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Am J Physiol Regul Integr Comp Physiol 291: R844-R853, 2006. First published April 13, 2006; doi:10.1152/ajpregu.00739.2005
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WATER AND ELECTROLYTE HOMEOSTASIS

Cloning and functional characterization of a second urea transporter from the kidney of the Atlantic stingray, Dasyatis sabina

Michael G. Janech,1 Wayne R. Fitzgibbon,1,2 Mark W. Nowak,3 Donald H. Miller,1,3 Richard V. Paul,2,4 and David W. Ploth1,2,4

1Marine Biomedicine and Environmental Sciences Center, Departments of 2Medicine and 3Pharmacology and Experimental Therapeutics, Medical University of South Carolina, and 4Ralph H. Johnson, Department of Veteran's Affairs Medical Center, Charleston, South Carolina

Submitted 19 October 2005 ; accepted in final form 10 April 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The cloning of cDNAs encoding facilitated urea transporters (UTs) from the kidneys of the elasmobranchs indicates that in these fish renal urea reabsorption occurs, at least in part, by passive processes. The previously described elasmobranch urea transporter clones from shark (shUT) and stingray (strUT-1) differ from each other primarily because of the COOH-terminus of the predicted strUT-1 translation product being extended by 51-amino acid residues compared with shUT. Previously, we noted multiple UT transcripts were present in stingray kidney. We hypothesized that a COOH terminally abbreviated UT isoform, homologous to shUT, would also be present in stingray kidney. Therefore, we used 5'/3' rapid amplification of cDNA ends to identify a 3'UTR-variant (strUT-1a) of the cDNA that encodes (strUT-1), as well as three, 3'UTR-variant cDNAs (strUT-2a,b,c) that encode a second phloretin-sensitive, urea transporter (strUT-2). The 5'UTR and the first 1,132 nucleotides of the predicted coding region of the strUT-2 cDNAs are identical to the strUT-1 cDNAs. The remainder of the coding region contains only five novel nucleotides. The strUT-2 cDNAs putatively encode a 379-amino acid protein, the first 377 amino acids identical to strUT-1 plus 2 additional amino acids. We conclude that 1) a second UT isoform is expressed in the Atlantic stingray and that this isoform is similar in size to the UT previously cloned from the kidney of the dogfish shark, and 2) at least five transcripts encoding the 2 stingray UTs are derived from a single gene product through alternative splicing and polyadenylation.

elasmobranch; euryhaline; alternative splicing; osmoregulation

MARINE ELASMOBRANCHS (sharks, skates, and rays) use a unique volume- and osmoregulatory strategy; in general, they maintain their body fluids hyperosmotic to the ambient environment (for a review, see Ref. 15; see also 29, 42). The high body fluid osmolality is achieved, in large part, by the retention of urea. At ocean salinities, plasma urea concentrations average 350 mmol/l, and urea contributes 30–50% of the plasma osmolality (for a review, see Ref. 15). The use of this strategy for body fluid volume regulation necessitates the efficient reabsorption of most of the filtered load of urea by the kidneys. For marine elasmobranchs maintained at ocean salinities, fractional urea reabsorption is 90–98% (9, 14, 34).

Some species of elasmobranch are found in estuaries, as well as offshore environments, and a small number of species are able to reside in marine, estuarine, riverine, and even freshwater habitats (5). These elasmobranchs remain ureosmotic even in freshwater habitats, in large part, by renal conservation of urea (for reviews, see Refs. 18 and 19).

The mechanisms by which urea is reabsorbed by the elasmobranch nephron have yet to be clarified. The elasmobranch nephron is anatomically far more complex than the mammalian nephron (21–23). Because urea concentration in urine is markedly less than that in plasma, it has been proposed that urea is actively reabsorbed against a concentration gradient (33, 34, 37). However, on the bais of the anatomical relationships between segments of the nephron (6, 43) and putative low interstitial urea concentration near the terminal segment, Boylan (3) suggested that urea could also be passively reabsorbed down localized concentration gradients (3). This proposal is supported by the presence of phloretin-sensitive, but not ouabain-sensitive, urea flux across nephron segments located within the peritubular sheath (10). The recent cloning of cDNAs encoding phloretin-sensitive, facilitated urea transporters from the kidneys of a number elasmobranch species has provided a molecular mechanism for the passive movement of urea across the tubular epithelia (13, 16, 18, 36).

Although only single cDNAs encoding unique urea transporters have been cloned from the kidneys of elasmobranchs, a number of unidentified transcripts homologous to the cloned urea transporters are expressed in the kidneys and extra-renal tissues of these fish (13, 16, 18, 24, 36). These findings indicate that multiple urea transporters isoforms could be expressed in the elasmobranch kidney.

Comparison of the cDNAs and putative protein sequences of the urea transporters from the spiny dogfish shark, winter skate, and Atlantic stingray, led us to suggest that strUT-1, skUT, and shUT represent isoforms encoded by a single elasmobranch urea transporter gene (18). We therefore hypothesized that a COOH terminally truncated shUT and skUT-like isoform would be expressed in the kidneys of the Atlantic stingray. To test this hypothesis and to further elucidate the molecular identity of the transporters underlying putative passive mechanisms for elasmobranch urea reabsorption, we used PCR-based cloning techniques to identify and characterize additional stingray urea transporter isoforms.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
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Animals. The experiments described in this manuscript were conducted with approval of the Medical University of South Carolina (MUSC) Institutional Animal Care and Use Committee and in accordance with the procedures and practices in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Male Dasyatis sabina (Lesueur, 1824) were kindly supplied by the South Carolina Department of Natural Resources, Fort Johnson, Charleston, SC. Stingrays were treated according to protocols described previously by Janech et al. (18) and maintained in holding tanks at the Marine Biomedicine and Environmental Sciences Center, MUSC, located within the Marine Research Complex at Fort Johnson.

The animals were allowed at least 1 wk to adapt to the holding conditions before euthanasia by placement in buffered (pH 8.0–8.3) seawater containing MS-222 (ethyl 3-aminobenzoate methane-sulfonate salt, Sigma, St. Louis, MO). After euthanasia, tissues were quickly removed and snap frozen in liquid nitrogen.

RT-PCR. Total RNA was isolated from whole frozen kidney using Trizol reagent (Invitrogen, Carlsbad, CA) and reverse transcribed (Superscript II reverse transcriptase, Invitrogen) using oligo(dT). Primers for this and subsequent PCR reactions are listed in Table 1. Gene-specific primers to the 5' and 3' untranslated regions (UTR) of strUT-1 cDNA, (5'UTR1 and SP6EXT2, respectively; Table 1) were used to amplify stingray kidney cDNA using the Elongase amplification system in a 50-µl reaction containing the following reagents [expressed as final concentrations]: 5 µl buffer A, 5 µl buffer B, 0.2 mM dNTP mix, 0.2 µM forward primer, and 0.2 µM reverse primer. Products were amplified according to the following parameters: initial denaturing for 30 s at 94°C, followed by 35 cycles of 30 s at 94°C, 1 min at 55°C, and 5 min at 68°C. The final round was followed by extension for an additional 10 min at 72°C. Two PCR products (1.8 kb and 2.8 kb) were gel-purified and sequenced at the MUSC Biotechnology Resource Laboratory. The 1.8-kb product was the same size and had the same sequence as a partial strUT-1 cDNA. In contrast, the 2.8-kb product contained a novel 971-base pair sequence containing a stop codon between nucleotides (nt) 1204 and 1205 of the strUT-1 cDNA and, as such, appeared to represent a second urea transporter isoform.


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  		Table 1. Sequences of the primers utilized for 5'/3'RACE, Northern probes and RT-PCR primers

 
5'/3' rapid amplification of cDNA ends- 5'RACE. SMART 5' rapid amplification of cDNA ends (RACE; Clontech, Palo Alto, CA) was used to identify the 5'UTR and confirm the coding region of this second isoform. Poly(A)+ RNA was isolated from stingray kidney total RNA and was reverse transcribed with Superscript II reverse transcriptase (Invitrogen) using the RACE cDNA synthesis primer and SMART II oligonucleotide. 5' RACE was performed using a gene-specific reverse primer (4KBAS2, Table 1, Fig. 1) unique to the 3' UTR of this putative second isoform. PCR was optimized for a 50-µl reaction: 10 x universal primer mix, reverse primer (0.4 µM), 3.3 x XL buffer, 0.8 mM dNTP mix, 2.25 mM Mg(OAc)2, and 2 units rTth DNA polymerase XL (Applied Biosystems, Foster City, CA). Amplification was carried out as follows: 1 min at 94°C followed by 38 cycles of 5 s at 94°C, 5 s at 65°C, 10 min at 68°C. Amplification was followed by an additional 10-min extension at 68°C. A single PCR product of ~2 kb was gel purified and sequenced. A small aliquot of this product was reamplified using the same conditions. Reamplification was repeated until a suitable amount of DNA could be attained for in vitro transcription. All cDNA was gel purified and treated with phenol/chloroform/isoamyl alcohol before precipitating with 7.5 M ammonium acetate (1.8 M final concentration) and ethanol. All cDNA was then dissolved in RNase-free water.


Figure 1
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  		Fig. 1. A: schematic representation of the proposed sequence of the stingray (Dasyatis sabina) urea transporter (strUT) pre-mRNA (based on the comparison of the nucleotide sequences of the transcripts encoding strUT-1 and strUT-2). Although we have not sequenced the elasmobranch urea transporter gene, we conclude that the putative strUT pre-mRNA is composed of at least six blocks (or cassettes) of nucleotide sequences and that the transcripts encoding the 2 urea transporter isoforms are produced by alternate splicing and alternate polyadenylation. The total number of nucleotides enclosed in each cassette is presented within each of the lettered boxes. The short vertical line represents the location within the putative pre-mRNA of the start codon, whereas the short vertical arrows represent the locations of the stop codons. B: schematic presentation of the arrangement of each of the cassettes of nucleotide sequences within the transcripts encoding strUT-1 and strUT-2. We propose that splicing the nucleotides contained in cassette I with those in cassette III would produce transcripts that encode strUT-1. In contrast, splicing the nucleotides in cassette I with those in cassette II would produce transcripts that encode strUT-2. We further propose that the 3'UTR variants of strUT-1 and strUT-2 result from alternate polyadenylation. Predicted initiation and termination codons are indicated by the vertical lines or arrows, respectively. (A+) indicates a Poly(A)+ tail.

 
3'RACE. Full-length 3' cDNA ends corresponding to a second isoform were also identified using the SMART RACE system (Clontech). 3' RACE was performed using a gene-specific forward primer (4KBSRACE, Table 1, Fig. 1) unique to the 3'UTR of the second isoform. PCR was performed using 10 x universal primer mix, the forward primer (0.8 µM final concentration), and reagents and protocols in AdvanTAge PCR cloning kit (Clontech). Amplification was carried out as follows: 1 min at 94°C followed by 30 cycles of 10 s at 94°C, 5 min at 67°C. After amplification products were allowed to extend further for 10 min at 67°C. Four PCR products corresponding to 1.6 kb, 1.9 kb, 2.1 kb, and 3.5 kb were gel purified and cloned into pGEM-TA (Promega, Madison, WI) for sequencing.

Identification of additional strUT 3' UTRs. A second forward primer (STS284, Table 1, Fig. 1) located within the common coding region of both strUT-1 and strUT-2 was further used to identify additional 3' cDNA ends not recognized by 4KBSRACE. PCR was performed using HotStarTaq (Qiagen, Düsseldorf, Germany). Amplification was conducted in a 50-µl reaction with the following reagents: 10 x universal primer mix, 0.4 µM forward primer, and HotstartTaq master mix (25 µl; plus an additional 0.5mM MgCl2). Amplification was carried out as follows: 15 min at 94°C followed by 34 cycles of 5 s at 94°C, 30 s at 60°C, 6 min at 72°C. After amplification, products were allowed to extend further for 10 min at 72°C. Two PCR products corresponding to 2.3 kb and 1.4 kb were gel purified and cloned into a sequencing vector (pGEM-TA, Promega).

Northern blot analysis of isoforms. Poly(A)+ RNA (3 µg) from kidney was separated, blotted, and hybridized using high-stringency conditions as reported previously (18). To detect UT transcripts that contain a nt cassette specific to strUT-2 mRNA, a cDNA corresponding to nt 1226–1577 was amplified with HotstartTaq (Qiagen) with primers STRUT2RTAS and STRUT2S2 (Table 1). An antisense, [{alpha}-32P]UTP-labeled riboprobe was generated using T7 RNA polymerase (Maxiscript kit, Ambion, Austin, TX) from the gel-purified PCR product. Hybridization was conducted in ULTRAhyb (Ambion) overnight at 68°C. A second kidney RNA blot was run in parallel and probed using a [{alpha}-32P]UTP-labeled riboprobe common to the open-reading frame of both strUT-1 and strUT-2 (nts 154 to 436), as previously described (16). Hybridized probe was visualized by autoradiography after exposing to film (Kodak-OMAT) for 96 h at –80°C.

Tissue distribution of strUT-2 by RT-PCR. Tissue distribution of strUT-2 mRNA transcripts and corresponding 3'RACE products were investigated using RT-PCR. Kidney, gill, liver, spiral valve, brain, testes, muscle, erythrocytes, and rectal gland were taken from a male Atlantic stingray, according to protocols described previously (18). Total RNA was isolated from each tissue by Trizol and treated with DNase 1 for 30 min. DNase was removed from the sample by phenol/chloroform, and RNA was precipitated with sodium acetate/isopropanol. Samples were reconstituted in distilled water. Total RNA (5 µg) was reverse transcribed (Superscript First-Strand Synthesis System for RT-PCR, Invitrogen) using oligo(dT). For all PCR reactions, 2 µl of each tissue-specific cDNA reaction mixture was amplified in a 50-µl reaction using HotstartTaq master mix (Qiagen) and 0.2 µM forward primer and 0.2 µM reverse primer. Control reactions were conducted on DNase-free, total RNA samples (1 µg) not treated with reverse transcriptase to assess DNA contamination.

The following primers (Table 1) were used to amplify partial cDNAs specific to transcripts corresponding to strUT-2 (STRUT2RTS and STRUT2RTAS), strUT-2b (4KBSRACE and 4KB5RACE1), strUT-2c (4KBSRACE and STRUT2CAS), and GAPDHS1 and GAPDHAS1. The GAPDH primers were based on a partial sequence of the GAPDH cDNA that we had previously identified and characterized from the kidney of the Atlantic stingray (GenBank accession No. AY277618).

Functional characterization. Both strUT-1 and strUT-2 cRNA were transcribed directly from 5' SMART RACE products using the T7 promoter included within the universal primer. Xenopus laevis oocytes were injected with 25 ng of cRNA according to protocols described in (18).

Uptake was determined at room temperature for individual oocytes placed in 250 µl Barth's solution containing 2.7 µCi/ml (0.44 mM) [14C]urea (NEN Life Science Products, Boston, MA) and 0.56 mM unlabeled, deionized urea. Urea uptake was terminated after 90 s by the addition of 2-ml ice-cold Barth's solution containing 1.1 mM deionized urea. Individual oocytes were further washed three times with 2 ml of ice-cold Barth's media containing 1.1 mM-deionized urea. Oocytes were solubilized in 10% SDS (0.5 ml) in 4 ml of scintillation fluid at 20°C for 1 h with repeated vortexing. [14C]urea uptake was determined by scintillation counting (Beckman Coulter LS6500, Fullerton, CA).

To determine whether urea uptake was dependent on the presence of sodium and/or chloride in the external medium, oocytes were preincubated for 1 h in a modified Barth's medium in which the sodium chloride was replaced by 180 mM mannitol. The urea uptake by individual oocytes in mannitol-Barth's medium was determined by incubation for 90 s.

Phloretin sensitivity of the urea transporter-mediated [14C]urea uptake was determined by preincubating strUT-2 cRNA oocytes in Barth's media containing 0.5 mM phloretin for 20 min and then by incubation in the uptake solution containing radio-labeled urea and 0.5 mM phloretin for 90 s.

The effect of urea analogs on UT-mediated [14C]urea uptake was determined by incubating strUT-1 or strUT-2 cRNA injected oocytes in a modified mannitol-Barth's uptake medium, where 150 mM of the mannitol was replaced with 150 mM urea or one the following urea analogs: acetamide, thiourea, methylurea, and 1,3 dimethylurea. Further, the effect of trimethylamine oxide (TMAO) on [14C]urea uptake was also tested by replacement of 150 mM mannitol in the uptake buffer with 150 mM TMAO. All uptake solutions were adjusted to 210 mosmol/kgH2O using additional mannitol if required. Briefly, oocytes were held in mannitol-Barth's medium for 1 h and then preincubated at room temperature for 3 min in the appropriate uptake solution. Uptake of radiolabeled urea by individual oocytes was determined after incubation for 90 s in the appropriate uptake solution containing 14[C]urea.

Effect of low salinity on urea transporter transcript abundance. Four stingrays were placed in each of two, 950-liter filtered seawater tanks containing water pumped from Charleston harbor (850 mosmol/kgH2O, high salinity). After 1 wk of equilibration, buffered, conditioned tap water was added to one of the tanks to dilute the medium to 50% harbor water (425 mosmol/kgH2O, low salinity). Buffered harbor water was added to the control tank. The animals were held for an additional 24 h, under conditions similar to those described previously (19). Animals were then anesthetized by placement in buffered (pH 8.0–8.3) sea water containing MS-222 (aminobenzoic acid ethyl ester, Sigma, St. Louis, MO). The stingrays were then placed ventral side up on a dissection table and the ventral aorta was exposed by longitudinal incision down the cardiac cavity between the gills. Whole animal perfusion with ice-cold elasmobranch Ringer solution (pH = 7.3) was accomplished by inserting a polyethylene catheter (PE-60) into the conus arteriosus and advanced cephalad into the ventral aorta. The Ringer solution was introduced at a rate of 0.5 ml/s. Perfusion was observed in the kidney through a ventral incision in the peritoneal cavity. Once complete, the left kidney was clamped using a hemostat and removed. The kidneys were frozen in liquid nitrogen and stored at –80°C.

Northern blot analysis of kidney Poly(A)+ RNA (3 µg) was performed as described above. Expression of GAPDH was also measured using the same membrane, following stripping of the strUT RNA probe. A [32P]UTP-labeled riboprobe was constructed from a GAPDH PCR product that was cloned into a pCRII vector and linearized by restriction digest (Invitrogen). Intensity was quantified by densitometry using a GS-670 densitometer and Molecular Analyst software (Bio-Rad, Hercules, CA).

Statistical analysis. For the functional study, data were tested for outliers before analysis to eliminate misinjected and damaged oocytes from the comparison. Outliers were defined as those data points greater or lower than 1.5 times the interquartile range. Suspect outliers were not included in further statistical analyses. Statistical analysis was performed using a one-way ANOVA, and post hoc comparisons were tested using Duncan's multiple range test. If data from the functional characterization studies were not distributed normally (Kolmogorov-Smirnov test), data were then log-transformed before statistical analysis. Statistical significance was achieved when P < 0.05.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
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Using 5'RACE, we obtained a single product, 1896 nt in length, comprising a 72-nt 5'UTR, a 1,137 nt open reading frame (ORF), and 687 nt of the 3'UTR. This newly identified cDNA was designated strUT-2. Except for a single nucleotide polymorphism (SNP, nucleotide substitution reading from strUT-1 to strUT-2; nt 369, A to G), the first 1,204 nt (the 72 nt of the 5'UTR and the first 1,132 nt of the ORF) of this cDNA are identical to those of the previously identified strUT-1 cDNA. However, the remaining portion of the ORF is composed of only 5 nts, indicating the newly identified cDNA encodes a shorter protein than that encoded by the strUT-1 cDNA.

Alternative 3'UTRs belonging to this strUT-2 cDNA were found by using optimized 3' RACE amplification. 3' RACE amplification with a primer specific to the strUT-2 transcript resulted in amplification of three distinct PCR products (1.9 kb, 2.1 kb, and 3.5 kb in length) whose sequences overlapped with the 5' RACE product. These products comprised alternate 3' cDNA ends of the strUT-2 cDNA. For each of the 3' RACE products, full-length cDNA sequences were reconstructed based on overlapping sequences between the 5' and 3' RACE products and were supported by overlapping with the sequence of the 2.8-kb product identified in the initial RT-PCR experiment. The shortest full-length transcript was designated strUT-2a (GenBank accession no. AF503513). This cDNA has a total of 3,616 nts before the poly(A)+ tail. A slightly longer transcript, strUT-2b (GenBank accession no. AY277794) is 3,851 nt in length and consists of the same nt sequence as strUT-2a with an additional 235 nt inserted before the Poly(A)+ tail. A third transcript, 5,179 nt in length was designated strUT-2c (GenBank accession no. AY277795). This transcript consists of an almost identical nt sequence as strUT-2b but with an additional 1,328 nt in the 3'UTR before the poly(A)+ tail. Interestingly, in addition to the polyadenylation signal immediately upstream from the poly(A)+ tail, each of the transcripts contain other potential polyadenylation signals (ATTAAA and AATAAA) throughout the 3'UTRs.

A second series of 3' RACE was used to identify possible alternative 3'UTRs for strUT-1. Using a forward primer common to the coding regions of strUT-1 and strUT-2, two discrete PCR products, 1.2 kb and 2.4 kb in length, were amplified. The 2.4 kb PCR product was identical to the previously identified strUT-1 cDNA. The smaller-sized cDNA was also identical to the strUT-1 cDNA except for the presence of a truncated 3'UTR. This new cDNA was found to be 1,509 nt in length [72 nt 5'UTR, 1,293 nt ORF, and 144 nt 3'UTR before the poly(A)+ tail]. This transcript appeared to be a 3'UTR variant of the previously identified strUT-1 mRNA. To maintain the nomenclature system for 3'UTR variants used for the strUT-2 cDNAs, the longer, previously identified cDNA was redesignated as strUT-1b (GenBank accession no. AF443781), and the shorter cDNA was designated strUT-1a (GenBank accession no. AY277793).

Alignment of all five transcripts revealed an interesting pattern of splicing. Each transcript can be divided into two or more "cassettes" of unique nt sequences. These sequence cassettes are presented schematically in Fig. 1. Because we did not identify the urea transporter genomic sequence, these sequence cassettes are not exons but may be composed of a single exon or groups of exons. The first 1,204 nts of each transcript are represented by cassette I. Cassette II represents the next 971 nts of the strUT-2 cDNAs. The stop codon starts at the sixth nt of this cassette. In contrast, the strUT-1 cDNAs do not contain the nt sequence represented by cassette II. The 3' end of the ORF of the strUT-1 isoform is encoded by nts contained in cassette III. The stop codon for this cDNA starts at nt 165 within this 305-nt cassette. The strUT-1a cDNA is polyadenylated at the end of cassette III, while the 3'UTR of strUT-1b cDNA contains an additional 1,138 nts (represented by cassette IV) before polyadenylation. The nt sequence of cassette III is the same for both strUT-1 and strUT-2 transcripts. In contrast, the sequence of cassette IV of strUT-1b cDNA is two nts longer than that within the strUT-2 cDNAs (thymines present at nt 1,817 and 1,905 of the strUT-1b cDNA are absent from the strUT-2 cDNAs). Further, several SNPs are also present: (nt substitutions read as strUT-1b to strUT-2; C [nt, 1,944] to T [nt, 2,913], G [nt, 2,381] to A [nt, 3,350]; nt, C [nt, 2,482] to T [nt, 3,451]). The strUT-2a cDNA is polyadenylated at the end of cassette IV, while the strUT-2b cDNA contains an additional 235 nts (represented by cassette V) before polyadenylation. The strUT-2c contains an additional 1,563 nts (represented by cassettes V [235 nts] and VI [1,328] nts) in the 3'UTR before polyadenylation. A SNP was present in cassette V (nt substitutions read as strUT-2b to strUT-2c; G [nt, 3,449] to T [nt, 3,449]).

The first ORF of the strUT-2 cDNA is the longest, beginning at nt 73 and ending at 1,209. The deduced ORF encodes a 379-amino acid protein, which we have designated, strUT-2. As expected from the similarity of the nucleotide sequences, the first 377 amino acids of strUT-2 and strUT-1 are identical (Fig. 2). However, the strUT-2 isoform is ~52 amino acids shorter than the strUT-1 isoform (Fig. 2). Interestingly, strUT-2 is 82% identical to the predicted urea transporter cloned from the dogfish shark, Squalus acanthias (shUT; Ref. 36) and contains nearly the same number of amino acid residues. Similar to other urea transporters in lower vertebrates and mammalian UT-A family, strUT-2 contains a repeated LP motif (amino acids 165–172 and 327–333) and lacks the ALE domain common to UT-B. Consensus site analysis of strUT-2 did not reveal features that would indicate that it is functionally different from that strUT-1.


Figure 2
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  		Fig. 2. Alignment of the amino acid sequences of the strUT-2 (GenBank accession no. AY277794), strUT-1 (GenBank accession no. AAM46683) and shark [Squalus acanthius urea transporter (shUT)], GenBank accession no. AAF66072). Identical residues are indicated by the shaded areas. The numbers indicate the number of amino acids predicted for each UT. Sequences were aligned using the ClustalV feature of the DNASTAR software package. The strUT-2 was deduced to be a 379-amino acid protein with the NH2 terminus identical to that of strUT-1 but with the COOH-terminus abbreviated by 51 amino acids and containing two unique amino acids. The strUT-2 had a high degree of sequence identity to shUT (82%).

 
High-stringency Northern blot analysis using a strUT-2 specific riboprobe indicated that a number of transcripts were present in kidney (Fig. 3, left). Two distinct renal transcripts of 3.8 kb and 5.5 kb were easily identifiable. The 3.8-kb band was the most abundant and was close to the predicted molecular sizes of the strUT-2a and strUT-2b cDNAs. The 5.5-kb band was similar in size to that expected for the strUT-2c cDNA. Although strUT-2 cDNAs that are less than 3 kb in length were not identified by RACE, smaller strUT-2 transcripts appear to be expressed in the stingray kidney. A large band was detected between 2.3 kb and 3.0 kb. The size of this band corresponds well with putative polyadenylation signals at nts 2,372, 2,697, 2,753, and 3,083 of strUT-2. We also performed a high-stringency Northern blot analysis with a riboprobe designed to hybridize to both strUT-1 and strUT-2 transcripts. This riboprobe corresponds to nts 154–436. Three bands similar in size to that detected by the strUT-2-specific riboprobe, as well as an additional 4.5-kb transcript could be identified (Fig. 3, right). The band at 2.6–3.0 kb corresponds to the size of the 2.7-kb transcript for strUT-1b (16), as well as the putative 2.7- and 3.1-kb transcripts for strUT-2. The 4.5-kb band does not correspond in size to any of the strUT isoforms identified so far. Interestingly, the strUT-1/strUT-2 probe did not detect a transcript at 1.8 kb (strUT-1a). The inability to detect a transcript corresponding to strUT-1a may be due to the low abundance of this transcript in kidney relative to the other strUT-1 and strUT-2 mRNA. No transcripts were detected in extrarenal tissues by high-stringency Northern blot analysis with either probe (data not shown).


Figure 3
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  		Fig. 3. Kidney tissue expression of strUT mRNA determined using Northern blot analysis using either a strUT-2-specific riboprobe (cassette B, nucleotides 1,226–1,577; left) or a riboprobe common to both strUT-1 and strUT-2 (cassette A, nucleotides 154–436; right) Transcripts at ~3.8 kb and 5.5 kb corresponding to the three strUT-2 transcripts (strUT-2a, b, and c) were identified using the strUT-2-specific probe. A large band spanning ~2.3 to 3.0 kb in length was also identified. This band corresponds to other possible alternatively polyadenylated strUT-2 transcripts and/or novel urea transporter transcripts. Using the riboprobe common to the strUT mRNAs, transcripts 2.8 kb, 3.8 kb, 4.5 kb, and 5.5 kb in size were identified. The band at 4.5 kb may indicate an unidentified strUT-1 splice variant or a unique strUT isoform. Expression of a transcript corresponding to strUT-1a was not detected by Northern blot analysis.

 
RT-PCR was used to detect strUT-2 transcripts in renal and extrarenal tissues (Fig. 4). Amplification of GAPDH mRNA served as a positive control (Fig. 4D). No products were amplified from negative controls (data not shown). Amplicons of a 568-nt product common to the three strUT-2 cDNAs were obtained from all the tissues examined, including erythrocytes, although the expression varied markedly between tissues (Fig. 4A). Amplicons of a 2-kb product indicative of strUT-2b and/or strUT-2c (the cDNAs containing the cassette V sequence) were obtained from all tissues with the exception of erythrocytes (Fig. 4B). In contrast, amplicons indicative of strUT-2c (a 3.2-kb product) were identified only from kidney, brain, and testes (Fig. 4C). As expected from the Northern blot analysis, the strUT-2 mRNAs were highly expressed in kidney. The strUT-2a and strUT-2b transcripts were expressed at high levels, and strUT-2c at moderate levels in brain and testes. The expression of the strUT-2a and strUT-2b was low in all the other extrarenal tissues, except that only strUT-2a was detected in erythrocytes.


Figure 4
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  		Fig. 4. Tissue expression of strUT-2 determined using reverse-transcriptase-PCR. Primers designed to part of the nucleotide sequence shared by 3'UTR variants of strUT-2 amplified a single 568 nt product in all tissues examined (A). A 2-kb product corresponding to the shared sequence between strUT-2b and strUT-2c was amplified from renal and extrarenal tissues but not erythrocytes (B). Expression of strUT-2c was limited to kidney, brain, and testes (C). A single amplicon representing stingray GAPDH was expressed in all tissues (D).

 
Heterologous expression of strUT-2 in Xenopus oocytes resulted in a marked increase in [14C]urea uptake (Fig. 5). Oocytes injected with strUT-2a cRNA exhibited a 17-fold elevation in [14C]urea uptake over water-injected oocytes (P < 0.05). Preincubation with phloretin (0.5 mM) completely attenuated strUT-2 induced [14C]urea uptake (P < 0.05, Fig. 5). To determine whether [14C]urea uptake was dependent on the presence of NaCl in the uptake medium, the NaCl was replaced by 150 mM mannitol. Replacement of the NaCl did not significantly alter [14C]urea uptake by oocytes injected with strUT-2 (Fig. 5), that is, strUT-2 induced phloretin-sensitive urea uptake was independent of sodium and chloride transport.


Figure 5
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  		Fig. 5. Effect of phloretin (0.5 mM) or replacement of NaCl in the uptake medium with mannitol (150 mM) on [14C]urea uptake by oocytes injected with 25 ng strUT-2a cRNA. The stingray urea transporter induced a marked elevation in [14C]urea uptake. Preincubation with phloretin completely attenuated strUT-2-induced [14C]urea uptake. Substitution of the NaCl in the uptake medium with mannitol did not significantly alter strUT-2-induced [14C]urea uptake. *P < 0.05 vs. water-injected oocytes. {dagger}P < 0.05 vs. strUT-2 injected oocytes. n = 5 to 8 oocytes.

 
To determine whether the difference in the carboxy terminal sequence between the two isoforms results in a functional difference in urea uptake, we measured the effect of a number of urea analogs on urea uptake induced by the two isoforms. Oocytes injected with either strUT-1 or strUT-2 cRNA were incubated in NaCl-free uptake solutions containing either unlabeled urea or the urea analogs (acetamide, thiourea, methylurea, 1,3 dimethylurea) or trimethylamine oxide. [14C]urea uptake induced by strUT-1 or strUT-2 was not significantly inhibited by excess unlabeled urea or 150 mM acetamide (Fig. 6). In contrast, thiourea, methylurea, and 1,3 dimethylurea inhibited strUT-1-induced urea uptake by 64%, 52%, and 85% and strUT-2-induced urea uptake by 74%, 62%, and 88% respectively (Fig. 6). Although these findings are qualitative (we did not normalize uptake for the amount of isoform expressed), they indicate that the profile of inhibition of urea transport (whether an analog inhibited urea transport and, if so, the degree of inhibition) was similar for strUT-1 and strUT-2. TMAO, a prominent protein-stabilizing solute in elasmobranch fluids, augmented strUT-2-mediated urea uptake.


Figure 6
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  		Fig. 6. Effect of unlabeled urea (150 mM), urea analogs (150 mM), and trimethylamine-N-oxide (TMAO, 150 mM) on [14C]urea uptake by either strUT-2a cRNA-injected (A) or strUT-1b cRNA-injected (B) oocytes. The urea analogs, thiourea, methylurea, and 1,3 dimethylurea significantly inhibited [14C]urea uptake compared with oocytes preincubated in mannitol. *P < 0.05 vs. strUT-1 in the presence of mannitol alone; {dagger}P < 0.05 vs. strUT-2 in the presence of mannitol alone. n = 5–8 oocytes.

 
Four distinct bands at 2.8, 3.8, 4.5, and 5.5 kb, corresponding to strUT transcripts, were detected using Northern blot analysis of kidney poly(A)+RNA from stingrays either maintained in harbor water (high salinity) or exposed to 50% harbor water (low salinity) for 24 h (Fig. 7A). GAPDH was detected in each lane as a single band at ~1.5 kb. The relative abundance of the 5.5 kb (strUT-2c) transcript was 25% lower for stingrays exposed to low salinity than those rays maintained at high salinity (strUT/GAPDH; 0.56 ± 0.06 vs. 0.42 ± 0.04 for high and low salinity, respectively, P < 0.05, Fig. 7B). There were no differences in relative expression of the bands at 3.8 kb (strUT-2a and strUT-2b), 2.8 kb (strUT-1b and other strUT-2 transcripts), and 4.5 kb (unidentified strUT transcript) between the rays in high salinity or in low salinity.


Figure 7
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  		Fig. 7. Northern blot analysis of strUT RNA abundance in rays maintained in harbor water or exposed to a 50% reduction in external salinity for 24 h. A: representative blot obtained using a probe designed to the coding regions of strUT-1 and strUT-2. Transcripts corresponding to the cloned isoforms were detected at 2.7 kb (strUT-1b), 3.8 kb (strUT-2a and strUT-2b), and 5.5 kb (strUT-2c). B: relative strUT RNA abundance normalized to GAPDH RNA abundance in kidneys from stingrays maintained in harbor water (solid bars) or exposed to a 50% harbor water (open bars) derived from Northern blot analysis. Only the expression of the 5.5-kb transcript (strUT-2c) was lower in the stingrays subjected to environmental dilution. *P < 0.05 (Student's t-test), n = 4 for each group.

 

   DISCUSSION
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We report the cloning of 3 cDNAs that encode a second stingray urea transporter designated as strUT-2, as well as an additional cDNA (designated as strUT-1a) encoding the previously identified strUT-1. The three strUT-2 cDNAs (strUT-2a, strUT-2b and strUT-2c) that were identified in this study vary only in their 3'UTRs. The strUT-1a transcript is a 3'UTR variant of the previously cloned transcript (redesignated strUT-1b). The 5'UTR and most of the coding region of the strUT-2 cDNAs are identical to that of the strUT-1 cDNAs. However, the presence of a unique sequence containing a stop codon at positions 1,210–1,212 of the strUT-2 cDNAs putatively results in translation of a shorter urea transporter isoform (strUT-2, 379 amino acids), that is, although the NH2 terminus of this isoform is predicted to be identical to that of strUT-1, the COOH-terminus is abbreviated by 51 amino acids and terminates with 2 unique amino acids. Further, the strUT-2 has a high level of sequence identity to and is almost identical in size with shUT, the spiny dogfish shark urea transporter (36).

The various transcripts that encode the strUT-1 or strUT-2 proteins result from alternative splicing. The deletion of a 971-nt sequence from the premRNA produces a transcript with an open-reading frame that encodes strUT-1. In contrast, retention of these nt results in the insertion of a more proximal stop codon, producing a transcript with a shorter open reading frame that encodes strUT-2.

Using consensus sequence analysis, we could not identify any likely sites for differential posttranslational modification (e.g., glycosylation, phosphorylation, or myristylation) that would suggest differences in functional regulation between the two proteins. Further, after heterologous expression in Xenopus oocytes, strUT-1 and strUT-2 had almost identical functional responses to urea analogs, indicating comparable substrate specificities. In contrast, the mammalian urea transporter splice variants exhibit differences in specificity to urea transport inhibition by urea analogs (26, 35, 44).

Alternative splicing and polyadenylation provide mechanisms by which structural and functional diversity, and tissue-specific control of expression can be derived from a single gene product. These posttranscriptional modifications are common features of the mRNAs encoding mammalian urea transporters, as well as renal cotransporters in other vertebrates (1, 11, 12, 20). Differences in 3'UTR length influence subcellular localization and mRNA storage (30, 39), which, in turn, affect translational efficiency. Furthermore, the binding of cytosolic regulatory proteins to the 3'UTR alters the stability and turnover of mRNA (32, 40, 41). In elasmobranchs, as well as mammals, differences in 3'UTR length are associated with functional diversity. For example, P450 aromatase cloned from the Atlantic stingray undergoes polyadenylation at two different sites and as a result is encoded by two transcripts that contain different 3'UTRs (17). Cells transfected with the longer transcript had lower P450 aromatase activity than the cells transfected with the shorter transcript (17).

In the nephron, differential processing of transporter pre-RNAs provides a potential mechanism for regulation of tubular transport (for a review, see Ref. 12). Although we could not identify functional differences between strUT-1 and strUT-2, the finding that the expression of only strUT-2c was lower in rays exposed to a 50% reduction in salinity (discussed below) indicates that the two transporter isoforms could play different roles in tubular reabsorption of urea. Different roles of strUT-1 and strUT-2 in renal urea reabsorption might arise from expression in different nephron segments, which is the case for various isoforms of the UT-A transporters within the mammalian kidney (25, 38) or trafficking to different membrane domains in polarized epithelia. Further, similar regulatory mechanisms could contribute to the selective expression of one or more of the 3'UTR variants of the strUT-1 and strUT-2 isoforms at different sites along the nephron.

The expression of urea transporters in extrarenal tissues would be expected for animals that are ureosmotic, as these proteins would allow the rapid distribution of urea throughout the body, as well as the rapid equilibration of tissue and plasma urea concentrations when these animals move between environments of differing salinities. Interestingly, transcripts homologous to strUT-1 and strUT-2 could not be detected in extrarenal tissues of D. sabina by high-stringency Northern blot analysis. In contrast, transcripts encoding strUT-2 were detected in the extrarenal tissues by RT-PCR. These findings are in agreement with our previous findings (18). This difference in the ability to detect message for strUT is most probably due to the difference in sensitivity between these two techniques of mRNA detection.

In a previous study, we used primers to the coding region of strUT-1 mRNA to detect its presence in erythrocytes and liver by RT-PCR (18). However, the findings of the present study indicate that the primers used in the previous study cannot distinguish between the two strUT-1 and the three strUT-2 transcripts. Further, the present findings indicate that of the strUT-2 mRNAs, only strUT-2a could be detected in erythrocytes and liver. Although, the question of the presence of strUT-1a and/or strUT-1b mRNAs in erythrocytes and liver remains to be clarified, it is not unreasonable to predict that one of these two transcripts is also present in these tissues. Despite the presence of strUT-2a and possibly strUT-1a/strUT-1b mRNAs, phloretin-inhibitable, facilitated transport does not appear to contribute to urea movement across the membranes of elasmobranch erythrocytes (4, 31, 45) or hepatocytes (45). Therefore, the physiological roles of the strUT-1/strUT-2 urea transporters in liver and erythrocytes remain to be elucidated. However, the relatively high levels of the strUT-1/strUT-2 mRNAs in brain and testes compared with the other extrarenal tissues could indicate a specific role for these transporter proteins in the function of these organs. Interestingly, in mammals, facilitated urea transporters are also prominently expressed in brain and testes. UT-B is expressed in astrocytes of the rat brain where it has been proposed that it regulates urea levels secondary to the production of polyamines from ornithine (2). UT-B is expressed in the Sertoli cells and seminiferous tubules of the rat testis (44), and UT-A5 (a novel isoform encoded by the UT-A gene) is expressed in the seminiferous tubules of the mouse testis (8). These urea transporters may play a role in spermatogenesis (44) by allowing urea to move across the blood:testis barrier (8).

Some elasmobranchs are able to exploit estuarine habitats (marginally euryhaline elasmobranchs), and a small number of species can exploit marine, estuarine, riverine, and even freshwater habitats (euryhaline elasmobranchs). The large increase in glomerular filtration rate after exposure of euryhaline and marginally euryhaline elasmobranchs to low salinity results in a marked increase in the filtered load of urea ([data recalculated from 14, 28, 34], 19). Although, this increase in the filtered load is to a large extent balanced by increased tubular reabsorption, fractional reabsorption of urea is lower in elasmobranchs exposed to low salinity (66–84%). The finding that kidney urea transporter RNA abundance was lower in marginally euryhaline skates, Raja erinacea exposed to low salinity for 5 days than that in the kidneys of skates at high salinity, has led to the proposal that decreased expression of facilitated urea transporter gene expression contributes at least in part, to the decrease in fractional urea reabsorption (24). We have previously observed that the euryhaline, Atlantic stingrays exposed to a 50% reduction in salinity for 24 h have lower fractional urea reabsorption (84%) compared with rays maintained at high salinity (96%). In the present study, we tested the above proposal by determining if strUT transcript abundance was also altered by acute exposure of Atlantic stingrays to low salinity. We detected transcripts that corresponded to message for strUT-1, strUT-2a, strUT-2b, and strUT-2c. For the stingrays exposed to low salinity, only the 5.5-kb strUT-2c transcript had a lower expression compared with that in the control rays. The lower expression of the 5.5-kb strUT-2c transcript indicates that the long 3'UTR splice variant of strUT-2 may be selectively downregulated during acute dilution. Although there are discontinuities between UT RNA and protein abundance (1, 46), if strUT protein abundance changes in parallel with strUT RNA levels, the acute decrease in fractional urea reabsorption reported for stingrays in dilute seawater (19) could result, at least in part, from downregulation of strUT-2 expression following a decrease in strUT-2c mRNA abundance.

Our finding that the expression of only one of the strUT-2 transcripts was lower after exposure to low salinity contrasts to the finding that the expression of all three transcripts homologous to a skate urea transporter were lower in the kidneys of little skates exposed to 50% seawater compared with those in 100% seawater (24). Interestingly, renal urea transporter expression was not different between spiny dogfish sharks exposed to 75% seawater or maintained in 100% seawater (27). This latter finding suggests that in some species of elasmobranchs a decrease in whole kidney urea transporter gene expression may not underlie the increase in absolute or the decrease in fractional urea reabsorption induced by exposure to low salinity. Therefore, the role of urea transporter gene expression in the increase in absolute or the decrease in fractional urea reabsorption induced by exposure to low salinity remains to be clarified.

The current findings support our previous proposal that a single gene encodes the urea transporters that have to this date been cloned from the kidneys of elasmobranchs (18). Furthermore, a comparison of the cDNAs and predicted protein sequences support the proposal that the two stingray urea transporters and the spiny dogfish urea transporter (36) originate from a common translational start site. After an analysis of the three possible ORF initiation sequences in the cDNA encoding shUT, Smith and Wright (36) concluded that the second site had the highest consensus with a typical Kozak sequence (AATCCATTCATGG, where if the nt at position –3 is not a purine then the G at position 4 is essential) (36). Interestingly, the first ORF initiation sequence of the stingray urea transporters is identical to the initiation sequence of the spiny dogfish shark.

The existence of strUT-2, a close homolog of shUT, suggests evolutionary pressure for conservation of this isoform between two species belonging to sister clades that appear to have diverged early in elasmobranch evolution (7). In preliminary studies, we cloned cDNAs that putatively encode close homologs of shUT and strUT-2 from the kidneys of the stenohaline, winter skate, Leucoraja ocellata (13), and the marginally euryhaline, bluntnose stingray, Dasyatis sayi (unpublished data). These findings suggest that this isoform is an important component of the passive mechanisms involved in elasmobranch renal urea reabsorption. Interestingly, a COOH terminally extended isoform, analogous to strUT-1, has not yet been identified from these stenohaline and marginally euryhaline elasmobranchs. Further studies may reveal whether the presence of the strUT-1 isoform is associated with euryhaline physiology.


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 ABSTRACT
 METHODS
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REFERENCES
 
This research was supported by Division of Nephrology Research funds and a grant from Dialysis Clinics W. R. Fitzgibbon was supported in part by a Veteran's Affairs Merit Award granted to David W. Ploth. This work was performed using equipment and resources funded, in part, by the Department of Veteran's Affairs. This work was presented in part at the XXXIV Congress of the International Union of Physiological Sciences, Christchurch, New Zealand, July 2001, Abstract #1712.

Present addresses: M. G. Janech, Div. of Nephrology, Dept. of Medicine, Medical Univ. of South Carolina, 96 Jonathan Lucas St., P.O. Box 250623, Charleston, SC, USA, 29425; R. V. Paul, Piedmont Nephrology, 1899 Tate Blvd. SE, Ste. 2101, Hickory, NC 28602; and M. W. Nowak, Neurion Pharmaceuticals, 180 North Vinedo Ave, Pasadena, CA.


   ACKNOWLEDGMENTS
 
We thank Bill Roumillat and the Inshore Fisheries Group of the South Carolina Department of Natural Resources for the very kind and generous support in providing the stingrays used in this study; and J. Raymond, M. Budisavljevic, and B. Tholanikunnel, for expert advice regarding the molecular approaches to these studies.


   FOOTNOTES
 

Address for correspondence: W. R. Fitzgibbon, Div. of Nephrology, Dept. of Medicine, Medical Univ. of South Carolina, 96 Jonathan Lucas St., P.O. Box 250623, Charleston, SC, (E-mail: fitzgiwr{at}musc.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.


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