From the
Department of Pharmacology and
Toxicology, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka,
Tokyo, 181-8611, Japan, the ||Department of Oral
Physiology, Chosun University College of Dentistry, Gwangju 501-759, Korea,
and the
Department of Physiology, Faculty
of Science, Mahidol University, Bangkok 10400, Thailand
Received for publication, March 28, 2003 , and in revised form, May 8, 2003.
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ABSTRACT |
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INTRODUCTION |
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MRP2, an ABC (ATP-binding cassette) transporter, was suggested to be one of the exit paths of PAH in proximal tubular cells. Studies of membrane vesicles from MRP2-expressing HEK or Sf9 cells have indicated that MRP2 transports PAH (20, 21) in an ATP-dependent manner. Recently, it was shown that the human sodium-dependent phosphate cotransporter type 1 (NPT1) present at the apical membrane of proximal tubules transports organic anions including PAH. The PAH transport by NPT1 expressed in HEK293 was Cl sensitive (22). Although NPT1 is a candidate of the exit path for PAH at the apical membrane of proximal tubules, the reported properties of NPT1, in particular its K+-dependence, is not consistent with those of the voltage-driven PAH transporter at the apical membrane. In the present study, to identify the transporter responsible for the exit of PAH through the apical membrane, we have performed expression cloning using pig kidney cortex poly(A)+ RNA and identified a novel transporter present at the apical membrane of proximal tubules that mediates voltage-driven facilitated diffusion.
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EXPERIMENTAL PROCEDURES |
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Preparation of Size-fractionated Pig Kidney Poly(A)+
RNA and Expression CloningFour hundred micrograms of pig
kidney poly(A)+ RNA was size-fractionated using preparative gel
electrophoresis (Bio-Rad, model 491 Prep cell)
(6,
2628).
The isolated poly(A)+ RNA fractions were examined for
[14C]PAH (20 µM) transport activity by expression in
X. laevis oocytes in the high potassium uptake solution. The positive
fraction showing the greatest activity of [14C]PAH uptake was used
to construct a directional cDNA library
(2729).
Screening of the cDNA library was performed as described previously
(6,
2729).
cRNA synthesized in vitro from pools of 500 clones was injected
into Xenopus oocytes. A positive pool showing the [14C]PAH
uptake activity in the high potassium uptake solution was sequentially
subdivided and analyzed until a single clone (OATV1) was
identified. The cDNA was sequenced in both directions by the dye terminator
cycle sequencing method using an ABI PRISM 3100 Genetic Analyzer (PE
Biosystems).
Functional CharacterizationTwenty-five nanograms of OATV1 cRNA synthesized in vitro using T7 RNA polymerase from the OATV1 cDNA in pSPORT1 linearized with NotI were injected into defolliculated Xenopus oocytes. Two or 3 days after injection the uptake of radiolabeled substrates was measured in various uptake solutions (differing in ion composition) to examine its dependence on sodium or chloride. In sodium-free solution, LiCl or choline-Cl were used to replace NaCl in the standard uptake solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, pH 7.4). For the high potassium uptake solution, NaCl in the standard uptake solution was replaced by KCl. For Cl-free medium, sodium gluconate, potassium gluconate, calcium gluconate, and MgSO4 were used to replace NaCl, KCl, CaCl2, and MgCl2, respectively. To prepare the uptake solution at pH 5.5 and 6.5, MES was used instead of HEPES for the buffer system (30). The uptake measurements were performed for 60 min at room temperature (2225 °C). The radioactivity was counted by liquid scintillation spectrometry, and the values are expressed as femtomole per oocyte per min.
To examine the effects of the extracellular concentration of K+ and Cl on the OATV1-mediated [14C]PAH uptake, the concentrations of K+ or Cl were varied over the range of 0 to 100 mM (0, 5, 10, 25, 50, and 100 mM). The concentrations of K+ in the uptake solution were varied in Na+-free solution in which [KCl] plus [choline-Cl] was equal to 100 mM. The concentration of Cl was varied in the high potassium uptake solution in which [KCl] plus [K-gluconate] was equal to 100 mM.
Kinetic StudyBecause the uptake of substrates in high potassium uptake solution was linear longer than 2 h, the uptake was measured for 30 min for kinetic studies. The concentrations of PAH, urate, and estrone sulfate were varied from 10 µM to 10 mM, from 10 µM to 5 mM, and from 1 to 500 µM, respectively. OATV1-mediated substrate uptake was calculated as the difference between the values of uptake into cRNA-injected oocytes and those of control oocytes (without cRNA injection). The Km values were determined with the Eadie-Hofstee equation.
To measure the Ki values for the transport, oocytes expressing OATV1 were incubated for 30 min in high potassium uptake solution with various concentrations of [14C]PAH with or without addition of inhibitors. The Ki values were determined by double-reciprocal plot analysis in which 1/uptake rate of [14C]PAH was plotted against 1/[14C]PAH concentration. The Ki values were calculated from the following equation when competitive inhibition was observed: Ki = (Kt[I])/(Ka Kt), where Ka, Kt, and [I] are Km of PAH with inhibitor, Km of PAH without inhibitor, and concentration of inhibitor, respectively (31).
Efflux MeasurementFifty nanoliters of [14C]PAH
(2.5 nCi, 1 mM), [14C]urate (2.5 nCi,
1
mM), or [3H]estrone sulfate (25 nCi,
20
mM) was injected into oocytes with a fine-tipped glass micropipette
as described elsewhere (32,
33). The individual oocytes
were incubated in ice-cold standard uptake solution (96 mM NaCl, 2
mM KCl, 1.8 mM CaCl2, 1 mM
MgCl2, 5 mM HEPES, pH 7.4) for 5 min and then
transferred to the standard uptake solution kept at room temperature and
incubated at room temperature for 2, 5, 15, 30, 60, 90, 120, or 150 min. Then,
the incubation solution was collected to determine the efflux of substrates
from the oocytes into bath solution at the end of the incubation period. The
radioactivity remaining in the oocytes was also measured. The efflux value was
expressed as % radioactivity calculated from the radioactivity in the bathing
solution x 100%/(the radioactivity in oocyte plus the radioactivity in
bathing solution). The dependence of [14C]PAH efflux on inorganic
ions was determined by comparing the [14C]PAH uptake in the
standard uptake solution and that in the solutions in which NaCl was replaced
by KCl, sodium gluconate, potassium gluconate, LiCl, or choline-Cl as
mentioned above. The pH dependence of [14C]PAH efflux was measured
in the standard uptake solution with varied pH to 5.5, 6.5, 7.4, and 8.5. The
efflux was measured for 15 min over which the linear efflux value was
obtained. To examine the trans-stimulation of the efflux of PAH,
[14C]PAH-injected oocytes were incubated in the standard uptake
solution with or without non-radiolabeled PAH (0.01, 0.1, 1, 5, or 10
mM) for 15 min at room temperature.
For the uptake and efflux measurements in the present study, 810 oocytes were used for each data point. The values are expressed as mean ± S.E. The reproducibility of the results was confirmed by three separate experiments with different batches of oocytes. The results from representative experiments are shown in figures.
Electrophysiological MeasurementsThe electrical currents induced by 5 mM PAH in both control and OATV1-cRNA-injected X. laevis oocytes were recorded during voltage clamp at 60 to +40 mV in the standard uptake solution (24, 34, 35). At each step (20 mV interval) of voltage clamp, the baseline electrical current was first determined and then 5 mM PAH was added to the bath medium for 2 min. The solution was then changed back to the standard uptake solution and the record was continued until the electrical current was returned to the baseline level.
Northern AnalysisPoly(A)+ RNA (4 µg/lane) isolated from pig tissues was electrophoresed on 1% agarose, 2.2 M formaldehyde gel and transferred to nitrocellulose filter (Schleicher & Schuell). The filter was hybridized with 500-bp OATV1 cDNA labeled with [32P]dCTP at 42 °C overnight and washed finally in 0.1x SSC, 0.1% SDS at 65 °C (25).
Anti-peptide AntibodyA rabbit polyclonal antibody against a keyhole limpet hemocyanin-conjugated synthetic peptide, EVQDWAKER-QNTYL, corresponding to 14 amino acid residues near the carboxyl terminus (454467 of the amino acid sequence) of OATV1 was generated and affinity purified as described elsewhere (36, 37).
Western Blot AnalysisPig kidney epithelial membranes were prepared as described previously (38) and subjected to SDS-polyacrylamide gel electrophoresis (39). The separated proteins were transferred electrophoretically to a Hybond-P polyvinylidene difluoride transfer membrane (Amersham Biosciences). The membranes were treated with diluted affinity purified anti-OATV1 antibody (1:5,000) overnight at 4 °C. Thereafter, horseradish peroxidase-conjugated anti-rabbit IgG was used as the secondary antibody (Jackson ImmunoResearch Laboratories, Inc). The signals were detected using the ECL Plus system (Amersham Biosciences). The specificity of immunoreaction was confirmed by an absorption experiment in the presence of antigen peptide (100 µg/ml).
ImmunohistochemistryThree-micrometer paraffin sections of pig kidney were processed for light microscopic immunohistochemical analysis as described previously (36). The kidney sections were incubated with affinity purified anti-OATV1 antibody (1:1,000) at 4 °C overnight and treated with Envision (+) rabbit peroxidase (DAKO) for 30 min. The immunoreactions were detected with diaminobenzidine (0.8 mM) (40). For absorption experiments, the serial kidney sections were treated with the primary antibody in the presence of antigen peptide (100 µg/ml).
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RESULTS |
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Tissue DistributionThe Northern blot using the
OATV1 cDNA fragment as a probe showed a strong signal in pig kidney
and liver (Fig. 1a).
The OATV1 transcript was not detected in brain, lung, small
intestine, and skeletal muscle. The major transcript detected in liver and
kidney was 3.2 kb. In addition, a faint band at
1.8 kb was also
detected in both kidney and liver. The 4.4-kb faint band was detected only in
liver.
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In Western blot analysis, the antibody raised against the COOH terminus peptide of OATV1 recognized a band of 60 kDa in the membrane protein prepared from total pig kidney and the brush border membrane of pig kidney cortex (Fig. 1b). This band was consistent with a predicted molecular mass of OATV1 protein (51 kDa). The band disappeared in the presence of antigen peptide in the absorption test, confirming the specificity of the immunoreaction (Fig. 1b).
Transport Activity of
OATV1Because the luminal facilitated
transport system in pig and rabbit was reported to be markedly dependent on
membrane potential (15,
16), we examined the effect of
raising external K+ on the OATV1-mediated transport. The
elevation of external K+ depolarizes the plasma membrane of
Xenopus oocyte (42).
As shown in Fig. 2, the uptake
values of [14C]PAH, [14C]urate, [3H]estrone
sulfate, and [3H]estradiol-17-glucuronide by OATV1
expressing oocytes were significantly higher in high potassium solution (98
mMK+) compared with those incubated in standard uptake
solution (2 mM K+), whereas control oocytes showed no
difference between these conditions, suggesting that OATV1-mediated
transport is dependent on membrane voltage. The uptake of [14C]PAH
was saturable and followed Michaelis-Menten kinetics
(Fig. 3a) with a
Km value of 4.38 ± 0.96 mM
(mean ± S.E. of three separate experiments)
(Fig. 3b).
Km values for [14C]urate and
[3H]estrone sulfate were >5 and 0.212 mM,
respectively (data not shown).
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Characteristic of OATV1-mediated Organic Anion TransportThe PAH uptake by OATV1-expressing oocytes was also measured in various uptake solutions with different ionic contents. Replacement of Na+ with Li+ or choline+ did not affect [14C]PAH uptake (Fig. 4a), indicating that OATV1-mediated [14C]PAH transport was not dependent on Na+ in the uptake solution. The replacement of Na+ with K+ or Rb+ increased the OATV1-mediated transport (Figs. 2 and 4a). Fig. 4b further shows the dependence of OATV1-mediated [14C]PAH uptake on K+ concentration, indicating that raising extracellular K+ increased PAH transport in a concentration-dependent manner. Fig. 4a also shows the effect of the replacement of Cl with other anions. PAH uptake was higher in OATV1-expressing oocytes incubated in potassium gluconate solution than that in KCl uptake solution. PAH uptake was also higher in sodium gluconate, NaBr, or NaF solution than that in NaCl solution. As shown in Fig. 4c, PAH transport was dependent on Cl concentration. In the chloride-free solution and the solution containing 5 mM chloride, OATV1 expressing oocytes showed a significant elevation of PAH uptake (Fig. 4c). The PAH uptake did not show any remarkable pH dependence within the pH range of 5.5 and 8.5 (Fig. 4d). We also found that urate transport by OATV1 showed the same properties as those of PAH transport (data not shown).
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Voltage-dependent PAH TransportFig. 4 showed that PAH uptake by OATV1-expressing oocytes was affected by the changes of K+ or Cl concentration in the uptake solution. It seemed that PAH uptake is dependent on membrane voltage change generated by the alteration of K+ and Cl concentrations. To confirm the voltage dependence of PAH transport by OATV1, we performed electrophysiological measurement of PAH-induced currents under the voltage clamp condition. PAH application to the bath medium induced outward current in the oocytes expressing OATV1, whereas no significant current was elicited by 5 mM PAH in the control oocytes. The PAH-induced current was dependent on membrane voltage (Fig. 5). The electric currents induced by 5 mM PAH were increased when the holding potential was elevated from 60 to +40 mV as shown in Fig. 5.
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Characteristic of OATV1-mediated PAH EffluxAccording to the voltage-driven transport of PAH, in the physiological condition PAH is supposed to be transported from the intracellular compartment to the extracellular compartment along the electrical gradient. As shown in Fig. 6a, OATV1-expressing oocytes preloaded with [14C]PAH showed time-dependent efflux of radioactivity when incubated in the standard uptake solution. The voltage dependence of PAH transport was also observed for the efflux experiments. The OATV1-expressing oocytes showed a reduction of PAH efflux from the oocytes to the extracellular medium when the oocytes were incubated in high potassium uptake solution, which generates inside positive potential (Fig. 6b). The effect of pH on [14C]PAH efflux was examined in the standard uptake solution with varied pH. The efflux of [14C]PAH from the oocytes expressing OATV1 was dependent on extracellular pH. [14C]PAH efflux was elevated by incubating the oocytes in the uptake solution with higher pH, whereas it was reduced by lowering the pH of the uptake solution (Fig. 6c). To determine whether OATV1 is an exchanger or a facilitated transporter, the efflux of radioactivity from the oocytes preloaded with [14C]PAH was compared in the absence and presence of extracellular PAH. The efflux of radioactivity was not affected by the extracellularly applied PAH (Fig. 6d), consistent with the property of the transporters mediating facilitated diffusion. The efflux of [14C]urate and [3H]estrone sulfate also showed the identical properties compared with that of [14C]PAH (data not shown).
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Substrate Selectivity of OATV1The substrate selectivity of OATV1 was investigated by inhibition experiments in which the uptake of 20 µM [14C]PAH was measured in the presence of high concentrations of inhibitor. The concentrations of inhibitors were 1 and 5 mM except for ochratoxin A (0.1 mM) and 5-nitro-2-(3-phenylpropylamino)benzoate (0.1 and 1 mM). The PAH uptake was highly inhibited by estrone sulfate, probenecid, 5-nitro-2-(3-phenylpropylamino)benzoate, bumetanide, and furosemide but not by D- and L-lactate, glutarate, and tetraethylammonium (Fig. 7a). Some sulfate and glucuronide conjugates also inhibited OATV1-mediated [14C]PAH uptake. The inhibitory effect on [14C]PAH uptake was stronger for sulfate conjugates than that for glucuronide conjugates (Fig. 5, b and c). The inhibition of [14C]PAH uptake by unlabeled PAH was shown to be competitive in a double reciprocal plot analysis with a Ki value of 0.83 mM. Urate and estrone sulfate also competitively inhibited PAH uptake with Ki values of 1.87 mM and 17.13 µM, respectively (data not shown). Because OATV1 exhibited sequence similarity to NPT1 that was reported to transport both phosphate and organic anions, we examined whether OATV1 transports [32P]phosphate. In the condition in which we detected the [32P]phosphate (100 µM) uptake by rat NaPi type IIa, Na+/phosphate transporter, and OATV1-expressing oocytes did not show a detectable amount of [32P]phosphate uptake (data not shown).
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Immunohistochemical AnalysisThe immunohistochemical analysis of pig kidney revealed that OATV1 protein was localized in the apical membrane of proximal tubules. The immunoreactivity was detected only in the cortex but not in the medulla portion of pig kidney (Fig. 8). The immunostaining at the apical membrane of proximal tubule completely disappeared in the absorption experiments in which the tissue sections were treated with primary antibody in the presence of antigen peptide (Fig. 8c), confirming the specificity of immunoreactions.
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DISCUSSION |
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Although OATV1-mediated PAH transport was Na+-independent, it was affected by altering extracellular K+ and Cl (Fig. 4). When the extracellular concentration of K+ was increased or that of Cl was decreased, the PAH uptake mediated by OATV1 was elevated. This suggests that the PAH transport was altered by the action of membrane voltage. Based on the Goldman-Hodgkin and Katz equation, Vm = (RT/F)-log[(PNa[Na+]o + PK[K+]o + PCl[Cl]i)/(PNa[Na+]i + PK[K+]i + PCl[Cl]o)], the increase in extracellular K+ or decrease in extracellular Cl results in the elevation of membrane potential (toward the positive direction). This membrane depolarization is proposed to promote the uptake of negatively charged PAH via OATV1 once the net charge of the fully loaded carrier is negative (24). Moreover, the results from electrophysiological measurements showed that the membrane depolarization increased the outward current associated with the PAH uptake into the oocytes expressing OATV1 (Fig. 5), supporting the idea that the PAH transport by OATV1 is a voltage-driven transport.
To prove that the transport via OATV1 is a facilitated transport of organic anions, the properties of the efflux of PAH mediated by OATV1 was examined. It was shown in the present study that the efflux of PAH was not stimulated by the application of PAH in the bath solution (extracellular medium), suggesting that PAH transport via OATV1 is not trans-stimulated. Therefore, together with the results from electrophysiological measurements demonstrating the voltage dependence of the outward current associated with PAH transport (Fig. 5), it is suggested that OATV1-mediated transport involves electrogenic facilitated diffusion driven by membrane voltage, rather than the exchange of substrate organic anions.
We found that PAH efflux by OATV1 expressed in oocytes was increased by elevating extracellular pH and decreased by lowering extracellular pH (Fig. 6c). This may be consistent with the idea of PAH/OH exchange that has been proposed for the PAH exit path through the apical membrane of renal proximal tubules (12, 13). We attempted to clarify whether OATV1 mediates the PAH/OH exchange by incubating the oocytes expressing OATV1 in the medium containing sodium acetate or NH4Cl that causes intracellular acidification (4446). PAH uptake was, however, not altered significantly by these maneuvers (data not shown). There are two possible explanations for this observation; the first is that OATV1 exclusively mediates facilitated transport and a PAH/OH exchange mode is not involved for OATV1. The second is that the voltage-driven PAH transport is much more predominant than the PAH/OH exchange mode, so that it masked the PAH/OH exchange mode in the experimental conditions. Further investigation is necessary to understand the action of extracellular pH on PAH efflux via OATV1.
OATV1 mediated the transports of PAH, urate, sulfate, and
glucuronide conjugates of steroid hormones in a sodium-independent manner. In
addition, OATV1 interacted with various anionic compounds such as
nonsteroidal anti-inflammatory drugs and diuretics. Therefore,
OATV1 is a multispecific organic anion transporter, overlapping
substrate selectivity with members of the OAT family. Most of the substrates
of OAT1 (6,
7,
47) except
-ketoglutarate and glutarate interacted with OATV1. In
addition, the substrates of OAT3
(48) and OAT4
(49) mostly interacted with
OATV1. It is interesting to know what structural trait is
responsible for such similar substrate selectivity among these structurally
distinct organic anion transporters. For OAT1, the presence of a negatively
charged moiety and an appropriately sized hydrophobic core is proposed to be
an important requirement to be a substrate
(50,
51). Such a simple requirement
would also be applicable to OATV1. Consistent with this, sulfate
conjugates inhibited OATV1-mediated uptake, whereas
Na2SO4 did not show any effects
(Fig. 7). The presence of the
anionic moiety and the backbone hydrophobic core is proposed to be an
important requirement to be accepted by the binding site of
OATV1.
Based on the voltage-driven facilitated transport mechanisms of OATV1 and its localization at the apical membrane of proximal tubules, OATV1 is presumed to be well suited as an exit path for organic anions via the apical membrane of renal proximal tubules. Organic anions would be transported across apical membrane via OATV1 along the electrochemical potential gradient. In fact, it is reported that PAH and organic anions driven by membrane voltage are secreted from the epithelial cells into the proximal tubule lumen. OATV1 is proposed to play an important role for this process. It is well known that PAH and many endogenous and exogenous organic anions are taken up from the blood across the basolateral membrane into proximal tubule cells via organic anion transporters OAT1 and OAT3 in exchange for intracellular dicarboxylates (69). OAT1 is highly expressed at the basolateral side of the S2 segment of the proximal tubule (40), whereas OAT3 is expressed throughout the proximal tubule (52). In this study, OATV1 was shown to be expressed in the apical membrane of all the segments of the proximal tubule. Therefore, we propose that organic anions are taken up by the epithelial cells of renal proximal tubules via OAT1 and OAT3 and then exit from the epithelial cell via OATV1. Large organic anions with bulky moieties that are not substrates of OATV1 may be transported out of the epithelial cells via MRP2, an ABC transporter, using energy from ATP hydrolysis. It was previously reported that the secretion of PAH via the apical membrane of the proximal tubule might also be mediated by a PAH/glutarate (18) or PAH/OH exchanger (12, 13). Therefore, in addition to OATV1, other unidentified transporters may also participate in the excretion of organic anions through the epithelium of the renal proximal tubules.
We have identified a novel Na+-independent voltage-driven organic anion transporter, OATV1. It localizes at the apical membrane of the proximal tubule. This transporter might play an important role in the renal excretion of drugs, xenobiotics, and their metabolites in the form of organic anions.
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FOOTNOTES |
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* This work was supported in part by grants from the Ministry of Education,
Culture, Sports, Science and Technology of Japan, the Japan Society for the
Promotion of Science, the Promotion and Mutual Aid Corporation for Private
Schools of Japan, the Japan Health Sciences Foundation, the Uehara Memorial
Foundation, and Health and Labor Sciences Research Grants for Research on
Advanced Medical Technology: Toxicogenomics Project. The costs of publication
of this article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Research fellow supported by the Labor Sciences Research Grants for
Research on Advanced Medical Technology: Toxicogenomics Project.
¶ Research fellow of the Japan Society for Promotion of Science.
** Supported by Japan Health Sciences Foundation.
Supported by the Uehara Memorial Foundation.
¶¶ To whom correspondence should be addressed: Dept. of Pharmacology and Toxicology, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, Japan. Tel.: 81-422-47-5511 (ext. 3451); Fax: 81-422-79-1321; E-mail: endouh{at}kyorin-u.ac.jp.
1 The abbreviations used are: PAH, p-aminohippurate; OAT,
organic anion transporter; OATV1, voltage-driven organic anion
transporter 1; MES, 4-morpholineethanesulfonic acid.
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ACKNOWLEDGMENTS |
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REFERENCES |
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