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Volume 272, Number 48, Issue of November 28, 1997
pp. 30088-30095
(Received for publication, July 30, 1997, and in revised form, September 19, 1997)
From the Laboratory of Pharmacology and Chemistry, NIEHS, National
Institutes of Health, Research Triangle Park,
North Carolina 27709
ABSTRACT Expression cloning in Xenopus laevis
oocytes was used to isolate an organic anion transport protein from rat
kidney. A cDNA library was constructed from size-fractionated
poly(A)+ RNA and screened for probenecid-sensitive
transport of p-aminohippurate (PAH). A 2,227-base pair
cDNA clone containing a 1,656-base pair open reading frame coding
for a peptide 551 amino acids long was isolated and named ROAT1.
ROAT1-mediated transport of 50 µM [3H]PAH
was independent of imposed changes in membrane potential. Transport was
significantly inhibited at 4 °C, or upon incubation with other
organic anions, but not by the organic cation tetraethylammonium, by the multidrug resistance ATPase inhibitor cyclosporin A, or by
urate. External glutarate and INTRODUCTION Renal organic anion transport has been widely studied for more
than a century, both as a prototypic transport process and as a primary
means for removal of xenobiotics from the body. Because many foreign
chemicals, including plant and animal toxins, drugs, and pesticides,
are organic anions or are metabolized to organic anions, the renal
organic anion secretory system plays a critical role in limiting or
preventing their toxicity. Over the last decade, a great deal of
progress has been made toward understanding the physiology of this
system, particularly its coupling to metabolic energy. Thus, it is now
well established that organic anion secretion is a complex process
involving distinctly different proteins at the apical and basolateral
membranes of the proximal tubule (Fig. 1;
for review, see Ref. 1). Transport across the basolateral membrane is
energetically uphill. It is accomplished by a tertiary active process
in which (a) Na+,K+-ATPase
establishes the out > in Na+ gradient, (b)
Na+/
[View Larger Version of this Image (16K GIF file)]
In contrast to the physiology of the organic anion transport system,
precise information about the structural properties of the transport
proteins that make up this system are not yet available. However,
considerable progress has recently been made for a variety of other
transport proteins through the application of expression cloning
techniques, leading to increased understanding of the regulation of
their expression, identification of substrate binding sites, and a much
more complete appreciation of their mechanisms of action (9-11). We
report here the successful isolation and characterization of a cDNA
encoding the organic anion transporter from rat kidney, ROAT1.
EXPERIMENTAL PROCEDURES Total RNA was isolated from rat kidney using guanidinium
thiocyanate extraction followed by cesium chloride gradient
centrifugation according to the protocol of Gasser et al.
(12). Poly(A)+ RNA was separated by running the total RNA
fraction over an oligo(dT)-cellulose column twice and eluting with
water. The poly(A)+ RNA was size-fractionated by
centrifugation through a linear sucrose gradient (5-25% w/v) using a
modification of the method of Hagenbuch et al. (13). The
isolated polyadenylated RNA fractions were checked for organic anion
transport activity by expression assay in Xenopus oocytes
(14). The fraction with the greatest activity (corresponding to 11.1%
sucrose) was used to make a cDNA library.
The cDNA library was
constructed using the SuperScript Plasmid System kit (Life
Technologies, Inc.). The synthesized cDNA was ligated into pSPORT1
vector with the start site of RNA transcription positioned downstream
from a T7 RNA polymerase promoter present in the vector, so that the
cDNA insert could be transcribed into cRNA in an in
vitro synthesis reaction. The cDNA library plasmids were
transformed into MAX Efficiency DH5 Ambion's T7 mMessage mMachine in
vitro transcription kit (Ambion, Inc., Austin, TX) was used to
synthesize capped cRNA from library plasmid DNA linearized with
BamHI. The cRNA products were quantitated in a
spectrophotometer and diluted before injection to allow delivery of 20 ng of cRNA/oocyte in 15 nl with a 10-s injection.
Adult female Xenopus
laevis (Xenopus One, Ann Arbor, MI) were anesthetized by
hypothermia and decapitated. Ovaries were then removed and stored in
Barth's buffer (88 mM NaCl, 1 mM KCl, 330 µM Ca(NO3)2, 410 µM
CaCl2, 820 µM MgSO4, 2.4 mM NaHCO3, 10 mM HEPES, pH 7.4).
Stage V and stage VI oocytes were manually dissected free of the ovary
and the follicles removed by treatment with collagenase A (Boehringer
Mannheim) modified from the protocol of Pajor et al. (15).
Briefly, oocytes were placed in collagenase solution (5 mg/ml
collagenase A, 1 mg/ml trypsin inhibitor type III-O (Sigma) in
Barth's) and gently rocked for 1 h at room temperature. After
collagenase treatment, the oocytes were rinsed five times with Barth's
containing 1 mg/ml BSA1 and
placed in a phosphate/BSA solution for 1 h (100 mM
K2HPO4·3H2O with 1 mg/ml BSA, pH
6.5). Oocytes were agitated every 15 min to remove the follicle and
then rinsed five times with Barth's with BSA. The oocytes were
maintained at 18 °C in Barth's containing 0.05 mg/ml gentamycin
sulfate, 2.5 mM sodium pyruvate, and 5% heat inactivated
horse serum. The oocytes were allowed to recover overnight before
injection.
Two or three days after
injection with 20 ng of cRNA, the oocytes were divided into
experimental groups (containing 10 oocytes each) and incubated at
18-22 °C for 10 or 60 min in oocyte Ringer's 2 (OR-2; in
mM: 82.5 NaCl, 2.5 KCl, 1 Na2HPO4,
3 NaOH, 1 CaCl2, 1 MgCl2, 1 pyruvic acid, 5 HEPES, pH 7.6) containing 50 µM
[3H]p-aminohippurate (4 µCi/ml) in the
absence or presence of 1 mM probenecid, a known inhibitor
of organic anion transport in the kidney. After uptake, oocytes were
rapidly rinsed three times with ice-cold OR-2 and placed into
individual scintillation vials containing 0.5 ml of 1 M
NaOH, incubated at 65 °C for 20 min, and neutralized with 0.5 ml 1 M HCl. Finally, 4.7 ml of Ecolume (ICN Biomedical,
Cleveland, OH) was added and oocyte radioactivity measured in
disintegrations per minute in a Packard 1600TR liquid scintillation
counter with external quench correction. PAH uptake was calculated in
pmol/oocyte, i.e. from dpm/oocyte and medium specific
activity. Water-injected or uninjected oocytes were included as
negative controls in every experiment.
The cDNA clone was sequenced at the
University of North Carolina Automated DNA Sequencing Facility on a
model 373A DNA sequencer (Applied Biosystems, Foster City, CA) using
the Taq DyeDeoxy Terminator Cycle Sequencing Kit (Applied
Biosystems). The full-length sequence of both strands was obtained
using M13/pUC forward and reverse primers, as well as synthetic
oligonucleotide primers based on the previously determined sequence
(Life Technologies, Inc.). All DNA sequence comparisons and data base
searches were done with the Wisconsin Package software with default
settings (16).
The amino acid sequence was analyzed
using five different modeling programs for predicting potential
transmembrane domains: 1) Kyte-Doolittle (17), 2) TMPred (18), 3) DAS
(19), 4) PHDhtmTop (20), and 5) TopPred 2 (21), all with default
parameters.
Approximately 1 µg of plasmid DNA from
several library fractions was linearized with the restriction enzyme
BamHI and separated by electrophoresis on a 1% agarose gel
in TBE buffer at 150 V for 2 h. The gel was transferred to a
Hybond N+ nylon membrane under alkaline conditions (0.4 M NaOH). The blot was probed with a 643-bp fragment
(positions 782-1425) from the liver organic anion transporter, oatp
(22), and with a 1,368-bp fragment (positions 186-1554) from ROAT1,
both amplified by PCR from cDNA clones. Primers used for PCR were:
r-liv OATcw (5 The PCR products were gel-isolated and recovered using a Qiaquick gel
extraction kit (Qiagen, Inc.). The probes were labeled using the ECL
kit (Amersham) and hybridized at 42 °C. The blot was washed under
conditions of high stringency (0.1 × SSC) and the probes detected
with the ECL kit reagents.
The rat multiple tissue Northern blot was
purchased from CLONTECH Laboratories, Inc. (Palo
Alto, CA) and probed with the 1,368-bp ROAT1 probe. The probe was
labeled using the Gene Images random prime labeling kit (Amersham) and
hybridized at 65 °C. The probe was detected with the Gene Images
CDP-Star detection kit (Amersham). The blot was stripped in boiling
0.1% SDS and reprobed with the human Data are presented as mean ± S.E.
Differences in mean values were considered to be significant when
p [3H]PAH (3.7 Ci/mmol) was obtained
from NEN Life Science Products. [14C]Glutarate (55 mCi/mmol) and [14C]tetraethylammonium (TEA; 55 mCi/mmol)
were obtained from American Radiolabeled Chemicals, Inc. (St. Louis,
MO). Unlabeled PAH, probenecid, glutarate, methylsuccinate, and TEA
were obtained from Sigma. All other chemicals were obtained from
commercial sources and were of the highest grade available.
RESULTS Screening of cDNA Library
Total poly(A)+ RNA was isolated from rat kidney and
size-fractionated on a linear sucrose gradient (14). Fractions
corresponding to 12.3% and 11.1% sucrose were shown to support
probenecid-sensitive uptake of 50 µM
[3H]PAH that was 2-3-fold higher than that observed for
the original unfractionated starting material when injected into
X. laevis oocytes (Fig.
2A). The 11.1% sucrose
fraction mRNA was used as template for the construction of a
cDNA library, which was screened by expression cloning in
Xenopus oocytes, yielding a single purified clone that
supported PAH uptake (Fig. 2B). In the presence of 1 mM probenecid, PAH uptake by oocytes expressing the cloned
transporter was always reduced to the level observed for water-injected
oocytes. Electrophoresis of the in vitro cRNA synthesis
product showed a band ~2.3 kb in size, which corresponds with the
previously reported size range for kidney mRNA that supported PAH
uptake (14). We refer to this cDNA clone as renal
organic anion transporter 1, or ROAT1.
[View Larger Version of this Image (19K GIF file)]
Molecular Characterization of ROAT1
The complete DNA sequence of both strands of ROAT1 was determined,
and the sense strand sequence is presented in Fig.
3A. The ROAT1 cDNA is
2,227 bp long, including 253 bp of 5
[View Larger Version of this Image (58K GIF file)]
A BLAST search (24) of the GenBank data base (25) identified a single
DNA sequence, NKT (accession number U52842; Ref. 26), with significant
homology to ROAT1. NKT was isolated from mouse kidney and described as
a gene product related to the organic cation transporter family (26). A
Smith and Waterman (27) alignment of the two DNA sequences showed a
95% identity, whether the 5
[View Larger Version of this Image (59K GIF file)]
The only other known cloned organic anion transporter is oatp from rat
liver (22). Comparison of ROAT1 and oatp revealed no homology between
the two at the DNA or peptide level (27, 28). Comparison between the
peptide sequence of ROAT1 and those of the organic cation transporters
OCT1 (11) and OCT2 (29), and another liver transporter of unknown
substrate specificity, NLT (30), showed some homology: ROAT1/OCT1, 40%
similarity and 33% identity; ROAT1/OCT2, 41% similarity and 31%
identity; ROAT1/NLT, 48% similarity and 38% identity. No obvious
regions of highly conserved sequence were identified, even in the
putative membrane-spanning domains. Rather, the peptides seem to share
a low level of identity throughout their sequence. There are, however,
three motifs conserved among ROAT1, NKT, OCT1, OCT2, and NLT: the
positioning of four cysteine residues and a protein kinase C consensus
site within the extracellular loop between predicted membrane-spanning
domains 1 and 2, and two casein kinase II consensus sites (Table
I). Whether any of these features is
involved in transporter function is unknown at this time.
Table I.
Conserved peptide motifs in cloned organic anion and cation
transporters
Expression Cloning and Characterization of ROAT1
THE BASOLATERAL ORGANIC ANION TRANSPORTER IN RAT KIDNEY*
and
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-ketoglutarate (1 mM),
both counterions for basolateral PAH exchange, also inhibited
transport, suggesting that ROAT1 is functionally similar to the
basolateral PAH carrier. Consistent with this conclusion, PAH uptake
was trans-stimulated in oocytes preloaded with
glutarate, whereas the dicarboxylate methylsuccinate, which is not
accepted by the basolateral exchanger, did not
trans-stimulate. Finally, ROAT1-mediated PAH transport was
saturable, with an estimated Km of 70 µM. Each of these properties is identical to those
previously described for the basolateral -ketoglutarate/PAH
exchanger in isolated membrane vesicles or intact renal tubules.
-ketoglutarate cotransport driven by the movement of
Na+ down its concentration gradient, in concert with
intracellular metabolic -ketoglutarate generation, sustains an out < in dicarboxylate gradient, and (c) dicarboxylate/organic
anion exchange moves the organic anion substrate into the cell (2, 3).
This cascade of events indirectly links organic anion transport to
metabolic energy and the Na+ gradient, allowing entry of
negatively charged substrate against both its chemical concentration
gradient and the electrical potential of the cell. Once inside the
cell, organic anions are subject to intracellular binding and to
sequestration within vesicular structures (4, 5). Finally, luminal exit
is thought to occur by anion exchange and/or facilitated diffusion
(6-8).
Fig. 1.
Model for organic anion transport in renal
proximal tubule. Organic anion entry across the basolateral
membrane is driven by indirect coupling to the sodium gradient through
sodium/-ketoglutarate cotransport followed by organic
anion exchange (1). Organic anions are sometimes sequestered
in vesicles within the cytoplasm (2?). Luminal exit is
presumed to occur via exchange for hydroxyl ions (3) or
through a facilitated diffusion process (4) down the
electrochemical gradient across the brush border membrane.
-competent cells (Life Technologies, Inc.). Transformed bacteria were plated onto Hybond N
nylon filters (Amersham), which were overlaid on Luria-Bertani (LB)
plates containing 100 µg/ml ampicillin and incubated overnight at
37 °C. To obtain purified plasmid DNA, whole filters (or filter subsections) were placed in 200 ml of LB broth with 100 µg/ml ampicillin and shaken at 225 rpm overnight at 37 °C. The bacteria were pelleted by centrifugation and plasmid DNA was isolated using a
Qiagen plasmid kit (Qiagen, Inc., Chatsworth, CA).
-CCGGTACCGACCATAACACCCAGTG-3) and r-liv OATccw
(5-CCGGTACCTTGAGCAGCTACACCTT-3) to generate the oatp probe; Anion3cw
(5-GTCCTGCACTGTATCTGGC-3) and Anion3ccw (5-TCTGGAAGCCTGGCTGC-3) to
generate the ROAT1 probe.
-actin control probe included
with the blot.
0.05 as determined by one sample or paired
Student's t test, as indicated in figure legends. The
degree of significance was indicated as follows: * denotes
p 0.05; ** denotes p 0.01; and
*** denotes p 0.001.
Fig. 2.
Expression cloning of a cDNA encoding an
organic anion transporter. A rat kidney cDNA library was
screened for the ability to support PAH uptake. cRNA transcribed from
library plasmid DNA was injected into Xenopus oocytes (20 ng/oocyte) and allowed to express. Two days after injection, the
oocytes were incubated for 1 h in 50 µM
[3H]PAH in the absence (No Inhibitor) or
presence (+ Probenecid) of 1 mM probenecid. Each
column shows the mean value ± S.E. for 2 animals (10 oocytes/treatment/animal). A, evaluation of rat kidney
poly(A)+ RNA sucrose gradient fractions; B,
response from a single positive clone (ROAT1).
-untranslated region, a single,
large open reading frame 1,656 bp long, and 318 bp of 3-untranslated
sequence. The ATG at position 254 has the strongest correlation with
Kozak's consensus sequence for translation initiation (23), giving a
predicted protein length of 551 amino acids (Fig. 3A).
Hydropathy analysis with five different modeling programs yielded four
different profiles for potential -helical membrane-spanning domains
(Fig. 3B). Several regions were identified by all the
modeling algorithms, and a few less well defined regions identified by
some. However, all predictions agree that there is a large
extracellular loop at approximately amino acid residues 40-136. Within
this domain are several potential modification sites, including
possible N-linked glycosylation sites at Asn-39, Asn-56,
Asn-92, and Asn-113; four cysteine residues at positions 49, 78, 105, and 128 that could participate in disulfide bond formation; and a
possible protein kinase C site at Ser-129. In addition, there are four
more protein kinase C consensus sites located at Ser-271, Ser-278,
Thr-284, and Thr-334 and potential casein kinase II sites at Ser-325,
Thr-515, and Ser-544. These latter consensus sites may or may not be
located intracellularly, depending on the model used.
Fig. 3.
DNA and predicted amino acid sequence of
ROAT1. Both strands of the ROAT1 cDNA were sequenced
completely. A, nucleotide sequence of the sense strand of
ROAT1. The predicted amino acid sequence of the single large open
reading frame is given. B, Kyte-Doolittle hydropathy
analysis. Proposed transmembrane domains are indicated by
numbers above the hydropathy plot. The analysis package used to obtain each prediction is given.
- and 3-untranslated regions were
included or not. Needleman and Wunsch (28) analysis of the predicted
amino acid sequences for the two proteins yielded a 96% similarity and
95% identity. The two peptides differ at 27 positions, and there is a
six-amino acid gap in NKT corresponding to residues 85-90 in ROAT1
(Fig. 4).
Fig. 4.
Amino acid sequence alignment of ROAT1 and
NKT. The peptide sequence alignment of these two proteins
illustrates their high degree of homology. The two sequences differ at
27 positions, as well as a six-amino acid gap present in NKT
corresponding to residues 85-90 in ROAT1. These differences are
indicated by boxes.
cDNA
Cysteine
residues
PKC consensus sites
CKII consensus sites
ROAT1
49, 78, 105,
128
Ser-271
Ser-325, Thr-515
NKT
49, 78, 99,
122
Ser-265
Ser-319, Thr-509
OCT1
50, 89, 122,
143
Ser-286
Ser-334, Thr-525
OCT2
50, 89, 122,
143
Ser-286
Ser-334, Thr-525
NLT
49, 80, 113,
136
Ser-279
Ser-331, Thr-521
Southern and Northern Analysis
A Southern blot of cDNA library fractions performed during the
course of the screen demonstrated that the positive library fractions
containing ROAT1 did not contain any sequence homologous to a 643-bp
probe from oatp (22), the only other known organic anion transporter
(Fig. 5, left panel).
Subsequent reprobing of the same blot with a 1,368-bp ROAT1 probe
confirmed that the ROAT1 probe bound only to the library fractions
known to support PAH uptake, and did not bind to oatp (Fig. 5,
right panel). Therefore, ROAT1 was determined to be unique
from oatp. This was confirmed by DNA sequence analysis (see above).
Fig. 5. Southern blot of cDNA library fractions. Left panel, the blot was probed with a 643-bp fragment from the liver organic anion transporter oatp (22). As shown, the probe recognizes itself, but nothing in any of the kidney cDNA library fractions. Right panel, the same blot was probed with a 1,368-bp ROAT1 fragment, demonstrating that it does not recognize the liver transporter, nor any library fractions that did not support PAH transport (i.e. ALf1, ALf9.2.4.3.2, and ALf9.2.4.3.3).
[View Larger Version of this Image (25K GIF file)]
The same ROAT1 probe was used for a Northern blot and detected a strong
signal in rat kidney (Fig. 6). ROAT1
transcript was not observed in rat heart, brain, spleen, lung, liver,
skeletal muscle, or testis. The major transcript detected in kidney was ~2.4 kb in size; however, a second, far less abundant transcript ~4.2 kb in size was also seen in longer exposures. The blot was stripped and reprobed with a human -actin probe, confirming that there was viable mRNA present in each lane of the blot (data not shown).
Fig. 6. Northern blot analysis of ROAT1 tissue distribution. A rat multiple tissue mRNA blot was probed with the 1,368-bp ROAT1 probe. A highly expressed 2.4-kb transcript was detected in kidney, as well as a much less abundant 4.2-kb transcript. There was no signal detected in rat heart, brain, spleen, lung, liver, skeletal muscle, or testis. The blot was stripped and reprobed with a human
-actin control probe, confirming the presence of viable
mRNA in all lanes of the blot (data not shown).
[View Larger Version of this Image (31K GIF file)]
Functional Characterization of ROAT1 Transport
Potential DependenceAs a first step in determining the
identity of ROAT1 (see Fig. 1), the effect of altered membrane
potential on PAH transport in ROAT1-injected oocytes was assessed. The
luminal facilitated diffusion system is markedly dependent upon
membrane potential (7, 8), whereas both the basolateral and luminal
exchangers are not (3, 31). Potential was altered by raising external K+, a condition previously shown to depolarize the plasma
membrane of the oocyte (11). When ROAT1-expressing oocytes were
incubated in OR-2 with a high potassium ion concentration (102.5 mM), there was no reduction in PAH transport (Fig.
7). As a positive control, oocytes
injected with OCT2 cRNA, an organic cation transporter known to have a
large potential-sensitive transport component (32), showed a 67% drop
in transport of [14C]TEA when exposed under the same
conditions (Fig. 7). Water-injected oocytes showed no uptake under
either condition. Therefore, ROAT1-mediated PAH transport is
independent of membrane potential.
Fig. 7. Effect of membrane potential on substrate uptake. The 60-min uptake of 50 µM [3H]PAH by water- or ROAT1 cRNA-injected oocytes was compared under normal (2.5 mM K+) and membrane depolarizing conditions (102.5 mM K+; (11)), in the absence (No Inhibitor) or presence (+ Probenecid) of 1 mM probenecid. We have documented the influence of potential on both ROAT1 and OCT2 in 6 animals; however, the data presented here are from a representative animal because the experiment has only been done with both transporters in the same animal on two occasions. Columns represent mean values ± S.E. from 10 oocytes.
[View Larger Version of this Image (21K GIF file)]
Inhibition Profile
The effects of various compounds and
reduced temperature on PAH uptake were also examined (Fig.
8). Incubation at 4 °C reduced uptake
to just 3% of that seen at room temperature. The organic anions
probenecid (1 mM), -ketoglutarate (-KG; 1 mM), bromcresol green (1 mM), and excess
unlabeled PAH (1 mM) each reduced [3H]PAH
uptake by 80-90%. In addition, like basolateral PAH/-KG exchange
(3, 33), but in contrast with luminal exchange (31), ROAT1-mediated PAH
uptake was not inhibited by urate (1 mM).
Fig. 8. ROAT1 substrate specificity. cRNA from ROAT1 was injected into oocytes and [3H]PAH transport was measured after 60 min in the presence of several organic anions and cations, and at reduced temperature. Data are presented as percent of control uptake. Values are mean ± S.E. for 4-6 animals (10 oocytes/treatment/animal). ** denotes p
0.01, and
*** denotes p 0.001. BCG, bromcresol
green.
[View Larger Version of this Image (21K GIF file)]
Transport was also unaffected by the P-glycoprotein inhibitor cyclosporin A (CSA; 10 µM). Unlike urate and CSA, which gave values essentially identical to control, the cation TEA appeared to inhibit slightly, albeit at a high concentration (5 mM). Lower concentrations of TEA were not inhibitory and, at concentrations from 0.05 to 1 mM, actually stimulated uptake 20-40% (data not shown). To establish whether these modest effects might indicate that TEA was a substrate for ROAT1, the uptake of 200 µM [14C]TEA by ROAT1 cRNA and water-injected oocytes was measured. No difference in uptake between the two groups was observed (data not shown).
The ability of -KG to cis-inhibit PAH uptake (Fig. 8) is
potentially diagnostic in that the basolateral dicarboxylate/organic anion exchanger should be inhibited by external -KG, whereas luminal
PAH carriers should not be inhibited by -KG. Glutarate is also an
effective counterion for this exchanger (3). When ROAT1 cRNA-injected
oocytes were incubated in OR-2 containing 0-1 mM
glutarate, a clear, dose-dependent inhibition of PAH uptake was observed, with significant inhibition at 200 µM
glutarate and above (Fig. 9).
Fig. 9. Effect of external glutarate on PAH uptake. Two days after injection, oocytes were incubated for 60 min in OR-2 with 50 µM [3H]PAH and 0-1 mM glutarate. Uptake is expressed as percent of uptake when no compound was present, i.e. 0 mM glutarate. Data are mean ± S.E. values for 4 animals (10 oocytes/treatment/animal). * denotes p
0.05, and ***
denotes p 0.001.
[View Larger Version of this Image (18K GIF file)]
trans-Stimulation
If ROAT1 is the basolateral dicarboxylate/organic anion exchanger,
increasing the intracellular concentration of -KG (or glutarate)
should induce trans-stimulation of PAH uptake (see Fig. 1).
For this determination, glutarate is the preferred counterion since, in
contrast to -KG, it is not extensively metabolized (34). Preliminary
experiments showed substantial accumulation of 1 mM
[14C]glutarate within uninjected Xenopus
oocytes over time (170 pmol/oocyte after 90 min; data not shown).
Incubating the oocytes in 1 mM glutarate for 90 min before
exposure to PAH (i.e. preloading) significantly stimulated
PAH uptake in ROAT1-expressing oocytes, as compared with non-preloaded
oocytes (Fig. 10). Moreover, glutarate preloading had no effect on water-injected oocytes. Thus, glutarate induced trans-stimulation of ROAT1-mediated PAH uptake.
Fig. 10. trans-Stimulation of PAH uptake. Water-injected or ROAT1 cRNA-injected oocytes, either non-preloaded (control) or preloaded by a 90-min incubation in 1 mM glutarate, were washed with glutarate-free medium and exposed to 50 µM [3H]PAH for 60 min in the absence (No Inhibitor) or presence (+ Probenecid) of 1 mM probenecid. Data are mean ± S.E. values from a representative animal (10 oocytes/treatment). The experiment was repeated four times and data calculated as percent of control uptake. Uptake by preloaded, cRNA-injected oocytes was 204 ± 19% (p
0.01). * denotes p 0.05, using
paired Student's t test.
[View Larger Version of this Image (23K GIF file)]
The specificity of trans-stimulation was assessed by
comparing the effects of glutarate with the poorly metabolized
dicarboxylate methylsuccinate, which cannot substitute for -KG or
glutarate on the -KG/PAH exchanger (35, 36). As shown in Fig.
11, increasing the preloading
concentration of glutarate from 0 to 5 mM increased glutarate's stimulatory effect on PAH uptake in a
dose-dependent fashion, reaching an impressive 250%
increase over non-preloaded oocytes at 5 mM. In contrast,
methylsuccinate failed to stimulate PAH uptake over the same
concentration range. Indeed, at 5 mM, methylsuccinate
inhibited PAH uptake by 70% (p 0.01).
Fig. 11. Dose response of glutarate trans-stimulation. Oocytes injected with ROAT1 cRNA were preloaded for 90 min in 0-5 mM glutarate or methylsuccinate, washed briefly in dicarboxylate-free medium, and incubated with 50 µM [3H]PAH for 60 min. Uptake is expressed as percent of uptake with no preloading, i.e. 0 mM glutarate or methylsuccinate. Data are mean ± S.E. values for 4-6 animals (10 oocytes/treatment/animal). * denotes p
0.05, and **
denotes p 0.01.
[View Larger Version of this Image (19K GIF file)]
Kinetics
PAH uptake by ROAT1-injected oocytes increased steadily with time
and was linear for about 1 h (data not shown). Using 10 min to
approximate initial rate, the kinetics of ROAT1-mediated PAH transport
were assessed by incubating oocytes expressing ROAT1 in medium
containing 0.05-1 mM PAH (Fig.
12A). A double-reciprocal plot of the saturation data was constructed and linear regression analysis was performed to obtain estimates for Km of 70 µM and for Vmax of 6 pmol/oocyte/10 min (Fig. 12B). It should be noted that, in
the oocyte expression assay system, Vmax
reflects the degree of cRNA expression rather than a true measure of
maximal uptake rate for a transporter in its native tissue.
Fig. 12. Kinetic analysis of ROAT1-mediated PAH uptake. ROAT1 cRNA-injected oocytes were exposed to increasing concentrations of PAH for 10 min. A, saturation analysis. Total PAH uptake was corrected for diffusion by subtracting a diffusion correction factor of 9.18 ± 0.85 pmol/µM PAH. This factor was obtained by plotting the PAH uptake at 1 mM and 3 mM for three animals and calculating the mean change in uptake. B, double-reciprocal plot of the diffusion corrected data with linear regression analysis performed. Km was estimated to be 70 µM. The data shown are mean values ± S.E. from 3-4 animals (10 oocytes/treatment/animal).
[View Larger Version of this Image (18K GIF file)]
DISCUSSION
Many toxic anions, whether of endogenous or environmental origin, are eliminated from the body by the organic anion secretory system of the renal proximal tubule. This system has been actively investigated for more than 100 years, in part because of its effectiveness, which can clear the renal plasma of a good substrate like PAH in a single pass, and in part because of its critical role in protecting against the toxic effects of anionic xenobiotics through their rapid excretion via the urine (1). Recent emphasis has been on the specificity of the basolateral carrier, which accepts a remarkably broad spectrum of agents, requiring only a hydrophobic backbone and negative or partial negative charges optimally separated by 6-7 Å (37), and on the energetics of transport, which is driven by a complex tertiary coupling to metabolic energy (1). As a first step toward understanding the molecular basis for these features, we have used expression cloning in X. laevis oocytes to isolate a cDNA that encodes a novel renal organic anion transporter, ROAT1, which mediates transport of the model organic anion, PAH (Fig. 2).
Sequence analysis revealed that the recently described mouse kidney gene NKT (26) is 96% similar and 95% identical to ROAT1 at the amino acid level (Fig. 4). However, Lopez-Nieto et al. (26) were unable to show any transport activity for NKT and, therefore, concluded NKT was related to the organic cation transporter family based on sequence homology. We believe that NKT is the mouse counterpart of ROAT1. Our Northern tissue distribution results indicated significant quantities of ROAT1 message are expressed only in the kidney (Fig. 6). In contrast to NKT (26), no transcript was detected in the lane containing rat brain mRNA. Additionally, a second band (~4.2 kb) was visible in the kidney mRNA lane, albeit much fainter than the 2.4-kb band. Whether this band represents an isoform of ROAT1, incompletely processed ROAT1 message, or a completely different transporter with significant homology to ROAT1 is unknown. Southern blot analysis indicated that the liver organic anion transporter oatp (22) and ROAT1 are not the same protein and do not represent tissue-specific isoforms of the same transporter (Fig. 5).
The moderate level of homology shared by all of the kidney organic ion transporters identified so far, whether components of the organic anion or organic cation transport systems, may be indicative of some basic properties common to these renal transporters. However, no regions of high homology (e.g. nearly identical sequences in the putative transmembrane domains) were observed. Instead, there was a low level of homology throughout the entire length of the peptides. Interestingly, oatp (22) did not share any homology with ROAT1, despite their similar function (38). This lack of homology may be related to the differing substrate specificities of these two anion transporters (this work and Ref. 22); however, if so, this makes the identity shared between ROAT1 and the cation transporters OCT1 and OCT2 all the more perplexing.
Clearly, ROAT1 is an organic anion transport protein. It mediates
probenecid-sensitive PAH transport, which is strongly inhibited by
other organic anions, but not by the organic cation TEA, or by CSA,
which inhibits the multidrug resistance transporter (Fig. 8). However,
as summarized in Fig. 1, several renal organic anion transport proteins
have been described. Thus, ROAT1 could code for the basolateral
-KG/PAH exchanger (2, 3), either of the luminal carriers (the
potential driven carrier (8) and the anion exchanger (31)), or the as
yet uncharacterized system responsible for accumulation of organic
anions within intracellular organelles (5). To differentiate between
these possibilities, the functional properties of ROAT1 were
investigated in cRNA-injected Xenopus oocytes. As shown in
Fig. 7, ROAT1-mediated PAH transport was independent of changes in
membrane potential. This observation demonstrated that the cloned
transporter is not the luminal facilitated diffusion carrier, since
that carrier shows marked potential dependence (8). Likewise, ROAT1's
lack of inhibition by urate (Fig. 8) would appear to distinguish it
from the luminal anion exchanger characterized by Blomstedt and Aronson
(31). Thus, the most likely candidate for ROAT1 is the basolateral
dicarboxylate/organic anion exchanger. Indeed, all properties of ROAT1
that were examined using the oocyte expression system, including its
insensitivity to membrane potential and its lack of inhibition by
urate, were entirely consistent with previously documented
characteristics of the basolateral exchanger. Its interactions with the
dicarboxylates -KG, glutarate, and methylsuccinate were particularly
diagnostic (Figs. 8, 9, 10, 11). A variety of evidence from membrane vesicles and intact renal tissue documents that both glutarate and -KG may
act as counterions for the basolateral anion exchanger (2, 3, 35, 39).
Thus, the presence of either dicarboxylate on the same side of the
membrane as PAH will cis-inhibit PAH transport, and an
outwardly directed gradient of either will accelerate
(trans-stimulate) PAH accumulation. As shown in Figs. 8, 9, 10, 11,
ROAT1-mediated uptake behaves exactly like the basolateral system,
being both cis-inhibited and trans-stimulated by
these compounds. Furthermore, as shown in Fig. 11, methylsuccinate,
which is not accepted by the basolateral exchanger (35), failed to
trans-stimulate ROAT1-mediated PAH uptake as well. Finally,
the Km for ROAT1-mediated PAH transport was shown to
be 70 µM (Fig. 12), which is in good agreement with the
value of 80 µM obtained by Ullrich et al. (40) in the intact renal tubules of the rat.
In summary, functional data indicate that ROAT1 is the basolateral dicarboxylate/organic anion exchanger of the renal proximal tubule. Furthermore, both DNA and protein sequence comparisons suggest that ROAT1 and the recently identified mouse gene, NKT, are species counterparts of the same transporter, although in the absence of functional data on NKT this remains to be conclusively shown. Identification of ROAT1 and its possible mouse homologue provide an important first step in the detailed characterization of the biochemical properties and control mechanisms which determine the functional state of this important excretory protein in the intact tissue.
FOOTNOTES
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF008221.
Current address: Dept. of Physiology, University of
Göttingen, 20400 Göttingen, Germany.
§ To whom all correspondence should be addressed: Laboratory of Pharmacology and Chemistry, NIH/NIEHS, Mail Drop F1-03, P. O. Box 12233, Research Triangle Park, NC 27709. Tel.: 919-541-4054; Fax: 919-541-5737; E-mail: pritchard{at}niehs.nih.gov.
1 The abbreviations used are: BSA, bovine serum albumin; PAH, p-aminohippurate; TEA, tetraethylammonium;
-KG, -ketoglutarate; CSA, cyclosporin A; OR-2, oocyte Ringer's
2; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase
pair(s).
ACKNOWLEDGEMENTS
We thank Destiny Sykes and Ramsey Walden for their expert technical support. We also gratefully acknowledge the assistance of Dr. Judith Stenger with the computer modeling analysis.
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S. Soodvilai, V. Chatsudthipong, K. K. Evans, S. H. Wright, and W. H. Dantzler Acute regulation of OAT3-mediated estrone sulfate transport in isolated rabbit renal proximal tubules Am J Physiol Renal Physiol, November 1, 2004; 287(5): F1021 - F1029. [Abstract] [Full Text] [PDF]
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T. Imaoka, H. Kusuhara, S. Adachi-Akahane, M. Hasegawa, N. Morita, H. Endou, and Y. Sugiyama The Renal-Specific Transporter Mediates Facilitative Transport of Organic Anions at the Brush Border Membrane of Mouse Renal Tubules J. Am. Soc. Nephrol., August 1, 2004; 15(8): 2012 - 2022. [Abstract] [Full Text] [PDF]
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G. L. Youngblood and D. H. Sweet Identification and functional assessment of the novel murine organic anion transporter Oat5 (Slc22a19) expressed in kidney Am J Physiol Renal Physiol, August 1, 2004; 287(2): F236 - F244. [Abstract] [Full Text] [PDF]
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 S. H. Wright and W. H. Dantzler Molecular and Cellular Physiology of Renal Organic Cation and Anion Transport Physiol Rev, July 1, 2004; 84(3): 987 - 1049. [Abstract] [Full Text] [PDF]
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M. Ljubojevic, C. M. Herak-Kramberger, Y. Hagos, A. Bahn, H. Endou, G. Burckhardt, and I. Sabolic Rat renal cortical OAT1 and OAT3 exhibit gender differences determined by both androgen stimulation and estrogen inhibition Am J Physiol Renal Physiol, July 1, 2004; 287(1): F124 - F138. [Abstract] [Full Text] [PDF]
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S. A. Eraly, J. C. Monte, and S. K. Nigam Novel slc22 transporter homologs in fly, worm, and human clarify the phylogeny of organic anion and cation transporters Physiol Genomics, June 17, 2004; 18(1): 12 - 24. [Abstract] [Full Text] [PDF]
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Y. Kobayashi, N. Ohshiro, A. Tsuchiya, N. Kohyama, M. Ohbayashi, and T. Yamamoto RENAL TRANSPORT OF ORGANIC COMPOUNDS MEDIATED BY MOUSE ORGANIC ANION TRANSPORTER 3 (MOAT3): FURTHER SUBSTRATE SPECIFICITY OF MOAT3 Drug Metab. Dispos., May 1, 2004; 32(5): 479 - 483. [Abstract] [Full Text] [PDF]
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D. Sykes, D. H. Sweet, S. Lowes, S. K. Nigam, J. B. Pritchard, and D. S. Miller Organic anion transport in choroid plexus from wild-type and organic anion transporter 3 (Slc22a8)-null mice Am J Physiol Renal Physiol, May 1, 2004; 286(5): F972 - F978. [Abstract] [Full Text] [PDF]
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A. Bahn, C. Ebbinghaus, D. Ebbinghaus, E. G. Ponimaskin, L. Fuzesi, G. Burckhardt, and Y. Hagos EXPRESSION STUDIES AND FUNCTIONAL CHARACTERIZATION OF RENAL HUMAN ORGANIC ANION TRANSPORTER 1 ISOFORMS Drug Metab. Dispos., April 1, 2004; 32(4): 424 - 430. [Abstract] [Full Text] [PDF]
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Y. Nozaki, H. Kusuhara, H. Endou, and Y. Sugiyama Quantitative Evaluation of the Drug-Drug Interactions between Methotrexate and Nonsteroidal Anti-Inflammatory Drugs in the Renal Uptake Process Based on the Contribution of Organic Anion Transporters and Reduced Folate Carrier J. Pharmacol. Exp. Ther., April 1, 2004; 309(1): 226 - 234. [Abstract] [Full Text]
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C. Sauvant, D. Hesse, H. Holzinger, K. K. Evans, W. H. Dantzler, and M. Gekle Action of EGF and PGE2 on basolateral organic anion uptake in rabbit proximal renal tubules and hOAT1 expressed in human kidney epithelial cells Am J Physiol Renal Physiol, April 1, 2004; 286(4): F774 - F783. [Abstract] [Full Text] [PDF]
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S. A. Eraly, K. T. Bush, R. V. Sampogna, V. Bhatnagar, and S. K. Nigam The Molecular Pharmacology of Organic Anion Transporters: from DNA to FDA? Mol. Pharmacol., March 1, 2004; 65(3): 479 - 487. [Abstract] [Full Text] [PDF]
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C. Kneuer, K. U. Honscha, and W. Honscha Sodium-dependent methotrexate carrier-1 is expressed in rat kidney: cloning and functional characterization Am J Physiol Renal Physiol, March 1, 2004; 286(3): F564 - F571. [Abstract] [Full Text]
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 N. Shikano, Y. Kanai, K. Kawai, N. Ishikawa, and H. Endou Transport of 99mTc-MAG3 via Rat Renal Organic Anion Transporter 1 J. Nucl. Med., January 1, 2004; 45(1): 80 - 85. [Abstract] [Full Text] [PDF]
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M. D. Blaufox Transport of 99mTc-MAG3 via Rat Renal Organic Anion J. Nucl. Med., January 1, 2004; 45(1): 86 - 88. [Full Text] [PDF]
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A. Lungkaphin, V. Chatsudthipong, K. K. Evans, C. E. Groves, S. H. Wright, and W. H. Dantzler Interaction of the metal chelator DMPS with OAT1 and OAT3 in intact isolated rabbit renal proximal tubules Am J Physiol Renal Physiol, January 1, 2004; 286(1): F68 - F76. [Abstract] [Full Text] [PDF]
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C. Sauvant, H. Holzinger, and M. Gekle Short-Term Regulation of Basolateral Organic Anion Uptake in Proximal Tubular Opossum Kidney Cells: Prostaglandin E2 Acts via Receptor-Mediated Activation of Protein Kinase A J. Am. Soc. Nephrol., December 1, 2003; 14(12): 3017 - 3026. [Abstract] [Full Text] [PDF]
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 E. Beery, P. Middel, A. Bahn, H. S. Willenberg, Y. Hagos, H. Koepsell, S. R. Bornstein, G. A. Muller, G. Burckhardt, and J. Steffgen Molecular Evidence of Organic Ion Transporters in the Rat Adrenal Cortex with Adrenocorticotropin-Regulated Zonal Expression Endocrinology, October 1, 2003; 144(10): 4519 - 4526. [Abstract] [Full Text] [PDF]
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A. Aslamkhan, Y.-H. Han, R. Walden, D. H. Sweet, and J. B. Pritchard Stoichiometry of organic anion/dicarboxylate exchange in membrane vesicles from rat renal cortex and hOAT1-expressing cells Am J Physiol Renal Physiol, October 1, 2003; 285(4): F775 - F783. [Abstract] [Full Text] [PDF]
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J. E. Gibbs, T. Rashid, and S. A. Thomas Effect of Transport Inhibitors and Additional Anti-HIV Drugs on the Movement of Lamivudine (3TC) across the Guinea Pig Brain Barriers J. Pharmacol. Exp. Ther., September 1, 2003; 306(3): 1035 - 1041. [Abstract] [Full Text] [PDF]
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 G.-H. Kim, K. Y. Na, S.-Y. Kim, K. W. Joo, Y. K. Oh, S.-W. Chae, H. Endou, and J. S. Han Up-regulation of organic anion transporter 1 protein is induced by chronic furosemide or hydrochlorothiazide infusion in rat kidney Nephrol. Dial. Transplant., August 1, 2003; 18(8): 1505 - 1511. [Abstract] [Full Text] [PDF]
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G.-H. Kim, K. Y. Na, S.-Y. Kim, K. W. Joo, Y. K. Oh, S.-W. Chae, H. Endou, and J. S. Han Up-regulation of organic anion transporter 1 protein is induced by chronic furosemide or hydrochlorothiazide infusion in rat kidney Nephrol. Dial. Transplant., August 1, 2003; 18(88): 1505 - 1511. [Abstract] [Full Text]
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P. Jutabha, Y. Kanai, M. Hosoyamada, A. Chairoungdua, D. K. Kim, Y. Iribe, E. Babu, J. Y. Kim, N. Anzai, V. Chatsudthipong, et al. Identification of a Novel Voltage-driven Organic Anion Transporter Present at Apical Membrane of Renal Proximal Tubule J. Biol. Chem., July 18, 2003; 278(30): 27930 - 27938. [Abstract] [Full Text] [PDF]
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M. Hasegawa, H. Kusuhara, H. Endou, and Y. Sugiyama Contribution of Organic Anion Transporters to the Renal Uptake of Anionic Compounds and Nucleoside Derivatives in Rat J. Pharmacol. Exp. Ther., June 1, 2003; 305(3): 1087 - 1097. [Abstract] [Full Text] [PDF]
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D. H. Sweet, L. M. S. Chan, R. Walden, X.-P. Yang, D. S. Miller, and J. B. Pritchard Organic anion transporter 3 (Slc22a8) is a dicarboxylate exchanger indirectly coupled to the Na+ gradient Am J Physiol Renal Physiol, April 1, 2003; 284(4): F763 - F769. [Abstract] [Full Text] [PDF]
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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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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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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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 F. Knauf, B. Rogina, Z. Jiang, P. S. Aronson, and S. L. Helfand Functional characterization and immunolocalization of the transporter encoded by the life-extending gene Indy PNAS, October 29, 2002; 99(22): 14315 - 14319. [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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S. M. Grassl Urate/alpha -ketoglutarate exchange in avian basolateral membrane vesicles Am J Physiol Cell Physiol, October 1, 2002; 283(4): C1144 - C1154. [Abstract] [Full Text] [PDF]
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A. Enomoto, M. F. Wempe, H. Tsuchida, H. J. Shin, S. H. Cha, N. Anzai, A. Goto, A. Sakamoto, T. Niwa, Y. Kanai, et al. Molecular Identification of a Novel Carnitine Transporter Specific to Human Testis. INSIGHTS INTO THE MECHANISM OF CARNITINE RECOGNITION J. Biol. Chem., September 20, 2002; 277(39): 36262 - 36271. [Abstract] [Full Text] [PDF]
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Y. Kato, K. Kuge, H. Kusuhara, P. J. Meier, and Y. Sugiyama Gender Difference in the Urinary Excretion of Organic Anions in Rats J. Pharmacol. Exp. Ther., August 1, 2002; 302(2): 483 - 489. [Abstract] [Full Text] [PDF]
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C. Sauvant, H. Holzinger, and M. Gekle Short-Term Regulation of Basolateral Organic Anion Uptake in Proximal Tubular OK cells: EGF Acts via MAPK, PLA2, and COX1 J. Am. Soc. Nephrol., August 1, 2002; 13(8): 1981 - 1991. [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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 Y. L. Zhao, X. B. Cen, M. Ito, K. Yokoyama, K. Takagi, K. Kitaichi, M. Nadai, M. Ohta, K. Takagi, and T. Hasegawa Shiga-Like Toxin II Derived from Escherichia coli O157:H7 Modifies Renal Handling of Levofloxacin in Rats Antimicrob. Agents Chemother., May 1, 2002; 46(5): 1522 - 1528. [Abstract] [Full Text] [PDF]
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S. C. N. Buist, N. J. Cherrington, S. Choudhuri, D. P. Hartley, and C. D. Klaassen Gender-Specific and Developmental Influences on the Expression of Rat Organic Anion Transporters J. Pharmacol. Exp. Ther., April 1, 2002; 301(1): 145 - 151. [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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R. Kojima, T. Sekine, M. Kawachi, S. H. Cha, Y. Suzuki, and H. Endou Immunolocalization of Multispecific Organic Anion Transporters, OAT1, OAT2, and OAT3, in Rat Kidney J. Am. Soc. Nephrol., April 1, 2002; 13(4): 848 - 857. [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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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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 G. Lee, S. Dallas, M. Hong, and R. Bendayan Drug Transporters in the Central Nervous System: Brain Barriers and Brain Parenchyma Considerations Pharmacol. Rev., December 1, 2001; 53(4): 569 - 596. [Abstract] [Full Text] [PDF]
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F. Islinger, M. Gekle, and S. H. Wright Interaction of 2,3-Dimercapto-1-propane Sulfonate with the Human Organic Anion Transporter hOAT1 J. Pharmacol. Exp. Ther., November 1, 2001; 299(2): 741 - 747. [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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A. Takeuchi, S. Masuda, H. Saito, T. Abe, and K.-i. Inui Multispecific Substrate Recognition of Kidney-Specific Organic Anion Transporters OAT-K1 and OAT-K2 J. Pharmacol. Exp. Ther., October 1, 2001; 299(1): 261 - 267. [Abstract] [Full Text] [PDF]
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N. A. WOLFF, B. GRUNWALD, B. FRIEDRICH, F. LANG, S. GODEHARDT, and G. BURCKHARDT Cationic Amino Acids Involved in Dicarboxylate Binding of the Flounder Renal Organic Anion Transporter J. Am. Soc. Nephrol., October 1, 2001; 12(10): 2012 - 2018. [Abstract] [Full Text] [PDF]
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N. Morita, H. Kusuhara, T. Sekine, H. Endou, and Y. Sugiyama Functional Characterization of Rat Organic Anion Transporter 2 in LLC-PK1 Cells J. Pharmacol. Exp. Ther., September 1, 2001; 298(3): 1179 - 1184. [Abstract] [Full Text] [PDF]
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D. H. Sweet, K. T. Bush, and S. K. Nigam The organic anion transporter family: from physiology to ontogeny and the clinic Am J Physiol Renal Physiol, August 1, 2001; 281(2): F197 - F205. [Abstract] [Full Text] [PDF]
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S. H. Cha, T. Sekine, J.-i. Fukushima, Y. Kanai, Y. Kobayashi, T. Goya, and H. Endou Identification and Characterization of Human Organic Anion Transporter 3 Expressing Predominantly in the Kidney Mol. Pharmacol., April 16, 2001; 59(5): 1277 - 1286. [Abstract] [Full Text]
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P. M. Gerk, C. Y. Oo, E. W. Paxton, J. A. Moscow, and P. J. McNamara Interactions between Cimetidine, Nitrofurantoin, and Probenecid Active Transport into Rat Milk J. Pharmacol. Exp. Ther., January 1, 2001; 296(1): 175 - 180. [Abstract] [Full Text]
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A. Shuprisha, S. H. Wright, and W. H. Dantzler Method for measuring luminal efflux of fluorescent organic compounds in isolated, perfused renal tubules Am J Physiol Renal Physiol, November 1, 2000; 279(5): F960 - F964. [Abstract] [Full Text] [PDF]
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Y. Uwai, H. Saito, Y. Hashimoto, and K.-I. Inui Interaction and Transport of Thiazide Diuretics, Loop Diuretics, and Acetazolamide via Rat Renal Organic Anion Transporter rOAT1 J. Pharmacol. Exp. Ther., October 1, 2000; 295(1): 261 - 265. [Abstract] [Full Text]
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S. Wada, M. Tsuda, T. Sekine, S. H. Cha, M. Kimura, Y. Kanai, and H. Endou Rat Multispecific Organic Anion Transporter 1 (rOAT1) Transports Zidovudine, Acyclovir, and Other Antiviral Nucleoside Analogs J. Pharmacol. Exp. Ther., September 1, 2000; 294(3): 844 - 849. [Abstract] [Full Text]
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L. Li, P. J. Meier, and N. Ballatori Oatp2 Mediates Bidirectional Organic Solute Transport: A Role for Intracellular Glutathione Mol. Pharmacol., August 1, 2000; 58(2): 335 - 340. [Abstract] [Full Text]
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R. A. M. H. Van Aubel, R. Masereeuw, and F. G. M. Russel Molecular pharmacology of renal organic anion transporters Am J Physiol Renal Physiol, August 1, 2000; 279(2): F216 - F232. [Abstract] [Full Text] [PDF]
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G. Burckhardt and N. A. Wolff Structure of renal organic anion and cation transporters Am J Physiol Renal Physiol, June 1, 2000; 278(6): F853 - F866. [Abstract] [Full Text] [PDF]
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A. Pavlova, H. Sakurai, B. Leclercq, D. R. Beier, A. S. L. Yu, and S. K. Nigam Developmentally regulated expression of organic ion transporters NKT (OAT1), OCT1, NLT (OAT2), and Roct Am J Physiol Renal Physiol, April 1, 2000; 278(4): F635 - F643. [Abstract] [Full Text] [PDF]
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G. You, K. Kuze, R. A. Kohanski, K. Amsler, and S. Henderson Regulation of mOAT-mediated Organic Anion Transport by Okadaic Acid and Protein Kinase C in LLC-PK1 Cells J. Biol. Chem., March 31, 2000; 275(14): 10278 - 10284. [Abstract] [Full Text] [PDF]
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H. Uchino, I. Tamai, H. Yabuuchi, K. China, K.-i. Miyamoto, E. Takeda, and A. Tsuji Faropenem Transport across the Renal Epithelial Luminal Membrane via Inorganic Phosphate Transporter Npt1 Antimicrob. Agents Chemother., March 1, 2000; 44(3): 574 - 577. [Abstract] [Full Text]
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S. A. Terlouw, O. Tanriseven, F. G. M. Russel, and R. Masereeuw Metabolite Anion Carriers Mediate the Uptake of the Anionic Drug Fluorescein in Renal Cortical Mitochondria J. Pharmacol. Exp. Ther., March 1, 2000; 292(3): 968 - 973. [Abstract] [Full Text]
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E. S. HO, D. C. LIN, D. B. MENDEL, and T. CIHLAR Cytotoxicity of Antiviral Nucleotides Adefovir and Cidofovir Is Induced by the Expression of Human Renal Organic Anion Transporter 1 J. Am. Soc. Nephrol., March 1, 2000; 11(3): 383 - 393. [Abstract] [Full Text] [PDF]
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S. H. Cha, T. Sekine, H. Kusuhara, E. Yu, J. Y. Kim, D. K. Kim, Y. Sugiyama, Y. Kanai, and H. Endou Molecular Cloning and Characterization of Multispecific Organic Anion Transporter 4 Expressed in the Placenta J. Biol. Chem., February 11, 2000; 275(6): 4507 - 4512. [Abstract] [Full Text] [PDF]
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