cells. The coding region of the hUT-A1 cDNA was amplified by RT-PCR by using primers 5'-TGGAAGTTCCATCGATAGAAGAGTGG-3' and 5'-GGAGAGAGTCACAGAACAGACCAAAG-3' and subcloned into the ApaI/NotI-digested pcDNA3 vector (Invitrogen, Carlsbad, CA). HEK-293 cells were used for analysis of hUT-A1 protein expression after transient transfection, because these cells do not express any known urea transporter. Transfection was performed by using the FuGene6 reagent (Boehringer-Mannheim) with 50% confluent cells growing in 10-cm culture dishes (Corning). The FuGene6 reagent was combined with 1.0-5.0 µg of pcDNA3 DNA and added to the plate containing the cells. Transfection was performed in normal isotonic growth medium (minimum essential medium with L-glutamine and 5% FCS). Cells were harvested 48 h after transfection.

Functional analysis in oocytes. Functional activity of the hUT-A1 transporter was determined by measuring urea uptake in Xenopus laevis oocytes. After linearization of the hUT-A1 pcDNA3 construct, cRNA was synthesized by using T7 polymerase provided in the mMessage mMachine (Ambion, Austin, TX), precipitated with lithium chloride, and injected into collagenase-treated X. laevis oocytes (1 ng/nl, 5 or 50 ng/oocyte). Control oocytes were injected with a similar volume of water. Injected oocytes were incubated at 18°C in modified Barth's medium for 48 h before assay for urea uptake. Uptake of urea was determined in individual oocytes incubated in 200 µl of Barth's solution containing 8 µCi/ml (1.4 mM) [14C]urea (NEN Life Science Products, Boston, MA) at room temperature for 90 s. Uptake was terminated by the addition of 2-ml ice-cold Barth's medium containing 1.4 mM deionized urea, followed by three washes. After solubilization in 10% SDS, urea content was measured by liquid scintillation counting. The effect of phloretin was determined in oocytes preincubated in 0.5 mM phloretin (Sigma, St. Louis, MO) for 20 min. Competitive inhibition by thiourea (Sigma) was analyzed in oocytes preincubated for 30 min in the presence of 50 mM mannitol/150 mM thiourea or 200 mM mannitol added to the Barth's solution. The effect of cAMP was studied by adding 0.05 mM forskolin, 0.5 mM 8-Br-cAMP, and 0.5 mM IBMX (Sigma) to the preincubation medium (1 h) and to the uptake medium. Statistical analysis of the urea uptake measurements was evaluated by one-way ANOVA, and pairwise, post hoc comparisons were made by using the Tukey-Kramer method. Statistical significance was determined when P < 0.05.

Western analysis of cell and tissue extracts. The tissue samples or transfected cell samples were homogenized in isolation buffer (10 mM triethanolamine, 250 mM sucrose, 1 µg/ml leupeptin, 0.1 mg/ml phenylmethylsulfonyl fluoride, pH 7.6, using 0.025-0.1 g tissue/ml isolation buffer). The cell or tissue extracts were size separated by SDS-polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene difluoride membranes (Gelman Scientific, Ann Arbor, MI). Blots were incubated with a primary rabbit polyclonal antibody generated toward the COOH-terminal portion of rat UT-A1 or preimmune serum from the same rabbit, as previously described (6).

Immunohistochemistry. Sections (5 µm) of formalin-fixed, paraffin-embedded human renal tissue were stained with the anti-UT-A1 antibody at 1:400-800 dilution. Positive antigen-antibody interaction was detected with an avidin-biotinylated enzyme complex kit (Signet Laboratories, Dedham, MA), employing the capillary gap staining technique with the automated TechMate 1000 immunostaining system (Ventana Medical, Tucson, AZ) according to the manufacturer's instructions.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cloning of hUT-A1 cDNA. The cDNA sequence of hUT-A1 was identified by analysis of overlapping cDNA fragments obtained by RT-PCR, 5'- RACE, and 3'-RACE. The 5'-end of the transcript was determined by both 5'-RACE and primer extension. The hUT-A1 cDNA included 4,153 bp (GenBank accession no. AF349446). The largest open reading frame of 2,760 bp encodes a peptide of 920 amino acids, with a predicted molecular mass of 101 kDa, which is 89% identical to the rat UT-A1 peptide (Fig. 1) and 64% identical to hUT-B peptide. The Kyte-Doolittle plot of hUT-A1 and rUT-A1 suggests that the two proteins have essentially the same structural configuration (Fig. 2A). The hUT-A1 protein has four consensus sequences for cAMP-dependent phosphorylation (S490, T515, S554, S909), one for tyrosine-kinase (Y509) and five for protein kinase C (S15, S71, T439, S485, S549), localized within the intracellular domains.


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Fig. 1.   Predicted amino acid sequence of human homolog of the rat largest urea transporter of the UT-A family (hUT-A1; h) aligned with the predicted amino acid sequence of rat UT-A1 (r). Identical amino acid sequences between human and rat UT-A1 are shown in boxes.



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Fig. 2.   A: Kyte-Doolittle plots of hUT-A1 and rUT-A1, aligned. B: Western analysis of hUT-A1 translation products in HEK cells transiently transfected with pcDNA3-hUT-A1 (hUT-A1), pcDNA3-rUT-A1 (rUT-A1), and pcDNA3 without insert (C). A protein with a molecular mass consistent with UT-A1 (arrow) is present in the cells transfected with hUT-A1 and rUT-A1 cDNA but is not evident in cells tranfected with vector only.

The translation product of hUT-A1 cDNA was examined in HEK-293 cells, which had been transiently transfected with the hUT-A1-pcDNA3 construct containing the entire open reading frame. Extracts from the transfected cells and from sham-transfected controls were examined by Western analysis with an antibody specific for rat UT-A1 and showed the hUT-A1 protein as an ~97-kDa band evident in transfected cells but not in sham-transfected cells (Fig. 2B).

Functional analysis of hUT-A1 protein. The function of hUT-A1 was tested in oocytes injected with hUT-A1 cRNA, which demonstrated significant uptake of [14C]urea compared with water-injected controls (Fig. 3A). The uptake was inhibited by 0.5 mM phloretin (55% decrease, Fig. 3B). In oocytes injected with 50 ng cRNA, urea uptake was not stimulated by cAMP or agonists, was not Na dependent, and was inhibited by 150 mM thiourea (73% decrease, Fig. 4A). However, when the amount of cRNA injected was reduced to 5 ng, a small but significant stimulation by cAMP was observed (Fig. 4B). The reason for this different response to cAMP is unclear.


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Fig. 3.   A: time course of urea uptake by oocytes injected with 5 ng hUT-A1cRNA, 50 ng hUT-A1 cRNA, or water. B: inhibition of urea uptake by 0.5 mM phloretin in oocytes injected with 50 ng hUT-A1 cRNA. Values are means ± SE. *P <=  0.05.



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Fig. 4.   A: in oocytes injected with 50 ng hUT-A1 cRNA, urea uptake is not affected by cAMP, forskolin, and IBMX. Replacing NaCl with 200 mM mannitol in the uptake medium does not affect urea uptake. Significant inhibition is observed in the presence of 150 mM thiourea. B: in oocytes injected with 5 ng hUT-A1 cRNA, a modest but significant stimulation by cAMP, forskolin, and IBMX is observed. Values are means ± SE. *P <=  0.05.

Expression of hUT-A1 in human kidney. By Northern hybridization with a cDNA probe specific for UT-A1, we detected three transcripts of 6.5, 4.4, and 2.2 kb in the inner medulla but not in the cortex of a human kidney (Fig. 5A). The hUT-A1 mRNA appears as a 4.4-kb band, and the 2.2-kb band is likely to represent the human homologous mRNA of rat UT-A3 (2). The 6.5-kb signal does not correspond to any known UT-A mRNA isoform and may indicate the existence of a novel isoform or different polyadenylation. The expression of an ~100-kDa hUT-A1 protein in the human inner medulla was demonstrated by Western hybridization with the COOH-terminal antibody previously described (Fig. 5B). By immunohistochemistry on sections of human kidney using the same antibody, expression of hUT-A1 is localized to IMCDs (Fig. 5C).


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Fig. 5.   A. Northern analysis of UT-A1 mRNA expression in human kidney, with a hUT-A1-specific cDNA probe, reveals hUT-A1 as an ~4.4-kb mRNA in the inner medulla (IM) but not in the cortex (C). The 2.2-kb mRNA is consistent with expression of the UT-A3 isoform in the human IM. A 6.5-kb mRNA is also detected and may represent a novel UT-A human isoform. B: Western analysis of UT-A expression in human renal medulla using the antibody to the COOH terminus of rat UT-A1 shows expression of hUT-A1 as an ~100-kDa protein and a smaller ~50-kDa protein, probably representing hUT-A2. The 2 proteins are not detected by preimmune (preimm.) serum. C: expression of hUT-A1 is localized to inner medullary collecting duct (IMCD) tubules of human kidney (arrows) by immunohistochemistry using the same antibody.

Genomic organization of hUT-A1. By matching the hUT-A1 cDNA sequence with genomic sequences deposited in the available human genome databases, we determined that the entire hUT-A1 cDNA is distributed in 20 exons spanning ~67.5 kb of chromosome 18 (Table 1). We could not assign to chromosome 18 the initial 182 bp at the 5'-end of the hUT-A2 cDNA [GenBank accession no. X96969 (7)]; the remainder of hUT-A2 is transcribed from exon 12 to 20 (Fig. 6). Therefore, we could not find a human counterpart of rat exon 13, which includes the beginning of rUT-A2 5'-UTR and is transcribed only in the rUT-A2 isoform (4). Compared with the rat gene (~300 kb), the human gene is shorter, and its 5'-UTR is included almost exclusively in the first exon, whereas in the rat it spans three widely spaced exons (4). Analysis of the first 1,000 bp upstream from exon 1 reveals no TATA box. Two CAAT motifs are present within 100 bp from the transcription start site, but we could not find consensus sequences for the Tonicity Enhancer motif (TonE), which mediates tonicity-responsive gene transcription.

                              
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Table 1.   Intron-exon organization of the human UT-A1 urea transporter encoded by the Slc14a2 gene



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Fig. 6.   Organization of the human Slc14a2 gene (A) from which hUT-A1 and hUT-A2 are transcribed, and of the rat Slc14a2 gene (B) from which rUT-A1 and rUT-A2 are transcribed. The exons including the coding region and the predicted start of translation (Transl) are shown in each gene for both transcripts.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study we report cloning the human urea transporter hUT-A1. We describe the functional characterization, expression in human kidney, and genomic organization of hUT-A1.

The hUT-A1 protein is shorter than rUT-A1, although the two proteins are highly homologous. By Western analysis, expression of hUT-A1 is detected in renal inner medulla, and by immunohistochemistry, hUT-A1 is localized in IMCD tubules. The intensity of staining is generally not as strong as in rat IMCDs (Bagnasco S, unpublished observations), suggesting that the human kidney may have lower hUT-A1 protein abundance compared with rodents. In the rat, UT-A1 is thought to be the vasopressin-regulated urea transporter that is responsible for urea reabsorbption across the apical membrane of the IMCD (8). Only four consensus sequences for cAMP-dependent phosphorylation are present in hUT-A1 compared with seven in rUT-A1. This may contribute to the very low sensitivity to cAMP stimulation of hUT-A1 functional activity observed in oocytes. It is possible that in human IMCD, vasopressin may not be as important as in rat IMCD for the short-term regulation of urea transport by UT-A1. Such a possibility may be consistent with humans normally excreting less concentrated urine than rats, which could require lower interstitial urea concentrations in the human inner medulla and lower urea transport rates in the human IMCD, compared with in rodents.

Northern analysis shows hUT-A1 mRNA expression in the human renal inner medulla but not in the cortex. In addition, the 2.2-kb transcript identified by the probe specific for the initial half of hUT-A1 cDNA suggests that the UT-A3 isoform may be expressed in the human kidney. The identity of the larger 6.5-kb mRNA species detected with the same probe remains to be clarified.

We were able to define the organization of the human Slc14a2 gene on chromosome 18. Using sequences available in the human genome database, we mapped the exons and introns from which hUT-A1 and hUT-A2 are transcribed. Our analysis indicates that the gene encoding the human UT-A transporter is larger than the human Slc14a1 gene encoding the UT-B urea transporter/Kidd antigen (3) but smaller than the rat Slc14a2 gene (4). The UT-A coding region appears relatively well conserved between the human and the rat gene (4). However, differences are evident in the region encoding the 5'-UTR of UT-A1, which in rats spans 3 exons distributed over ~260 kb, and in humans is almost entirely included in exon 1. The 5'-UTR of rat UT-A2 starts in exon 13, which is unique to this rat isoform but does not have a counterpart in human UT-A2.

Analysis of the initial 1 kb of the 5'-flanking region of exon 1 does not reveal any consensus sequence for TonE, which in the rat promoter was found at -377. However, it is possible that TonE sequences may be present further upstream in the human gene. The TonE-TonE binding protein (TonEBP) pathways regulate tonicity-responsive transcription of rat UT-A1 and UT-A3 (5) and may contribute to increase expression of these two isoforms in rat kidney (1). The relative importance of tonicity-responsive transcription in the long-term regulation of human UT-A transporter expression remains to be established because, compared with rats, humans excrete less concentrated urine and may have lower tonicity in their inner medulla. Further study will be necessary to characterize the mechanisms regulating UT-A expression and urea transport in the human kidney.


    ACKNOWLEDGEMENTS

We thank Mark W. Nowak and Wayne Fitzgibbons for assistance and support. We also thank Janet D. Klein and Germain Rousselet for helpful suggestions. We are grateful to Sharon D. Langley for help in sequencing.


    FOOTNOTES

This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants R01-DK-53917 and R01-DK-41707 and a grant from the Department of Veterans Affairs. Part of this work was presented and published in abstract form at the annual Experimental Biology 2001 Meeting, March 31-April 4, 2001, Orlando FL (FASEB J A852, 2001).

Address for reprint requests and other correspondence: S. M. Bagnasco, Dept. of Pathology, Emory Univ. School of Medicine,WMB Rm. 7105 A, 1639 Pierce Dr., NE, Atlanta, GA 30322 (E-mail: sbagnas{at}emory.edu).

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.

Received 8 March 2001; accepted in final form 30 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bagnasco, SM, Peng T, Nakayama Y, and Sands JM. Differential expression of individual UT-A urea transporter isoforms in rat kidney. J Am Soc Nephrol 11: 1980-1986, 2000[Abstract/Free Full Text].

2.   Karakashian, A, Timmer RT, Klein DJ, Gunn RB, Sands JM, and Bagnasco SM. Cloning and characterization of two new isoforms of the rat kidney urea transporter: UT-A3 and UT-A4. J Am Soc Nephrol 10: 230-237, 1999[Abstract/Free Full Text].

3.   Lucien, N, Sidoux-Walter F, Olives B, Moulds J, Le Pennec PY, Cartron JP, and Bailly P. Characterization of the gene encoding the human Kidd blood group/urea transporter protein. J Biol Chem 273: 12973-12980, 1998[Abstract/Free Full Text].

4.   Nakayama, Y, Naruse M, Karakashian A, Peng T, Sands JM, and Bagnasco SM. Cloning of the rat Slc14a2 gene and genomic organization of the UT-A urea transporter. Biochim Biophys Acta 1518: 19-26, 2001[ISI][Medline].

5.   Nakayama, Y, Peng T, Sands JM, and Bagnasco S. The TonE/TonEBP pathway mediates tonicity-responsive regulation of UT-A urea transporter expression. J Biol Chem 275: 38275-38280, 2000[Abstract/Free Full Text].

6.   Naruse, M, Klein JD, Ashkar ZM, Jacobs JD, and Sands JM. Glucocorticoids downregulate the rat vasopressin-regulated urea transporter in rat terminal inner medullary collecting ducts. J Am Soc Nephrol 8: 517-523, 1997[Abstract].

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12.   Terris, J, Knepper MA, and Wade JB. UT-A3: localization and characterization of an additional urea transporter isoform in the IMCD3. Am J Physiol Renal Physiol 280: F325-F332, 2001[Abstract/Free Full Text].


Am J Physiol Renal Fluid Electrolyte Physiol 281(3):F400-F406
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