AJP - Regu Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Regul Integr Comp Physiol 282: R1490-R1500, 2002; doi:10.1152/ajpregu.00323.2001
0363-6119/02 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (8)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Janech, M. G.
Right arrow Articles by Ploth, D. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Janech, M. G.
Right arrow Articles by Ploth, D. W.
Vol. 282, Issue 5, R1490-R1500, May 2002

Molecular and functional characterization of a urea transporter from the kidney of a short-finned pilot whale

Michael G. Janech1, Ruihua Chen2, Janet Klein3, Mark W. Nowak4, Wayne McFee5, Richard V. Paul2,6, Wayne R. Fitzgibbon2, and David W. Ploth2,6

1 Department of Marine Biomedicine and Environmental Sciences, Medical University of South Carolina, Charleston 29412; 2 Division of Nephrology, Department of Medicine, 4 Department of Pharmacology and Experimental Therapeutics, Medical University of South Carolina, and 5 Center for Coastal Environmental Health and Biomolecular Research, US Department of Commerce/National Oceanographic and Atmospheric Administration/National Ocean Service, and 6 Ralph H. Johnson Veterans Affairs Medical Center, Charleston 29425; and 3 Renal Division, Department of Medicine, Emory University, Atlanta, Georgia 30322


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cetaceans (whales and dolphins) always excrete urine with an osmolality markedly higher than that of plasma. Although the mechanisms by which cetaceans concentrate urine have not been elucidated, data support a role for medullary urea accumulation in this process, as is the case for terrestrial mammals. Therefore, we hypothesized that facilitated urea transporters are present in the kidney of cetaceans. Using 5'/3'-rapid amplification of cDNA ends, we cloned a 2.7-kb cDNA from the kidney of the short-finned pilot whale Globicephala macrorhynchus. The putative open-reading frame encoded a 397-amino acid protein [pilot whale urea transporter A2 (whUT-A2)] that has 94% amino acid sequence identity to the A2 isoform of the human urea transporter (hUT-A2). Heterologous expression of whUT-A2 cRNA in Xenopus oocytes induced phloretin-inhibitable urea transport. Although Northern analysis and RT-PCR indicated that whUT-A2 was exclusively expressed in kidney, Western blotting using a polyclonal antibody to rat UT-A1/UT-A2 detected various immunoreactive proteins in kidney and other tissues. Furthermore, RT-PCR analysis suggested the presence of alternatively spliced UT-A transcripts in the kidney as well as extrarenal tissues. We conclude that renal urea transporters are highly conserved among mammals inhabiting terrestrial and pelagic environments. A urea-based concentrating mechanism, presumably evolved to meet the demands of an arid terrestrial environment, may have contributed a fortuitous preadaptation that enabled the ancestors of cetaceans to reinvade the sea.

urea reabsorption; cetaceans; osmoregulation; urinary concentration


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CETACEANS INHABIT AN ENVIRONMENT (1,100 mosmol/kgH2O) where fresh water is not accessible and the osmotic gradient favors water loss (plasma osmolality = 330-360 mosmol/kgH2O) (27, 34). Several physiological adaptations for water conservation in cetaceans have been identified, including reduced rate of respiratory water loss (11), the absence of sweat glands (17), and the production of concentrated urine (34). Water is obtained through food (fish and marine invertebrates), metabolism of body fat, and/or consumption of seawater (18, 34). Although controversial, other findings have suggested that water movement across the skin may also contribute to water balance (1, 18).

Because of their large size and the protection afforded cetaceans under treaties and laws, the number of studies investigating water and electrolyte balance has been limited. Because no extrarenal mechanism has been identified for the excretion of a salt load, the concentrating ability of the cetacean kidney has been considered sufficient for maintaining water and solute balance under conditions where water is obtained from metabolism of body fat and consumption of food only (22, 26, 34) or under conditions of an extra salt load after consumption of 0.5-2 l/day of seawater (34). In delphinid cetaceans, the osmolality of urine is always higher than that of plasma and usually markedly exceeds that of the surrounding ocean (except during fasting). Urine osmolalities are typically 1,300-2,000 mosmol/kgH2O and, under fasting conditions, have been reported to be 800-900 mosmol/kgH2O (28, 34).

On the basis of fossil evidence, mammals reinvaded the marine environment during the early Eocene, nearly 55 million years ago (2, 35, 45). Because present-day cetaceans do not appear to possess extrarenal organs for salt excretion and have only a small number of anatomic changes in the kidney (33), it is probable that the aquatic ancestors of modern cetaceans relied on physiological mechanisms already present in the kidney of their terrestrial counterparts to maintain water and electrolyte homeostasis in seawater. For terrestrial mammals, urinary concentration and water homeostasis are dependent on accumulation of urea in the renal medulla (3). Medullary accumulation of urea is, in turn, dependent on urea reabsorption across the epithelium of the inner medullary collecting duct and recycling of urea between the ascending vasa recta and the descending thin loops of Henle, between ascending and descending vasa recta, and between ascending and descending branches of the loops of Henle (24).

Urea moves rapidly across epithelia through urea transporter proteins. Two genes are known to encode for urea transporters (UT) in the mammalian kidney [UT-B (Slc14a1) and UT-A (Slc14a2); National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.niv.gov)].1 Four UT-A isoforms are expressed in the medullary tubular epithelia of the kidney (21, 31, 39, 42). UT-B is expressed in erythrocytes and in endothelial cells of the vasa recta (47, 51).

The isoforms of UT-A are highly conserved across the terrestrial mammalian species examined to date (37, 40). Given the degree of sequence conservation and the close relationship of cetaceans to terrestrial mammals, we proposed that urea transporters homologous to UT-A would also be present in the kidney of marine mammals. We report here the cloning of a functional urea transporter cDNA from the kidney of a short-finned pilot whale. The cloned cDNA had 91% nucleotide sequence identity with the human UT-A2 cDNA. The predicted protein encoded by this cDNA [whale urea transporter (whUT-A2)] is 94% identical to that of the predicted human UT-A2.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animal. A male short-finned pilot whale (Globicephala macrorhynchus), 424 cm long, was stranded alive on Sullivan's Island, near Charleston, SC, on 25 June 1998. After evaluation of its condition, the animal was euthanized and transported to the National Ocean Service Laboratory on James Island, Charleston, SC, for necropsy. Tissues used for this study were quickly dissected at the time of necropsy (~3 h after death) and stored at -80°C until processed. In addition, formalin-preserved and frozen samples were forwarded to the Armed Forces Institute of Pathology (Washington, DC) for histological analysis. Results from the evaluation performed by the Armed Forces Institute of Pathology indicated myocardial necrosis consistent with an infarct and diffuse fibrosis of the myocardium from previous heart muscle damage as a possible cause of death. Pulmonary and hepatic congestion also reflected acute heart failure.

RT-PCR. Kidney total RNA was isolated from whole tissue (cortex and medulla) using TRIzol reagent (GIBCO BRL, Gaithersburg, MD). Total RNA (5 µg) was reverse transcribed using a degenerate primer based on rat UT-A1 nt 3150-3129 (39) (GenBank accession no. U77971): 5'-GGA TCC GC(T/C) TG(A/G) TA(T/C) TTT GT(A/G/T) ATC ATC G-3' and Superscript II RNase H- reverse transcriptase (GIBCO BRL). PCR was performed using the above reverse primer and a forward primer based on nt 2672-2694 of the rat UT-A1: 5'-GAA TTC GG(A/G/T/C) TG(T/C) GA(T/C) AA(T/C) CC(A/G/T/C) TGG AC(A/G/T/C) GG-3'. First-strand-synthesized cDNA was amplified by Taq polymerase (1 unit; GIBCO BRL) in a 50-µl reaction volume containing the following reagents (expressed as final concentration): 5 µl of 10× Taq amplification buffer, 1.5 mM MgCl2, 0.2 mM dNTP mix, 0.2 µM forward primer, and 0.2 µM reverse primer. Products were amplified using a thermal DNA cycler (model 9700, Perkin-Elmer, Norwalk, CT) with initial denaturing for 5 min at 94°C and then 30 cycles of amplifications as follows: 60 s at 94°C, 60 s at 55°C, 2 min at 72°C, and final extension for 10 min at 72°C. The PCR product was run on an agarose gel, and a single band (500 bp) was gel purified and sequenced at the Biotechnology Resource Laboratory at the Medical University of South Carolina using an automated DNA sequencer (model ABI 377, PE Biosystems, Foster City, CA).

5'/3'-Rapid amplification of cDNA ends. 5'/3'-Rapid amplification of cDNA ends (RACE) was performed using the Marathon cDNA amplification kit (Clontech, Palo Alto, CA). Poly(A)+ RNA, isolated from total kidney RNA using Oligotex resin (Qiagen, Valencia, CA), was reverse transcribed using an oligo(dT) primer. Second-strand synthesis and adapter ligation were performed as described by Clontech (protocol PT1115-1, version PR8Y870). Forward and reverse gene-specific primers were designed on the basis of the nucleotide sequence of the pilot whale kidney RT-PCR product to conduct 5'/3'-RACE: 5'-TGC CAG GTG ATG ACG TAG AAC ATG C-3' (5'-RACE reverse primer) and 5'-CTC ACT CTC GCG ACA CCC TTT GAC T-3' (3'-RACE forward primer).

5'/3'-RACE was conducted using the 5'-RACE reverse primer or the 3'-RACE forward primer and the Marathon adapter primer (AP1): 5'-CCA TCC TAA TAC GAC TCA CTA TAG GGC-3'. Double-stranded, adapter-ligated cDNA was amplified in a 50-µl reaction volume as described above using Taq polymerase (GIBCO BRL) and cycled with initial denaturing for 30 s at 94°C followed by 30 cycles of amplifications as follows: 5 s at 94°C, 4 min at 68°C, and final extension for 10 min at 72°C. 5'-RACE (1,400 bp) and 3'-RACE (1,300 bp) PCR products were gel purified and separately subcloned into the pCRII plasmid (Invitrogen, Carlsbad, CA) for sequencing. We then utilized the sequence comparison resources available through BLAST (http://www.ncbi.nlm.nih.gov:80/BLAST/) to compare the test sequences with other known cDNAs and the CLUSTALW algorithm of the DNAStar software package to compare putative protein sequences.

Northern analysis. Poly(A)+ RNA (3 µg) from kidney, liver, muscle, lung, adrenal gland, and blubber was separated by electrophoresis on a 2.2 M formaldehyde-1% agarose gel and blotted onto a positively charged nylon membrane (Hybond-N+, Amersham, Arlington Heights, IL). Poly(A)+ RNA was immobilized by ultraviolet cross-linking (1 min) and prehybridized in Quikhyb (Stratagene, La Jolla, CA) for 1 h. We utilized a 3'-RACE PCR product (corresponding to nt 1417-2086) as a cDNA probe by labeling with [alpha -32P]dCTP using the Prime-It II random primer labeling kit (Stratagene). Hybridization was conducted in Quikhyb for 1 h at 65°C and washed: twice for 15 min at room temperature in 2× saline-sodium citrate buffer + 0.1% SDS and then for 30 min at 62°C in 0.1× saline-sodium citrate buffer + 0.1% SDS. The blot was visualized by exposure to autoradiographic film (Kodak-OMAT) for 10 and 72 h at -70°C.

Multitissue RT-PCR. Total RNA (5 µg) from kidney, liver, muscle, lung, adrenal gland, and blubber was reverse transcribed using an oligo(dT) primer and Superscript II RNase H- reverse transcriptase (GIBCO BRL). PCR was performed using the following primers: forward primer 1391 (corresponding to nt 1391-1415, 5'-CTC ACT CTC GCG ACA CCC TTT GAC T-3') and reverse primer 2472 (nt 2445-2472, 5'-CCC AAG TCT GGA GAA CAT CTC ATG ACT C-3') for set 1 (3' product; Fig. 1B) or forward primer 250 (corresponding to nt 250-274, 5'-GTT GCC AAG TGT GCA GCA AAT TCA A-3') with reverse primer 861 (corresponding to nt 839-861, 5'-GAG ATG GCC CAC CAG GGG TTC TG-3') for set 2 (5' product; see Fig. 1C). First-strand-synthesized cDNA was amplified using 0.6 units of HotStarTaq DNA polymerase (Qiagen) in a 50-µl reaction volume containing the following reagents (expressed as final concentration): 5 µl of HotStarTaq 10× amplification buffer (containing 15 mM MgCl2), 0.2 mM dNTP mix, 0.4 µM forward primer, and 0.4 µM reverse primer. Products were amplified by a thermal DNA cycler (model 9700, Perkin-Elmer) with initial denaturing for 15 min at 94°C and then 34 cycles of amplifications as follows: 60 s at 94°C, 30 s at 60°C, 2 min at 72°C, and final extension for 10 min at 72°C. PCR products were run on a 1% agarose gel. Product size was determined using a 1-kb DNA ladder (GIBCO BRL). PCR products were gel purified and sequenced by the Biotechnology Resource Laboratory at the Medical University of South Carolina using an automated DNA sequencer (model ABI 377, PE Biosystems). We then utilized the sequence comparison resources available through BLAST to compare the test sequences with whUT-A2 and other known cDNAs.


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 1.   Schematic representation of regions of whale urea transporter (whUT-A2) cDNA to which the cDNA probe for Northern analysis (A) and primers for RT-PCR (B and C) were designed. A: cDNA probe was generated from gel-purified cDNA derived from the 3'-rapid amplification cDNA end (RACE) product in pCRII cut with EcoRI and ApaI, resulting in a segment of the RACE product spanning nt 1417-2086. After random labeling with 32P, this product was used as a cDNA probe for Northern analysis. This probe would be expected to hybridize to whUT-A2 (and other transcripts homologous to the 3' region of whUT-A2). B: primers were designed for PCR amplification of a 1,081-nt product spanning nt 1391-2472 of the whUT-A2 cDNA. C: primers were designed for PCR amplification of a 611-nt product spanning nt 250-861 of the whUT-A2 cDNA. ORF, open reading frame.

Western analysis. Tissue samples were homogenized in isolation buffer (10 mM triethanolamine, 250 mM sucrose, 1 µg/ml leupeptin, 0.1 mg/ml phenylmethylsulfonyl fluoride, pH 7.6, 0.025-0.1 g tissue/ml isolation buffer). Concentrated SDS was added to the homogenized samples to achieve a final concentration of 1%, and then the samples were sheared by passage through a 28-gauge needle and centrifuged for 15 min at 14,000 g. Protein concentration was determined in the supernatant fractions using a protein assay kit (DC kit, Bio-Rad, Richmond, CA). After they were boiled in Laemmli sample buffer, proteins were separated on 10% SDS-polyacrylamide gels and then transferred to a polyvinylidene difluoride membrane. Membranes were blocked with 5% Carnation instant milk in Tris-buffered saline (TBS), pH 7.5, for 30 min at room temperature and then incubated at 4°C overnight with primary antibody (affinity-purified polyclonal anti-UT-A1 antibody prepared against the COOH-terminal portion of UT-A1/UT-A2/UT-A4) (20) in TBS with 0.5% Tween 20 (TBS-Tween) in a sealed bag. Membranes were washed twice for 15 min each with TBS-Tween and then incubated for a further 2 h at room temperature with horseradish peroxidase-linked anti-rabbit IgG (Amersham). After two washes with TBS-Tween, immunoreactive proteins were visualized by enhanced chemiluminescence (ECL, Amersham, Arlington Heights, IL).

In vitro transcription. cRNA was transcribed from a PCR-amplified cDNA containing the putative open-reading frame (ORF) encoding for a pilot whale urea transporter. A T7 phage promoter was added to the 5' end of the PCR product, 49 nt upstream of the putative ATG start site, using the following sense primer: 5'-TAA TAC GAC TCA CTA TAG GTG GGA GCT TCT TGC TCG TC-3'. PCR amplification was carried out using a reverse primer corresponding to nt 2472 (5'-CCC AAG TCT GGA GAA CAT CTC ATG ACT C-3'). Initial denaturation was for 1 min at 94°C and then 30 cycles of amplifications as follows: 1 min at 94°C, 30 s at 55°C, 2 min at 72°C, and 10 min at 72°C. A single product was gel purified and sequenced to confirm the addition of the T7 promoter sequence before transcription.

In vitro transcription was performed using the mMessage mMachine kit (Ambion, Austin, TX). cRNA was precipitated with LiCl overnight and dissolved in 20 µl of RNase-free water. RNA quality was assessed by electrophoresis on a 2.2 M formaldehyde-1% agarose gel, and concentration was determined by optical density at 260 nm.

Functional characterization. Oocytes were surgically removed from gravid Xenopus laevis and treated with collagenase type 1A-S (2 mg/ml; Sigma, St. Louis, MO) in calcium-free OR-2 solution (in mM: 82.5 NaCl, 2 KCl, 1 MgCl2, 5 HEPES, pH 7.4). Collagenase-treated oocytes were microinjected with 50 nl of water or 40 ng of cRNA in 50 nl of water. Injected oocytes were incubated at 18°C in modified Barth's medium [in mM: 88 NaCl, 1 KCl, 0.33 Ca(NO3)2, 0.41 CaCl2, 0.82 MgSO4, 2.4 NaHCO3, 10 HEPES, pH 7.5] adjusted to 200 mosmol/kgH2O with mannitol and treated with 50 µg/ml gentamicin. Oocytes were incubated at 18°C for 72 h before assay for urea uptake.

Uptake was determined in individual oocytes incubated in 200 µl of Barth's solution containing 8 µCi/ml (1.3 mM) [14C]urea (NEN Life Science Products, Boston, MA) at room temperature. Uptake was terminated after 300 s (49) by the addition of 2 ml of ice-cold Barth's solution containing 1.4 mM deionized urea. Individual oocytes were further washed three times with 2 ml of ice-cold Barth's (1.3 mM urea) medium. Oocytes were solubilized in 10% SDS (0.5 ml) and 4 ml of scintillation fluid at 20°C for 1 h with repeated vortexing. [14C]urea uptake was determined by liquid scintillation counting (model LS6500 liquid scintillation counter, Beckman, Fullerton, CA). The effect of phloretin on urea uptake was studied by preincubating oocytes in Barth's medium containing 0.5 mM phloretin (Sigma) for 20 min before uptake. Phloretin is a potent inhibitor of facilitated urea transport in tissues (25) and, at 0.2-1 mM, of facilitated [14C]urea uptake by urea transporter cRNA-injected Xenopus oocytes (12, 14, 32, 39, 40, 42, 43, 47, 49).

Statistical analysis. Data from the functional characterization studies were not distributed normally (Kolmogorov-Smirnov test). Therefore, the data were logarithmically transformed before statistical analysis using one-way ANOVA. Post hoc comparisons were tested using the Tukey-Kramer method. Statistical significance was set at P < 0.05. Nontransformed values (and logarithmically transformed data in parentheses) are means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Using degenerate primers designed to the rat UT-A1 nucleotide sequence, we amplified a 480-bp PCR product from pilot whale kidney mRNA that was 86% identical to the human UT-A2. 5'/3'-RACE using gene-specific primers was then used to obtain two PCR products, ~1,500 and 2,000 bp long, corresponding to overlapping 5' and 3' ends of the putative pilot whale urea transporter cDNA. Reconstruction of the full-length cDNA (Fig. 2; GenBank accession no. AY061881) revealed a putative ORF of 1,194 bp, a 580-bp 5'-untranslated region (5'-UTR), and an 873-bp 3'-UTR, not including the poly(A)+ tail. A polyadenylation signal sequence (ATTAAA) was located at nt 2629-2634 in the 3'-UTR.


View larger version (88K):
[in this window]
[in a new window]
  		Fig. 2.   Nucleotide sequence for the cDNA cloned from the pilot whale, which putatively encodes a 397-amino acid protein, whUT-A2, homologous to renal UT-A proteins from terrestrial mammals. The putative ATG start is preceded by 580 nucleotides containing several ATG codons (italicized). A polyadenylation signal is found between nucleotides 2629-2634. UTR, untranslated region.

The putative ORF of the pilot whale urea transporter was compared with the human and rat urea transporter nucleotide sequences located in GenBank. The putative ORF of the pilot whale urea transporter was found to be most similar in identity and size to the UT-A2 isoform (sequence identity was 91 and 88% for human and rat UT-A2, respectively). In contrast, although the ORF of the pilot whale urea transporter was similar in size to the rat UT-A3, UT-A4, and UT-B isoforms, it was considerably less similar in sequence identity (62, 70, and 67% identical to the UT-A3, UT-A4, and UT-B isoforms, respectively).

The ORF encodes for a putative protein containing 397 amino acids (Fig. 3). The molecular size of this protein was calculated to be 43 kDa. The amino acid sequence deduced from the cDNA sequence for the pilot whale urea transporter (whUT-A2) was found to be very similar to the human (94%), rabbit (91%), and rat (92%) UT-A2 isoforms. Consensus site analysis of whUT identified two putative glycosylation sites at amino acids 210-213 (NITW) and 288-291 (NSTL). Unlike human, rat, and rabbit UT-A2, whUT-A2 contains only a single putative protein kinase A consensus phosphorylation site at the COOH terminus (amino acid 383-386, RRAS). Two protein kinase C consensus phosphorylation sites were also identified at amino acids 26-28 (SGK) and 136-138 (SDK).


View larger version (90K):
[in this window]
[in a new window]
  		Fig. 3.   Sequence alignment between the predicted amino acid sequence for whUT-A2, human UT-A2 (hUT-A2), rabbit UT-A2 (oUT-A2), rat UT-A2 (rUT-A2), and rat UT-B1 (rUT-B1). Blocked sequences represent nonconserved amino acid substitutions determined by a consensus of all 5 sequences. whUT-A2 is most identical to hUT-A2 (94%).

Xenopus oocytes injected with 40 ng of whUT-A2 cRNA exhibited an 18-fold elevation in [14C]urea uptake compared with water-injected controls: 76.5 ± 15.3 (1.8 ± 0.1) vs. 4.3 ± 0.5 (0.6 ± 0.05) pmol · oocyte-1 · 300 s-1 (P < 0.05; Fig. 4). Preincubation with 0.5 mM phloretin inhibited [14C]urea uptake by 81% in whUT-injected oocytes [76.5 ± 15.3 (1.8 ± 0.1) vs. 14.7 ± 6.9 (0.9 ± 0.2) pmol · oocyte-1 · 300 s-1, P < 0.05] but did not alter [14C]urea uptake in water-injected control oocytes [4.3 ± 1.2 (0.6 ± 0.1) vs. 4.3 ± 0.5 (0.6 ± 0.05) pmol · oocyte-1 · 300 s-1].


View larger version (10K):
[in this window]
[in a new window]
  		Fig. 4.   Uptake of [14C]urea by oocytes injected with 40 ng of whUT-A2 cRNA (solid bars) or water (open bars). whUT-A2-injected oocytes exhibited an elevation in urea permeability 18-fold over control. Preincubation of whUT-A2-injected oocytes with 0.5 mM phloretin for 20 min abolished [14C]urea uptake. * P < 0.05 vs. water-injected controls. dagger  P < 0.05 vs. whUT-injected oocytes.

Northern analysis under high-stringency conditions resulted in the detection of two mRNA transcripts (4 and 2.7 kb) in kidney (Fig. 5). In contrast, no transcripts were detected in liver, muscle, lung, adrenal gland, or blubber (Fig. 5). It was possible that because the autoradiographic film was exposed to the blot for only 10 h, we failed to detect low-abundance transcripts in the extrarenal tissues. Therefore, the blot was reexposed to film for another 62 h. However, longer exposure did not result in detection of transcripts in the extrarenal tissues (data not shown).


View larger version (79K):
[in this window]
[in a new window]
  		Fig. 5.   Tissue expression of whUT-A2 mRNA determined using Northern blot analysis. Poly(A)+ RNA (3 µg) isolated from several tissues was hybridized at high stringency (62°C) with a random-primed [32P]dCTP cDNA probe specific to the whUT-A2 3'-RACE product. The blot was exposed to autoradiographic film for 10 h at -70°C. Two transcripts (4.0 and 2.7 kb) were detected in kidney. No transcripts were detected in other tissues.

Northern analysis was performed with a randomly labeled 3'-RACE probe that represented the cDNA sequence commencing at nt 1417 (within the ORF) and terminating at nt 2086 (within the 3'-UTR; Fig. 1A). The finding that whUT-A2 message was not detectable in extrarenal tissues suggested that transcripts homologous to whUT-A2 message are expressed only in kidney or the abundance of the whUT-A2 mRNA in the extrarenal tissues was too low for detection by Northern analysis. To address this latter concern, we utilized RT-PCR to examine the expression of whUT-A2 in kidney, liver, muscle, lung, adrenal, and blubber using primers designed to amplify a 1,081-nt-long product (corresponding to the ORF and most of the 3'-UTR encompassed by the 3'-RACE cDNA probe used in the Northern analysis; Fig. 1B). The findings are presented in Fig. 6A. A single PCR product of the expected length was amplified only from kidney. Sequence analysis of this product indicated that a segment of whUT-A2 cDNA had been amplified. In a second RT-PCR experiment, whUT-A2 gene-specific primers (primer set 2) were designed to amplify a 611-nt-long product that spanned a region that included a portion of the 5'-UTR, the predicted start site, and a portion of the 5'-ORF, i.e., a region upstream from that amplified by primer set 1 (Fig. 1C). The findings are presented in Fig. 6B. A product of the expected size was amplified only from kidney. This product had a sequence identical to the same 5' region of whUT-A2. In addition, several longer-than-expected products (1,100, 1,300, and 1,600 nt long) were amplified from kidney, and a single product (~1,300 nt long) was amplified from liver, muscle, and, to a lesser extent, adrenal gland. The 1,300-nt products from kidney, liver, and muscle were sequenced. These products were identical to each other and appear to represent a novel urea transporter isoform.


View larger version (46K):
[in this window]
[in a new window]
  		Fig. 6.   Tissue expression of whUT-A2 determined using RT-PCR. Products amplified after 34 cycles of PCR were run on agarose gels. Lane 1, kidney; lane 2, liver, lane 3, muscle; lane 4, lung; lane 5, adrenal gland; lane 6, blubber. First and last lanes contained DNA size markers. A: a single ~1,000-nt-long product identical to the 3' end of whUT-A2 was amplified from kidney. No products were amplified from other tissues. B: products obtained from RT-PCR of whale RNA using primers spanning a segment of the 5' sequence of whUT-A2. A product of the expected size (~600 nt) and sequence was amplified only from kidney. Additionally, several longer-than-expected urea transporter products were amplified from kidney, and a single urea transporter product (~1,300 nt long) was amplified from liver, muscle, and adrenal gland.

Taken together, the results from the Northern analysis and the RT-PCR experiments suggest that whUT-A2 is expressed in kidney but not in extrarenal tissues. Additionally, transcripts of novel UT-As appear to be expressed in kidney and extrarenal tissues.

The polyclonal antibody used in this study has previously been shown to be specific to UT-A1, UT-A2, and UT-A4 isoforms, since no immunoreactive proteins were detected in rat kidney using preimmune serum or the antiserum preincubated with immunizing peptide (21, 23, 30). Western analysis of the various pilot whale tissues revealed a number of immunoreactive proteins (Fig. 7). The pilot whale kidney samples reflected immunoreactive proteins present in whole kidney homogenate, not subsections. A broad band centered at 55 kDa was observed in the sample from the pilot whale kidney. This UT-A2-like band had the diffuse appearance representative of a glycosylated protein. Furthermore, a faint, discrete, higher-molecular-weight band was observed at 76 kDa. Interestingly, in rat kidney, the UT-A1 from inner medulla exists as two glycosylated variants (97 and 117 kDa) of a single 88-kDa parent protein (5), while rat outer medulla contains a diffuse 55-kDa UT-A2 band (48).


View larger version (47K):
[in this window]
[in a new window]
  		Fig. 7.   Western analysis of pilot whale tissues. Tissue homogenates (10 µg/lane) were separated using SDS-PAGE and probed with an antibody to the COOH terminus of rat urea transporter that should recognize UT-A1, UT-A2, and UT-A4 isoforms. Diffuse bands at 55 kDa were detected in all tissues except blubber. A prominent 37-kDa band (liver) as well as a 76-kDa band (liver and lung) may represent additional UT-A isoforms in these extrarenal tissues.

Strongly immunoreactive 41-, 53-, 76-, and 180-kDa proteins were obtained from pilot whale liver (Fig. 7). The muscle and lung from the pilot whale show diffuse bands centered at 55 kDa (similar to that observed for kidney; Fig. 7). In lung, there is also evidence of a tight, discrete, higher-molecular-weight band at 76 kDa (similar to that seen in the liver and, to a lesser extent, kidney). In contrast to the other tissues, UT-A-immunoreactive proteins did not appear to be present in blubber.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The mechanisms by which cetaceans maintain water and solute homeostasis are only partially understood. However, as with terrestrial mammals, urinary concentration is an important mechanism involved in the regulation of water and solute balance in cetaceans. It appears that cetaceans rely on physiological mechanisms that were present in the kidney of their terrestrial ancestors to maintain water and electrolyte homeostasis in seawater. Urea accumulation in the medulla underlies the urinary concentration process in terrestrial mammals. Urea transporter proteins (products of the Slc14a1 and Slc14a2 genes) regulate urea accumulation in the renal medulla. The UT-A family facilitates urea transport across tubular epithelia, while UT-B functions to return urea taken up by the medullary circulation to the medullary interstitium. Therefore, we proposed that urea transporters homologous to UT-A would also be present in the kidney of marine mammals.

We utilized the technique of 5'/3'-RACE to clone a cDNA with a putative ORF encoding a 397-amino acid urea transporter homolog from the kidney of the short-finned pilot whale. The cDNA and the putative urea transporter protein showed a high degree of sequence identity to mammalian UT-A2, especially the human isoform. We have designated this urea transporter whUT-A2.

On the basis of the predicted amino acid sequence, UT-A2 appears to be highly conserved between cetaceans and eutherian mammals relative to other proteins reported from cetaceans (Table 1). Considering the phylogenetic alignment of cetaceans within the artiodactyl clade (29) and the high level of sequence conservation of UT-A2 among eutherian mammals, it is predicted that a similar level of UT-A2 sequence conservation exists between the artiodactyls and other mammalian orders.

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Comparison of amino acid identity between cetacean and terrestrial mammal proteins deduced from cDNA

Heterologous expression of whUT-A2 cRNA in Xenopus oocytes resulted in an increase in urea permeability comparable to that obtained for other mammalian urea transporters (42, 52). Furthermore, whUT-A2-induced urea transport was inhibited by phloretin, a finding characteristic of all facilitated urea transporters (12, 14, 21, 39, 42, 43 49).

High-stringency Northern analysis of poly(A)+ RNA from pilot whale tissues indicated kidney-specific expression of two bands at 4.0 and 2.7 kb. The pattern of kidney urea transporter mRNA expression was almost identical to that reported for UT-A1 and UT-A2 mRNA transcripts from other mammals (21, 42, 52). For example, Northern blot analysis detected two separate bands when RNA from the kidneys of Sprague-Dawley rats was hybridized with UT-A2 cDNA probes. The lower-molecular-weight transcripts (2.9-3.0 kb) appear to code for UT-A2, while the larger transcripts (4.0 kb) appear to code for UT-A1 (42). In the present study, the molecular size of the lower band corresponded almost exactly to that determined for whUT-A2 cDNA. Thus the presence of the lower-molecular-weight transcript confirmed that whUT-A2 is expressed in pilot whale kidney. Furthermore, the finding of a 4-kb mRNA transcript suggests that a larger urea transporter isoform, possibly UT-A1, may also be expressed in pilot whale kidney.

The kidney-specific expression of whUT-A2 message was confirmed by RT-PCR. Using gene-specific primers to the 5' or 3' region of whUT-A2, we were able to amplify cDNAs of the predicted sizes from kidney but not from the other tissues studied.

The antibody used to perform the Western analyses was prepared to the COOH-terminal peptide sequence of rat UT-A1/UT-A2 (30). This antibody has been found to also cross react with rat UT-A4 [inasmuch as this urea transporter also shares the same COOH-terminal sequence with UT-A1 and UT-A2 (19)] and UT-A isoforms from other mammalian species but does not appear to recognize UT-B gene products (23, 30). Because the putative COOH terminus of whUT-A2 was very similar to that of the other mammalian urea transporters, we expected that the antibody would detect the presence of whUT-A2 as well as other UT-A isoforms in whale tissues. In kidney, a diffuse immunoreactive band was detected at ~55 kDa. This molecular size was larger than the predicted size of whUT-A2 (43 kDa) but could be expected for a 397-amino acid protein that had undergone glycosylation and was similar to the size (55 kDa) reported for rat UT-A2 (48).

A 76-kDa immunoreactive protein (detected as a discrete band on Western analysis) was observed in the kidney of the pilot whale. It is possible that this protein is UT-A1. However, it is smaller in size than other mammalian UT-A1 proteins; e.g., in the rat kidney, UT-A1 is expressed as two glycosylated isoforms (97 and 117 kDa) of a common 88-kDa protein (5).

Although message for whUT-A2 could be detected only in kidney, using PT-PCR, we detected transcripts for novel UT-A isoforms in extrarenal tissues (Fig. 6B). Therefore, we used Western analysis to determine whether UT-A-immunoreactive proteins are expressed extrarenally. We observed a number of UT-A-immunoreactive proteins in liver, muscle, and lung. A protein similar in size to whUT-A2 (55 kDa) was expressed in muscle and lung. Furthermore, a 76-kDa protein, similar in size to that observed in kidney, was heavily expressed in liver, muscle, and lung. Interestingly, a strong set of bands at 41 and 53 kDa were observed in liver. These proteins are similar in size to the 36- and 49-kDa UT-A isoforms reported to be present in rat liver (23). In contrast, the 76- and 180-kDa proteins observed in the liver of the pilot whale do not appear to be present in liver from terrestrial mammals (23). The 180-kDa protein may represent an isoform that is novel to cetaceans or a urea transporter protein complex similar to the 206-kDa UT-A1 complex observed in rat kidney (5). Taken together, these findings indicate that urea transporters belonging to the UT-A family are expressed in extrarenal tissues in the pilot whale and that these proteins are encoded by splice variants of Slc14a2 other than whUT-A2. Isolation of the complete cDNAs for these splice variants will require further studies.

Interestingly, there have been conflicting reports regarding UT-A gene expression in extrarenal tissues of other mammals. In the original report of the cloning and characterization of the facilitated urea transporters, You and co-workers (52) reported that, under high-stringency conditions, a UT-A2 (UT2) cDNA probe hybridized to 3- and 4-kb transcripts from rabbit kidney and colon. Furthermore, under low-stringency conditions, they observed additional hybridization to 3- and 4-kb transcripts from liver and lung (52). In contrast, Northern analyses under high-stringency conditions using a full-length UT-A2 (UT2) probe or specific probes to UT-A1 or UT-A2 did not detect transcripts in extrarenal tissues from the human (32) or rat (21, 39, 42). In contrast to these latter findings, the antibody used in the present study has detected urea transporterimmunoreactive proteins in the liver of rats (23). The findings obtained by Northern analysis using specific cDNA probes have also been conflicting. Specific cDNA probes that recognize UT-A1/UT-A3/UT-A4 isoforms have been used to detect ~1.7-kb transcripts in a number of extrarenal tissues (testes, heart, brain, liver, and skeletal muscle), and a specific probe to UT-A3 has been used to detect a ~3.4-kb transcript in testes (19). In contrast to these findings, Northern analysis using a probe to UT-A1/UT-A3/UT-A4 did not detect transcripts to UT-A3 in extrarenal tissues (40). Despite these conflicting results, the recent cloning of a novel UT-A isoform (UT-A5) from testes (14) indicates that UT-A gene expression does occur in extrarenal tissues.

Extrarenal urea transporters may have specific functions depending on the organ in which they are expressed. Because the liver is the main site of ureagenesis, urea transporters would allow the rapid secretion of urea into the circulation (23). Urea cycle enzymes in the liver of marine mammals have activities similar to those of equivalent enzymes in the liver of terrestrial mammals (6). Therefore, urea transporters in the liver of marine mammals would be expected to play a similar role in terrestrial mammals. Although a functional urea cycle does not appear to be present in extrahepatic tissues, arginase, which catalyzes the hydrolysis of arginine to ornithine and urea, is expressed in extrahepatic tissues. In terrestrial mammals, there are two isoforms of this enzyme (20): arginase I is a cytosolic enzyme that catalyzes the final reaction of the urea cycle and is highly expressed in liver and in many other cell types (20), and arginase II is an extrahepatic, mitochondrial enzyme that is expressed in numerous tissues, including muscle and lung (8, 16, 20), and in endothelial cells (7). Although arginase II participates in arginine homeostasis (41), the functional significance of the extrahepatic arginases is not completely understood. Because ornithine is the precursor of proline, glutamate, and the polyamines, the extrahepatic arginases may play an important role in the synthesis of these amino acids and/or polyamines (50). Thus the "A" family of urea transporters in the extrahepatic, extrarenal tissues may function to excrete urea, formed as a by-product of the synthesis of proline, glutamate, and/or polyamines, into the interstitium or directly into the circulation (47). Urea transporters may also function to transport urea across fluid-secreting epithelia and, thus, facilitate urea-dependent fluid movement; e.g., fluid movement into seminiferous tubules may occur as a result of urea excretion by Sertoli cells (14). Although the presence of arginase in extrarenal, extrahepatic tissues of marine mammals has yet to be determined, the finding of the present study that a number of UT-A-immunoreactive proteins are present in whale tissues supports the suggestion that, in marine mammals, urea is formed extrahepatically. The extrarenal, extrahepatic urea transporters would be expected to have functions similar to those in terrestrial mammals.

The ability of mesic mammals (those in arid environments but with standing water available) to generate concentrated urine is proportional to the medullary thickness corrected for body weight (4). In contrast, marine mammals have thinner outer and inner medullary regions than would be expected for their size but are able to concentrate their urine to a degree similar to that of large mammals in an arid environment (4). Although novel morphological features such as the sporta perimedullaris musculosa (a layer of muscle surrounding the medullary pyramid) (9) and medullary vasa recta bundling (33) have been described for the kidneys of whales, the anatomic features or physiological mechanisms that have evolved to enable sustained urinary concentrating ability in the face of a proportionally smaller medullary thickness have not been elucidated. An interesting corollary of the ability of cetaceans to concentrate their urine to a degree similar to that of large mammals in an arid environment is that they do not appear to be able to excrete dilute urine. In fasted dolphins given 4 liters of distilled water directly into the stomach, urine concentration decreased, but urine osmolality remained markedly higher than plasma osmolality (28, 34). In terrestrial mammals, regardless of the state of water balance, tubular fluid undergoes dilution on passage through the cortical thick ascending limb of the loop of Henle and the early distal tubule as a result of NaCl reabsorption with only minimal water reabsorption. Marine mammals have a very thin cortex compared with terrestrial mammals of comparable body mass (4). It is possible to infer from this finding that, for marine mammals, the length of the cortical thick ascending limb of the loop of Henle and the early distal tubule along which NaCl reabsorption can occur would be proportionally shorter than for terrestrial mammals. As a consequence, tubular fluid may not undergo significant dilution as it passes through the cortex. Thus the markedly lower relative thickness of the cortex may account, at least part, for the inability of cetaceans to dilute urine to an osmolality near that of plasma.

Although the physiological role of the extrarenal urea transporters remains uncertain, the function of renal whUT-A2 appears to be essential for life in the ocean. In terrestrial mammals, urinary concentration is dependent on accumulation of urea in the renal medulla, which in turn is dependent on urea transporter proteins. The finding of a highly conserved UT-A isoform in the kidney of the short-finned pilot whale, the requirement for sustained urinary concentrating ability, and the recent data confirming the artiodactyl ancestry of cetaceans (15, 46) support the conclusion that a urea-based renal concentrating mechanism constituted a fortuitous preadaptation that enabled the ancestors of cetaceans to reinvade the sea.

Consensus site analysis of the putative UT-A2 proteins suggests that there are differences in functional domains between terrestrial and marine mammals. The apparent molecular size of the mammalian UT-A2 appears to be due to glycosylation of the native protein. Interestingly, although the 397-amino acid whUT-A2 appears to be similar in molecular size (55 kDa) to the 397-amino acid UT-A2 (55 kDa), the whUT-A2 appears to have only two glycosylation sites, while, in contrast, terrestrial mammals appear to contain three (rat and rabbit) or four (human) such sites (determined from sequence data available in GenBank). Also, in contrast to human, rat, and rabbit UT-A2s, which have two cAMP/cGMP-dependent phosphorylation consensus sites (one at the NH2- and one at the COOH-terminal portion), whUT-A2 has only a single cAMP/cGMP-dependent protein kinase phosphorylation site at amino acids 383-386; i.e., the NH2-terminal site is absent in whUT-A2. The importance of these differences in functional domains between the terrestrial and marine mammal UT-A2 is unknown. It is intriguing to speculate that the missing protein kinase A phosphorylation site in whUT-A2 indicates that this urea transporter may be regulated differently from the renal UT-A2 of terrestrial mammals. Testing of this speculation awaits comparative functional analysis of whUT-A2 and other mammalian urea transporters.


    ACKNOWLEDGEMENTS

We thank the South Carolina Department of Natural Resources and South Carolina Marine Mammal Network volunteers for their response to this stranded whale and Dr. J. Ohlandt and the Town of Sullivan's Island and Sullivan's Island Police Department for logistical support at the stranding site. We also thank J. Sands for scientific input and critical reading of the manuscript, J. Raymond, M. Budisavljevic, and B. Tholanikunnel for expert advice regarding the molecular approaches to these studies, and G. Rousselet and L. Ball for valuable advice regarding the functional characterization of the cloned transporter.


    FOOTNOTES

Sample collection was made possible through the responsibility of the National Oceanographic and Atmospheric Administration under the Marine Mammal Health and Stranding Response Act (1993).

This research was supported in part by Division of Nephrology Research funds and from funds provided by Dialysis Clinics. M. G. Janech is a student in the Marine Biomedicine and Environmental Sciences Graduate Program and was supported by a Dean's Graduate Fellowship from the Department of Graduate Studies, Medical University of South Carolina.

This work was presented in part at Experimental Biology 2000, San Diego, CA, and published in abstract form (FASEB J 14: 277.22, 2000).

Present address of R. Chen: FMC, Route 1 and Plainsboro Rd., Princeton, NJ 08543.

1 Assignment of gene name has been reversed by the NCBI from that indicated in Refs. 13 and 40. We will use the updated assignment of gene name from NCBI.

Address for reprint requests and other correspondence: W. R. Fitzgibbon, Div. of Nephrology, Dept. of Medicine, Medical University of South Carolina, 96 Jonathan Lucas St., Charleston, SC 29425 (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.

10.1152/ajpregu.00323.2001

Received 7 June 2001; accepted in final form 14 January 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Andersen, SH, and Nielsen E. Exchange of water between the harbor porpoise, Phocoena phocoena, and the environment. Experientia 39: 52-53, 1983[ISI][Medline].

2.   Bajpai, S, and Gingerich PD. A new Eocene archaeocete (Mammalia, Cetacea) from India and the time of origin of whales. Proc Natl Acad Sci USA 95: 15464-15468, 1998[Abstract/Free Full Text].

3.   Bankir, L. Urea and the kidney. In: The Kidney (3rd ed.), edited by Brenner BM, and Rector FC.. Philadelphia, PA: Saunders, 1996, p. 571-606.

4.   Beuchat, CA. Structure and concentrating ability of the mammalian kidney: correlations with habitat. Am J Physiol Regulatory Integrative Comp Physiol 271: R157-R179, 1996[Abstract/Free Full Text].

5.   Bradford, AD, Terris JM, Ecelbarger CA, Klein JD, Sands JM, Chou CL, and Knepper MA. 97- and 117-kDa forms of collecting duct urea transporter UT-A1 are due to different states of glycosylation. Am J Physiol Renal Physiol 281: F133-F143, 2001[Abstract/Free Full Text].

6.   Brown, GW, Jr, and Mayhall WS. On urea formation in marine mammals. Experientia 33: 591-592, 1977[ISI][Medline].

7.   Buga, GM, Singh R, Pervin S, Rogers NE, Schmitz DA, Jenkinson CP, Cederbaum SD, and Ignarro LJ. Arginase activity in endothelial cells: inhibition by NG-hydroxy-L-arginine during high-output NO production. Am J Physiol Heart Circ Physiol 271: H1988-H1998, 1996[Abstract/Free Full Text].

8.   Carraway, MS, Piantadosi CA, Jenkinson CP, and Huang YC. Differential expression of arginase and iNOS in the lung in sepsis. Exp Lung Res 24: 253-268, 1998[ISI][Medline].

9.   Cave, AJE, and Aumonier FJ. The reninculus of Tursiops truncatus, Stenella longirostris and other cetaceans. J R Microsc Soc 86: 323-342, 1967[Medline].

10.   Cooper, CB, Winkfein RJ, Szerencsei RT, and Schnetkamp PP. cDNA cloning and functional expression of the dolphin retinal rod Na-Ca + K exchanger NCKX1: comparison with the functionally silent bovine NCKX1. Biochemistry 38: 6276-6283, 1999[Medline].

11.   Coulombe, HN, Ridgway SH, and Evans WE. Respiratory water exchange in two species of porpoise. Science 149: 86-88, 1965[Abstract/Free Full Text].

12.   Couriaud, C, Leroy C, Simon M, Silberstein C, Bailly P, Ripoche P, and Rousselet G. Molecular and functional characterization of an amphibian urea transporter. Biochim Biophys Acta 1421: 347-352, 1999[Medline].

13.   Fenton, RA, Hewitt JE, Howorth A, Cottingham CA, and Smith CP. The murine urea transporter genes Slc14a1 and Slc14a2 occur in tandem on chromosome 18. Cytogenet Cell Genet 87: 95-96, 1999[ISI][Medline].

14.   Fenton, RA, Howorth A, Cooper GJ, Meccariello R, Morris ID, and Smith CP. Molecular characterization of a novel UT-A urea transporter isoform (UT-A5) in testis. Am J Physiol Cell Physiol 279: C1425-C1431, 2000[Abstract/Free Full Text].

15.   Gingerich, PD, Haq M, Zalmout IS, Khan IH, and Malkani MS. Origin of whales from early artiodactyls: hands and feet of eocene protocetidae from Pakistan. Science 293: 2239-2242, 2001[Abstract/Free Full Text].

16.   Gotoh, T, Sonoki A, Nagasaki K, Terada K, Takiguchi M, and Mori M. Molecular cloning of cDNA for nonhepatic mitochondrial arginase (arginase II) and comparison of its induction with nitric oxide synthase in a murine macrophage-like cell line. FEBS Lett 395: 119-122, 1996[ISI][Medline].

17.   Harrison, RJ, and Thurley KW. Fine structural features of delphinid epidermis. J Anat 111: 498-500, 1972[ISI][Medline].

18.   Hui, CA. Seawater consumption and water flux in the common dolphin Delphinus delphis. Physiol Zool 55: 430-440, 1981.

19.   Inoue, Y, Itou T, Jimbo T, Sakai T, Ueda K, Imajoh-Ohmi S, and Iida T. Molecular cloning and identification of bottle-nosed dolphin flavocytochrome bgp91phox and p22phox subunits. Vet Immunol Immunopathol 76: 137-150, 2000[ISI][Medline].

20.   Jenkinson, CP, Grody WW, and Cederbaum SD. Comparative properties of arginases. Comp Biochem Physiol B Biochem Mol Biol 114: 107-132, 1996[Medline].

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

22.   Kirschner, LB. Extrarenal mechanisms in hydromineral and acid-base regulation in aquatic vertebrates. In: Handbook of Physiology. Comparative Physiology. Bethesda, MD: Am. Physiol. Soc, 1997, sect. 13, chapt. 9, p. 577-622.

23.   Klein, JD, Timmer RT, Rouillard P, Bailey JL, and Sands JM. UT-A urea transporter protein expressed in liver: upregulation by uremia. J Am Soc Nephrol 10: 2076-2083, 1999[Abstract/Free Full Text].

24.   Knepper, MA, and Roch-Ramel F. Pathways of urea transport in the mammalian kidney. Kidney Int 31: 629-633, 1987[ISI][Medline].

25.   Knepper, MA, and Star RA. The vasopressin-regulated urea transporter in renal inner medullary collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol 259: F393-F401, 1990[Abstract/Free Full Text].

26.   Krogh, A. Osmotic Regulation in Aquatic Animals. Cambridge, UK: University Press, 1939, p. 242.

27.   Lambertsen, RH, Birnir B, and Bauer JE. Serum chemistry and evidence of renal failure in the North Atlantic fin whale population. J Wildl Dis 22: 389-396, 1986[Abstract].

28.   Malvin, RL, and Rayner M. Renal function and blood chemistry in Cetacea. Am J Physiol 214: 187-191, 1968.

29.   Milinkovitch, MC, Berube M, and Palsboll PJ. Cetaceans are highly derived artiodactyls. In: The Emergence of Whales. Evolutionary Patterns in the Origin of Cetacea, edited by Thewissen JGM. New York: Plenum, 1998, p. 113-131.

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

31.   Nielsen, S, Terris J, Smith CP, Hediger MA, Ecelbarger CA, and Knepper MA. Cellular and subcellular localization of the vasopressin-regulated urea transporter in rat kidney. Proc Natl Acad Sci USA 93: 5495-5500, 1996[Abstract/Free Full Text].

32.   Olives, B, Martial S, Mattei MG, Matassi G, Rousselet G, Ripoche P, Cartron JP, and Bailly P. Molecular characterization of a new urea transporter in the human kidney. FEBS Lett 386: 156-160, 1996[ISI][Medline].

33.   Pfeiffer, CJ. Renal cellular and tissue specializations in the bottlenose dolphin (Tursiops truncatus) and beluga whale (Delphinapterus leucas). Aquatic Mammals 23: 75-84, 1997.

34.   Ridgeway, SH. Homeostasis in the aquatic environment. In: Mammals of the Sea: Biology and Medicine, edited by Ridgeway SH.. Springfield, IL: Thomas, 1972, p. 590-747.

35.   Roe, LJ, Thewissen JGM, Quade J, O'Neil JR, Bajpai S, Shani A, and Hussain ST. Isotopic approaches to understanding the terrestrial-to-marine transition of the earliest cetaceans. In: The Emergence of Whales. Evolutionary Patterns in the Origin of Cetacea, edited by Thewissen JGM. New York: Plenum, 1998, p. 399-422.

36.   Romano, TA, Ridgway SH, Felten DL, and Quaranta V. Molecular cloning and characterization of CD4 in an aquatic mammal, the white whale Delphinapterus leucas. Immunogenetics 49: 376-383, 1999[ISI][Medline].

37.   Sands, JM. Regulation of renal urea transporters. J Am Soc Nephrol 10: 635-646, 1999[Abstract/Free Full Text].

38.   Semba, U, Shibuya Y, Okabe H, Hayashi I, and Yamamoto T. Whale high-molecular-weight and low-molecular-weight kininogens. Thromb Res 97: 481-490, 2000[ISI][Medline].

39.   Shayakul, C, Steel A, and Hediger MA. Molecular cloning and characterization of the vasopressin-regulated urea transporter of rat kidney collecting ducts. J Clin Invest 98: 2580-2587, 1996[ISI][Medline].

40.   Shayakul, C, Tsukaguchi H, Berger UV, and Hediger MA. Molecular characterization of a novel urea transporter from kidney inner medullary collecting ducts. Am J Physiol Renal Physiol 280: F487-F494, 2001[Abstract/Free Full Text].

41.   Shi, O, Morris SM, Jr, Zoghbi H, Porter CW, and O'Brien WE. Generation of a mouse model for arginase II deficiency by targeted disruption of the arginase II gene. Mol Cell Biol 21: 811-813, 2001[Abstract/Free Full Text].

42.   Smith, CP, Lee WS, Martial S, Knepper MA, You G, Sands JM, and Hediger MA. Cloning and regulation of expression of the rat kidney urea transporter (rUT2). J Clin Invest 96: 1556-1563, 1995.

43.   Smith, CP, and Wright PA. Molecular characterization of an elasmobranch urea transporter. Am J Physiol Regulatory Integrative Comp Physiol 276: R622-R626, 1999[Abstract/Free Full Text].

44.   St-Laurent, G, and Archambault D. Molecular cloning, phylogenetic analysis and expression of beluga whale (Delphinapterus leucas) interleukin 6. Vet Immunol Immunopathol 73: 31-44, 2000[ISI][Medline].

45.   Thewissen, JGM Phylogenetic aspects of cetecean origins: a morphological perspective. J Mamm Evol 2: 157-183, 1994.

46.   Thewissen, JGM, Williams EM, Roe LJ, and Hussain ST. Skeletons of terrestrial cetaceans and the relationship of whales to artiodactyls. Nature 413: 277-281, 2001[Medline].

47.   Tsukaguchi, H, Shayakul C, Berger UV, Tokui T, Brown D, and Hediger MA. Cloning and characterization of the urea transporter UT3: localization in rat kidney and testis. J Clin Invest 99: 1506-1515, 1997[ISI][Medline].

48.   Wade, JB, Lee AJ, Liu J, Ecelbarger CA, Mitchell C, Bradford AD, Terris J, Kim GH, and Knepper MA. UT-A2: a 55-kDa urea transporter protein in thin descending limb of Henle's loop whose abundance is regulated by vasopressin. Am J Physiol Renal Physiol 278: F52-F62, 2000[Abstract/Free Full Text].

49.   Walsh, PJ, Heitz MJ, Campbell CE, Cooper GJ, Medina M, Wang YS, Goss GG, Vincek V, Wood CM, and Smith CP. Molecular characterization of a urea transporter in the gill of the gulf toadfish (Opsanus beta). J Exp Biol 203: 2357-2364, 2000[Abstract].

50.   Wu, G, and Morris SM, Jr. Arginine metabolism: nitric oxide and beyond. Biochem J 336: 1-17, 1998.

51.   Xu, Y, Olives B, Bailly P, Fischer E, Ripoche P, Ronco P, Cartron JP, and Rondeau E. Endothelial cells of the kidney vasa recta express the urea transporter HUT11. Kidney Int 51: 138-146, 1997[ISI][Medline].

52.   You, G, Smith CP, Kanai Y, Lee WS, Stelzner M, and Hediger MA. Cloning and characterization of the vasopressin-regulated urea transporter. Nature 365: 844-847, 1993[Medline].

Am J Physiol Regul Integr Comp Physiol 282(5):R1490-R1500


This article has been cited by other articles:


Home page
 Am. J. Physiol. Renal Physiol.Home page
J. C. Q. Velez, A. M. Bland, J. M. Arthur, J. R. Raymond, and M. G. Janech
Characterization of renin-angiotensin system enzyme activities in cultured mouse podocytes
Am J Physiol Renal Physiol, July 1, 2007; 293(1): F398 - F407.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
M. G. Janech, W. R. Fitzgibbon, R. Chen, M. W. Nowak, D. H. Miller, R. V. Paul, and D. W. Ploth
Molecular and functional characterization of a urea transporter from the kidney of the Atlantic stingray
Am J Physiol Renal Physiol, May 1, 2003; 284(5): F996 - F1005.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
S. M. Bagnasco
Gene structure of urea transporters
Am J Physiol Renal Physiol, January 1, 2003; 284(1): F3 - F10.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
J. M. Sands
Molecular Approaches to Urea Transporters
J. Am. Soc. Nephrol., November 1, 2002; 13(11): 2795 - 2806.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed