Molecular and functional characterization of a ureatransporter from the kidney o - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:35 |只看该作者 |倒序浏览 |打印

作者：Michael G.Janech, Wayne R.Fitzgibbon, RuihuaChen, MarkW.Nowak, Donald H.Miller, Richard V.Paul,  David W.Ploth,作者单位：Departments of Marine Biomedicine andEnvironmental Sciences, Medicine, and Pharmacology and Experimental Therapeutics, MedicalUniversity of South Carolina, and Ralph H. JohnsonVeterans Affairs Medical Center, Charleston, South Carolina 29525 * f2 i! i8 J- d8 W* A( O


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      【摘要】( \1 H: T# t6 E' U2 U
   In general, marineelasmobranch fishes (sharks, skates, and rays) maintain body fluidosmolality above seawater, principally by retaining large amounts ofurea. Maintenance of the high urea concentration is due in large partto efficient renal urea reabsorption. Regulation of renal ureareabsorption also appears to play a role in maintenance of fluidhomeostasis of elasmobranchs that move between habitats of differentsalinities. We identified and cloned a novel 2.7-kb cDNA from thekidney of the euryhaline Atlantic stingray Dasyatissabina (GenBank accession no. AF443781 ). This cDNA putativelyencoded a 431-amino acid protein (strUT-1) that had a high degree ofsequence identity (71%) to the shark kidney facilitated ureatransporter (UT). However, the predicted COOH-terminal region ofstrUT-1 appears to contain an additional sequence that is unique amongcloned renal UTs. Injection of strUT-1 cRNA into Xenopus oocytes induced a 33-fold increase in [ 14 C]urea uptakethat was inhibited by phloretin. Four mRNA bands were detected inkidney by Northern blot: a transcript at 2.8 kb corresponding to theexpected size of strUT-1 mRNA and bands at 3.8, 4.5, and 5.5 kb.Identification of a facilitated UT in the kidney of the Atlanticstingray provides further support for the proposal that passivemechanisms contribute to urea reabsorption by elasmobranch kidney.
      【关键词】 euryhaline ‘/‘rapid amplification of cDNA ends osmoregulation
   INTRODUCTION/ r' T, `; Q, O5 C- j& J
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IN GENERAL, MARINE ELASMOBRANCHS (sharks, skates, and rays) maintain body fluidshypertonic to the ambient environment. In the ocean, plasma osmolalityis ~1,050 mosmol/kgH 2 O (for review, see Ref. 19 ), which is 2-10% higher than the osmolality ofthe seawater. This high plasma osmolality is achieved, in part, by theretention of urea ( 40 ); i.e., elasmobranchs areureosmotic. Plasma urea concentrations average 350 mmol/l (range209-453 mmol/l), and urea contributes 30-50% of the plasmaosmolality (for review, see Ref. 19 ). Clearance of ureafrom the body is low, in large part because of reabsorption of90-98% of the filtered load of urea by the kidneys ( 12, 15, 40 ).. I' e/ v0 [5 O; ^" Q6 f6 j4 T
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Although most marine elasmobranchs are stenohaline (i.e., their rangeis restricted to marine habitats), a number of species are marginallyeuryhaline (i.e., they are also found in brackish environments, such asestuaries and river mouths) ( 7 ). Interestingly, arelatively small number of species (the euryhaline elasmobranchs) areable to exploit marine, estuarine, riverine, and even freshwater habitats. Most euryhaline elasmobranchs appear to migrate between habitats on a seasonal basis ( 35, 42 ). However, at leastone species (Atlantic stingray) can reproduce and complete its life cycle in freshwater, whereas another (bull shark) has the potential todo so ( 22, 43 ).( q# X+ Z9 l  \0 d
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Because the gills of elasmobranchs are freely permeable to water( 5 ), in the ocean the small osmotic gradient betweenseawater and the extracellular fluid leads to an influx of free waterinto the fish. The kidneys maintain volume homeostasis by excreting urine hyposmolar to plasma (i.e., the osmotically driven uptake ofwater is balanced by the excretion of solute-free water).
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A very high plasma osmolality and the free movement of water across thegills present a challenge to volume homeostasis for marginallyeuryhaline and euryhaline elasmobranchs when they move betweenenvironments of different salinities or are resident in low-salinityhabitats. In response to this challenge, two strategies are employed:plasma osmolality is varied in the same direction as the change inexternal salinity by altering the plasma concentrations of urea and/orNa and Cl ( 8, 10, 15, 16, 21, 30, 34, 41, 42, 48 ) and by altering renal excretory function inverselywith changes in environmental salinity so that fluid efflux counteractsthe osmotically driven water influx ( 15, 21, 28, 34, 48 ). However, for marginally euryhaline and euryhaline elasmobranchs, changes in internal osmolality are not directly proportional to changesin the external osmolality. Thus, when these fish exploit low-salinityenvironments, there is an increase in the external-internal osmoticgradient ( 29, 30, 42 ). This osmotic gradient can beremarkable; for euryhaline elasmobranchs in freshwater, it is in therange 600-650 mosmol/kgH 2 O ( 29, 42 ). Theincreased gradient leads to increased osmotically driven water uptake( 6, 8, 10, 15, 41 ). Fluid balance is returned toward the predilution level by a marked increase in renal excretory function, including increased glomerular filtration rate, urinary flow rate, freewater clearance, and urea and electrolyte excretion ( 15, 16, 21, 28, 34, 48 ). The increase in urea excretion exceeds that of theother solutes, so that urea becomes the predominant urinary solute( 21, 28, 48 ). The increase in urea excretion results froman increase in filtered load and a decrease in fractional reabsorption( 21, 28, 34 ).5 O( o+ g9 }5 ^8 i4 u, l( \

Interestingly, elasmobranchs remain ureosmotic in low-salinityenvironments. Urea remains a major osmolyte, contributing 30-40% to the extracellular fluid osmolality ( 8, 10, 15, 21, 34, 43, 48 ), and the kidneys remain the primary site for urea retention.The amount of urea reabsorbed by the renal tubules is markedlyincreased after exposure to a low-salinity environment; i.e., despite alower fractional reabsorption of urea (decreasing from 90-98% to66-84%), the increase in the filtered load of urea coupled withthe high reabsorptive capacity of the renal tubules results in a largeincrease in the absolute amount of urea reabsorbed (data recalculatedfrom Refs. 15, 28, and 34; seealso Ref. 21 ). Thus the mechanisms that regulate renalurea reabsorption are important in the osmoregulatory andvolume-regulatory processes of marine elasmobranchs.
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The countercurrent arrangement of the marine elasmobranch nephron( 23 ) supports the role of passive mechanisms in renal ureareabsorption ( 3, 13 ). A phloretin-sensitive, facilitated urea transporter has been cloned from the kidney of the spiny dogfishshark, a marginally euryhaline elasmobranch ( 39 ). Because this urea transporter was highly expressed in the kidney, the authorsproposed that it and possibly other homologous facilitated ureatransporters contributed to tubular urea reabsorption. In a recentstudy, we observed that Atlantic stingrays held in harbor waterreabsorbed 96% of the filtered load of urea, indicating that thisspecies displays a high degree of renal urea conservation, even in anestuarine salinity ( 21 ). Unlike many dasyatid rays in thisregion, this species is commonly caught in oligohaline-to-freshwater habitats (17, 22, 29; W. Roumillat, personal communication). We therefore hypothesized that if phloretin-sensitive facilitated ureatransporters played a role in tubular urea reabsorption in theelasmobranch kidney, urea transporters homologous to the spiny dogfishurea transporter would also be present in the kidneys of the Atlanticstingray, a phylogenetically distinct marine elasmobranch that,unlike the spiny dogfish, exploits habitats that cover a remarkablerange of environmental salinities.
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METHODS+ C( X. w) ?! Y: H$ o8 g3 p
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Animals1 G1 Z6 W( X& @# v& P9 e3 E! m

The experiments were conducted with approval of the MedicalUniversity of South Carolina (MUSC) Institutional Animal Care and Use Committee.5 B1 r- q7 S1 ^, y) R

Male Atlantic stingrays ( Dasyatis sabina, Lesueur,1824 ) were kindly supplied by the South Carolina Departmentof Natural Resources (Fort Johnson, Charleston, SC). The stingrayswere caught in Charleston Harbor and neighboring estuaries by the SouthCarolina Department of Natural Resources during scheduled surveys oflocal fisheries. The stingrays were placed in transportation tanksuntil the survey vessel returned to Fort Johnson. The rays were then placed in a filtered, closed-circulating, 15,000-liter holding tank atthe Department of Marine Biomedicine and Environmental Sciences, MUSC,located within the Marine Research Complex at Fort Johnson.

The salinity of the water in the holding tank was maintained at 28 (~850 mosmol/kgH 2 O), water temperature was maintained between 20 and 24°C, and pH was monitored and, if necessary, adjusted to 8.0-8.3 with a seawater buffer (Marine Buffer, SeaChem, Stone Mountain, GA). The stingrays were fed a diet of shrimp during theholding period. The animals were allowed 1 wk to adapt to the holdingconditions before euthanization by placement in buffered (pH8.0-8.3) seawater containing aminobenzoic acid ethyl ester (MS-222, Sigma, St. Louis, MO). After euthanasia, tissues were quicklyremoved and snap-frozen in liquid nitrogen.( _9 R' r5 J% p2 \3 a  F. C

RT-PCR: x8 E5 J4 p5 d4 ?

Total RNA from whole kidney was isolated using TRIzol reagent(GIBCO BRL, Gaithersburg, MD). The RNA was reverse transcribed usingSuperscript II reverse transcriptase (GIBCO BRL) with an antisenseprimer (5'-AGC CAC CAG TAC CAG TCT CC-3') corresponding to amino acids133-139 of the Squalus acanthias urea transporter protein sequence (GenBank accession no. AAF66072; shUT) ( 39 ). Degenerate sense primers for PCR were derived tocorrespond to amino acids 60-66 [5'-GC(A/G/C/T) CAG GTC ATG TTTGT(G/C) AAC-3'] and 81-86 [5'-CA(A/G) AAC CC(A/C) TGG TGGGC-3'] of the shUT protein sequence. cDNA was amplified in twoseparate reactions using Taq polymerase (GIBCO BRL) in a50-µl reaction containing the following reagents (expressed as finalconcentrations): 5 µl of 10× Taq amplification buffer,1.5 mM MgCl 2, 0.2 mM dNTP mix, 0.2 µM forward primer, and0.2 µM reverse primer. Products were amplified according to thefollowing parameters: initial denaturing for 5 min at 94°C followedby 32 cycles of 1 min at 94°C, 1 min at 55°C, and 2 min at 72°C.The final round was followed by extension for an additional 10 min at72°C. Two PCR products of predicted length (147 and 237 bp) were gelpurified and sequenced at the Biotechnology Resource Laboratory (MUSC).The sequence of the 237-bp product was used to design gene-specificprimers for cloning using rapid amplification of cDNA ends(5'/3'-RACE).% C7 T$ G, p) }9 I0 F1 ~5 a' S4 V
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5'/3'-RACE

Initial cloning. 5'/3'-RACE was performed using reagents and the protocol provided inthe Marathon cDNA amplification kit (Clontech, Palo Alto, CA). Briefly,poly(A) RNA was reverse transcribed, and second-strandcDNA was synthesized using the provided reagents. 5'-RACE was performedon Marathon adaptor-ligated cDNA using a gene-specific reverse primer(5'-CGA CCA ATA TGC CAT TGT ACC CAG C-3'). PCR was performed usingreagents described above, and amplification was carried out as follows: 1 min at 94°C followed by 30 cycles of 10 s at 94°C and 5 min at 68°C. Products were allowed to extend for 10 min at 72°C. A single PCR product of ~500 bp was amplified, gel purified, and inserted into pCRII-TOPO vector (Invitrogen, Carlsbad, CA). A second,gene-specific forward primer (5'-CTC GGG ACA GTG TTT GCA ACT TTG G-3')was used to perform 3'-RACE. Amplification was carried out as follows:1 min at 94°C followed by 30 cycles of 10 s at 94°C, 30 sat 60°C, and 4 min at 72°C. Products were allowed to extend for 10 min at 72°C. A PCR product of ~2 kb was identified, gel purified,and inserted into a pCRII-TOPO vector.
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Competent Escherichia coli (One-shot, Invitrogen) weretransformed with the pCRII-TOPO vector insert and plated.Positive colonies were selected, and the plasmids were isolated.Plasmids containing the 5'- or 3'-RACE insert were sequenced asdescribed above by primer walking. Overlapping 5'- and 3'-RACE products allowed the reconstruction of the almost full-length putative ureatransporter cDNA.6 o3 v3 z. f+ |0 m- _* R& k. I
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Confirmatory cloning. SMART RACE (Clontech) technology was utilized to independently confirmthe Marathon RACE product and to identify additional base pairs in the5'-untranslated region (UTR). Poly(A) RNA was isolatedfrom the kidney of a second stingray and reverse transcribed withSuperscript II reverse transcriptase (GIBCO BRL) using the 5'-RACE cDNAsynthesis primer and SMART II oligonucleotide. 5'-RACE was performedusing a gene-specific reverse primer (5'-TAA CAC TGT GCC ATG CAA GGTTCA G-3') complementary to nucleotides 1864-1888 within the3'-UTR. PCR was performed using the reverse primer (0.8 µM finalconcentration) and reagents and protocols in the AdvanTAge PCR cloningkit (Clontech). Amplification was carried out as follows: 1 min at94°C followed by 35 cycles of 5 s at 94°C, 10 s at65°C, and 4 min at 68°C. Products were allowed to extend for 10 minat 68°C. A PCR product of ~1.8 kb was gel purified and sequenced. Asmall aliquot of this product was reamplified under identicalconditions. Reamplification was repeated until a suitable amount of DNAcould be attained for in vitro transcription. All cDNA was gel purifiedand treated with phenol-chloroform-isoamyl alcohol before precipitationwith 7.5 M ammonium acetate (1.8 M final concentration) and ethanol.All cDNA was then dissolved in RNase-free water." B  [" a5 |% I  I1 O2 d6 w

Northern Analysis, k) z3 y  |; D

Poly(A) RNA (3 µg) from kidney, gill, liver,spiral valve, brain, testes, heart, and whole blood cells and total RNA(10 µg) from rectal gland were separated by electrophoresis, blotted, and cross-linked onto a positively charged nylon membrane (Hybond-N , Amersham, Arlington Heights, IL) and then prehybridized in ULTRAhyb (Ambion, Austin, TX). To detect tissue-specific distribution of ureatransporter mRNA, a hybridization probe corresponding to nucleotides154-436 of the cloned urea transporter was constructed using theMarathon 5'-RACE product subcloned into a pCR4-TOPO vector (Invitrogen)and linearized by restriction digestion with Acc I. Anantisense [ - 32 P]UTP-labeled riboprobe was generatedusing the Maxiscript kit (Ambion). Hybridization was conducted inULTRAhyb overnight at 68°C (high stringency) or 63°C (lowstringency). The membrane was washed twice for 5 min at roomtemperature in 2× saline-sodium citrate buffer 1% SDS (lowstringency) and then twice for 15 min at 68°C in 0.1× saline-sodiumcitrate buffer 1% SDS (high stringency). The hybridized probewas visualized after incubation at 80°C for 72 h (highstringency) or 1 wk (low stringency).
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Multiple-Tissue RT-PCR
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A third stingray was placed in buffered (pH 8.0-8.3)seawater containing aminobenzoic acid ethyl ester until swimmingmovements had ceased but sustained movement of the gill muscles wasmaintained (to allow adequate ventilation). The animal was placeddorsal side and head down on a slant board (~30°) so that thespiracles were completely under the water. The conus arteriosis wasexposed by a ventral incision proximal to the heart. The conusarteriosis was cannulated, and the animal was perfused at low pressurewith ice-cold elasmobranch Ringer solution. Tissues were quicklyharvested, snap-frozen in liquid nitrogen, and stored at 80°C until processed.
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A fourth stingray was anesthetized as described above. Whole bloodwas collected using a 21-gauge needle attached to a heparinized syringefrom the caudal haemal arch and centrifuged at 800 g for 1 min. Plasma and blood cells residing at the layer above the erythrocytes were removed. The erythrocytes were then resuspended inelasmobranch Ringer solution. This process was repeated twice to ensurecomplete removal of nonerythrocytes. Erythrocytes were then transferredto a 50-ml polyethylene tube, snap-frozen in liquid nitrogen, andstored at 80°C until processed.: L- e5 w# N1 K; H
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Total RNA was isolated from the tissues and erythrocytes using TRIzolreagent as described above. The RNA samples were pretreated with DNaseI (Ambion), extracted with phenol-chloroform-isoamyl alcohol, andprecipitated with sodium acetate-isopropanol. Treated RNA (3 µg) fromeach tissue was reverse transcribed with an oligo(dT) primer asdescribed above. PCR was conducted using stingray urea transporter-specific primers designed to regions that flank the nucleotide sequence of the riboprobe used for Northern analysis: 5'-ACAAAA TCC ATT CAT GGA GCA-3' (forward) and 5'-TCT TCT CCG GGT ACG TCA CTTCGC ATA G-3'(reverse). Degenerate primers to a conserved region ofglyceraldehyde-3-phosphate dehydrogenase were used for positivecontrols: 5'-GAG TCC ACT GG(AT) GTC TTC ACC ACC A-3' (sense primer) and5'-GGA TGA CCT TGC C(AC)A CAG CCT TGG C-3' (reverse primer).Amplification of cDNA was conducted using HotStarTaq master mix(Qiagen, Valencia, CA). Products were amplified according to thefollowing parameters: initial denaturing for 14 min at 94°C followedby 30 or 35 cycles of 30 s at 94°C, 30 s at 56°C, and 3 min at 72°C. The final round was followed by extension for anadditional 10 min at 72°C.
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In Vitro Transcription
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cRNA was transcribed directly from the 5' SMART RACE productusing the T7 promoter included in the universal primer. In vitro transcription was performed using the mMessage mMachine kit (Ambion). cRNA was precipitated with LiCl overnight and dissolved in 25 µl ofRNase-free water. RNA quality was assessed by electrophoresis on a 2.2 M formaldehyde-1% agarose RNA gel, and concentration was determined byoptical density at 260 nm.

Functional Characterization

Oocytes were surgically removed from gravid Xenopuslaevis and prepared for [ 14 C]urea uptake studies asdescribed previously ( 20 ). Uptake was determined forindividual oocytes by incubation in 200 µl of Barth's solutioncontaining 8 µCi/ml (1.3 mM) [ 14 C]urea (NEN LifeScience Products, Boston, MA) at room temperature. For the time seriesexperiments, urea uptake was terminated between 30 s and 60 min bythe addition of 2 ml of ice-cold Barth's solution containing 1.4 mMdeionized urea. For all other experiments, uptake was terminated after90 s by the addition of 2 ml of ice-cold Barth's solutioncontaining 1.4 mM deionized urea. Individual oocytes were furtherwashed three times with 2 ml of ice-cold Barth's medium containing 1.4 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 repeatedvortexing. [ 14 C]urea uptake was determined byscintillation counting (Coulter LS6500, Beckman, Fullerton, CA).

Phloretin sensitivity of the urea transporter-mediated[ 14 C]urea uptake was determined by preincubation ofoocytes in Barth's medium containing 0.5 mM phloretin for 20 min andthen by incubation in the uptake solution containing radiolabeled ureaand 0.5 mM phloretin.& X9 Q- u# Y# N1 e
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To determine whether urea uptake was dependent on the presence ofNa and/or Cl in the external medium, oocyteswere preincubated for 1 h in a modified Barth's medium containing8 µCi/ml (1.3 mM) [ 14 C]urea in which the NaCl wasreplaced by sufficient mannitol to bring the osmolality of the solutionto 200 mosmol/kgH 2 O (~180 mM mannitol).[ 14 C]urea uptake by individual oocytes in thismannitol-Barth's medium was determined by incubation for 90 s.
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The effect of urea analogs on stingray urea transporter(strUT)-1-mediated [ 14 C]urea uptake was determined byincubating oocytes in a modified mannitol-Barth's uptake medium, where150 mM mannitol was replaced with 150 mM urea or one the following ureaanalogs: acetamide, thiourea, methylurea, or 1,3-dimethylurea. Theeffect of trimethylamine oxide (TMAO) on [ 14 C]urea uptakewas also tested by replacement of the mannitol in the uptake bufferwith 150 mM TMAO. All uptake solutions were adjusted to 210 mosmol/kgH 2 O using additional mannitol if required. Briefly, oocytes were held in mannitol-Barth's medium for 1 h andthen preincubated at room temperature for 3 min in the appropriate uptake solution. Uptake of radiolabeled urea by individual oocytes wasdetermined after incubation in the appropriate uptake solution containing [ 14 C]urea.

Statistical Analysis4 b! H/ I7 d( D8 n

Data from the functional characterization studies were notdistributed normally (Kolmogorov-Smirnov test). Therefore, the datawere logarithmically transformed before statistical analysis usingone-way ANOVA. If data did not fit a normal distribution aftertransformation, one-way ANOVA on ranks was used to determine significance. Post hoc comparisons were tested using the Tukey-Kramer method (parametric) or Dunn's multiple comparison test(nonparametric). Statistical significance was achieved when P means ± SE.
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RESULTS1 h1 _3 e& \+ T" T9 c; ?

We used RT-PCR and 5'/3'-RACE to obtain a full-length cDNAsequence for strUT-1. The strUT-1 cDNA is 2,670-bp long, with a putative open-reading frame (ORF) of 1,296 bp(73-1,368), a 5'-UTR of 72 bp, and a 3'-UTR includingthe poly(A) tail that is 1,302-bp long (Fig. 1;GenBank accession no. AF443781 ). Twosequential polyadenylation signals (AATAAA) at nucleotide positions1250-1255 and 1258-1263 are located just upstream of thepoly(A) tail.0 M5 `/ T* F' T) J# H+ Z1 `! K& F

Fig. 1. Nucleotide sequence of strUT-1 cDNA cloned from kidney of theAtlantic stingray (GenBank accession no. AF443781 ). UTR,untranslated region; ORF, open-reading frame.
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The first ORF of the strUT-1 cDNA is the longest and encodes a putativeprotein 431 amino acids long (strUT-1; Fig. 2 ). The putative protein is 50 aminoacids longer than the shUT; i.e., strUT-1 has a longer and unique COOHterminus. The molecular weight of strUT-1 was calculated to be 48,000. The putative protein is 71% identical to shUT and 53% identical tothe frog kidney urea transporter (Fig. 2 ). However, if only thepredicted amino acids 1-377 are considered, strUT-1 is 79%identical to shUT. Compared with the mammalian urea transporterisoforms, strUT-1 has a slightly higher sequence identity to rat UT-A2(53%) and human UT-A2 (53%) than either rat UT-A3 (45%) or UT-A4(47%) and shares 48% sequence identity with UT-B1.
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Fig. 2. Alignment of amino acid sequences of the stingray ureatransporter ( Dasyatis sabina, strUT-1), shark ureatransporter ( Squalus acanthias, shUT, GenBank accession no.AF66072), frog urea transporter ( Rana esculenta, fUT,GenBank accession no. CAA73322 ), and rat UT-A2. Identical residues areindicated by the boxed areas. Numbers at right indicatenumber of amino acids predicted for each urea transporter. Sequenceswere aligned using Clustal feature of the DNASTAR software package.strUT-1 was deduced to be a 431-aa protein that showed a high degree ofsequence identity to shUT (71%) and fUT (53%) but has a longer anddistinct COOH-terminal sequence.0 e( f! a9 j& F/ I

The putative protein contained a repeated LP motif (aa 165-172 and327-333), a feature characteristic of urea transporters. Furthermore, strUT-1 lacks the ALE domain, a feature present in UT-B1and UT-B2 but absent from other urea transporters. Consensus siteanalysis of strUT-1 identified a single glycosylation site (NITW) atamino acids 203-206. Several protein kinase CK2 phosphorylation sites were also noted: TIVE (aa 6-9), TWPD (aa 205-208), andTYPE (aa 360-363). In contrast to shUT, strUT-1 contains a protein kinase C (PKC) phosphorylation site (SDK) at amino acids 129-131. As with other urea transporters from the lower vertebrates and ratUT-A3, strUT-1 contains a multicopper oxidase signature(GLWSYNSVLACIAVGGMFYAL, aa 276-296). Interestingly, rat UT-A2 hasa number of amino acid changes in this region and thus does not containthis multicopper oxidase signature.
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High-stringency Northern analysis of poly(A) RNA using a5'-RACE riboprobe specific to strUT-1 detected four transcripts in kidney. In contrast, no transcripts were detected in other tissues (Fig. 3 ). The renal transcripts hadmolecular sizes of 2.8, 3.8, 4.5, and 5.5 kb. The 2.8-kb transcript wasvery close to the predicted molecular size of strUT-1 cDNA. The findingthat multiple transcripts are present indicates that urea transportershomologous to strUT-1 may also be expressed in kidney. Underlow-stringency conditions, a 2.8-kb transcript was detectable only inliver (data not shown). In contrast, no transcripts were detected inmuscle, gill, blood, spiral valve, brain, rectal gland, or testes, evenafter exposure of the blots to film for 1 wk at 80°C.

Fig. 3. Tissue expression of strUT-1 mRNA determined usingNorthern blot analysis. Poly(A) RNA (3-10 µg)isolated from several tissues was hybridized at high stringency(68°C) with a [ 32 P]UTP-labeled riboprobe specific tostrUT-1 5' region. Blot was exposed to autoradiographic film for72 h at 80°C. Four transcripts (2.8, 3.8, 4.5, and 5.5 kb insize) were detected in the kidney. The 2.8-kb transcript was very closeto the predicted molecular size of the strUT-1 cDNA.
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We utilized RT-PCR to further investigate the tissue specificity ofstrUT-1 expression. A single PCR product of the predicted size ofstrUT-1 was detectable only in kidney after 30 cycles (data not shown).However, after 35 cycles of PCR, a single product the same size as theproduct obtained for kidney was detected in all the extrarenal tissuesexamined and in erythrocytes (Fig. 4 ).PCR containing DNase I-treated total RNA that had not been reversedtranscribed did not produce any detectable product (Fig. 4 ), indicatingthat the detection of strUT-1 in extrarenal tissues was not due to DNAcontamination of any of the total RNAs. These findings indicate thatstrUT is expressed in extrarenal tissues but at markedly lower levelsthan in kidney.
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Fig. 4. Tissue expression of strUT determined using RT-PCR. TotalRNA was reverse transcribed, and PCR products were amplified usingprimers designed to strUT-1 (specifically, regions that flank thenucleotide sequence of the riboprobe used for Northern analysis) or tostingray glyceraldehyde-3-phosphate dehydrogenase (GAPDH). As anegative control, PCR using the strUT-1 primers was performed withtotal RNA that had not been reverse transcribed as the template. After35 cycles of PCR, expression of strUT mRNA could be detected in alltissues as well as erythrocytes. Expression of strUT was markedly lowerin extrarenal tissues than in kidney.
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Heterologous expression of strUT-1 in Xenopus oocytesinduced a marked increase in [ 14 C]urea uptake (Fig. 5 ). [ 14 C]urea uptake byoocytes injected with 25 ng of strUT cRNA was logarithmic, with maximaluptake after 40 min (data not shown). Characterization of thestrUT-1-induced [ 14 C]urea uptake was determined at theinitial rate of urea uptake, i.e., 90 s after the oocytes wereexposed to the uptake medium. Oocytes injected with strUT-1 cRNAexhibited a 33-fold elevation in [ 14 C]urea uptake overwater-injected oocytes (68.1 ± 5.7 vs. 2.1 ± 1.3 pmol · oocyte 1 · 90 s 1, P 5 ). Preincubationwith 0.5 mM phloretin completely attenuated strUT-1-induced[ 14 C]urea uptake ( P 5 ).Replacement of the NaCl did not significantly alter[ 14 C]urea uptake by oocytes injected with 25 ng of strUTcRNA (Fig. 5 ). This finding indicates that strUT-1-induced urea uptakewas independent of external Na and Cl.
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Fig. 5. Effect of 0.5 M phloretin or replacement of NaCl inuptake medium with 150 mM mannitol on [ 14 C]urea uptake byoocytes injected with 25 ng of strUT-1 cRNA. Stingray ureatransporter induced a marked elevation in [ 14 C]ureauptake. Preincubation with phloretin completely attenuatedstrUT-1-induced [ 14 C]urea uptake. Substitution of NaCl inuptake medium with mannitol did not significantly alter strUT-1-induced[ 14 C]urea uptake. Data were analyzed by 1-way ANOVA onranks, and then comparisons between means were tested using Dunn'smultiple comparison test. * P n = 5-8 oocytes. dH 2 O, distilled water.

The [ 14 C]urea uptake induced by strUT-1 was notsignificantly inhibited by 150 mM urea, acetamide, or TMAO (Fig. 6 ). In contrast, the urea analogsthiourea, methylurea, and 1,3-dimethylurea at 150 mM markedly inhibitedurea uptake by 75, 74, and 92%, respectively (Fig. 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[ 14 C]urea uptake by strUT-1 cRNA-injected (25 ng)oocytes. Urea analogs, thiourea, methylurea, and 1,3-dimethylurea,significantly inhibited [ 14 C]urea uptake compared withoocytes preincubated in mannitol. In contrast, excess unlabeled urea,acetamide, and TMAO, a prominent organic component of elasmobranchplasma, did not significantly alter strUT-1-induced urea uptake.* P n = 7-8 oocytes.+ }( N% @: w) b% r) `

DISCUSSION
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Whether marine elasmobranchs are in the ocean or in estuaries orrivers, renal reabsorption of urea appears to be one of the centralmechanisms underpinning their osmoregulatory strategy (i.e., tomaintain body fluid osmolality higher than that of the surroundingmedium). The mechanisms involved in and the tubular site(s) ofelasmobranch renal urea reabsorption have yet to be identified. Severalmechanisms have been proposed by which urea can be reabsorbed from thetubular fluid of elasmobranchs. These mechanisms have involved passivereabsorption of urea down localized concentration gradients viafacilitated urea transporters and/or active reabsorption viaNa -urea cotransporters ( 3, 18, 34, 46 ).1 ]8 D9 z% v7 N
, z5 p0 {3 v" U0 V" f/ |! @" [  z
We hypothesized that if phloretin-sensitive facilitated ureatransporters played a role in tubular urea reabsorption in the elasmobranch kidney, urea transporters homologous to that identified inthe kidney of S. acanthias would also be present in thekidneys of D. sabina, a myliobatiform marine elasmobranchthat exploits habitats across a remarkable range of environmentalsalinities. Using 5'/3'-RACE techniques, we identified a novel ureatransporter from the kidney of the Atlantic stingray. We designatedthis transporter strUT-1. This urea transporter was functional,displaying phloretin-inducible urea transport comparable to thatobserved for other lower vertebrate and mammalian urea transporters( 1, 9, 20, 36, 39, 44, 45, 49 ).
# r; b" ]& a+ l- ?  ^
The stingray urea transporter strUT-1 was predicted to be 431 aminoacids long, making it 51 amino acids longer than the shark ureatransporter. The difference in length is due to an extension of thehydrophilic COOH-terminal region of strUT-1. This COOH-terminal extension represents a unique sequence not present in other lower vertebrate or mammalian urea transporters. Inasmuch as no putative consensus regulatory sites are predicted to reside within this COOH-terminal extension, its functional significance remains to be determined.% ~$ H7 e& L4 a/ a% r

Except for the COOH-terminal extension, the stingray and shark ureatransporters have a high degree of sequence identity. Furthermore,several consensus regulatory sites (glycosylation, protein kinase CK2,and multicopper oxidase) are located at similar positions in bothtransporters. However, there are some differences between the twotransporters in the number and type of consensus sites in theNH 2 -terminal region. The stingray urea transporter hasa putative protein kinase CK2 regulatory site close to the NH 2 terminus, a feature that is absent from the shark ureatransporter. The shark urea transporter has two potential glycosylationsites in the NH 2 -terminal region that are absent fromstrUT-1. Interestingly, the stingray urea transporter contains aPKC consensus site within its NH 2 terminus, again a featurethat is absent from the shark renal urea transporter. The presence of aPKC consensus site in strUT-1 leads us to propose that PKC may act todirectly alter the function of the stingray, but not the shark,urea transporter.

Recent analysis of the mammalian urea transporter genes indicates thatthey have developed by a series of duplications of a common ancestralmodule that have resulted in two genes [UT-A ( Slc14a2 ) andUT-B ( Slc14a1 )] ( 11, 26, 27 ). Four UT-Aprotein isoforms are expressed in the medullary tubular epithelia ofthe kidney ( 33 ). Interestingly, each isoform is generatedby several splice variants ( 2 ). Transcription of three ofthe isoforms (UT-A1/A3/A4) is under the control of one promoter,whereas the other (UT-A2) is under the control of another promoter( 26 ).

The gene(s) encoding urea transporters from the lower vertebrates hasyet to be sequenced. However, comparison of the cDNAs of strUT-1 andshUT provides some insight into the elasmobranch urea transporter gene.Although D. sabina and S. acanthias are representative of phylogenetically distinct marine elasmobranchs, thereis a high degree of sequence identity within the ORFs of the cDNAs forstrUT-1 and shUT (with the exception of the most 3' region of the ORF).Thus we propose that strUT-1 and shUT are orthologous products of acommon elasmobranch urea transporter gene and that transcription ofthese products may be under the control of a single promoter (based onsimilarities in the sequence and length of the 5'-UTRs).8 b0 D4 p# ]* ^, G
" q: D% a# W" N- {. }' o
Elucidation of the cDNA and putative protein sequences for the ureatransporters from the lower vertebrates has provided preliminary information about the possible links between the lower vertebrate andmammalian genes. On the basis of the observations that the dogfishshark urea transporter had a slightly higher putative protein sequenceidentity to UT-A2 than to UT-B2 and lacked the ALE domain (a motifpresent only in UT-B1/UT-B2), Smith and Wright ( 39 )suggested that shUT is a phylogenetic ancestral form of UT-A2. However,a marked degree of evolutionary divergence appears to have occurredbetween Slc14a2 and the gene encoding strUT-1 and shUT. TheUT-A2s from rabbit, rat, mouse, human, and whale have a very highdegree of sequence identity, and only UT-A2 is encoded by the exonsdistal to the second promoter in Slc14a2 ( 20, 27, 38, 49 ). In contrast, strUT-1 and shUT appear to represent twodistinct isoforms derived from a common elasmobranch urea transportergene. Taking the findings from the present study into account and therecent observations that the cloned urea transporters from the gills ofteleost fishes have a slightly higher sequence identity to UT-A2 thanto UT-B and lack the ALE domain ( 44, 45 ), we propose thatthe urea transporters from elasmobranch and teleost fish and themammalian UT-A2s are derived from a common ancestral form. A corollaryof this proposal is that, of the mammalian urea transporter genes, theUT-A2 component of the mammalian UT-A gene is the most representativeof the common ancestral form.

Preliminary evidence from our laboratory indicates that a shUT-likeisoform may be present in the kidneys of other elasmobranchs ( 14 ). Thus shUT appears to be representative of a commonurea transporter isoform present in the kidneys of phylogenetically distinct elasmobranch groups. In contrast, it has yet to be determined whether an isoform equivalent to strUT-1 is expressed in the kidneys ofelasmobranchs other than the Atlantic stingray. The renal urea transporters appear to play an important role in the regulation oftubular urea reabsorption, which, in turn, appears to contribute to themaintenance of water homeostasis after movement between habitats ofdifferent salinities. The identification of those elasmobranch speciesthat express the strUT-1 isoform may help clarify the role of this ureatransporter. It is possible that this isoform may be unique to D. sabina. However, it is interesting to speculate that thestrUT-1-like isoform may be expressed in the kidneys of euryhalineelasmobranchs but not in the kidneys of the stenohaline and/ormarginally euryhaline elasmobranchs.7 x' A4 Q4 p& i3 e& X# [' j, J/ G

In the mammalian kidney, the various products of the UT-A gene havespecific tubular locations and specific functions in the urinaryconcentrating process: urea reabsorption from the lumen of themedullary collecting duct or recycling between the medullary interstitium and the tubular fluid ( 32 ). The complexdistribution of cell types in the elasmobranch nephron, the bundling ofproximal and distal nephron segments in a countercurrent arrangementwithin a peritubular sheath, the meandering arrangement of proximal and distal nephron segments in the sinus zone ( 23, 24 ), andzones of differing interstitial urea concentration within the kidney ( 18 ) indicate that urea reabsorption may occur at severalsites along the nephron. Thus it is possible that different ureatransporter isoforms may have specific tubular locations or playdifferent roles in urea reabsorption by the elasmobranch kidney.3 o# I( F1 @+ ]/ Z/ o1 A
4 `! I6 W. c; F& v8 `
A number of urea analogs (at 150 mM) have been found to inhibitfacilitated urea transporter-mediated urea uptake by oocytes ( 9, 36-38, 49 ). The most potent inhibitors, thiourea and1,3-dimethylurea, markedly attenuate facilitated ureatransporter-mediated urea uptake, including that induced by strUT-1 inthe present study. However, there are differences among the ureatransporter isoforms in the effectiveness of acetamide blockade of ureauptake. Acetamide inhibits UT-A3-induced urea uptake ( 37 )and partially inhibits the uptake of urea induced by the gill ureatransporter from the teleost fish, the Lake Magadi tilapia( 44 ), but does not alter urea uptake induced by UT-A2( 49 ). Because acetamide did not alter strUT-1-induced ureauptake, the stingray urea transporter shares a similar functionalcharacteristic with UT-A2.. G- {) K; V, [

Northern analysis indicated that strUT-1 as well as homologoustranscripts appeared to be almost exclusively expressed in kidneys ofAtlantic stingrays. Only in liver, under low-stringency conditions,was a low-molecular-weight transcript detected. This limited tissueexpression of urea transporter transcripts detectable by Northernanalysis contrasts with that observed for the dogfish shark, wheretranscripts homologous to shUT were detected in the kidney and brain of S. acanthias by high-stringency analysis and in all thetissues surveyed by low-stringency Northern analysis ( 39 ).Therefore, we further examined the apparent renal-specific strUTexpression by utilizing the more sensitive technique of RT-PCR todetermine whether strUT-1 or homologous transcripts were expressed inextrarenal tissues as well as in the kidney of D. sabina.Our findings indicated that strUT message was expressed in allextrarenal tissues examined, but at markedly lower abundance than inthe kidney. If we assume that the expression of urea transporter protein parallels transcript abundance, then the extrarenal expression of strUT message has interesting implications for the role of strUT-like urea transporters in a number of tissues. Expression ofstrUT transcripts in liver and erythrocytes would indicate that ureamovement across hepatocyte and erythrocyte membranes could occur, inpart, through a strUT-1 or similar facilitated urea transporterisoform. However, the presence of facilitated urea transport inelasmobranch erythrocytes has not, in general, been supported byobservations from physiological experiments. Although it was initiallyproposed that urea moves across elasmobranch red blood cell membranesby facilitated transport ( 25 ), a number of more recentstudies have found no evidence to support such a transport mechanism inthese cells ( 4, 31, 47 ). Similarly, low-stringencyNorthern analysis and/or RT-PCR indicated that an elasmobranch ureatransporter homolog is present in the liver of S. acanthias ( 39 ) and D. sabina (the present study).However, phloretin-inhibitable, facilitated transport does not appearto contribute to urea movement across the membranes of hepatocytes fromthe lesser spotted dogfish Scyliorhinus canicula or D. sabina ( 47 ). Therefore, the physiological roles ofthe extrarenal urea transporters remain to be clarified." ~/ y1 x+ k( h9 K' J

In summary, we have identified a novel facilitated urea transporter(strUT-1) from the kidney of the myliobatiform euryhaline elasmobranch,the Atlantic stingray. It is probable that strUT-1 and otherfacilitated urea transporter isoforms contribute, at least in part, tothe reabsorption of urea by the kidney. Furthermore, these facilitatedurea transporters may contribute to the regulation of tubular ureareabsorption during the movement of marginally euryhaline andeuryhaline elasmobranchs between habitats of different salinities.
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ACKNOWLEDGEMENTS1 a4 m# \! [7 W- p- o; c
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We thank B. Roumillat (Inshore Fisheries, South Carolina Departmentof Natural Resources) for the very kind and generous support inproviding the stingrays used in this study; J. Raymond, M. Budisavljevic, and B. Tholanikunnel for expert advice regarding themolecular approaches to these studies; and G. Rousselet and L. Ball forvaluable advice regarding the functional characterization of the cloned transporter.- Q% s+ y2 Y: k& S$ U9 D
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