Function and localization of urate transporter 1 in mouse kidney

ARTICLESFunction and Localization of Urate Transporter1inMouse KidneyMAKOTO HOSOYAMADA,*KIMIYOSHI ICHIDA,†ATSUSHI ENOMOTO,‡TATSUO HOSOYA,†and HITOSHI ENDOU**Department of Pharmacology and Toxicology,Kyorin University School of Medicine,Tokyo,Japan;†Division of Kidney and Hypertension,Department of Internal Medicine,Jikei University School of Medicine,Tokyo,Japan;‡Department of Clinical Preventive Medicine,Nagoya University School of Medicine,Aichi,Japan.Abstract.Mouse renal-specific transporter(RST)cDNA,the amino acid sequence of which has74%identity with that of human urate transporter1(hURAT1),is potentially the mouse homologue of hURAT1,the gene responsible for hereditary renal hypouricemia.The aim of this study is to determine the location and characteristics of RST molecule in mouse kidney and investigate urate transport by RST using the Xenopus oocyte expression system.RST transported14C-urate in a Michaelis-Menten manner.The K m and the V max values of RST-dependent urate transport were1213Ϯ222␮M and 268.8Ϯ38.0pmol/oocyte per hr,respectively(nϭ3).RST-dependent urate transport was cis-inhibited significantly by1 mM probenecid(68.7Ϯ9.4%),50␮M benzbromarone(67.9Ϯ6.4%),and10mM lactate(50.9Ϯ9.5%).However,1mM p-aminohippurate(PAH),1mM xanthine,and1mM oxonate did not inhibit RST-dependent urate transport.Substitution of Cl anion with gluconate in the external solution enhanced RST-dependent urate transport.Pre-injected pyrazinoic acid (PZA)or L-lactate trans-stimulated RST-dependent urate ing immunohistochemistry for mouse kidney,the brush border or intracellular membrane of proximal tubules was stained by an affinity-purified antibody that recognized mouse URAT1(mURAT1)expressed on Xenopus ing Western blotting,anti-mURAT1antibody detected70-kD and 62-kD protein bands.The70-kD protein was N-glycosylated and was identified as a Triton X-100insoluble brush border membrane protein.RST mRNA and protein levels were higher in male kidneys than female.RST transported urate similar to hURAT1and,therefore,appears to be mURAT1—the mouse homologue of hURAT1.Urate is metabolized by uricase for most mammals,and is an intermediate product of purine metabolism.Urate becomes the end product of purine metabolism for higher primates who have lost uricase activity.Therefore,it is important to under-stand urate handling mechanisms in the kidney because the underexcretion of urate has been implicated in the development of hyperuricemia that leads to gout.However,urate handling mechanisms are complicated;in that,urate is transported bidi-rectionally,being both reabsorbed and secreted in the kidney. Moreover,the differences in renal urate transport among dif-ferent species have made it difficult to analyze urate handling mechanisms in the kidney.Nevertheless,renal urate transport in various animals has been investigated for comparison and to elucidate the evolution of renal urate handling mechanisms. Pigs and rabbits excrete more urate than is filtered through the glomerulus.Birds,like humans,have lost uricase activity. However,birds don’t reabsorb urate in the kidney(1).In contrast,rats and mice reabsorb urate in their kidney,like humans,although uricase maintains their plasma urate at a lower level.The cloning and characterization of the urate transporter from mice is significant for understanding urate handling in the human kidney because the renal transport system of urate in mice was considered to be similar to that in humans(2).As a transporter molecule for urate reabsorption,we recently cloned urate transporter1(URAT1),located at the apical membrane in the proximal tubules of human kidney.We dem-onstrated that human URAT1(hURAT1)reabsorbs urate by showing that some patients with renal hypouricemia have a hURAT1gene(SLC22A12)abnormality(3).Because probe-necid and benzbromarone inhibit urate transport by hURAT1 expressed on Xenopus oocyte,it has been deduced that hU-RAT1is a transporter responsible for urate reabsorption in human kidney.However,we have no information about the localization of hURAT1in lower nephron segments of human kidney.It was also demonstrated that urate transport by hU-RAT1expressed on Xenopus oocyte was trans-stimulated by the preinjection of PZA into oocyte.Therefore,the antiurico-suric effect of PZA may not be caused by inhibition of urateReceived March14,2003.Accepted October21,2003.Correspondence to Dr.Makoto Hosoyamada,Department of Pharmacologyand Toxicology,Kyorin University School of Medicine,Shinkawa6-20-2,Mitaka,Tokyo,181-8611,Japan.Phone:81-422-47-5511ext.3453;Fax:81-422-79-1321;E-mail:hosoyamd@kyorin-u.ac.jp1046-6673/1502-0261Journal of the American Society of NephrologyCopyright©2004by the American Society of NephrologyDOI:10.1097/01.ASN.0000107560.80107.19J Am Soc Nephrol15:261–268,2004secretion,but by enhancement of urate reabsorption as sug-gested by vesicle studies(4,5).As the mouse homologue of hURAT1,Mouse RST has been shown to have significant identity in predicted amino acid sequence(Genbank accession No.AB005451).Similar to hU-RAT1,expression of RST was demonstrated in the renal prox-imal tubule by in situ hybridization(6).Nevertheless,there has been no information about urate transport function by RST. The purpose of this study is to clarify the urate transport function of RST and to verify RST as a urate transporter in mouse kidney.Moreover,the localization and characterization of mURAT1molecule in the kidney is investigated to under-stand urate handling in the kidney.Materials and MethodsConstruction of RST cDNARST cDNA was amplified by the Advantage HF-2PCR kit(BD Biosciences Clontech Palo Alto,CA)with mURAT1-05sense primer (5'-TCTGTGAAGTGGAAGCTGCGTGGTGG-3')and mURAT1-06R antisense primer(5'-TGTTCCTTCTCCAGGCAACTACAG CC-3')from the EST clone as Genbank accession number AW106663. The cDNA clone was rescued with the TOPO-TA sequencing vector kit(Invitrogen).The sequence of the isolated clone was confirmed by the BigDye Terminator cycle sequencing kit(Applied Biosystems, Foster City,CA).Functional Analysis of Urate Transport by RST RST cRNA was synthesized by the mMessage mMachine T7kit (Ambion,Austin,TX)and poly(A)ϩtailing kit.Synthesized cRNA was purified by the MEGAclear kit(Ambion).Fifty nanograms of RST cRNA was injected into Xenopus oocyte,which was defollicu-lated by1.5to2.0mg/ml collagenase(Sigma C-9891)treatment for1.5to2.0h in OR-2solution(in mM:82NaCl,2.5KCl,1.0MgCl2, 5.0HEPES)at room temperature.After2to3d cultures at18°C, uptake studies were performed at room temperature with10␮M 14C-urate,and cold urate for1h in ND96solution(in mM:96NaClor Na gluconate,2.0KCl,1.8CaCl2,1.0MgCl2,5.0HEPES,pH7.4) and various concentrations of cold urate.For the inhibition studies,1 mM probenecid,50␮M benzbromarone,10mM L-lactate,1mM PAH,1mM xanthine,and1mM oxonate were added to the uptake solution containing10␮M14C-urate.For the preinjection studies, uptake studies followed a50nl injection of100mM of potassium L-lactate(pH7.4)or potassium PZA(pH7.4).Uptake studies were stopped by adding ice-cold14C-urate free ND96solution,and oocytes were washed five times.Each oocyte,solubilized with200␮l of10% SDS,was mixed with2.5ml of Aquasol-2(Packard,Meriden,Con-necticut)for radioactivity determination using a scintillation counter (LC-3010,ALOKA,Japan).Immunofluorescence of mURAT1Expressed in Xenopus OocyteAnti-mURAT1polyclonal antibody was obtained as1.705mg/ml of IgG fraction by immunization of rabbit with KLH-conjugated synthetic peptide ELLDRVGGLGRF,corresponding to RST amino acid5-17,and by peptide affinity-purification.Xenopus oocytes, which were injected with50ng of RST cRNA and cultured for3d at 18°C,were fixed in4%paraformaldehyde in ND96overnight at4°C. Eight␮m-frozen sections were made by a Cryostat(MICROM HM500M,Carl Zeiss)and dried by cool air for30min.The sections were stained with anti-mURAT1antibody(1:100),followed by stain-ing with Cy5conjugated anti-rabbit IgG(1:200,Jackson ImmunoRe-search Laboratories,Inc.,West Grove,Pennsylvania).Images were visualized by an Olympus FLUOVIEW FW500confocal laser microscope.Immunohistochemistry of mURAT1in Mouse Kidney Ten-week-old male mice purchased from Saitama experimental animal supplier company(Saitama,Japan)were anesthetized by pen-tobarbital injection(50mg/kg intraperitoneally),perfused with4% paraformaldehyde/PBS through the heart,and their sliced kidneys were embedded in paraffin.The2-␮m sections were stained with anti-mURAT1antibody(1:500)followed by staining with Envision kit(DakoCytomation,Glostrup,Denmark).Nuclei were stained with hematoxylin.Images were visualized,atϫ40,ϫ100,andϫ400 magnification by an Olympus BX60microscope.Western Blotting of Mouse KidneySamples for Western blot analysis were prepared as follows.After adding4vol/wt of PBS containing protease inhibitors(Complete, Roche)to1vol/wt of mouse kidney tissue,the mixture was homog-enized in a Potter-Elvehjam homogenizer on ice.The homogenate was centrifuged at6000ϫg for15min at4°C,and the resultant super-natant was centrifuged at100,000ϫg for30min at4°C.The precipitate was resuspended with the PBS containing protease inhib-itors as a crude membrane fraction,or resuspended with extraction buffer(150mM NaCl,1%Triton X-100,50mM Tris-HCl,pH8.0) followed by30-min shaking on ice and100,000ϫg centrifugation for 30min at4°C.The resultant supernatant was used as the Triton X-100 extracts of mouse kidney.Protein concentrations of samples were determined with a BCA protein assay kit(Pierce Biotechnology, Rockford,IL).For preparation of brush border membrane,mouse kidney cortex was homogenized on ice in10vol/wt of homogenate solution(10mM mannitol and2mM Tris-HCl,pH7.1).The homogenate was centri-fuged for2min at200ϫg to remove unbroken cells.Solid MgCl2·6H2O was added to the resultant supernatant to give a con-centration of10mM and then shaken for15min in an ice bath.After centrifugation for12min at1500ϫg,the resultant supernatant was centrifuged for an additional12min at15,000ϫg.The precipitatewas resuspended in the homogenate solution with10mM MgCl2and centrifuged for12min at2200ϫg.The resultant supernatant was centrifuged for12min at15000ϫg.The precipitate was washed three times and resuspended in the homogenate solution withoutMgCl2as a brush border membrane fraction(7).Alkaline phosphatase activity of the samples was determined with the Alkaline Phospha K kit(Wako Pure Chemical Industries,Ltd.,Osaka,Japan).For the deglycosylation of crude membrane fraction,100␮g of crude membrane fraction,which had been boiled for1h in20mM phosphate buffer(pH7.4)containing0.25%SDS and125mM2-mer-captoethanol,was deglycosylated with5mU PNGase F and12% NP-40(Bio-Rad Laboratories,Hercules,CA)at37°C overnight.Western blotting analysis was performed as follows:10␮g of the samples,the crude membrane fraction,the Triton X-100extracts,the brush border membrane fraction,and the deglycosylated crude mem-brane fraction were separated with10%polyacrylamide gel by Lae-mmli method and semidry blotted on a nitrocellulose filter(Hybond-ECL,Amersham Biosciences UK Ltd,Buckinghamshire,UK).The blotted filter was shaken for1h at room temperature in blocking solution(5%blocking agent of the ECL kit,Amersham Biosciences, in the TBS with0.02%Tween20),and was washed three times with washing solution(150mM NaCl,10mM Tris-HCl,pH7.8,0.1%262Journal of the American Society of Nephrology J Am Soc Nephrol15:261–268,2004Tween20).The blocked filter was stained overnight at4°C using affinity-purified anti-mURAT1antibody(1:900)with or without10␮g/␮l antigen peptide in the TBS-T solution containing1%bovine serum albumin.The detection was performed according to the man-ufacturer’s instructions with the ECL kit(Amersham Biosciences). Sex Difference of the mURAT1mRNA andProtein LevelsThree10-wk male and female mice were anesthetized by pento-barbital injection(50mg/kg intraperitoneally),and their kidneys were excised.Ten micrograms of crude membrane fraction prepared from a kidney was analyzed by Western blotting.The total RNA was extracted from another kidney using ISOGEN kit(Nippon Gene Co. Ltd.,Tokyo,Japan)and purified with RNeasy kit(QIAGEN K.K., Tokyo,Japan)and RNase-free DNase.Two micrograms of total RNA was separated with denatured agarose gel and transfered to a positive charged nyron membrane(Hybond-Nϩ,Amersham Biosciences UK Ltd,Buckinghamshire,UK)by alkali blotting(3M NaCl,0.01N NaOH).Probes for mURAT1mRNA was prepared by PCR DIG probe synthesis kit(F.Hoffmann-La Roche Ltd,Basel,Switzerland) with mURAT1–07sense primer(5'-ATGACCTTGAACGCCTT-GGGCTTCAG-3')and the mURAT1–06R antisense primer(5'-TGT-TCCTTCTCCAGGCAACTACAGCC-3')from mouse RST cDNA. Probes for mouse␤-actin mRNA were prepared with mBACT-01 sense primer(5'-ACCTCATGAAGATCCTGACCG-3')and the mBACT-02R antisense primer(5'-TGCTTGCTGATCCA-CATCTGC-3')from mouse␤-actin cDNA that was cloned by RT-PCR from mouse kidney.Hybridization and0.1ϫSSC washing were conducted at65°C.Detection of mURAT1mRNA was performed with DIG Luminescent Detection kit(Roche).ResultsUrate Transport by RST/mURAT1Figure1A indicates that14C-urate uptake by mouse RST/ URAT1cRNA-injected oocytes increased linearly in a time-dependent manner for75min and was significantly different from intrinsic14C-urate uptake by non-injected oocytes after 15min.Therefore,we observed14C-urate uptake for either30 or60min in the following experiments.Figure1B indicates the typical concentration dependence of RST-dependent urate uptake determined by the subtraction of 14C-urate transport of non-injected oocytes from the14C-uratetransport of RST cRNA-injected oocytes.From this figure, RST-dependent urate uptake was determined to be Michaelis-Menten type,and the Eadie-Hofstee plot,shown in the inset, was linear.The average K m value from three independent experiments was1213Ϯ222␮M,and the average V max was 268.8Ϯ38.0pmol/oocyte per hr(meanϮSEM,nϭ8,nϭ3).These results demonstrated that RST transported urate and verified that RST was mURAT1,the mouse homologue of hURAT1.Figure2shows the inhibition profiles of urate secreting and retaining drugs,indicated as the percent of urate uptake ob-served in the presence of each drug,compared with mURAT1 uptake in the absence of drugs as the control.One mM probe-necid(68.7Ϯ9.4%),50␮M benzbromarone(67.9Ϯ6.4%), and10mM lactate(50.9Ϯ9.5%)significantly cis-inhibited mURAT1-dependent urate transport.On the other hand,1mM PAH,xanthine,and oxonic acid did not inhibit urate transportby mURAT1.Figure3shows that urate transport by mURAT1was trans-stimulated by Cl anion,L-lactate,and PZA.Substitution of NaCl in ND96solution with Na gluconate enhanced urate transport by mURAT1.This suggests that mURAT1is a urate/ Cl-exchanger like hURAT1.Preinjected L-lactate and PZA trans-stimulated urate influx by mURAT1.Therefore,mU-RAT1appears to be a urate/lactate and urate/PZA exchanger just like hURAT1.Immunohistochemistry of mURAT1in the Kidney Figure4A is an immunofluorescence of mURAT1stained with mURAT1antibody.Because noninjected oocyte was not stained by this antibody(Figure4B),the antibody was verified to recognize mURAT1protein.Figure1.(A)Time course of10␮M14C-urate and100␮M cold urate uptake in non-injected(open circles)or mouse RST/URAT1RNA-injected(filled circles)Xenopus oocyte under ND96(pH7.4).Points shown are meansϮSEM(nϭ8).(B)Typical concentration depen-dence of urate transport by mouse RST/URAT1expressed in Xenopus oocyte under ND96solution.The Eadie-Hofstee plot is shown in the inset.Points shown are meansϮSEM(nϭ8).From three indepen-dent experiments,the Kmvalue was1213Ϯ222␮M,and averageVmaxwas268.8Ϯ38.0pmol/oocyte per hr(meanϮSEM,nϭ8,n ϭ3).J Am Soc Nephrol15:261–268,2004Function and Localization of Mouse URAT1263Figures 4C to 4F are immunohistochemistries of mouse kidney using the affinity-purified mURAT1antibody.Staining of mURAT1was restricted to the proximal tubule and is not present in the medulla (Figure 4C),and the staining was absorbed by 100␮g/␮l antigen peptide (Figure 4D).Localiza-tion of mURAT1was observed from the exit of Bowman ’s capsule and mixed in the proximal tubule cells,with dotlike patterns in the cytoplasm and linear patterns in the brush border membrane (Figure 4,E and F).Therefore,it was difficult to indicate the relationship between the localization pattern of mURAT1and the segments of proximal tubule.Western Blotting of mURAT1Figure 5shows the western blot analysis of mouse kidney using the affinity-purified mURAT1antibody.Two bands of 70-kD and 62-kD protein were detected in the crude membrane sample (lane 1).These bands completely disappeared follow-ing the addition of 10␮g/␮l of antigen peptide (lane 5).The 62-kd protein was in the Triton X-100soluble fraction (lane 2),and the band was completely eliminated by the addition of 10␮g/␮l of antigen peptide (lane 6).Because the upper 70-kD band disappeared in the Triton X-100extracts from the crude membrane sample,the 70-kD protein was in the TritonX-100Figure 2.Inhibition of mouse RST/URAT1dependent 10␮M 14C-urate transport under ND96solution by 1mM probenecid,50␮M benzbromarone,10mM L-lactate,1mM PAH,1mM xanthine,and 1mM oxonic acid.From three independent experiments,values are shown as means ϮSEM of percent urate uptake by mouse RST/URAT1in the presence of drugs compared with mURAT1uptake in the absence of drugs as the control (n ϭ8,1.00Ϯ0.10pmol/oocyte per hr).**P Ͼ0.01.Figure 3.Enhancement of mouse RST/URAT1dependent 10␮M 14C-urate transport by 5nmol preinjected L-lactate and pyrazinoic acid (PZA)under ND96-NaCl and that of mouse RST/URAT1dependent 10␮M 14C-urate uptake under ND96-Na gluconate.From three independent experiments,values shown are means ϩSE of %uptake of mouse RST/URAT1dependent urate uptake without preinjection under ND96-NaCl as a control (n ϭ8,0.37Ϯ0.07pmol/oocyte per 30min).**P Ͼ0.01.264Journal of the American Society of Nephrology J Am Soc Nephrol 15:261–268,2004insoluble fraction.However,only the upper 70-kD band was detected in the brush border membrane fraction (lane 3),the alkaline phosphatase activity of which was 25.5times higher than that of the initial homogenate.The upper 70-kD band relocated to the lower 62-kD band following deglycosylation of crude membrane fraction using PNGase F (lane 4).There-fore,the N-glycosylated 70-kD form of mURAT1was sorted in the brush border membrane,and the native 62-kD form of mURAT1was soluble with Triton X-100.Sex Difference of mURAT1ExpressionFigure 6shows the sex difference of mURAT1protein (6A)and mRNA (6B)expression.There was a significant sex dif-ference in body weight (male,37.7Ϯ0.33g;female,27.3Ϯ0.17g)and kidney weight (male,0.57Ϯ0.05g;female,0.40Ϯ0.01g);and male mURAT1protein levels in 10␮g crude membrane fraction were higher than those in -paring mouse ␤-actin mRNA levels,male mURAT1mRNAlevels in 2␮g total RNA from kidney were also higher than those of females.The male/female ratio of the intensity of mURAT1mRNA band was 2.3,normalized by the intensity of mouse ␤-actin mRNA band.Therefore,mURAT1transcription was sex-dependent,being greater in males than females.DiscussionStructure of mURAT1RST was reported as a 553-amino-acid protein with 30%identity with the rat organic cation transporter 1at the amino acid level.RST cDNA and hURAT1cDNA contained 1659-bp coding regions that were 78.1%identical at the nucleotide level and 74.0%identical at the expected amino acid level.In comparison,human organic anion transporter 4(OAT4)was only 37.4%identical with RST at the expected amino acid level.Therefore,RST was verified as the mouse homologue of hURAT1based on molecularstructures.Figure 4.Immunofluorescence of mouse URAT1cRNA-injected oocyte (A)and non-injected oocyte (B)using affinity-purified anti-mURAT1antibody.2.55␮g/ml antibodies were applied at 4°C overnight and stained in the red fluorescence with Cy5conjugated anti-rabbit IgG.Immunohistochemistry of mouse kidney using affinity-purified anti-mURAT1antibody in 40ϫ(C),100ϫ(E),and 400ϫ(F)magnification.0.85␮g/ml antibodies were applied at 4°C overnight and stained in dark brown with diaminobentizine.(D)antibody absorption test using 100␮g/ml of antigen peptide corresponding to panel C.J Am Soc Nephrol 15:261–268,2004Function and Localization of Mouse URAT1265The identity between RST and hURAT1at the expected amino acid level was lower than the 87.8%identity between rat OAT1(8)and human OAT1(9),which has been reported to be a urate transporter (10).Mouse Slc22a12,contained in a genomic clone from mouse chromosome 19(Genbank acces-sion No.AC124394),was separated to ten exons by nine introns at the same exon-intron boundaries of human SLC22A12(Table 1).The nucleotide identities between each exon of human SLC22A12and that of mouse gene were dis-tributed from 67.2%(exon 10)to 83.8%(exon 9).Some N -glycosylation sites and protein kinase A-dependent phos-phorylation sites are identical between two cDNA clones in-dicated as Figure 7.Urate Handling in Mouse KidneyPlasma concentrations of urate in mice of the DBA/2N strain and ddY strain were reported as 0.192Ϯ0.017mg/dl and 0.173Ϯ0.016mg/dl,respectively (11).These values corre-spond to 10␮M urate and are much lower than the K m value of mURAT1.Therefore,the plasma urate level is not depen-dent on the urate transport activity of mURAT1in normal mice,but rather on the urate breakdown by uricase.However,the plasma urate concentration of uricase-deficient hyperurice-mia model mice (11.0Ϯ1.7mg/dl)closely approximated the K m value of mouse URAT1(12).Consequently,mURAT1may regulate the plasma urate level in uricase-deficient mice,similar to hURAT1.The fractional excretions of urate (CUA/CCr)in mice of the DBA/2N and ddY strains were 0.278Ϯ0.020and 0.382Ϯ0.021,respectively (11).Therefore,the mouse is one of the mammals that are categorized as net urate-reabsorption ani-mals,like humans,rats,and dogs.Like hURAT1,urate trans-port by mURAT1was trans-stimulated by L-lactate.Therefore,the physiologic concentration gradient between the lower con-centration of L-lactate in the luminal urine and the higher intracellular concentration of the proximal tubule cell can function as a driving force for reabsorption of urate.Benzbromarone and probenecid inhibited urate transport by mouse URAT1expressed on Xenopus oocytes.The uricosuric activity of benzbromarone and probenecid was demonstrated by in vivo studies with mice (11).Luminal perfusion of pro-benecid partially inhibited net uric acid reabsorption in mi-croperfusion studies with the mouse proximal tubule (13).Thus,mURAT1could function as a transporter for urate reab-sorption in mouse kidney.Localization of mURAT1was ob-served from the exit of Bowman ’s capsule coinciding with physiologic studies of the reabsorption site of urate in early proximal tubule.Pyrazinamide is rapidly hydrolyzed to the active antiurico-suric agent PZA.Subsequently PZA is oxidized by xanthine oxidase to 5-hydroxy PZA,which is devoid of antiuricosuric effects (14).Because pyrazinamide in the bath solution abol-ished the secretion of urate in the microperfusion studies with mouse proximal tubule (13),PZA generated in proximal tubule cells might trans-stimulate urate transport by mURAT1.Al-though trans-stimulation of urate transport by mouse and hu-man URAT1advocates that the anti-uricosuric effect of pyra-zinamide is through enhancement of urate reabsorption in kidney,UAT expressed at the brush border membrane was also reported to have a PZA-sensitive urate channel activity (15,16).Therefore,the possibility of inhibition of urate secretion by PZA must be investigated in vivo .Oxonate has been used as an uricase inhibitor to produce a hyperuricemia model in rodents (17,18).Since oxonate did not inhibit urate transport by mURAT1,oxonate-treated mice were suitable for studying the function of mURAT1in vivo .URAT1Expression in Mouse KidneyIt was demonstrated that the RST gene was expressed as a 2.3-kb transcript only in the kidney using Northern blotting.Moreover,in situ hybridization analysis using a cRNAprobeFigure 5.Western blot analysis of 10␮g of protein prepared from mouse kidney with 0.19␮g/ml affinity-purified ne 1,crude membrane fraction of mouse ne 2,Triton X-100extract from the crude membrane fraction of mouse ne 3,brush border membrane fraction from mouse ne 4,deglycosylated sample of the Triton X-100extract by PNGase nes 5and 6,antibody absorption tests by the 10␮g/ml antigen peptide corresponding to lanes 1and 2,respectively.Figure 6.Sex difference of the mURAT1expression.(A)Western blot analysis of 10␮g of protein of crude membrane fractions pre-pared from three male and three female mouse kidneys with 0.19␮g/ml affinity-purified anti-mURAT1antibody.(B)Northern blot analysis of 2␮g of total RNA purified prepared from three male and three female mouse kidneys using mURAT1probe and mouse ␤-actin probe.The ratio of band intensity of mURAT1mRNA/␤-actin mRNA was 1.17Ϯ0.05in male and 0.50Ϯ0.10in female (mean ϮSD).266Journal of the American Society of Nephrology J Am Soc Nephrol 15:261–268,2004constructed from a 1.5-kb 5'-RACE fragment showed that RST gene expression is restricted to the renal proximal tubule (6).It was also demonstrated that some proximal tubule cells ex-pressed mURAT1at the brush border membrane similar to human URAT1(3).Moreover,other proximal tubule cells expressed mURAT1as a dotlike pattern in the cytoplasm,which indicated that mURAT1localized on the intracellular organelles.From Western blotting,the N -glycosylated 70-kD form corresponds to mURAT1at the brush border membrane,and the native 62-kD form corresponds to the mURAT1attheFigure 7.Deduced amino acid sequence of mURAT1compared with that of hURAT1.Dots in the hURAT1sequence represent identity to mOAT1.*Predicted N -glycosylation sites;#predicted protein kinase A-dependent phosphorylation sites.Predicted membrane spanning domain are underscored.GenBank accession numbers:AB005451,RST/mURAT1;AB071863,hURAT1.Table 1.Sequence homology between mouse and human SLC22A12Mouse SLC22A12(bp)Human SLC22A12(bp)Nucleotide Identity (%)Amino Acid SequenceAmino Acid Identity (%)Exon 1a 40240277.61–13473.1Intron 1118782049Exon 210410481.7135–16879.4Intron 233052250Exon 315515580.0170–22078.4Intron 3777567Exon 416916978.1222–27671.9Intron 41529471228Exon 512412473.4278–31867.5Intron 514316853Exon 611611671.6319–35665.8Intron 656475254Exon 721521577.2358–42871.8Intron 741247656Exon 810910983.5430–46482.9Intron 817625953Exon 920420483.8466–53283.6Intron 954454963Exon 10646467.2534–55350.0aFrom initiation codon.J Am Soc Nephrol 15:261–268,2004Function and Localization of Mouse URAT1267intracellular organelles.The molecular weight of the deglyco-sylated62-kD protein was very close to61kD,the expected molecular weight from the amino acid sequence of mURAT1. Because the N-glycosylated70-kD form was in the Triton X-100insoluble fraction,mURAT1protein might be localized in the raft component in the brush border membrane.Although some intestinal brush border proteins were demonstrated to localize in the Triton X-100insoluble fraction(19),renal brush-border proteins in vivo have not been demonstrated in the Triton X-100insoluble fraction yet.The brush border protein Naϩ/Hϩexchanger3was reported to be expressed primarily in the detergent-insoluble fraction in lipid rafts of the apical surface of proximal tubule-derived OK cells(20). Sex differences in urate levels in human blood is one of the typical sex differences in endocrinological data,and is caused by a sex-dependent difference in the probenecid-sensitive urate reabsorption in human kidney(21).Because URAT1is respon-sible for the probenecid-sensitive urate reabsorption,the sex-dependent difference of mURAT1expression level is coinci-dent with pharamacological data from humans.Further investigation is needed to detail hormonal regulation of URAT1transcription and promoter analysis.In summary,RST transported urate(based on characteristics of molecular structure,transport function,and cellular local-ization)is apparently the mouse homologue of hURAT1.The 70-kD N-glycosylated form of mURAT1protein was ex-pressed in the brush border membrane of proximal tubule. Moreover,a sex-dependent difference of mURAT1transcrip-tion level was demonstrated.Therefore,mouse URAT1is an appropriate experimental model for studying the mechanism of membrane sorting of the URAT1protein and the hormonal regulation of its expression.AcknowledgmentsThe authors thank Ms.Akie Toki for her technical assistance.This study was supported in part by Grants-in-Aid from the Ministry of Education,Science,Sports,and Culture(#s1*******and13770048). 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