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Vol. 59, Issue 5, 1277-1286, May 2001
Department of Pharmacology and Toxicology (S.H.C., T. S., Y. K., H. E.) and Second Department of Surgery (J.F., Y.K., T.G.), Kyorin University School of Medicine, Shinkawa, Mitaka, Tokyo, Japan
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Abstract |
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 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
References
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A cDNA encoding a multispecific organic anion transporter 3 (hOAT3) was isolated from a human kidney cDNA library. The hOAT3 cDNA consisted of 2179 base pairs that encoded a 543-amino-acid residue protein with 12 putative transmembrane domains. The deduced amino acid sequence of hOAT3 showed 36 to 51% identity to those of other members of the OAT family. Northern blot analysis revealed that hOAT3 mRNA is expressed in the kidney, brain, and skeletal muscle. When expressed in Xenopus laevis oocytes, hOAT3 mediated the transport of estrone sulfate (Km = 3.1 µM), p-aminohippurate (Km = 87.2 µM), methotrexate (Km = 10.9 µM), and cimetidine (Km = 57.4 µM) in a sodium-independent manner. hOAT3 also mediated the transport of dehydroepiandrosterone sulfate, ochratoxin A, PGE2, estradiol glucuronide, taurocholate, glutarate, cAMP and uric acid. Estrone sulfate did not show any trans-stimulatory effects on either influx or efflux of [3H]estrone sulfate via hOAT3. hOAT3 interacted with chemically heterogeneous anionic compounds, such as nonsteroidal anti-inflammatory drugs, diuretics, sulfobromophthalein, penicillin G, bile salts and tetraethyl ammonium bromide. The hOAT3 protein was shown to be localized in the basolateral membrane of renal proximal tubules and the hOAT3 gene was determined to be located on the human chromosome 11q12-q13.3 by fluorescent in situ hybridization analysis. These results suggest an important role of hOAT3 in the excretion/detoxification of endogenous and exogenous organic anions in the kidney.
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Introduction |
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Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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The
kidney, as well as the liver, plays a primary role in the excretion of
drugs and drug metabolites (Moller and Sheikh, 1982
; Pritchard and
Miller, 1993; Ullrich and Rumrich, 1993; Meier, 1995; Muller and
Jansen, 1997). In addition to glomerular filtration, the kidney
excretes charged drugs via carrier-mediated pathways, which are organic
anion and organic cation transport pathways, in renal proximal tubular
cells (Sperber, 1959; Weiner and Mudge, 1964; Ullrich and Rumrich,
1988; Pritchard and Miller, 1991). In particular, the organic anion
transport pathway has been shown to mediate the elimination of various drugs.
Transepithelial transport of organic anions in proximal tubules is
carried out by two distinct transporters; first, organic anions are
transported from the peritubular plasma by basolateral organic anion
transporter(s) and subsequently effluxed into the tubular lumen by
luminal transporter(s). Until early 1990s, renal organic anion
transport was thought to be carried out by a few carrier proteins that
showed wide substrate specificity. Various anionic drugs have been
indicated to be taken up into the proximal tubular cells by the classic
p-aminohippurate (PAH) transporter. For example, 
-lactam
antibiotics were suggested to be rapidly extracted by the kidney via
the PAH transporter, and the concomitant use of probenecid, which is a
typical inhibitor of the system reduces renal elimination of
-lactams (Ullrich et al., 1989). Recently, we have cloned the
classic PAH transporter expressed in the basolateral membranes
of proximal tubular cells from the rat kidney, and designated it as rat
organic anion transporter 1 (rOAT1) (Sekine et al., 1997). OAT1 is a
PAH/dicarboxylate exchanger and mediates the high-affinity transport of
PAH in a sodium-independent manner. Analysis of heterologous expression
systems of rOAT1 in oocytes and culture cells revealed that rOAT1 has
the ability to transport anionic drugs, such as -lactam antibiotics,
NSAIDs, methotrexate, and antiviral drugs, as well as various
endogenous organic anions and exogenous substances. The transport
properties of rOAT1 are nearly identical to those of the classic PAH
transporter. In addition, we have identified several OAT isoforms:
rOAT2 (Sekine et al., 1998), rOAT3 (Kusuhara et al., 1999), and hOAT4
(Cha et al., 2000). All of these isoforms are commonly expressed in the kidney, and their potential roles in renal handling of organic anions
have been indicated.
Although the number of the OAT isoforms is growing rapidly, characterization of each isoform is very limited. Moreover, information on human homologs is sparse. Among the OAT isoforms, rOAT3, as well as rOAT1, exhibits a markedly wide substrate selectivity, and its human homolog is considered a key molecule in the renal handling of organic anions. Because hOAT1 has been suggested to show rather limited capacity for organic anion transport, hOAT3 might play a large role in the human kidney.
Here, we report on the molecular cloning, functional characterization, and localization of hOAT3. The results indicate that hOAT3 shows a very wide substrate specificity and is localized in basolateral membranes of proximal tubular cells.
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Experimental Procedures |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Materials. The materials used were purchased from following sources: [14C]p-aminohippurate (2.00 GBq/mmol), [3H]cAMP (1.2 TBq/mmol), [3H]DHEA sulfate (520 GBq/mmol), [3H]estradiol-glucuronide (1.6 TBq/mmol), [3H]estrone sulfate (2.0 TBq/mmol), [3H]PGE2 (7.4 TBq/mmol), [14C]salicylate (2.0 GBq/mmol), [14C]succinate (2.18 GBq/mmol), [3H]taurocholate (111 GBq/mmol), and [14C]TEA (0.13 GBq/mmol) were from PerkinElmer Life Science Products (Boston, MA); [3H]glucuronic acid (185 GBq/mmol), [14C]glutarate (2.0 GBq/mmol), [3H]ibuprofen (18.5 TBq/mmol), [14C]uric acid (1.85 GBq/mmol), and [14C]valproic acid (2.1 GBq/mmol) were from American Radiolabeled Chemicals, Inc. (St. Louis, MO); [3H]acyclovir (333 GBq/mmol), [3H]methotrexate (555 GBq/mmol), and [3H]ochratoxin A (547.6 GBq/mmol) were from Moravek Biochemical Inc. (Brea, CA); [3H]cimetidine (673 GBq/mmol) was from Amersham Pharmacia Biotech (Uppsala, Sweden); DHEA sulfate, estrone sulfate, N-methyl-D-glucosamine, p-aminohippuric acid, sulfobromophthalein, ibuprofen, furosemide, bumetanide and azidothymidine were from Sigma (St. Louis, MO). All other chemicals and reagents used were of analytical grade and obtained from commercial sources.
Reverse Transcription-PCR and Isolation of hOAT3.
EST
(expressed sequence tag) database were searched for "query rOAT3",
and an EST clone (H20345) was identified. Primers were designed based
on the nucleotide sequence of H20345: forward primer,
5'-AAGTTCATCACCATCCTCTC-3'; reverse primer, 5'-GATCCCGTAAGATGATATTG-3'. Using this set of primers, we performed reverse transcription-PCR using
the human kidney poly(A)+ RNA. The protocol for
PCR was as follows: 94°C for 10 s, 57°C for 30 s, 72°C
for 30 s, 35 cycles. The 32P-labeled dCTP
probe was synthesized from the PCR clone and used for the screening of
a human kidney cDNA library. A nondirectional cDNA library was prepared
from human kidney poly(A)+ RNA (CLONTECH, Palo
Alto, CA) using the Superscript Choice system (Life Technologies,
Gaithersburg, MD), and the cDNAs were ligated into 
ZipLox
EcoRI arms. Replicated filters of a phage library were
hybridized overnight at 37°C in a hybridization solution [50%
formamide, 5× standard saline citrate (SSC), 3× Denhardt's solution,
0.2% SDS, 10% dextran sulfate, 0.2 mg/ml denatured salmon sperm DNA,
2.5 mM sodium pyrophosphate, 25 mM MES, and 0.01% Antifoam B, pH
6.5], and washed at 37°C in 0.1× SSC and 0.1% SDS.
Sequence Determination. Specially synthesized oligonucleotide primers were used for the sequencing of the hOAT3 cDNA by the dye-termination method using ABI Prism 310.
cRNA Synthesis and Uptake Experiments using Xenopus
laevis Oocytes.
cRNA synthesis and uptake measurements
were performed as described previously (Kusuhara et al., 1999). The
capped cRNA was synthesized in vitro using T7 RNA polymerase from the
plasmid DNA linearized with XbaI. Defolliculated oocytes
were injected with 10 ng of the capped hOAT3 cRNA and incubated in
Barth's solution (88 mM NaCl, 1 mM KCl, 0.33 mM
Ca(NO3)2, 0.4 mM
CaCl2, 0.8 mM MgSO4, 2.4 mM
NaHCO3, and 10 mM HEPES) containing 50 µg/ml
gentamicin at 18°C. After 2 to 3 days of incubation, uptake and
efflux experiments were performed at room temperature in ND96 solution
(96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM
MgCl2 and 5 mM HEPES, pH 7.4) as described
elsewhere (Kusuhara et al., 1999). We repeated each experiment more
than two times to confirm the results. The representative results are
shown in Figs. 3 to 5. The kinetic parameters were obtained by an
iterative nonlinear least-squares method using a MULTI program (Yamaoka
et al., 1981).
Northern Blot Analysis. A commercially available hybridization blot containing poly (A)+ RNA from various human tissues (human 12-lane multiple tissue Northern blot; CLONTECH) was used for the Northern blot analysis for hOAT3. We used full-length of hOAT3 cDNA as a probe. The master blot filter was hybridized with the probe 1 h at 68°C according to the manufacture's instructions. The filter was washed finally in a high stringency condition (0.1× SSC and 0.1% SDS at 68°C).
Immunohistochemical Analysis. For immunohistochemical analysis, rabbits were immunized with a keyhole limpet hemocyanin-conjugated synthesized peptide, CRIPLQPHGPGLGSS, corresponding to cysteine and the 14 amino acids of the COOH terminus of hOAT3. Two-micrometer wax sections of nephrectomized human kidney were processed for light microscopic immunohistochemical analysis, using the streptavidin-biotin-horseradish peroxidase complex technique (LSAB kit; DAKO, Carpinteria, CA). The renal tissue was from a tumor patient and approved by the Kyorin University Institutional Review Board to be used for medical study. Sections were dewaxed, rehydrated, and incubated with 3% H2O2 for 10 min to eliminate endogenous peroxidase activity. After rinsing in 0.05 M Tris-buffered saline containing 0.1% Tween-20, sections were treated with 10 µg/ml of primary rabbit polyclonal antibodies (4°C overnight). Thereafter, the sections were incubated with the secondary antibody, biotinylated goat polyclonal antibody against rabbit immunoglobulin (DAKO), diluted 1:400 for 30 min with horseradish peroxidase-labeled streptavidin. This step was followed by incubation with diaminobenzidine and hydrogen peroxide. The sections were counterstained with hematoxylin and examined by light microscopy. For preabsorption experiment, the hOAT3 peptide (200 µg/ml) was added to the hOAT3-specific antibody solution and incubated overnight at 4°C. Using this preabsorbed antibody, the immunohistochemistry was performed as described above.
Fluorescent in Situ Hybridization Analysis.
Lymphocytes
isolated from human blood were cultured in a-minimal essential medium
(a-MEM) supplemented with 10% fetal calf serum and phytohemagglutinin
at 37°C for 68 to 72 h. The lymphocyte cultures were treated
with 0.18 mg/ml bromodeoxyuridine (Sigma) to synchronize the cell
population. The synchronized cells were washed three times with
serum-free medium to release the block and recultured at 37°C for
6 h in a-MEM with 2.5 mg/ml thymidine (Sigma). Cells were
harvested and slides were made using standard procedures including
hypotonic treatment, fixation, and air-drying. The hOAT3 probe was
biotinylated with dATP using the BRL BioNick labeling kit (15°C,
1 h) (Heng et al., 1992). The slides were then baked at 55°C for
1 h. After the RNase treatment, the slides were denatured in 70%
formamide in 2× SSC for 2 min at 70°C followed by dehydration with
ethanol. Proves were denatured at 75°C for 5 min in a hybridization
mixture consisting of 50% formamide and 10% dextran sulfate. Probes
were applied to the denatured chromosomal slides. After overnight
hybridization, the slides were washed and screened, as well as
amplified. The fluorescent in situ hybridization (FISH) signals and
4,6-diamidino-2-phenylindole (DAPI) banding patterns were recorded
separately by taking photographs, and the assignment of the FISH
mapping data to chromosomal bands was achieved by superimposing FISH
signals with DAPI-banded chromosomes (Heng and Tsui, 1993)
Statistical Analysis. Data are expressed as mean ± S.E.M. Statistical differences were determined using Student's t test. Differences were considered significant at the level of p < 0.05.
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Results |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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EST database search identified an EST clone, H20345, that showed
significant identity to rOAT3. Using H20345 as a probe, one positive
clone (hOAT3) was isolated by screening 240,000 plaques from the human
kidney cDNA library. Although the hOAT3 has been reported (Race et al.,
1999), it has no transport function. To compare with our hOAT3 gene, we
employed hOAT3* that was cloned by Race et al. hOAT3 cDNA
consisted of 2179 base pairs encoding a 543-amino-acid residue protein.
Figure 1 shows the deduced amino acid
sequence of hOAT3 in the alignment with those of hOAT3*, hOAT1, rOAT2,
and hOAT4. The amino acid sequence of hOAT3 showed 85, 51, 36, and 44%
identity with those of hOAT3*, hOAT1, rOAT2, and hOAT4, respectively.
hOAT3 also showed significant identity with human OCT1 (39%) and OCT2
(37%) (Gorboulev et al., 1997) and rat OCT3 (35%) (Kekuda et al.,
1998). The SOSUI analysis (Kyte and Doolittle, 1982) predicted 12 membrane-spanning domains in hOAT3 (hydropathy plot not shown). As in
the case of members of OAT and OCT families, N-glycosylation
sites (residues 54, 81, 86, and 102) and protein kinase C-dependent
phosphorylation sites (residues 266, 528, and 511) are suggested in the
sequence of hOAT3 (Fig. 1).
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The expression of hOAT3 mRNA in human tissues was investigated (Fig.
2). A strong mRNA band was detected in
the kidney (2.2 kilobase pairs) and weak bands were also detected in
the skeletal muscle and brain. No hybridization signals were detected
with mRNAs isolated from other tissues, including the heart, thymus, spleen, liver, small intestine, lung, and peripheral blood leukocytes.
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Using the Xenopus laevis oocyte expression system, we
investigated the transport characteristics using various substances. Figure 3 shows the transport properties
of estrone sulfate via hOAT3. The cell-associated count of
[3H]estrone sulfate increased linearly until
3 h of incubation in hOAT3-expressing oocytes. This result
indicates that hOAT3 not only binds but also translocates estrone
sulfate into the cytoplasm (Fig. 3A). The uptake rate of estrone
sulfate via hOAT3 was not affected by the replacement of the
extracellular sodium with lithium, choline, or
N-methyl-D-glucosamine (Fig. 3B). In
the experiment shown in Fig. 3C, the trans-stimulatory
effect of estrone sulfate on OAT3-mediated efflux of estrone sulfate
was examined. The efflux of estrone sulfate was not
trans-stimulated in the presence of extracellular estrone
sulfate (0.5, 5, and 50 µM).
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The concentration dependence of hOAT3-mediated uptake of
[3H]estrone sulfate,
[14C] PAH,
[3H]methotrexate, and
[3H]cimetidine was examined (Fig.
4). hOAT3-mediated uptake of these four
compounds showed saturable kinetics and followed the Michael-Menten equation. Nonlinear regression analyses yielded
Km values of 3.1 ± 0.8 µM,
87.2 ± 11.1 µM, 10.9 ± 1.7 µM, and 57.4 ± 10.9 µM and Vmax values of 5.7 ± 1.0 pmol/h/oocytes, 20.4 ± 3.6 pmol/h/oocytes, 2.8 ± 0.3 pmol/h/oocytes, and 92.2 ± 11.2 pmol/h/oocytes for estrone sulfate, PAH, methotrexate, and cimetidine, respectively.
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The influx of various organic tracers via hOAT3 was investigated (Table
1). The uptake rates of
[3H]estrone sulfate,
[3H]DHEA sulfate,
[3H]ochratoxin A,
[14C]p-aminohippurate,
[3H]methotrexate,
[3H]cimetidine,
[3H]prostaglandin E2,
[3H]estradiol glucuronide,
[3H]taurocholate, [14C]
glutarate, [14C] salicylate,
[3H]cAMP, and [14C]uric
acid in oocytes expressing hOAT3 were much higher than those of control
oocytes. No significant uptake of [14C]valproic
acid, [14C]succinate,
[3H]glucuronic acid,
[3H]ibuprofen,
[14C]TEA, and
[3H]acyclovir was detected.
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To investigate substrate selectivity of hOAT3, an inhibition study was
performed. The cis-inhibitory effect of various compounds (5 µM) on hOAT3-mediated [3H]estrone sulfate (50 nM) uptake was investigated (Fig. 5A).
Five micromolar unlabeled probenecid, DHEA-s, indomethacin, ibuprofen, diclofenac, furosemide, bumetanide, and cholate exhibited definite inhibitory potency. Salicylate, glutarate, penicillin G,
sulfobromophthalein, taurocholate, corticosterone (a neutral steroid
hormone), quinidine, and TEA bromide exhibited modest inhibitory
activity. In contrast, ouabain and guanidine did not show inhibitory
activity. The IC50 values of estrone sulfate,
MTX, PAH, and cimetidine were about 3.0 × 10
6 M, 4.2 × 105
M, 1.8 × 104 M, and 7.0 × 105 M, respectively (Fig. 5B).
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Light microscopy of 2-µm wax sections demonstrated that there was
specific immunostaining of hOAT3 in the proximal tubular cells (Fig.
6A). There was no staining of hOAT3 in
Bowman's capsule, glomerular cells, distal tubules, or cortical
collecting duct. Under high magnification, hOAT3 was located in the
basolateral membranes of the proximal tubules (Fig. 6C). hOAT3
immunoreactivity was not observed in the medulla (Fig. 6B). By
preincubation of the antibody with hOAT3 peptide, the immunoreactivity
was completely diminished (Fig. 6D). The specificity of the antibody
for hOAT3 was verified by these results.
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Under the conditions used, FISH detection efficiency was approximately
47% using this probe (among the 100 checked mitotic figures, 47 showed
hybridization signals on one pair of chromosomes). When DAPI banding
was used to identify the specific chromosome, the assignment between
signals from the probe and the long arm of chromosome 11 (Fig.
7A) was obtained. The detailed position was further determined based on 10 photos (Fig. 7B). Therefore, this
probe was mapped to the human chromosome 11, region q12-q13.3.
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Discussion |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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In the present study, we report on the isolation and characterization of multispecific hOAT3. hOAT3 encodes a 543-amino-acid residue protein, that shows 36 to 51% identity to hOAT1, rOAT2, hOCT1, hOCT2, and rOCT3.
In the previous studies using membrane vesicles, kidney slices, and in
situ microperfusion, most of the basolateral uptake of hydrophobic
organic anions have been attributed to the function of the PAH
transporter. In 1997, the PAH/decarboxylate exchanger, rOAT1, was
cloned (Sekine et al., 1997; Sweet et al., 1997), and subsequently
three isoforms (rOAT2, rOAT3, and hOAT4) were identified (Sekine et
al., 1998; Kusuhara et al., 1999; Cha et al., 2000). In addition to the
OAT family, two other multispecific organic anion transporter families
were identified, namely the Mrp (multidrug resistance associated
protein) family (Buchler et al., 1996; Ito et al., 1997) and the oatp
(organic anion transporting polypeptide) family (Jacquemin et al.,
1994; Noe et al., 1997). Despite the rapid progress in molecular
biology of organic anion transporters, the physiological role of each
molecule in renal organic anion transport remains to be elucidated.
Among these multispecific organic anion transporters, only rOAT1
(Nakajima et al., 2000), rOAT3 (R. Kojime, T. Sekine, M. Kauachi, S. H. Cha, Y. Suzuki, and H. Endou, submitted) and Mrp1 (Raggers et
al., 1999) are localized in basolateral membranes of proximal tubular
cells. Mrp1 mediates the extrusion of organic anions from cells, and it
does not function for the cellular uptake of organic anions. OAT1 and
OAT3 are now the only multispecific organic anion transporters
responsible for the basolateral uptake of organic anions from the
peritubular plasma.
For a comprehensive understanding, determination of the contribution of
each transporter in the renal organic anion transport system is
required. OAT1 has been considered the predominant organic anion
transporter in the basolateral membrane (Hosoyamada et al., 1999);
however, the role of OAT1 might be overestimated, particularly in the
human kidney. First, it should be noted that most of the characterization of renal organic anion transporters have been obtained
from the experiments in which PAH was used as a tracer, and substrate
selectivity was examined in terms of inhibitory effects of test
substances. The results of such experiments primarily reflect the
functional characteristics of an organic anion transporter, OAT1, which
mediates the high-capacity transport of PAH. Second, before the
identification of OAT isoforms, transport characteristics of OAT3 might
have not been determined. Third, there seems to exist differences in
species among OATs. hOAT1 is also a PAH/decarboxylate exchanger and
mediates the high-affinity transport of PAH; however, it was suggested
that the ability of hOAT1 to translocate organic anions might be
limited. Lu et al. (1999) reported that hOAT1 does not translocate
methotrexate or PGE2, both of which are transportable substrates of rOAT1. They suggested that hOAT1 shows narrow substrate selectivity for organic anions. A similar observation of difference in
species was reported in the oatp family, another multispecific organic
anion transporter family, in which the difference in species is more
apparent. In the rat liver, oatp1 and/or oatp2 are predominant isoforms; in contrast, the corresponding human homolog, OATP, is weakly
expressed in the human liver, where a distinct isoform LST1
(liver-specific transporter 1), seems to be the predominant isoform
(Abe et al., 1999). Multispecific organic anion transporters, which
function primarily in the detoxification and elimination of various
exogenous compounds and metabolites, may evolve differently depending
on the circumstance. Although immunoreactivity of OAT1 (S2) and OAT3
(S1 > S2 = S3) are observed in basolateral membranes of proximal
tubules, OAT1 and OAT3 show different Km
values for the same substrates. The Km
values of PAH for each of these transporters were: rOAT1, 14 µM;
rOAT3, 65 µM; hOAT1, 9 µM; and hOAT3, 87 µM. It suggests that
these transporters would have different contributions in tubular
secretion. Concerning the circumstance of renal blood circulation,
broad distribution of hOAT3 in renal proximal tubule increases the
opportunity to contact circulated organic anionic substrates
with hOAT3. The contributions of hOAT1 and hOAT3 in the renal organic
anion transport systems should be clarified using human kidney samples
and/or in vivo clearance studies with relatively specific inhibitors
for hOAT1 and hOAT3.
Recently, Race et al. (1999) have reported on the hOAT3* gene isolated
from the human kidney. The homology between rOAT3 and hOAT3* was only
68%. In particular, the C terminus of hOAT3* was longer than that of
rOAT3. In addition, they could not show any functional properties of
the tested PAH, urate oxalate (organic anionic substrates), and TEA
(organic cationic substrate). The deduced amino acid sequence of our
hOAT3 showed 85% identity with that of hOAT3*. The major difference in
amino acid sequence between hOAT3 and hOAT3* lies in the four regions
(residues 263-286, 334-341, 399-406, and 513-527 in hOAT3 residue;
see Fig. 1). In particular, hOAT3* possesses 25 more amino acid
insertions than hOAT3 in the near portion of the C terminus, which may
alter the conformation of the protein and interfere with the binding of
the substrate to hOAT3*. It remains to be elucidated whether hOAT3 and
hOAT3* are formed by alternative splicing or are encoded by different genes.
hOAT3 mediates the high-affinity transport of methotrexate (MTX)
(Km = 10.9 µM). MTX is an antineoplastic
agent used in the treatment of acute lymphoblastic leukemia (Balis et
al., 1998) and choriocarcinomas (Ohno et al., 1993). MTX is also used
in the treatment of nonneoplastic diseases, such as psoriasis
(Zonneveld et al., 1996), rheumatoid arthritis (Zonneveld et al.,
1996), systemic lupus erythematosus (Ravelli et al., 1998) and
dermatomyositis (Itoh et al., 1999). Because MTX manifests potentially
toxic effects, such as suppression of bone marrow (Iqbal and Ali, 1993)
and intestinal epithelial damage (Nakamaru et al., 1998) under high
plasma concentration, understanding of the pharmacokinetics of MTX is
required. In humans, MTX is mainly excreted in the urine in the
unchanged form via both glomerular filtration and tubular secretion. It
has been reported that concomitant use of MTX with acidic drugs, such
as nonsteroidal anti-inflammatory drugs and -lactam antibiotics, causes severe suppression of bone marrow. This seems to be the result
of competitive inhibition of the process of the renal organic anion
transport system. Because hOAT3 seems to be the major route for the
basolateral uptake of MTX, information on the substrate specificity of
hOAT3 is important for the use of MTX, particularly for the treatment
of patients with decreased renal function.
hOAT3 shows overlapping substrate selectivity with hOAT1. Phylogenetic
analysis reveals that hOAT3 is located nearest to hOAT1, which may
underlie the similar substrate selectivity. There are, however,
distinct differences in substrate recognition between hOAT1 and hOAT3.
Oocytes expressing hOAT3 mediates the transport of estrone sulfate,
estradiol-glucuronide. On the other hand, oocytes expressing hOAT1
showed little or no transport of PGE2, methotrexate, and
taurocholate (data not shown). hOAT3 can mediate the transport of
organic anions with bulky side groups, compared with hOAT1.
Interestingly, hOAT3 mediates the high-affinity transport of
cimetidine, a cationic substance. It is well known that cimetidine is a
bisubstrate type of organic anion and cation transporter. Ullrich et
al. (1993) studied the recognition of bisubstrates using a renal
organic anion transporter employing the stop-flow peritubular-capillary
microperfusion method. They showed the importance of hydrophobicity and
partial charge, but not that of net charges of the substrates. In
addition, TEA (5 µM) and quinidine showed significant inhibitory
effects on the hOAT3-mediated transport of estrone sulfate. Thus, the
charge recognition of hOAT3 seems to be not so stringent. Oatp1,
another isoform of the other multispecific organic anion transporter
family, has been also shown to mediate the transport of an organic
cation, and Eckhardt et al. (1999) proposed that oatp1 be called a
polyspecific transporter. Thus, the substrate selectivity of hOAT3 is
also similar to those of oatp families.
The present study indicates that hOAT3 mRNA is exclusively expressed in the kidney and weak bands could be seen in the skeletal muscle and brain. hOAT3 mRNA expression in skeletal muscle is quite different from rOAT3 mRNA expression. At present, it is not clear whether hOAT3 functions in nonsecreting tissue, such as skeletal muscle.
FISH analyses revealed that the hOAT3 gene is located at the locus of the human chromosome 11q12-q13.3. This chromosomal localization is slightly different from that of hOAT3*, which was reported to be located in chromosome 11q11.7.
In conclusion, we report on the identification and characterization of human OAT3. In the human kidney, hOAT3 seems to play important roles in the basolateral uptake of organic anions in proximal tubular cells and to be a key molecule determining the pharmacokinetics of anionic drugs in human.
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Footnotes |
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Received August 28, 2000; Accepted January 26, 2001
This work was supported in part by grants from the Japanese Ministry of Education Science, Sports and Culture, Grants-in-Aids for Scientific Research, and High-Tech Research Center from the Science Research Promotion Fund of the Japan Private School Promotion Foundation, and Research on Health Sciences focusing on Drug Innovation from the Japan Health Sciences Foundation.
The nucleotide sequence reported in this article has been submitted to the GenBank/EBI Data bank with accession number AB042505.
Send reprint requests to: Dr. Hitoshi Endou, Dept. of Pharmacology and Toxicology, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo, 181-8611, Japan. E-mail: endouh{at}kyorin-u.ac.jp
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hOAT, human organic anion transporter; PAH, para-aminohippurate; rOAT, rat organic anion transporter; DHEA, dehydroepiandrosterone; PG, prostaglandin; TEA, tetraethylammonium; PCR, polymerase chain reaction; EST, expressed sequence tag; SSC, standard saline citrate; FISH, fluorescent in situ hybridization; MES, 4-morpholineethanesulfonic acid; MTX, methotrexate; DAPI, 4,6-diamidino-2-phenylindole; oatp, organic anion transporting polypeptide; Mrp, multidrug resistance associated protein.
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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A. G. Aslamkhan, Y.-H. Han, X.-P. Yang, R. K. Zalups, and J. B. Pritchard Human Renal Organic Anion Transporter 1-Dependent Uptake and Toxicity of Mercuric-Thiol Conjugates in Madin-Darby Canine Kidney Cells Mol. Pharmacol., March 1, 2003; 63(3): 590 - 596. [Abstract] [Full Text] [PDF]
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B. C. Burckhardt, S. Brai, S. Wallis, W. Krick, N. A. Wolff, and G. Burckhardt Transport of cimetidine by flounder and human renal organic anion transporter 1 Am J Physiol Renal Physiol, March 1, 2003; 284(3): F503 - F509. [Abstract] [Full Text] [PDF]
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 C. E. Wood, K. E. Gridley, and M. Keller-Wood Biological Activity of 17{beta}-Estradiol-3-Sulfate in Ovine Fetal Plasma and Uptake in Fetal Brain Endocrinology, February 1, 2003; 144(2): 599 - 604. [Abstract] [Full Text] [PDF]
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C. E. Groves, L. Munoz, A. Bahn, G. Burckhardt, and S. H. Wright Interaction of Cysteine Conjugates with Human and Rabbit Organic Anion Transporter 1 J. Pharmacol. Exp. Ther., February 1, 2003; 304(2): 560 - 566. [Abstract] [Full Text] [PDF]
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B. Ugele, M. V. St-Pierre, M. Pihusch, A. Bahn, and P. Hantschmann Characterization and identification of steroid sulfate transporters of human placenta Am J Physiol Endocrinol Metab, February 1, 2003; 284(2): E390 - E398. [Abstract] [Full Text] [PDF]
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Y. Miki, T. Nakata, T. Suzuki, A. D. Darnel, T. Moriya, C. Kaneko, K. Hidaka, Y. Shiotsu, H. Kusaka, and H. Sasano Systemic Distribution of Steroid Sulfatase and Estrogen Sulfotransferase in Human Adult and Fetal Tissues J. Clin. Endocrinol. Metab., December 1, 2002; 87(12): 5760 - 5768. [Abstract] [Full Text] [PDF]
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S. Khamdang, M. Takeda, R. Noshiro, S. Narikawa, A. Enomoto, N. Anzai, P. Piyachaturawat, and H. Endou Interactions of Human Organic Anion Transporters and Human Organic Cation Transporters with Nonsteroidal Anti-Inflammatory Drugs J. Pharmacol. Exp. Ther., November 1, 2002; 303(2): 534 - 539. [Abstract] [Full Text] [PDF]
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A. Bahn, M. Knabe, Y. Hagos, M. Rodiger, S. Godehardt, D. S. Graber-Neufeld, K. K. Evans, G. Burckhardt, and S. H. Wright Interaction of the Metal Chelator 2,3-Dimercapto-1-propanesulfonate with the Rabbit Multispecific Organic Anion Transporter 1 (rbOAT1) Mol. Pharmacol., November 1, 2002; 62(5): 1128 - 1136. [Abstract] [Full Text] [PDF]
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A. S. Koh, T. A. Simmons-Willis, J. B. Pritchard, S. M. Grassl, and N. Ballatori Identification of a Mechanism by Which the Methylmercury Antidotes N-Acetylcysteine and Dimercaptopropanesulfonate Enhance Urinary Metal Excretion: Transport by the Renal Organic Anion Transporter-1 Mol. Pharmacol., October 1, 2002; 62(4): 921 - 926. [Abstract] [Full Text] [PDF]
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M. Takeda, S. Khamdang, S. Narikawa, H. Kimura, M. Hosoyamada, S. H. Cha, T. Sekine, and H. Endou Characterization of Methotrexate Transport and Its Drug Interactions with Human Organic Anion Transporters J. Pharmacol. Exp. Ther., August 1, 2002; 302(2): 666 - 671. [Abstract] [Full Text] [PDF]
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D. H. Sweet, D. S. Miller, J. B. Pritchard, Y. Fujiwara, D. R. Beier, and S. K. Nigam Impaired Organic Anion Transport in Kidney and Choroid Plexus of Organic Anion Transporter 3 (Oat3 (Slc22a8)) Knockout Mice J. Biol. Chem., July 19, 2002; 277(30): 26934 - 26943. [Abstract] [Full Text] [PDF]
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Y. Kobayashi, N. Ohshiro, A. Shibusawa, T. Sasaki, S. Tokuyama, T. Sekine, H. Endou, and T. Yamamoto Isolation, Characterization and Differential Gene Expression of Multispecific Organic Anion Transporter 2 in Mice Mol. Pharmacol., July 1, 2002; 62(1): 7 - 14. [Abstract] [Full Text] [PDF]
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A. Enomoto, M. Takeda, A. Tojo, T. Sekine, S. H. Cha, S. Khamdang, F. Takayama, I. Aoyama, S. Nakamura, H. Endou, et al. Role of Organic Anion Transporters in the Tubular Transport of Indoxyl Sulfate and the Induction of its Nephrotoxicity J. Am. Soc. Nephrol., July 1, 2002; 13(7): 1711 - 1720. [Abstract] [Full Text] [PDF]
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A. Enomoto, M. Takeda, M. Shimoda, S. Narikawa, Y. Kobayashi, Y. Kobayashi, T. Yamamoto, T. Sekine, S. H. Cha, T. Niwa, et al. Interaction of Human Organic Anion Transporters 2 and 4 with Organic Anion Transport Inhibitors J. Pharmacol. Exp. Ther., June 1, 2002; 301(3): 797 - 802. [Abstract] [Full Text] [PDF]
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Y. Nagata, H. Kusuhara, H. Endou, and Y. Sugiyama Expression and Functional Characterization of Rat Organic Anion Transporter 3 (rOat3) in the Choroid Plexus Mol. Pharmacol., May 1, 2002; 61(5): 982 - 988. [Abstract] [Full Text] [PDF]
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C. M. Breen, D. B. Sykes, G. Fricker, and D. S. Miller Confocal imaging of organic anion transport in intact rat choroid plexus Am J Physiol Renal Physiol, May 1, 2002; 282(5): F877 - F885. [Abstract] [Full Text] [PDF]
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H. Kimura, M. Takeda, S. Narikawa, A. Enomoto, K. Ichida, and H. Endou Human Organic Anion Transporters and Human Organic Cation Transporters Mediate Renal Transport of Prostaglandins J. Pharmacol. Exp. Ther., April 1, 2002; 301(1): 293 - 298. [Abstract] [Full Text] [PDF]
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H. Motohashi, Y. Sakurai, H. Saito, S. Masuda, Y. Urakami, M. Goto, A. Fukatsu, O. Ogawa, and K.-i. Inui Gene Expression Levels and Immunolocalization of Organic Ion Transporters in the Human Kidney J. Am. Soc. Nephrol., April 1, 2002; 13(4): 866 - 874. [Abstract] [Full Text] [PDF]
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M. Hasegawa, H. Kusuhara, D. Sugiyama, K. Ito, S. Ueda, H. Endou, and Y. Sugiyama Functional Involvement of Rat Organic Anion Transporter 3 (rOat3; Slc22a8) in the Renal Uptake of Organic Anions J. Pharmacol. Exp. Ther., March 1, 2002; 300(3): 746 - 753. [Abstract] [Full Text] [PDF]
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M. Takeda, S. Khamdang, S. Narikawa, H. Kimura, Y. Kobayashi, T. Yamamoto, S. H. Cha, T. Sekine, and H. Endou Human Organic Anion Transporters and Human Organic Cation Transporters Mediate Renal Antiviral Transport J. Pharmacol. Exp. Ther., March 1, 2002; 300(3): 918 - 924. [Abstract] [Full Text] [PDF]
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G. Hill, T. Cihlar, C. Oo, E. S. Ho, K. Prior, H. Wiltshire, J. Barrett, B. Liu, and P. Ward The Anti-Influenza Drug Oseltamivir Exhibits Low Potential to Induce Pharmacokinetic Drug Interactions via Renal Secretion---Correlation of in Vivo and in Vitro Studies Drug Metab. Dispos., January 1, 2002; 30(1): 13 - 19. [Abstract] [Full Text] [PDF]
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D. H. Sweet, D. S. Miller, and J. B. Pritchard Ventricular Choline Transport. A ROLE FOR ORGANIC CATION TRANSPORTER 2 EXPRESSED IN CHOROID PLEXUS J. Biol. Chem., November 2, 2001; 276(45): 41611 - 41619. [Abstract] [Full Text] [PDF]
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D. S. Miller Nucleoside Phosphonate Interactions with Multiple Organic Anion Transporters in Renal Proximal Tubule J. Pharmacol. Exp. Ther., November 1, 2001; 299(2): 567 - 574. [Abstract] [Full Text] [PDF]
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J. M. Pombrio, A. Giangreco, L. Li, M. F. Wempe, M. W. Anders, D. H. Sweet, J. B. Pritchard, and N. Ballatori Mercapturic Acids (N-Acetylcysteine S-Conjugates) as Endogenous Substrates for the Renal Organic Anion Transporter-1 Mol. Pharmacol., November 1, 2001; 60(5): 1091 - 1099. [Abstract] [Full Text]
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, putative
N-linked glycosylation sites in OAT3; 
, putative
protein kinase C phosphorylation sites. The probe (
) and OAT3-expressing oocytes (
