Roles of Slc13a1 and Slc26a1 sulfate transporters of eel kidney in sulfatehomeostasis and osmoregulation in freshwater

Tsutomu Nakada,1 Kambiz Zandi-Nejad,2 Yukihiro Kurita,1 Hisayuki Kudo,1 Vadjista Broumand,2

Charles Y. Kwon,2 Adriana Mercado,2 David B. Mount,2,3 and Shigehisa Hirose1

1Department of Biological Sciences, Tokyo Institute of Technology, Yokohama, Japan; 2RenalDivision, Brigham and Women’s Hospital, Harvard Medical School, Boston; and 3RenalDivision, Veterans Affairs Boston Healthcare System, Boston, Massachusetts

Submitted 25 October 2004; accepted in final form 29 March 2005

Nakada, Tsutomu, Kambiz Zandi-Nejad, Yukihiro Kurita, Hi-sayuki Kudo, Vadjista Broumand, Charles Y. Kwon, AdrianaMercado, David B. Mount, and Shigehisa Hirose. Roles of Slc13a1and Slc26a1 sulfate transporters of eel kidney in sulfate homeostasisand osmoregulation in freshwater. Am J Physiol Regul Integr CompPhysiol 289: R575–R585, 2005. First published March 31, 2005;doi:10.1152/ajpregu.00725.2004.—Sulfate is required for proper cellgrowth and development of all organisms. We have shown that therenal sulfate transport system has dual roles in euryhaline eel, namely,maintenance of sulfate homeostasis and osmoregulation of bodyfluids. To clarify the physiological roles of sulfate transporters inteleost fish, we cloned orthologs of the mammalian renal sulfatetransporters Slc13a1 (NaSi-1) and Slc26a1 (Sat-1) from eel (Anguillajaponica) and assessed their functional characteristics, tissue localiza-tion, and regulated expression. Full-length cDNAs coding forajSlc13a1 and ajSlc26a1 were isolated from a freshwater eel kidneycDNA library. Functional expression in Xenopus oocytes revealed theexpected sulfate transport characteristics; furthermore, both transport-ers were inhibited by mercuric chloride. Northern blot analysis, in situhybridization, and immunohistochemistry demonstrated robust apicaland basolateral expression of ajSlc13a1 and ajSlc26a1, respectively,within the proximal tubule of freshwater eel kidney. Expression wasdramatically reduced after the transfer of eels from freshwater toseawater; the circulating sulfate concentration in eels was in turnmarkedly elevated in freshwater compared with seawater conditions(19 mM vs. 1 mM). The reabsorption of sulfate via the apicalajSlc13a1 and basolateral ajSlc26a1 transporters may thus contributeto freshwater osmoregulation in euryhaline eels, via the regulation ofcirculating sulfate concentration.

freshwater adaptation; immunohistochemistry; sulfate transporter; re-nal proximal tubule

SULFATE IS ESSENTIAL for a variety of metabolic and cellularprocesses, including production of highly sulfated proteogly-cans by chondrocytes, detoxification, and elimination of xeno-biotics and endogenous compounds by sulfoconjugation in theliver and kidney, and biosynthesis of sulfated hormones suchas gastrin and cholecystokinin (27). Mechanisms regulating thelevels of plasma sulfate are therefore essential for the mainte-nance of normal physiology. In mammals, the regulation ofsulfate homeostasis is largely determined by the kidney, withthe major fraction of filtered sulfate being reabsorbed in theproximal tubule. Two sulfate transporters have been identifiedthat are involved in this reabsorption of sulfate from the

glomerular ultrafiltrate: solute carrier family 13a1 (Slc13a1;NaSi-1) and solute carrier family 26a1 (Slc26a1; Sat-1).Slc13a1 is an electrogenic Na�-dependent sulfate transporter(alternatively called Na�-SO4

2� cotransporter) that is located inthe apical membrane of renal proximal tubule cells and medi-ates entry of Na�-SO4

2� with a stoichiometry of 3:1 (5, 24).Slc26a1 is sulfate/anion exchanger mediating SO4

2� effluxacross the basolateral membrane in exchange for HCO3

� (17).Physiological significance of this regulatory system is wellestablished in mammals through targeted disruption of theSlc13a1 gene (9).

Physiological studies on sulfate homeostasis also have beenconducted in nonmammalian systems, including those of birds(12, 40, 44, 45), bivalve (11), and fish (10, 36, 37, 43, 46–49).However, little is known concerning their molecular mecha-nisms. We were interested in the regulation of sulfate ho-meostasis in euryhaline fish, which migrate between freshwaterand sulfate-rich seawater, and initiated cloning of eel orthologsof mammalian Slc13a1 and Slc26a1 as the first step towardclarification of molecular mechanisms involved in sulfate han-dling by euryhaline fish exposed to a wide range of environ-mental sulfate concentrations. Cloning of the relevant cDNAs,followed by functional analysis, Northern blot analysis, RNAin situ hybridization histochemistry, and immunohistochemis-try, indicated that a transport system very similar to that ofmammals is operating in freshwater eel kidneys that is rapidlyand dramatically downregulated under seawater conditions.Interestingly, the eel sulfate transport system, composed ofapical Slc13a1 and basolateral Slc26a1, turned out to play dualroles under freshwater conditions: 1) to prevent sulfate loss andmaintain sulfate homeostasis and 2) to accumulate and retainrelatively high concentrations of sulfate as an osmolyte. Ourfindings help explain not only the mechanism of maintainingsulfate homeostasis in freshwater but also why adult eelsmigrate to freshwater (32).

MATERIALS AND METHODS

Animals. Japanese eel (Anguilla japonica) were purchased from alocal dealer. They were reared in a freshwater tank for 2 wk (fresh-water eels). Some eels were transferred to a seawater tank andacclimated for 2 wk before use (seawater eels). The water temperaturewas maintained at 18–22°C. All eels were anesthetized by immersionin 0.1% ethyl m-aminobenzoate (MS-222 or tricaine) before beingkilled by decapitation. The tissues required for RNA extraction were

Address for reprint requests and other correspondence: S. Hirose, Dept.of Biological Sciences, Tokyo Institute of Technology, 4259-B-19 Naga-tsuta-cho, Midori-ku, Yokohama 226-8501, Japan (E-mail: shirose@bio.titech.ac.jp).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Am J Physiol Regul Integr Comp Physiol 289: R575–R585, 2005.First published March 31, 2005; doi:10.1152/ajpregu.00725.2004.

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dissected out, snap-frozen in liquid nitrogen, and stored at �80°C forfuture use. Artificial seawater (Rohtomarine) was obtained fromRei-Sea (Tokyo, Japan). Sulfate-free seawater was prepared by mix-ing the following chemicals (in g/100 l): 2,398 NaCl, 1,085MgCl2 �6H2O, 112 CaCl2, 67 KCl, 19.6 NaHCO3, 9.8 KBr, 4.13SrCl2 �6H2O, 2.66 H3BO3, 0.306 NaF, and 0.007 NaI. The animalprotocols and procedures were approved by the Institutional AnimalCare and Use Committee of Tokyo Institute of Technology andconform to the American Physiological Society’s “Guiding Principlesin the Care and Use of Laboratory Animals.”

RNA isolation. Total RNA was isolated from various tissues byperforming acid guanidinium thiocyanate-phenol-chloroform extrac-tion with Isogen (Nippon Gene, Tokyo, Japan). Tissues were homog-enized in Isogen (1 g tissue/10 ml Isogen) by using a Polytron tissuehomogenizer, followed by chloroform extraction, isopropanol precip-itation, and 75% (vol/vol) ethanol washing of precipitated RNA. Theobtained RNA was dissolved in diethyl pyrocarbonate-treated water,and its concentration was measured spectrophotometrically at 260 nm.

Molecular cloning. A fragment of ajSlc13a1 cDNA (aj for A.japonica) was isolated by performing RT-PCR using the 5�-rapidamplification of cDNA ends (RACE) method. The 5�-RACE cDNAwas synthesized with a First Choice RLM-RACE kit (Ambion, Aus-tin, TX) according to the manufacturer’s instructions. Briefly, 1 �g oftotal RNA from eel kidney was first treated with calf intestinalphosphatase (CIP) in 1� CIP buffer at 37°C for 1 h, phenol extracted,and then incubated with tobacco acid pyrophosphatase (TAP) in 1�TAP buffer at 37°C for 1 h. A single-stranded 5�-RACE adapter wasadded to the decapped mRNA with T4 RNA ligase at 37°C for 1 h,and modified RNA was stored at �20°C. Two microliters of adaptor-ligated RNA were then reverse-transcribed with oligo(dT) and Molo-ney murine leukemia virus (MMLV) reverse transcriptase (RT) in 1�RT buffer with dNTPs and RNase inhibitor at 42°C for 1 h to createthe first-strand cDNA. The synthesized cDNA was then subjected totwo rounds of PCR by using adaptor-specific primers with gene-specific primer (GSP) 1 (5�-GATGAGGTTGGTTGAGGTGCC-3�)and GSP 2 (5�-GCGATGCGTTTGTGGAGGTTC-3�). These GSPswere designed on the basis of the amino acid sequence alignment ofhuman (21, 23), rat (3, 28), and mouse Slc13a1 orthologs (1, 22) inaddition to that found in the fugu genomic database (http://genome.jgi-psf.org/fugu6/fugu6.home.html). The amplification product of�360 bp was subcloned into EcoRV-digested pBluescript II SK(�)(Stratagene, La Jolla, CA) and sequenced using a BigDye TerminatorCycle Sequencing Ready Reaction kit (Applied Biosystems, FosterCity, CA) and the ABI 310 DNA sequence system (Applied Biosys-tems). This clone was used as a probe for Northern blot analysis ofajSlc13a1. To obtain the 3�-end sequence of cDNA, 3�-RACE wasconducted with the same kit by following the manufacturer’s instruc-tions. Briefly, 1 �g of total RNA from eel kidney was reverse-transcribed with 3�-adapter and MMLV RT in 1 � RT buffer withdNTPs and RNase inhibitor at 42°C for 1 h to create the first-strandcDNA. The synthesized cDNA was then subjected to two rounds ofPCR using adaptor-specific primers GSP 1 (5�-CTGCTACTGCTGC-CTCTTCCAC-3�) (nucleotides 61–82), and GSP 2 (5�-CTGTTCT-TCCCCCTGTTCGGCAT-3�) (nucleotides 190–212). The amplifica-tion product of �2,200 bp was subcloned into pBluescript II SK(�)and sequenced. To determine full-length cDNA sequences, we carriedout direct sequencing of PCR products using primers that recognizethe 5�-flanking region (5�-GATATCAGTTGGTCCTGGGTAAG-3�)and 3�-flanking region (5�-CTACTTGGCACCATTTAGTCTGT-3�)of the cDNA. For confirmation of the sequence, PCR products weresubcloned into pZErO-2 (Invitrogen) and sequenced.

A fragment of ajSlc26a1 cDNA of �900 bp was isolated byRT-PCR from eel kidney RNA with the use of sense (5�-ATH-TATTTTTTNATGGGNAC-3�) and antisense (5�-GTNGTRAAR-CARTGNARRAA-3�) degenerate primers. The PCR product wassubcloned into pBluescript II SK(�) and sequenced. This clone wasused as a probe for Northern blot analysis of ajSlc26a1. Full-length

cDNA of ajSlc26a1 was obtained by 5�-RACE, 3�-RACE, and RT-PCR by using a strategy similar to that for ajSlc13a1 cloning asdescribed above. The primers used were 5�-CAGATAGTGAGACG-GTGAAGGC-3� (5�-RACE, GSP 1 antisense), 5�-AGCTCAGT-GGGCAAGGGGATCT-3� (5�-RACE, GSP 2 antisense), 5�-TCAT-GGTTGGTCAGGTGGTGGA-3� (3�-RACE, GSP 1 sense), 5�-GCT-GAAGATCCCTCGACACCAAG-3� (3�-RACE, GSP 2 sense), 5�-TGAGTGCGTTAGTGAGATAGAG-3� (5�-flanking region, sense),and 5�-GCTTTGTGCTGTTAGACTTCAT-3� (3�-flanking region,antisense).

Northern blot analysis. Total RNA (20 �g/lane) from a pool ofvarious tissues of freshwater and seawater eels was electrophoresedon formaldehyde-agarose (1%) denaturing gels in 10� MOPS run-ning buffer (20 mM MOPS, pH 7.0, 8 mM acetate, 1 mM EDTA) andthen transferred onto Hybond-N� nylon membranes (Amersham Bio-sciences, Piscataway, NJ) by capillary blotting. After transfer, mem-branes were baked for 2 h at 80°C and prehybridized for 2 h at 65°Cin PerfectHyb hybridization solution (Toyobo, Osaka, Japan). Theprobes were labeled with [�-32P]dCTP (3,000 Ci/mmol) with the useof a Ready-To-Go DNA labeling kit (Amersham Biosciences), and theunincorporated nucleotides were removed by passage through a Seph-adex G-50 column (Amersham Biosciences). The membranes werethen hybridized separately with each 32P-labeled probe in the samebuffer at 68°C for 16 h. The blots were subsequently washed withincreasingly stringent conditions (final wash: 1� SSC and 0.1% SDSfor 30 min at 60°C). Membranes were exposed to imaging plates (FujiFilm, Tokyo, Japan) in a cassette overnight. The results were analyzedusing a Fuji BAS2000 Bio-image analyzer (Fuji Film). A probe for eel�-actin (33) was used as a control to verify loading and RNAintegrity.

Expression of ajSlc26a1 and ajSlc13a1 in Xenopus laevis oocytes.The ajSlc26a1 and ajSlc13a1 constructs in pZErO-2 were linearized attheir 3�-end with the use of EcoR1 and BamH1 restriction enzymes,respectively, and cRNAs were transcribed in vitro using T7 RNApolymerase and mMESSAGE mMACHINE kit (Ambion). Oocyteswere surgically collected from anesthetized animals under 0.17%tricaine and incubated for 1–2 h with shaking in ND96 withoutcalcium (in mM: 96 NaCl, 2 KCl, 1 MgCl2, and 5 HEPES-Tris, pH7.4) and 2 mg/ml collagenase A (for chemical defolliculation), afterwhich oocytes were washed with ND96 with calcium (in mM: 96NaCl, 2 KCl, 2 CaCl2, 1 MgCl2, and 5 HEPES-Tris, pH 7.4) fourtimes and incubated in ND96 solution for 24 h. Stage V-VI defol-liculated oocytes (13) were then injected with 50 nl of water or 0.5�g/�l of the cRNA (25 ng/oocyte) solution with the use of aNanoliter-2000 injector (World Precision Instruments, Sarasota, FL).Oocytes were incubated at 16°C in ND96 supplemented with 2.5 mMsodium pyruvate and 5 mg/100 ml gentamicin for 48–72 h. Theincubation medium was changed every 24 h, including the day of theuptake experiment. All the uptake solutions had 10 �Ci/ml 35S (ICNBiomedicals, Costa Mesa, CA).

Oocytes injected with ajSlc26a1 cRNA were placed in an uptakesolution either without chloride [in mM: 100 N-methyl-D-glucamine(NMDG)-gluconate, 2 potassium gluconate, 1 calcium gluconate, 1magnesium gluconate, 10 HEPES-Tris, and 1 K2SO4] or with chloride(in mM: 100 choline chloride, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES-Tris, and 1 K2SO4) for the 1-h uptake. Inhibiting substances includedDIDS (1 mM), HgCl2 (100 �M), potassium thiosulfate (5 mM),oxalate (5 mM), or selenate (5 mM). For chloride depletion (see Fig.5C), oocytes were placed in a medium lacking sodium and chloride (inmM: 100 NMDG-gluconate, 2 potassium gluconate, 1 calcium glu-conate, 1 magnesium gluconate, and 10 HEPES-Tris) for 3 h beforeuptake studies were performed. For chloride uptake, oocytes wereplaced in an uptake medium containing (in mM) 90 NMDG-glu-conate, 2 potassium gluconate, 1 calcium gluconate, 1 magnesiumgluconate, 10 HEPES-Tris, and 10 [36Cl]KCl.

Oocytes injected with ajSlc13a1 were placed in an uptake solutioneither with sodium (in mM: 100 sodium gluconate, 2 potassium

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gluconate, 1 calcium gluconate, 1 magnesium gluconate, 10 HEPES-Tris, and 1 K2SO4) or without sodium (in mM: 100 NMDG-glu-conate, 2 potassium gluconate, 1 calcium gluconate, 1 magnesiumgluconate, 10 HEPES-Tris, and 1 K2SO4) for 1 h. Uptakes wereperformed in the presence and absence of HgCl2 (100 �M), potassiumthiosulfate (5 mM), oxalate (5 mM), or selenate (5 mM).

After uptake, the oocytes were washed three times in ice-colduptake solution (without radionuclide) and individually dissolved in10% SDS. Tracer activity was then determined using scintillationcounting. The uptake experiments all included 10–25 oocytes in eachexperimental group, statistical significance was defined as two tailed(P 0.05), and results are reported as means SE.

Antibody production and specificity. A 245-bp fragment encodinga part of an intracellular domain (amino acid residues 179–259) ofajSlc13a1 and a 303-bp fragment encoding a part of the intracellularCOOH terminus (amino acid residues 603–702) of ajSlc26a1 weresubcloned into the BamHI/EcoRI sites of the bacterial expressionvector pHAT10 (BD Biosciences Clontech, Palo Alto, CA). Therecombinant proteins were purified with Talon metal affinity resins(BD Biosciences Clontech) according to the manufacturer’s instruc-tions. Briefly, BL21 cells transformed with pHAT-ajSlc13a1 orpHAT-ajSlc26a1 were used to inoculate 1.5 liters of Luria-Bertanibroth containing 100 �g/ml ampicillin. The cultures were grown to anA600 of 0.5 at 37°C, and protein expression was induced by addingisopropyl-1-thio-D-galactopyranoside to a final concentration of 1 mMfor 3 h at 37°C. The cells were harvested from the cultures bycentrifugation and resuspended in 20 ml of extraction/wash buffer,disrupted by freezing-thawing and sonication. After centrifugation(10,000 g at 4°C), supernatants and pellets were saved as water-soluble and -insoluble fractions, respectively. In the case of pHAT-ajSlc13a1, the recombinant protein was purified from supernatantusing Talon metal affinity resin. In the case of pHAT-ajSlc26a1,however, the recombinant protein was recovered in the pellet and wassolubilized by resuspension in solution containing 8 M urea, 50 mMphosphate, and 300 mM NaCl, pH 7.6, for 2 h at 4°C. The mixture wasthen centrifuged at 10,000 g for 40 min to remove any insolublematerials. Urea-solubilized recombinant ajSlc26a1 was purified usingTalon metal affinity resin. After purification, ajSlc13a1 and ajSlc26a1were dialyzed against saline at 4°C. Polyclonal antibodies toajSlc13a1 and ajSlc26a1 were prepared in New Zealand White rabbitsby injecting �200 �g of purified recombinant proteins, emulsifiedwith the adjuvant TiterMax Gold (CytRx; 1:1), intramuscularly atmultiple sites. The rabbits were injected three times at 1-mo intervalsand bled 7 days after the third immunization.

Specificities of the antisera were established by Western blotting ofthe protein extracts of HEK-293T cells exogenously expressingajSlc13a1 or ajSlc26a1 (data not shown). The fragments correspond-ing to the open reading frames of ajSlc13a1 and ajSlc26a1 weresubcloned into the pcDNA3 vector. HEK-293T cells were cultured inDulbecco’s modified Eagle’s medium (Sigma-Aldrich Japan, Tokyo,Japan) containing 10% fetal bovine serum (Invitrogen Japan, Tokyo,Japan), 100 U/ml penicillin, and 100 �g/ml streptomycin. The cellswere transfected with the ajSlc13a1, ajSlc26a1, or mock plasmid byusing Lipofectamine 2000 (Invitrogen Japan) according to the man-ufacturer’s instructions. The cells were washed three times with PBSand solubilized with Laemmli buffer. The cell lysates were separatedby SDS-PAGE using a 7.5% polyacrylamide gel and electroblottedonto a polyvinylidene difluoride membrane. Nonspecific binding wasblocked with 5% nonfat skim milk in TBST (100 mM Tris-HCl, pH7.5, 150 mM NaCl, and 0.1% Tween 20) for 1 h at room temperature.The membranes were incubated with anti-ajSlc13a1, anti-ajSlc26a1,or preimmune serum at 1:10,000 dilution overnight at 4°C. Afterbeing washed with TBST, membranes were then reacted with horse-radish peroxidase-conjugated donkey anti-rabbit IgG at 1:30,000 di-lution for 1 h at room temperature. The bound secondary antibody wasvisualized by enhanced chemiluminescence detection using ECL-Plusreagent (Amersham Bioscience) according to the manufacturer’s in-

structions. Two specific protein bands of 60 and 120 kDa were stainedwith anti-ajSlc13a1 antiserum, which correspond to monomeric anddimeric forms of ajSlc13a1. Similarly, anti-ajSlc26a1 antiserum visu-alized 80-kDa monomer and 170-kDa dimer bands of ajSlc26a1 onWestern blotting of extracts of HEK-293 cells expressing ajSlc26a1.The sizes of ajSlc13a1 and ajSlc26a1, determined by Western blot-ting, are consistent with those predicted from the coding sequences oftheir cDNAs.

In situ hybridization. Kidneys from anesthetized freshwater eelsperfused and fixed with 10% buffered neutral formalin (Muto PureChemicals, Tokyo, Japan) were harvested, embedded in paraffin, andsectioned (4 �m). The following DNA templates were used for thepreparation of digoxigenin (DIG)-labeled riboprobes: a 431-bp frag-ment of ajSlc13a1 cDNA (nucleotides 215–645) and a 489-bp frag-ment of ajSlc26a1 cDNA (nucleotides 1831–2319). DIG RNA label-ing mix (Roche Diagnostics, Mannheim, Germany) was used forsynthesizing DIG-labeled sense and antisense probes. Alkaline phos-phatase-conjugated anti-DIG antibody and nitro blue tetrazolium/bromochloroindolyl phosphate substrates were used to visualize thesignal, followed by counterstaining with Kernechtrot (Muto PureChemicals).

Immunohistochemistry. Kidneys from freshwater eel were fixed in0.1 M phosphate buffer, pH 7.4, containing 4% (wt/vol) paraformal-dehyde for 1 h at 4°C. After incubation in 0.1 M phosphate buffer, pH7.4, containing 20% (wt/vol) sucrose for 16 h at 4°C, specimens werefrozen in Tissue Tek OCT compound on a cryostat holder. Sections (6�m) were prepared in �20°C cryostat and mounted on (3-aminopro-pyl)triethoxysilane-coated glass slides and air dried for 1 h. Afterbeing washed with PBS, sections were incubated for 2 h at roomtemperature with 2.5% (vol/vol) normal goat serum and then over-night at 4°C with anti-ajSlc13a1 antiserum (1:10,000) or anti-ajSlc26a1 antiserum (1:10,000). Sections were then washed with PBSand treated with methanol containing 0.3% (vol/vol) H2O2 for 20 minat room temperature. After being washed with PBS, the specimenswere treated with biotinylated secondary antibody (1:2,000) andavidin-peroxidase conjugate using a Vectastain ABC kit (VectorLaboratories, Burlingame, CA). Finally, bound antibodies were visu-alized by using 3,3�-diaminobenzidine tetrahydrochloride in 50 mMTris �HCl, pH 7.4, and counterstaining with hematoxylin.

Measurement of serum ionic composition. The blood was collectedfrom the ventricle of anesthetized eel and allowed to clot; the serumwas then prepared by centrifugation and stored at �40°C for futureuse. The serum osmolarity was measured using the cryoscopic method(38). Concentrations of Na�, K�, and Cl� were measured using theestablished electrode methods (25). Ca2� and Mg2� concentrationswere measured using the o-cresolphthalein complexone method (7)and xylysine blue method (26), respectively. These measurementswere conducted by SRL Laboratories (Tokyo, Japan). The concentra-tion of SO4

2� was measured using the ion chromatographic method (6)with PIA-1000 (Shimazu, Kyoto, Japan) and serially diluted serumsamples (5 �l). The standard curve was made by using definedconcentrations of K2SO4.

RESULTS

Molecular properties of ajSlc13a1 and ajSlc26a1. We iso-lated an eel homolog of mammalian Slc13a1 by using RT-PCRof freshwater eel kidney total RNA and sequence informationfor rat (29), human (21), and mouse Slc13a1 cDNA(1), inaddition to relevant sequence for the fugu genome. The com-plete coding sequence was obtained by 5�- and 3�-RACE anddeposited in the DDBJ/EMBL/GenBank database (accessionno. AB111926). The isolated cDNA was �2.3 kb, including 30bp of 5�-untranslated region (UTR) and 380 bp of 3�-UTR. Thecoding region predicts a protein of 619 amino acid residueswith a calculated molecular mass of 68 kDa (Fig. 1A). The

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protein contains putative phosphorylation sites (protein kinaseA: Ser170, protein kinase C: Ser9, Ser262, Ser348, Ser354, Thr360,and Thr445) and an N-glycosylation site (Asn615) (Fig. 1, A andD). As expected, the sequence is most closely related to thoseof known Slc13a1 orthologs among the members of the sulfatepermease family. For example, it shares high amino acidsequence identity with Slc13a1 from rat (54%), mouse (53%),human (54%), zebrafish (58%), fugu (55%), and Africanclawed frog (48%) (Fig. 1A). In addition, the consensus se-quence for Na�-coupled cotransporters (Prosite PS01271),known as the “Na�-sulfate signature,” also is present inajSlc13a1 (Fig. 1D). Furthermore, ajSlc13a1 is placed in theSlc13a1 cluster by a phylogenetic analysis (Fig. 1B) and istherefore termed the eel gene ajSlc13a1. Hydrophobicity anal-ysis of the ajSlc13a1 protein predicted a membrane topologywith 11 putative transmembrane spans, an intracellular NH2-terminal domain, and an extracellular COOH-terminal tail (Fig.1, C and D). The number of predicted transmembrane spansvaried from 8 (29) to 13 (2) depending on computer programsused for prediction. Because the rabbit Slc13a2 has beenconfirmed experimentally to contain an odd number of trans-membrane spans (35, 54), we tentatively assumed an 11-transmembrane model for ajSlc13a1.

The ajSlc26a1 ortholog was cloned by taking a similarapproach, using RT-PCR and 5�/3�-RACE reactions. The iso-lated cDNA was �3.2 kb long, including 0.2 kb of 5�-UTR and0.9 kb of 3�-UTR. It has high sequence homology to trout(75%), fugu (70%), mouse (52%), and human (52%) Slc26a1(Fig. 2A) and encodes a membrane protein of 702 amino acidresidues with a calculated molecular mass of 77 kDa. Theprotein contains putative protein kinase C phosphorylationsites (Thr38, Thr112, and Thr636) and N-glycosylation sites(Asn159, Asn165, and Asn170). Hydrophobicity analysis ofajSlc26a1 predicted a membrane topology with 12 putativetransmembrane spans and intracellular NH2-terminal andCOOH-terminal tails (Fig. 2, C and D). Like Slc26a1 of otherspecies, ajSlc26a1 contains the following three motifs charac-teristic of members of the Slc26 sulfate/anion exchanger family(34): the Prosite Slc26 sulfate transporter signature (PS01130)and sulfate transporter and anti-sigma factor domain (STAS;Pf01740) (Fig. 2D). These characteristics, in addition to itsposition in the phylogenetic tree (Fig. 2B), confirm thatajSlc26a1 is indeed the eel ortholog of mammalian Slc26a1; itssequence information has been deposited in the DDBJ/EMBL/GenBank database (accession no. AB111927).

Kidney- and freshwater-specific expression. Via Northernblot analysis, we next determined the tissue distribution ofajSlc13a1 mRNA using total RNA preparations from eight

different tissues of freshwater and seawater eels. A singletranscript with the expected size of �3 kb was detected veryspecifically in the kidney of freshwater eel (Fig. 3, top).Furthermore, kidney was the major site of expression forajSlc26a1 mRNA; a strong hybridization signal of �3.5 kbwas detected in freshwater kidney and a weak signal in sea-water kidney (Fig. 3, bottom). Low but detectable levels ofthe ajSlc26a1 message also were observed in the posteriorintestine.

Figure 4A depicts the time course of changes in the messagelevels of ajSlc13a1 and ajSlc26a1 when eels were transferredfrom seawater to freshwater or vice versa. Both ajSlc13a1 andajSlc26a1 transcript levels increased gradually during adapta-tion of eels to freshwater but declined very rapidly after theirtransfer to seawater (Fig. 4A).

Salinity-dependent regulation of message levels ofajSlc13a1 and ajSlc26a1. To determine whether the above-mentioned marked changes in the expression levels ofajSlc13a1 and ajSlc26a1 mRNA are dependent on the sulfateconcentrations of freshwater (2 mM) and seawater (�26mM), we prepared sulfate-supplemented freshwater (FW�sulfate)and sulfate-free seawater (SW�sulfate) and measured their ef-fects on the message levels of the two sulfate transporters.Unexpectedly, large changes were observed in their levels inthe kidney when freshwater eels were transferred to SW�sulfate,whereas no significant difference was observed in the degree ofchanges between seawater and SW�sulfate (Fig. 4B). Theseresults indicate that ajSlc13a1 and ajSlc26a1 mRNA levels inthe kidney are mainly regulated in a salinity-dependent man-ner, rather than in response to variation in ambient sulfate.

Functional properties. The functional and pharmacologicalcharacteristics of ajSlc13a1 and ajSlc26a1 were determined byheterologous expression in X. laevis oocytes, using publishedprotocols (53). The sulfate uptake by ajSlc13a1-injected oo-cytes was not statistically higher than that of control water-injected oocytes in the absence of sodium (Fig. 5A). However,there was a 10-fold increase in sulfate uptake, from 23.5 to261.2 pmol �oocyte�1 �h�1 (P 0.000002) in ajSlc13a1-ex-pressing oocytes in the presence of 100 mM Na� (Fig. 5A).There also was a modest but statistically significant Na�-dependent sulfate uptake in water-injected cells (from 17.1 to29.8 pmol �oocyte�1 �h�1, P 0.0005), consistent with theexpression of an endogenous Slc13a1 or Slc13a4 ortholog inXenopus oocytes. The effect of different inhibitors on thesulfate uptake by ajSlc13a1-injected oocytes is depicted in Fig.5A and is similar to that in previous studies with the rat andhuman orthologs (21, 31). Whereas all of the studied com-pounds inhibit Na�-dependent sulfate uptake, this effect is

Fig. 1. Structural features and phylogenetic tree of ajSlc13a1. A: alignment of amino acid sequences of ajSlc13a1 and members of the Slc13a1 family of otherspecies. Accession numbers are as follows: ajSlc13a1, A. japonica Slc13a1 (AB111926); drSlc13a1, Danio rerio Slc13a1 (BC058313); trSlc13a1, Takifugurubrips Slc13a1 (hypothetical protein from scaffold 3041 of the fugu genome database); hSlc13a1, human Slc13A1 (AF260824); and xSlc13a1, Xenopus laevisSlc13a1 (BC047132). Bold lines indicate transmembrane (TM) spans of ajSlc13a1 predicted from the Kyte-Doolittle hydropathy plot (20). Asterisks (�) anddollar signs ($) indicate putative PKC phosphorylation sites and PKA phosphorylation site, respectively; pound sign (#) indicates putative N-glycosylation site.Wavy underline indicates the region used as antigen. B: phylogenetic tree of ajSlc13A1 and Slc13 family members of other species. The phylogenetic tree wasconstructed using the ClustalW computer program. The scale bar represents a genetic distance of 0.1 amino acid substitutions per site. aj; Eel (A. japonica); h,human; r, rat, f, winter flounder; tr, fugu (T. rubrips); dr, zebrafish (D. rerio); and x, frog (X. laevis). Accession numbers are as follows: ajSlc13a1 (AB111926),drSlc13a1 (BC058313), xSlc13a1 (BC047132), hSlc13A1 (AF260824), rSlc13a1 (L19102), hSlc13A2 (NaDC-1, U26209), rSlc13a2 (NaDC-1, AB001321),drSlc13a2 (BC044437), xSlc13a2 (NaDC-2, U87318), hSlc13A3 (NaDC-3, AY072810), rSlc13a3 (NaDC-3, AF081825), fSlc13a3 (NaDC-3, AF102261),hSlc13A4 (SUT-1, AF169301), hSlc13A5 (NaCT-1, AY151833), and rSlc13a5 (NaCT-1, AF522186). C: Kyte-Doolittle hydropathy plot of ajSlc13a1. The plotwas constructed using the GENETYX-MAC computer program. D: secondary structure model of ajSlc13A1 with putative 11 TM spans. The regions used asantigens are indicated by bold lines.

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most robust with HgCl2. This finding may have further impli-cations, because mercury is a known environmental toxin (seeDISCUSSION).

As shown in Fig. 5B, ajSlc26a1 mediates sulfate transport inthe absence of extracellular Cl� and Na� (27.7 vs. 4.9pmol �oocyte�1 �h�1 in water-injected oocytes, P 0.0002).Sulfate uptakes in ajSlc26a1-expressing oocytes were not af-fected by substitution of NMDG-gluconate with 100 mMsodium gluconate (data not shown). However, sulfate uptakesincreased to 45.8 pmol �oocyte�1 �h�1 (P 0.0007) in thepresence of 100 mM extracellular choline chloride (Fig. 5B),similar to published values for mouse, human, and rat Slc26a1(22, 42, 52, 53) (see also DISCUSSION). Whereas ajSlc26a1 wassensitive to 1 mM DIDS in the absence of extracellular Cl�,the addition of 100 mM choline chloride increased sensitivityto this inhibitor (18.9 vs. 9.3 pmol �oocyte�1 �h�1 with orwithout Cl�, respectively, P 0.00015). As we previouslyreported for mouse Slc26a1 (see also DISCUSSION), we did notfind that ajSlc26a1 directly transports 36Cl� (Fig. 5C, withmouse Slc26a6 as a positive control). Like ajSlc13a1,ajSlc26a1 is sensitive to cis-inhibition by extracellular thiosul-fate, oxalate, and selenate (Fig. 5D). Furthermore, as reportedfor rat Slc26a1, ajSlc26a1 is also highly sensitive to 100 �MHgCl2 (45.3 vs. 14.0 pmol �oocyte�1 �h�1 in the presence ofHgCl2, P 0.00008) (Fig. 5E).

Polarized colocalization of ajSlc13a1 and ajSlc26a1 inrenal proximal tubule cells. Because Northern blotting indi-cated that the ajSlc13a1 and ajSlc26a1 genes are almost ex-

clusively expressed in freshwater eel kidney, we performedRNA in situ hybridization to determine their expression pat-terns in the kidney at the cellular level. Clear labeling wasdetected in proximal tubule cells of freshwater eel kidney withboth ajSlc13a1 and ajSlc26a1 cRNA probes (Fig. 6A). Colo-calization of ajSlc13a1 and ajSlc26a1 in renal proximal tubulecells was confirmed by immunostaining of two serial sections(Fig. 6B). Immunohistochemistry further demonstrated apicallocalization of ajSlc13a1 and basolateral localization ofajSlc26a1 (Fig. 6B). Consistent with the results of Northernblot analysis, little or no significant staining was obtained whensections of seawater eel kidney were stained with the antisera,suggesting downregulation of the two sulfate transporters alsoat the protein level (data not shown). Specificities of theantisera were established by Western blot analysis (data notshown).

Elevated levels of serum sulfate in freshwater eels. When wemeasured the serum concentration of sulfate in freshwater and

Fig. 3. Freshwater-specific expression of ajSlc13a1 and ajSlc26a1. Northernblot analysis was performed using 20 �g of total RNA from freshwater (F) orseawater (S) eel tissues. Eels were acclimated to freshwater or seawater for 2wk.

Fig. 4. Time course of salinity-dependent changes in mRNA levels ofajSlc13a1 and ajSlc26a1. A: total RNA was isolated from kidney at 0, 0.25, 1,3, 7, and 14 days after transfer of eels from seawater (SW) to freshwater (FW)(left) or from FW to SW (right) and subjected to Northern blot analysis. B:Northern blot showing that changes in mRNA levels of ajSlc13a1 andajSlc26a1 are mainly dependent on salinity rather than on sulfate concentra-tion. Sampling was performed at 0 and 14 days after transfer to sulfate (26mM)-supplemented freshwater (FW�sulfate) or to sulfate-free seawater(SW�sulfate).

Fig. 2. Structural features and phylogenetic tree of ajSlc26a1. A: alignment of amino acid sequences of ajSlc26a1 and members of the Slc26a1 family of otherspecies. Accession numbers are as follows: ajSlc26a1, A. japonica Slc26a1 (AB111927); omSlc26a1, Oncorhynchus mykiss Slc26a1 (AB111927); trSlc26a1, T.rubrips Slc26a1 (hypothetical protein from scaffold 281 of the fugu genome database); hSlc26a1, human Slc26A1 (AF297659); and mSlc26a1, mouse Slc26a1(AF349043). Bold lines indicate TM spans of ajSlc26a1 predicted from the Kyte-Doolittle hydropathy plot (20). Wavy underline indicates the region used asantigen. Asterisks and pound signs indicate putative PKC phosphorylation sites and putative N-glycosylation sites, respectively. B: phylogenetic tree of ajSlc26a1and Slc26 family members of other species. The phylogenetic tree was constructed using the ClustalW computer program. The scale bar represents a geneticdistance of 0.1 amino acid substitutions per site. aj, Eel (A. japonica); h, human; r, rat; tr, fugu (T. rubrips); om, trout (O. mykiss); and dr, zebrafish (D. rerio).Accession nos. are as follows: ajSlc26a1 (AB111927), omSlc26a1 (AY512964), hSlc26A1 (AF297659), rSlc26a1 (P45380), mSlc26a1 (AF349043), hSlc26A2(U14528), rSlc26a2 (O70531), ajSlc26a3 (AB111930), hSlc26A4 (O43511), rSlc26a4 (AF167412), hSlc26A5 (AF523354), rSlc26a5 (AJ303372), drSlc26a5(AY278118), hSlc26A6 (AF279265), ajSlc26a6a (AB084425), ajSlc26a6b (AB111928), ajSlc26a6c, (AB111929), hSlc26A7 (AF331521), hSlc26A8(AF331522), hSlc26A9 (AF331525), and hSlc26A11 (NM_173626). C: Kyte-Doolittle hydropathy plot of ajSlc26a1. The plot was constructed using theGENETYX-MAC computer program. D: secondary structure model of ajSlc26A1 with putative 12 TM spans. STAS, sulfate transporter anti-sigma factor. Boldlines indicate the regions used as antigens.

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seawater eels, we obtained an unexpected result. The serumsulfate concentration was much higher in freshwater eels (�19mM, or �38 meq) than in seawater eels (�1 mM, or �2 meq)(Table 1). This increase was accompanied by a marked de-crease in Cl� concentration in freshwater eel serum (from 120mM to 80 mM) to maintain plasma osmolarity within aphysiological range. This fact suggests, as discussed below indetail, dual physiological roles for the sulfate transport systemcomposed of ajSlc13a1 and ajSlc26a1: maintenance of sulfatehomeostasis and osmoregulation.

DISCUSSION

Over the last decade there have been major advances in ourunderstanding of the molecular and cellular basis for theregulation of sulfate homeostasis in mammals through theidentification (1, 3, 21, 22, 29, 42), molecular and functionalcharacterization (5, 28, 39), and cellular and subcellular local-ization studies (8, 17, 24) of two sulfate transporters, Slc13a1and Slc26a1 (Fig. 7) (for a review, see Ref. 27). In the presentstudy we applied a similar approach to the euryhaline eelsystem, cloning and characterizing the eel orthologs ajSlc13a1and ajSlc26a1. We thus have established that a very similarsulfate-transporting system is operative in renal proximal tu-

bule cells of freshwater eels, as schematically summarized inFig. 7. In freshwater conditions where sulfate is not readilyavailable, sulfate ions appear to be reabsorbed from the glo-merular filtrate to the blood, to a large extent by a combinedaction of ajSlc13a1 and ajSlc26a1, via the mechanisms illus-trated in Fig. 7. This model is supported by the robust expres-sion of these genes in the proximal tubule, as observed withNorthern blotting (Fig. 3), in situ hybridization (Fig. 6A), andimmunohistochemistry (Fig. 6B), in addition to the functionalattributes of the expressed transporters (Fig. 5).

The renal sulfate transport system of the eel seems to playimportant roles not only in maintaining sulfate homeostasis butalso in osmoregulation in freshwater habitats. Surprisingly,serum concentrations of sulfate were much higher in freshwa-ter eels than in seawater eels, and the increment was attenuatedby a concomitant decrease in chloride levels (Table 1). Thesecompensatory changes of sulfate and chloride ions appear to bean efficient strategy for freshwater adaptation, because theabsorption of chloride from freshwater to maintain body fluidosmolarity is energetically costly compared to the renal reab-sorption of sulfate ions derived, for example, from metabolismof sulfur-containing substances. Indeed, it is known thatCl�influx rates in eels are very low (4, 15, 16, 18); for

Fig. 5. Functional characterization of ajSlc13a1 and ajSlc26a1. A: [35S]SO42� uptake mediated by water-injected or ajSlc13a1-injected X. laevis oocytes in the

presence or absence of 100 mM Na� and in the presence of the indicated inhibitors. *P 0.000002 compared with ajSlc13a1-injected cells in the absence ofNa�. **P 0.01, compared with ajSlc13a1-injected cells in the presence of Na� alone. B: [35S]SO4

2� uptake mediated by water-injected or ajSlc26a1-injectedoocytes in the presence or absence of 100 mM choline chloride in the presence or absence of DIDS. *P 0.0001 compared with water-injected cells. **P 0.0007 compared with ajSlc26a1-injected cells in the absence of extracellular Cl�. C: [36Cl]Cl� uptake by ajSlc26a1-injected and mouse Slc26a6-injectedoocytes, including cells that were incubated for 3 h in the absence of extracellular Cl�, as indicated. *P 0.000001 compared with water-injected cells. D:cis-inhibitory effects of the indicated anions on [35S]SO4

2� uptake by ajSlc26a1-injected oocytes in the absence of extracellular Cl�. E: effect of 100 �M HgCl2on [35S]SO4

2� uptake by ajSlc26a1-injected oocytes. **P 0.00008 compared with ajSlc26a1-injected cells in the absence of HgCl2.

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example, in the European eel A. anguilla, it has been reportedto be only 0.05 mmol �kg�1 �h�1 compared with 116mmol �kg�1 �h�1 in the rainbow trout (16). Given such anextremely low rate of Cl� uptake, the mechanism for main-taining ionic homeostasis in freshwater eels was something of

a mystery before the current study was performed. The above-mentioned osmoregulatory role of the ajSlc13a1 and ajSlc26a1sulfate transporters is further consistent with the observationthat their expressions were strongly induced even in sulfate-supplemented freshwater (Fig. 4B). Furthermore, we observedrapid downregulation of the two transporters upon transfer toseawater, even when sulfate free (Fig. 4B); we hypothesize thatthis is necessary for achieving, upon seawater transfer, animmediate lowering of serum sulfate concentrations from �26mM to �1 mM, to compensate for passive accumulation ofchloride ions under seawater conditions. In addition to theosmoregulatory role of sulfate in freshwater, it is tempting tospeculate that sulfate, a multivalent anion, tends to hold waterand help prevent desiccation, enabling eels to move a consid-erable distance over land and colonize isolated ponds.

Differences in the degree and kinetics of induction or down-regulation of ajSlc13a1 and ajSlc26a1 are noteworthy (Fig. 4).The apical ajSlc13a1 exhibited more rapid and complete down-regulation than the basolateral ajSlc26a1 when eels were trans-ferred from freshwater to seawater. This differential regulationof expression is consistent with the previous notion that api-cally located Slc13a1 represents the major target for knownregulatory factors such as 1,25(OH)2 vitamin D3 (14), thyroidhormones (51), and glucocorticoids (50). It is also known that

Fig. 6. In situ hybridization and immunohistochemistry of ajSlc13a1 andajSlc26a1 in freshwater eel kidney. Left (a, c, e, and g), low-magnificationimages; right (b, d, f, and h), high-magnification images of boxed areas incorresponding images at left. A: proximal tubule cell expression of ajSlc13a1and ajSlc26a1 mRNA. Arrow in c points to brown melanin and hemosideringranules present in the intertubular hematopoietic tissue of teleost kidney. B:colocalization of ajSlc13a1 and ajSlc26a1 in renal proximal tubule cells.Polarized localization of ajSlc13a1 (apical) and ajSlc26a1 (basolateral) is alsoshown in f and h. D, distal tubule; G, glomerulus; and P, proximal tubule. Scalebars, 20 �m.

Table 1. Serum ionic composition of freshwater and seawater eels

Type of Eel n Serum Osmolarity, mosM Na�, meq K�, meq Ca2�, meq Mg2�, meq Cl�, meq SO42�, meq

FW eel 4 293.3 (11.6) 165.3 (8.3) 4.2 (1.0) 3.5 (0.3) 1.2 (0.4) 78.7 (4.6) 37.5 (5.6)SW eel 4 338.7 (17.0) 169.3 (2.8) 5.6 (0.9) 3.1 (0.1) 1.8 (0.6) 125.3 (2.8) 1.9 (0.03)

Values are means (SD). Freshwater eels show a marked increase in sulfate (SO42�) concentration and a concomitant decrease in Cl� concentration to maintain

the appropriate ionic balance to keep serum osmolarity in a physiological range. Eels were acclimated to freshwater (FW) or seawater (SW) for 2 wk.

Fig. 7. Model of renal sulfate reabsorption system in freshwater eel. Thesulfate transporters ajSlc13a1 and ajSlc26a1 are located in the apical andbasolateral membranes, respectively, of proximal tubule cells. Driven by theNa� gradient created by Na�-K�-ATPase, the apical ajSlc13a1 transports theluminal sulfate anion into the cells. The increment of sulfate anion concentra-tion generates an outward sulfate gradient, leading to sulfate exit across thebasolateral membrane. Stoichiometries of the ions transported by ajSlc13a1and ajSlc26a1 have not been determined but are supposed to be the same asthose determined for the mammalian counterparts (5). BBM, brush-bordermembrane; PTEC, proximal tubule epithelial cell; TJ, tight junction.

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mammalian Slc26a1 orthologs function as robust oxalate ex-changers (17, 42, 53) such that persistent expression ofajSlc26a1 in seawater may reflect other roles in the homeosta-sis of this and/or other anions. The salinity-dependent regula-tion of expression of ajSlc13a1 and ajSlc26a1 is an interestingphenomenon for which the molecular mechanisms have yet tobe clarified.

Heterologous expression of both ajSlc13a1 and ajSlc26a1 inXenopus oocytes revealed the expected functional attributes,i.e., Na�-dependent and Na�-independent transport of sulfate,respectively. Slc26a1 is unique among the 10 mammalianSlc26 paralogs (34) in that Cl� and other monovalent anions(halides, formate, and lactate) dramatically enhance sulfate andoxalate transport (53). We found a similar activating effect ofextracellular Cl� on ajSlc26a1 sulfate transport (Fig. 5C),although in contrast to the results of others (22, 42, 52), we diddetect statistically significant sulfate transport by ajSlc26a1-injected (Fig. 5B) and mouse Slc26a1-injected oocytes (53) inthe absence of Cl�. Furthermore, we found an absence ofdirect Cl� transport in oocytes expressing ajSlc26a1; althoughthis finding is consistent with our prior data for mouse Slc26a1,it differs from data reported by Markovich and colleagues (22,42), who found that the mouse and human orthologs mediateCl� transport in oocytes. Notably, the “cis-activation” ofextracellular Cl�, consistent across several reports (17, 22, 42,52, 53), is not consistent with direct SO4

2�-Cl� exchange, inwhich one would expect the Cl� cis-inhibition that is charac-teristic of other Slc26 exchangers that mediate both SO4

2� andCl� exchange. It also is reported in several species that oxalateand SO4

2� exchange in basolateral membrane vesicles of therenal proximal tubule, likely mediated by Slc26a1 (17), is notsensitive to extravesicular Cl� (19, 41, 44). In summary,although we do not rule out a direct role for Slc26a1 in Cl�

transport, we favor a modulatory effect of this anion ondivalent anion exchange; regardless of the mechanism(s) ofanion exchange mediated by Slc26a1, this appears to be aconserved attribute of this transporter in mammals and eel.

Finally, we found that mercury (HgCl2) is a potent inhibitorof both ajSlc13a1 and ajSlc26a1. Similar data have beenreported for the mammalian orthologs in the context of detailedstudy of their differential sensitivity to a number of heavymetals. The mechanisms of this sensitivity are not known butmay differ for rat Slc13a1 and Slc26a1 (30, 31). Regardless,the shared sensitivity of the eel orthologs indicates that thisenvironmental toxin has the potential to almost completelyinhibit transepithelial sulfate transport in the proximal tubule ofeuryhaline fish.

Perspectives

Eels have a fascinating life cycle. They breed in the sea,migrate to freshwater to grow, and spend most of their lives infreshwater before returning to the sea to spawn. The efficientsulfate recycling system reported in this article may be advan-tageous to eels in a race for survival in freshwater; by reducingthe serum Cl� concentration required for freshwater survival,at �80 mM in eels (Table 1) vs. �120 mM in freshwater fish(16), this system reduces the cost of maintaining anion ho-meostasis in freshwater by �30%. In this context, our findingsmay provide a molecular basis for the life cycle of eels by

explaining how this species survives in freshwater with aminimum of Cl� absorption.

ACKNOWLEDGMENTS

We thank Akira Kato, Shinji Honda, Taku Fukuzawa, and Keith Choe fordiscussion; Hiroshi Kaneko, Noriko Hasegawa, and Yutaka Tamaura for serumsulfate measurement; Yoichi Noguchi of Genostaff for technical assistance inin situ hybridization histochemistry; and Setsuko Sato for secretarial assis-tance.

GRANTS

This work was supported by Grant-in-Aid for Scientific Research 14104002from the Ministry of Education, Culture, Sport, Science and Technology ofJapan (MEXT) and the 21st Century Centers of Excellence Program of MEXT,National Institute of Diabetes and Digestive and Kidney Diseases GrantsDK-57708 (to D. B. Mount) and DK-065482 (to V. Broumand), a VeteransAffairs Career Development Award (to D. B. Mount), and American HeartAssociation Postdoctoral Fellowship Award 0225706T (to K. Zandi-Nejad).

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