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作者：Douglas H.Sweet, Lauretta M. S.Chan, RamseyWalden, Xiao-PingYang, David S.Miller, John B.Pritchard作者单位：1 Department of Pharmaceutical Sciences, MedicalUniversity of South Carolina, Charleston, South Carolina29425; and Laboratory of Pharmacology andChemistry, National Institute of Environmental Health Sciences,National Institutes of Health, Research Triangle Park, North Carolina27709
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【摘要】
6 B; x6 T+ R$ \ ~. h/ M7 u Basolateral uptake of organicanions in renal proximal tubule cells is indirectly coupled tothe Na gradient through Na -dicarboxylatecotransport and organic anion/dicarboxylate exchange. One memberof the organic anion transporter (OAT) family, Oat1, is expressedin the proximal tubule and is an organic anion/dicarboxylate exchanger.However, a second organic anion carrier, Oat3, is also highly expressedin the renal proximal tubule, but its mechanism is unclear. Thus wehave assessed Oat3 function in Xenopus laevis oocytes andrat renal cortical slices. Probenecid-sensitive uptake of p- aminohippurate (PAH, an Oat1 and Oat3 substrate) andestrone sulfate (ES, an Oat3 substrate) in rat Oat3-expressing oocytes was significantly trans -stimulated by preloading the oocyteswith the dicarboxylate glutarate (GA). GA stimulation of ES transport by oocytes coexpressing rabbit Na -dicarboxylatecotransporter 1 and rat Oat3 was significantly inhibited when thepreloading medium contained Li or methylsuccinate (MS) orwhen Na was absent. All these treatments inhibit theNa -dicarboxylate cotransporter, but not rat Oat3.Li , MS, and Na removal had no effect whenapplied during the ES uptake step, rather than during the GA preloadingstep. Concentrative ES uptake in rat renal cortical slices was alsodemonstrated to be probenecid and Na sensitive.Accumulation of ES was stimulated by GA, and this stimulation wascompletely blocked by probenecid, Li , MS, taurocholate,and removal of Na . Thus Oat3 functions as an organicanion/dicarboxylate exchanger that couples organic anion uptakeindirectly to the Na gradient. $ ?) Y: C* u# f7 g2 o' ^. C3 y* l
【关键词】 kidney proximal tubule transport Oat estrone sulfate9 g8 q( O- d& i9 r3 V; R0 j' k
INTRODUCTION
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) V' t0 Q, G+ X W b. wACTIVE EXCRETORY TRANSPORT by the kidney is an important determinant of theeffects of therapeutics and toxic chemicals. Such transport is mediatedby multiple organic cation and organic anion transporters (OATs),primarily in the proximal segment of the nephron. The first step inrenal organic anion secretion, i.e., uptake at the basolateral membraneof renal proximal tubule cells, is mediated, uphill, andNa dependent (for a review, see Refs. 38 and 47 ). Mechanistically, this step is driven byindirect coupling to the Na gradient throughNa -dicarboxylate cotransport and organicanion/dicarboxylate exchange (tertiary active transport; Fig. 1 ) ( 34, 36, 37, 42 ). Thereis some uncertainty about the nature of the final step in organic anionsecretion, i.e., efflux across the apical membrane.
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% {8 K* P9 ~" T/ [Fig. 1. Model for classic renal organic anion (OA )secretion. Basolateral OA uptake on the classic renalOA transport system is indirectly coupled to theNa gradient by what has been termed a tertiary activeprocess. This process derives energy from ATP hydrolysis whenNa -K -ATPase (ATP) pumps Na outof the cell. Energetically downhill movement of Na intothe cell drives an Na -dicarboxylate cotransporter (NaDC),which maintains an outwardly directed dicarboxylate gradient. Finally,the dicarboxylate is used as a counterion by adicarboxylate/OA exchanger (OAT) to drive entry of theOA substrate into the cell. It is uncertain whetherefflux across the apical membrane occurs via OA exchangeor membrane potential difference-driven facilitated diffusion. -KG, -ketoglutarate; Prob, probenecid; MS, methylsuccinate.6 @- s/ |. u9 A! @9 R! E8 H+ [& C
8 } a+ ]% i# _1 kOat1 was the first renal OAT to be cloned ( 25, 41, 49 ).Not only was Oat1 localized to the basolateral membrane of renal proximal tubules ( 45, 51 ), but also it was shown tosupport organic anion/dicarboxylate exchange ( 49 ). Thus itwas assumed that Oat1 was indeed the transporter that drove theclassic, Na -dependent renal organic anion system. However,basolateral organic anion transport appears to be considerably morecomplex and may also be driven by one of the other OATs that have beencloned recently and partially characterized: Oat2, Oat3, and Oat4( 10, 23, 40, 43 ). Together with Oat1, they comprise asubfamily of the amphiphilic solute transporter family( Slc22a ) within the major facilitator superfamily ( 7, 44, 47, 52 ).9 r; p7 _) E4 Y$ H4 A
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The mechanisms by which Oat2, Oat3, and Oat4 couple transport tocellular metabolism have not been defined. This lack of understanding is particularly important for Oat3, inasmuch as it is the most highlyexpressed OAT in kidney and choroid plexus ( 28, 46 ). Recent immunolocalization studies detected the rat and human Oat3 orthologs (rOat and hOAT, respectively) in the basolateral membrane ofproximal tubules ( 9, 22, 28 ). Functional analysis of anOat3 knockout mouse model confirmed that murine Oat3 was present in thebasolateral membrane of proximal tubule cells and in the apicalmembrane of choroid plexus epithelial cells (i.e., the correctlocations to mediate concentrative organic anion uptake) and that thetransporter mediates a substantial fraction of organic anion uptakeacross the basolateral membrane of the renal proximal tubule and at theapical membrane of choroid plexus ( 46 ). When Oat3 wasinitially cloned and expressed in Xenopus laevis oocytes, experiments indicated that Oat3-mediated organic anion uptake wasneither coupled to the Na gradient nor trans -stimulated by dicarboxylates ( 9, 23 ). Nomechanism explaining how Oat3 could mediate uphill OA transport wasdemonstrated, and it was argued that Oat3 is not energetically coupledto metabolism but, rather, is simply a facilitated-diffusion carrier( 9, 23 ). However, facilitated diffusion cannot drive organic anion uptake in the face of opposing electrical andconcentration gradients, as required in the renal proximal tubule andchoroid plexus.5 l2 C! i# v6 ?2 Z8 U5 b
6 z- S% L. q9 q4 i& o# BWe recently found reduced uptake of p -aminohippurate (PAH),the model substrate for the classic renal OAT system, in renal corticalslices from Oat3 knockout mice ( 46 ). Because in every species tested, from mammals to invertebrates, PAH uptake by renal tissue is concentrative and almost entirely Na dependent,these results argued for a reassessment of the mechanism ofOat3-mediated transport. In the present study, we have assessed theenergetics of Oat3 using X. laevis oocytes expressing rOat3 and rat renal cortical slices. Our experiments unequivocally show thatOat3, similar to Oat1, is an organic anion/dicarboxylate exchanger thatindirectly couples concentrative organic anion uptake to theNa gradient. Thus, on the basis of energetics, transporton Oat3 is indistinguishable from transport on Oat1.
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4 Y3 [" }/ h- u0 K0 q' OMATERIALS AND METHODS5 T% @8 K( e7 y+ x
% m) }) z' `' C& h$ tTransport assays. X. laevis oocytes were obtained from dissected ovaries bycollagenase A treatment. Substrate uptake assays were performed 3 daysafter injection with 20 ng of capped cRNA as previously described( 14, 45, 48 ). Oocytes were randomly divided into experimental groups of four to eight each and incubated for 10 or 60 min at room temperature in oocyte Ringer 2 (OR-2) medium (in mM: 82.5 NaCl, 2.5 KCl, 1 Na 2 HPO 4, 3 NaOH, 1 CaCl 2, 1 MgCl 2, 1 pyruvic acid, and 5 HEPES, pH7.6) containing 25 µM [ 3 H]PAH (1 µCi/ml) or 100 nM 3 H-labeled estrone sulfate (ES; 2 µCi/ml) in the absenceor presence of 1 mM probenecid, 5 mM LiCl, or 1 mM methylsuccinate(MS). Individual oocyte radioactivity was measured by liquidscintillation spectroscopy with external quench correction. Forexperiments involving expression of Oat1 and anNa -dicarboxylate cotransporter, cRNA synthesized fromrOat1 and rabbit Na -dicarboxylate cotransporter 1 (rbNaDC-1) cDNA ( 33 ) were coinjected at a ratio of 2:1.For trans- stimulation studies, oocytes were preloaded by 90 min of incubation in OR-2 medium containing 0-5 mM glutarate (GA)or MS and quickly rinsed in GA- or MS-free medium before initiation ofuptake. Na -free uptake was determined in OR-2 mediumisosmotically adjusted using N -methylglucamine.
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Rat renal tissue slice preparation and uptake assays were performedaccording to standard laboratory protocols ( 35, 37 ). Animals were euthanized by CO 2 inhalation, and kidneys weredissected into freshly oxygenated ice-cold saline. Tissue slices ( 0.5mm, ~5-10 mg wet wt) were maintained in ice-cold modified Crossand Taggart saline (in mM: 95 NaCl, 80 mannitol, 5 KCl, 0.74 CaCl 2, and 9.5 Na 2 HPO 4, pH 7.4).Substrate (100 nM [ 3 H]ES) uptake was determined after5-60 min of incubation in the presence and absence of inhibitorsor Na (Na -free medium was isosmoticallyadjusted using N -methylglucamine). After uptake wasdetermined, slices were rinsed, blotted, and weighed and then dissolvedin 1 ml of 1 M NaOH, neutralized with 1 ml of 1 M HCl, and assayed byliquid scintillation spectroscopy. Duplicate medium samples (50 µl)were also assayed. Uptakes were calculated as uncorrectedtissue-to-medium ratios [i.e., (dpm/mg tissue) (dpm/µl medium)].0 N3 z' X6 X' r0 Q
6 Z# P w! D+ ~2 Z7 GAll animal experiments were conducted under protocols approved by theNational Institute of Environmental Health Sciences Animal Care and Use Committee.
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Statistics. Means were compared using paired or unpaired Student's t -test. Differences in mean values between control and testgroups were considered significant when P 0.05.
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) ^0 `! q1 b n9 M6 N" @. nChemicals. [ 3 H]PAH (4 Ci/mmol) and [ 3 H]ES (40 Ci/mmol)were purchased from PerkinElmer Life Sciences (Boston, MA). UnlabeledPAH, ES, probenecid, MS, GA, and taurocholate (TC) were obtained fromSigma (St. Louis, MO). All other chemicals were obtained fromcommercial sources and were of reagent grade.
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RESULTS
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Oat3-mediated organic anion transport in X. laevis oocytes. Sixty-minute uptake of 25 µM [ 3 H]PAH or 100 nM[ 3 H]ES was measured in oocytes expressing rOat1 or rOat3(Fig. 2 ). Oocytes expressing rOat1 andrOat3 exhibited substantial probenecid-sensitive PAH uptakecompared with water-injected control oocytes, demonstrating expressionof specific OATs in both groups of injected oocytes (Fig. 2 ). Despitefunctional transporter expression, rOat1-expressing oocytes did nottake up ES, whereas rOat3-expressing oocytes exhibited significantprobenecid-sensitive ES uptake (Fig. 2 ). As demonstrated previously forthe basolateral organic anion/dicarboxylate exchanger Oat1 ( 14, 49 ), incubating rOat1-expressing oocytes in medium containing2.5 mM GA for 90 min before substrate exposure (GA preloading)significantly stimulated probenecid-sensitive PAH uptake (Fig. 2 ). Importantly, preloading with GA also significantly stimulateduptake of PAH and ES by rOat3-expressing oocytes (Figs. 2 and 3 ). This trans stimulation ofrOat3-mediated ES uptake increased in a GA concentration-dependentmanner, with 2.5 mM GA preloading causing a maximal increase overnonpreloaded oocytes (data not shown). In contrast, preincubatingrOat3-expressing oocytes with MS, a dicarboxylate that cannotsubstitute for -ketoglutarate ( -KG) or GA on Oat1 ( 11, 18, 49 ), did not increase PAH or ES uptake by rOat3 (Fig. 3 ).8 T* A' _9 g( B
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Fig. 2. OA uptake mediated by trans -stimulation ofrat OA transporters 1 and 3 (rOat1 and rOat3). Oocytes injected withrOat1 and rOat3 cRNA were preincubated for 90 min in oocyte Ringer 2 (OR-2) medium without (control) or with 2.5 mM dicarboxy glutarate (GA;GA Preload). After cells were washed briefly with GA-free medium, theywere incubated in OR-2 with 25 µM 3 H-labeled p -aminohippurate (PAH) or 100 nM estrone sulfate (ES) for 60 min in the absence or presence of 1 mM probenecid (Prob).Water-injected oocytes showed negligible nonmediated substrate uptake(data not shown). Experiment was repeated in oocytes from 3 animals,and values are means ± SE from a representative animal (4-8oocytes per treatment). * Significantly different from control(nonpreloaded), P t -test).9 M6 h) L+ N4 C) i( \
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Fig. 3. Specificity of rOat3 trans -stimulation.Oocytes expressing rOat3 were preloaded for 90 min in OR-2 mediumwithout (control) or with 2.5 mM GA or MS, washed briefly indicarboxylate-free medium, and incubated with 25 µM[ 3 H]PAH or 100 nM [ 3 H]ES for 60 min.Experiments were repeated in oocytes from 2-4 animals (6-8oocytes per treatment per animal), and values are means ± SE.* Significantly higher than control, P t -test).
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$ C. P9 u+ J* t$ f& J) PTo determine whether the GA trans -stimulation ofrOat3-mediated transport observed in Figs. 2 and 3 could be facilitatedby energetic coupling to an Na gradient, X. laevis oocytes were coinjected with cRNAs for rOat3 and a rabbitNa -dicarboxylate cotransporter (rbNaDC-1). Previousstudies utilizing renal slices and renal basolateral membrane vesiclesfirmly established that the Na -dicarboxylate cotransporter(but not the organic anion/dicarboxylate exchanger) is inhibited by 5 mM Li , 1 mM MS, and Na replacement ( 6, 34, 37 ). We therefore determined the effects of those treatmentson the ability of GA preloading to stimulate ES uptake in doublyinjected oocytes (Fig. 4 ). Cellsincubated in OR-2 medium containing 100 µM GA for 90 min before ESuptake showed a significant increase in ES accumulation (Fig. 4 ).Li and MS added to the preloading buffer completelyblocked the trans- stimulatory effect of GA, andpreincubation in Na -free medium significantly reducedstimulation by GA (Fig. 4 ). These treatments were without effect whenimposed during the 10-min ES uptake period, rather than the preloadingperiod (data not shown). Thus these oocyte experiments demonstratedthat Oat3 is an organic anion exchanger that could be coupled to theNa gradient through Na -dicarboxylatecotransport.
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Fig. 4. Mechanistic coupling of rOat3 andNa -dicarboxylate cotransporter activities. Uptake of 100 nM [ 3 H]ES was measured in Xenopus laevis oocytes coinjected with 2:1 rOat3-rbNaDC-1 cRNAs. Oocytes werepreloaded for 90 min in OR-2 medium without (control) or with 100 µMGA. Effects of the presence of 5 mM Li , 1 mM MS, orNa -free medium during the preloading period were alsoassessed. Experiment was repeated in 6 animals (6-8 oocytes pertreatment per animal), and values are means ± SE( n = 6 animals). * Significantly different fromcontrol, P t -test). Significantly different fromGA-stimulated uptake in the absence of inhibitors, P t -test).! U' F, P0 I' w+ N" o
* R. j. w' O9 P8 QES uptake in rat renal cortical slices. ES is well established to be an Oat3 (but not an Oat1) substrate (Fig. 2 ) ( 9, 23, 46 ). Thus, to determine whether transport onOat3 was indeed indirectly coupled to Na in the kidney, wemeasured ES accumulation in rat renal cortical slices, a preparationthat tracks basolateral transport into the tubules ( 5, 54 ). Figure 5 shows the time course of 100 nM ES accumulation byrenal slices. Uptake was linear for 15 min and then appeared toapproach steady state during the subsequent 45 min. Uptake was clearlyconcentrative, e.g., tissue-to-medium ratios at 60 min were~22. ES accumulation was markedly inhibited by 1 mM probenecidthroughout the time course. Removal of Na from the uptakemedium was as effective as probenecid in inhibiting ES uptake (Fig. 5 ). Because this result is consistentwith tertiary active, indirect coupling to Na , aspreviously shown for Oat1 ( 14, 49 ), the ability ofexternally applied GA to stimulate ES uptake by the slices wasevaluated. External GA stimulated ES accumulation in aconcentration-dependent manner, with peak stimulation at 20 µM GA(Fig. 6 A ). This GAconcentration was used in all subsequent experiments. As in the oocyteexperiments shown above (Figs. 2-4 ), 5 mM Li and 1 mMMS (inhibitors of Na -dicarboxylate cotransport)significantly inhibited ES uptake in the presence of 20 µM GA,effectively reducing its accumulation to levels observed in thepresence of 1 mM probenecid. Removal of Na from the uptakebuffer (Na -free) was equally effective (Fig. 6 B ).
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Fig. 5. Na -dependent ES uptake in rat renal corticalslices. Tissue slices were incubated for 5-60 min in mediumcontaining 100 nM [ 3 H]ES without (control) or with 1 mMprobenecid (Prob) or in Na -free medium. The experiment wasrepeated in 3 animals (3 slices per treatment per animal), and datawere calculated as tissue-to-medium ratio [T/M; i.e., (dpm/mgtissue) (dpm/µl medium)]. Values are means ± SE( n = 3 animals). * Significantly different frominhibited uptake (i.e., probenecid or Na -freegroups), P t -test).
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Fig. 6. ES uptake by rat renal cortical slices. Tissue sliceswere incubated for 60 min in medium containing 100 nM[ 3 H]ES without (control) or with indicated inhibitors;some slices were incubated in Na -free medium. A : GA dose response, i.e., ES accumulation in the presenceof 0-200 µM GA in external buffer. * Significantly differentfrom 0 µM GA control, P t -test). B : inhibition of GA-stimulated EStransport. Significantly different from control: * P P t -test). Significantly different fromGA-stimulated uptake in the absence of inhibitors, P t -test). C : effectof taurocholate on GA-stimulated ES uptake. Significantly differentfrom control: * P P P t -test). Significantly different from uptake inthe presence of 20 µM GA, Na , and probenecid (Prob), P t -test). All experiments were repeated in 3 animals (3 slices per treatment per animal), and values are means ± SE( n = 3 animals)./ U5 O& A# {$ V+ r! m! J
6 A( I: ?9 }$ h( O1 j( TNa removal and probenecid did not completely inhibitconcentrative ES transport by renal slices. To investigate theremaining component of transport further, TC was used as an inhibitorof ES uptake (Fig. 6 C ). TC is known to be a substrate forseveral OATs, including Oat3 and members of the organicanion-transporting polypeptide (Oatp) family ( 1, 8, 20, 53 ). TC reduced ES uptake in a concentration-dependent manner.At 100 µM, TC inhibited ES uptake to a greater extent than 1 mMprobenecid or Na removal (Fig. 6 C ). Whenapplied together, 1 mM probenecid 100 µM TC reduced uptake toa greater extent than probenecid alone, indicating the presence of aprobenecid-insensitive, TC-sensitive component to ES uptake in renal slices.
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DISCUSSION2 G! w( T! P3 }
+ U5 Q8 E1 z L+ l$ |; rUnderstanding renal OAT function at the molecular level isessential to our ability to improve drug design, delivery andtherapeutic strategies and to understand and intervene in diseaseprogression. hOAT1-mediated transport has been implicated in thenephrotoxicity associated with nucleoside phosphonate antiviral(adefovir and cidofovir) therapy ( 13, 14, 19 ). Indeed,inhibition of active drug uptake into kidney proximal tubule cells viacoadministration of an Oat1 substrate may mitigate their nephrotoxicitywithout compromising their therapeutic value ( 29 ). OATfunction may also be a key factor in the progression of diseaseresulting from exposure to increased levels of toxic endogenous orxenobiotic substances. During chronic renal failure, uremic toxinsaccumulate in the blood, and recent studies have shown that severaluremic toxins are transported by, or are inhibitors of, rOat1 and rOat3 ( 15, 17, 30, 31 ). Thus these transporters may control theprogression of nephrotoxicity as well as urinary excretion ofpotentially toxic anionic drugs and metabolites.3 y. v1 @2 O( F7 e$ K, K. E! }3 h3 u
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Categorizing the contributions of individual OAT family members tooverall kidney transport function, although clinically important, hasbeen experimentally difficult, because multiple transporter paralogs(i.e., Oat1, Oat2, Oat3, and Oat4) are expressed in theproximal tubule and appear to be responsible for renal organic anionexcretion. However, increased knowledge of individual OAT specificityand subcellular localization is expanding our ability to interprettransport studies along these lines. For example, PAH is transported byOat1 and Oat3, but not by Oat2 or Oat4 ( 9, 10, 23, 40, 41, 46, 49 ); renal transport of ES is mediated by Oat3 and Oat4, but notby Oat1 or Oat2 ( 9, 10, 23, 40, 46 ); and TC is transportedby Oat3, but not by Oat1, Oat2, or Oat4 ( 9, 10, 40, 46 ).Thus, on the basis of specificity, Oat2 should not play any role in therenal transport of any of these three substrates. rOat1 ( 45, 51 ) and, recently, mouse Oat3 ( 46 ) have beenlocalized to the basolateral membrane of renal proximal tubule cells.In contrast, hOAT4 has been observed in the apical membrane( 3 ). This suggests that transport of PAH across thebasolateral membrane of renal proximal tubule cells (e.g., in renal cortical slices) would be predominantly mediated by Oat1 andOat3 and that, for the OATs, uptake of ES and TC would serve as ameasure of transport mediated solely by Oat3. In agreement with thisinterpretation, when expressed in X. laevis oocytes, rOat1and rOat3 transported PAH (Fig. 2 ) ( 23, 49 ). On the otherhand, as shown previously for Oat1 and Oat3 ( 9, 23, 46 ),ES is transported by Oat3, but not by Oat1 (Fig. 2 ). Thus, in oocytesand renal slice [reflects only basolateral uptake by the tubules( 5, 54 )], measurement of ES transport allows us to focuson Oat3-mediated events./ k2 T3 Q6 ?. h# a E# o
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Previous reports on Oat3 function in the X. laevis oocyteexpression system found no evidence for energetic coupling of transport to Na or dicarboxylate gradients ( 9, 23 ). Inthe present experiments, careful control of imposed dicarboxylategradients and coexpression of an Na -dicarboxylatecotransporter along with Oat3 has revealed indirect coupling toNa . Our data clearly show that rOat3-mediated uptake ofPAH and ES is markedly trans -stimulated by GA (Figs. 2 and 3 ). This is precisely the behavior previously demonstrated for thebasolateral organic anion/dicarboxylate exchanger rOat1/hOAT1( 14, 49 ). In addition, similar to the trans- stimulatory effect of GA on rOat1/hOAT1, GAstimulation of rOat3 uptake was concentration dependent (data notshown), whereas MS did not stimulate rOat3 uptake (Fig. 3 ). Thesefindings strongly suggest that Oat3, similar to Oat1, functions as anorganic anion/dicarboxylate exchanger and could be energeticallycoupled to the Na gradient through anNa -dicarboxylate cotransporter that generates thedicarboxylate counterion gradient utilized for organic anion exchange.
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To directly test the indirect coupling hypothesis, ES uptake wasmeasured in oocytes coexpressing rbNaDC-1 and rOat3 (Fig. 4 ). Again,preloading with GA stimulated uptake. However, as expected for thecoupled system, preloading in the presence of Li or MS(inhibitors of renal Na -GA cotransport) or removal ofNa blocked stimulation by preventing uptake of GA byrbNaDC-1 during the preincubation step (Fig. 4 ). None of thesetreatments was inhibitory when applied during ES uptake (data notshown). Thus, when GA entry was prevented by inhibition of theNa -GA cotransporter, rOat3-mediated ES uptake was notstimulated by external GA. These findings in X. laevis oocytes demonstrate that rOat3 (similar to Oat1) is an organicanion/dicarboxylate exchanger that can be driven by indirect couplingto the Na gradient.8 e, ]6 O1 ~/ z. I& [. H. M
) l) P7 n1 w3 c/ A) K" Y0 pThe same indirect coupling mechanism was demonstrated for the Oat3substrate ES at the basolateral membrane of proximal tubular cells. ESuptake by rat renal cortical slices in vitro was clearly Na dependent (Fig. 5 ). Moreover, as seen inrOat3-expressing oocytes, ES uptake was stimulated by addition ofexternal GA, and this stimulation was prevented by Li , MS,and probenecid and by Na removal (Fig. 6 B). This patternof stimulation and inhibition is that expected for organic aniontransport indirectly coupled to the Na gradient throughNa -dicarboxylate cotransport. It is the same pattern seenpreviously for renal uptake of the Oat1 and Oat3 substrate PAH( 34, 35, 37, 49 ).
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These experiments also demonstrate that Oat3 is not the onlytransporter responsible for ES uptake by rat renal cortical slices. Results from inhibition experiments using another Oat3 substrate, TC,indicate the presence of an Na -independent,probenecid-insensitive organic anion transporter (i.e., non-OAT). Another family of transporters, Oatp ( Slc21a ), mediate Na -independent, probenecid-insensitive transportof ES and TC ( 1, 2, 8, 20, 21, 32 ). Of these familymembers, Oatp1 is expressed on the apical membrane in the kidney,whereas expression of Oatp2, Oatp3, and Oatp4 was not detected in thekidney ( 1, 4, 21, 24, 53 ). The remaining member clonedthus far, Oatp5, is highly expressed in the kidney; however, it has not been localized or functionally characterized ( 12, 24 ).Thus this residual transport activity may be attributable to Oatp5." x) h& C G# n" L/ p
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In conclusion, we demonstrate here the first data identifying themechanism of organic anion uptake by Oat3. When studied in the X. laevis oocyte expression system or in rat renal cortical slices,rOat3 proved to be an organic anion/dicarboxylate exchanger that couldbe energetically coupled to the Na gradient throughNa -dicarboxylate cotransport. Thus, on the basis ofenergetic considerations (not specificity), Oat1 and Oat3 appear to beindistinguishable. This conclusion has important implications inseveral areas. First, it will be important to reevaluate pastconclusions concerning the relative roles of these two transporters(and the other OATs) in transport of endogenous metabolites, toxins,and therapeutic drugs and in drug-drug or drug-xenobiotic interactions.Second, renal transport of small organic anions (PAH and fluorescein) is regulated by multiple signals, including hormones and protein kinases ( 27, 39, 55 ). Because transport wasNa dependent, it was assumed that the target of regulationwas Oat1. However, Oat1 and Oat3 appear to respond to protein kinase Cactivation ( 26, 50 ). Thus, in light of the presentfindings, it will be important to determine in intact renal tissue howsignaling affects each of the transporters. Finally, the presentresults raise the following question: Do other members of the OATfamily of transporters utilize a similar mechanism? Although thisquestion has not been addressed directly, Oat2, which appears to belocalized to the basolateral membrane in the kidney and liver,transports dicarboxylates, suggesting that it too may be an organicanion/dicarboxylate exchanger ( 16, 40, 43 ). It remains tobe demonstrated that this is indeed the case.
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9 L3 }0 y% i; ^8 D3 _ACKNOWLEDGEMENTS$ N9 ~; o# \, B' l
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We thank Laura Hall for assistance with the oocyte expression experiments., [) U5 A( q6 o, j' b5 g5 D
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* ?9 B0 T6 }& u4 _4 N( }/ `3. Babu, E,Takeda M,Narikawa S,Kobayashi Y,Enomoto A,Tojo A,Cha SH,Sekine T,Sakthisekaran D,andEndou H. Role of human organic anion transporter 4 in the transport of ochratoxin A. Biochim Biophys Acta 1590:64-75,2002 .* S3 d( H& v3 y R+ g9 j
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