![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan (M.H., H.K., Y.S.); Department of Pharmacology and Toxicology, Kyorin University School of Medicine, Tokyo, Japan (H.E.); and Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Tokyo, Japan (H.K., Y.S.)
Received November 12, 2002; accepted March 25, 2003.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
; Dresser et al.,
2001; Kusuhara and Sugiyama,
2002; Russel et al.,
2002). Cumulative evidence suggests that an exchanger with
intracellular dicarboxylates is involved in the renal uptake of organic anions
on the basolateral membrane of the proximal tubules
(Pritchard and Miller, 1993).
Rat organic anion transporter 1 (rOat1; Slc22a6) has been isolated
from the kidney as a candidate for the classical renal organic anion
transporter (Sekine et al.,
1997; Sweet et al.,
1997). rOat2 (Slc22a7) and rOat3 (Slc22a8) have
been isolated by homology screening of the database and brain cDNA library
(Sekine et al., 1998;
Kusuhara et al., 1999), and
human isoforms corresponding to rat isoforms have been already isolated and
characterized (Hosoyamada et al.,
1999; Lu et al.,
1999; Cha et al.,
2000). rOat2 is abundantly expressed in the liver and female
kidney, but only moderately in male kidney
(Buist et al., 2002;
Kobayashi et al., 2002). The
site of rOat2 expression in the kidney is the brush-border membrane of the
tubules in the medullary thick ascending loop of Henle and cortical and
medullary collecting ducts (Kojima et al.,
2002). However, human OAT2 (hOAT2) is localized on the basolateral
membrane of the proximal tubules in human kidney
(Enomoto et al., 2002). rOat3
is expressed in the liver, kidney, and brain but only weakly in the eye of
male rats (Kusuhara et al.,
1999). rOat3 and hOAT3 are localized on the basolateral membrane
of the proximal tubules (Cha et al.,
2001; Hasegawa et al.,
2002). In rats, basolateral localization of rOat1 and rOat3
indicates their involvement in the renal uptake of organic anions. Both rOat1
and rOat3 have broad substrate specificity, which overlaps to a degree
(Sekine et al., 2000;
Russel et al., 2002). PAH,
ochratoxin A, methotrexate, and 
-lactam antibiotics are their common
substrates (Russel et al.,
2002). rOat3 accepts amphipathic compounds, such as conjugated
steroids, 17-estradiol-D-17-glucuronide, estrone
sulfate, and an hydroxy-3-methylglutaryl-CoA reductase inhibitor, pravastatin,
as well as the organic cation cimetidine
(Kusuhara et al., 1999;
Sugiyama et al., 2001).
Recently, Sweet et al. (2003)
have demonstrated that rOat3 is indirectly coupled to the Na+
gradient through Na+/dicarboxylate cotransport and functions as an
organic anion/dicarboxylate exchanger as the case of rOat1. Their results
suggest that rOat3 is also a tertiary active transport system and that it is
responsible for the energy-dependent uphill uptake of organic anions via the
basolateral membrane of the proximal tubule. Our previous kinetic analyses
have demonstrated that rOat1 and rOat3 play a major role in the renal uptake
of PAH and pravastatin, respectively, in male rats
(Hasegawa et al., 2002).
Together with these previous results, we suggested that rOat1 and rOat3 are
responsible for the renal uptake of relatively small and hydrophilic organic
anions and relatively hydrophobic organic anions, respectively. Recently, the
mouse Oat3 (mOat3) knockout mouse has been established
(Sweet et al., 2002). The
uptake of taurocholate and estrone sulfate by kidney slices was markedly
reduced in the mOat3 knockout mice (Sweet
et al., 2002). In contrast to our results using rats, the uptake
of PAH by kidney slices from mOat3 knockout mice was also reduced to one-half
of that from the corresponding wild type, which may be ascribed to a species
difference (Sweet et al.,
2002).
Even if a compound is a good substrate, it is still unclear whether the
transporter plays a major role in the membrane transport of the compounds. The
contribution in the total membrane transport process should be examined to
identify the transporter, which plays an important role in the disposition of
drugs and endogenous substrates. The purpose of the present study is to
investigate the importance of rOat1 and rOat3 in the total renal uptake of
anionic compounds and nucleoside derivatives. In the present study, eight
compounds were selected: temocaprilat, benzylpenicillin (PCG),
2,4-dichlorophenoxyacetate (2,4-D), salicylate, acyclovir (ACV),
3'-azido-3'-deoxythymidine (AZT), estrone sulfate, and
dehydroepiandrosterone sulfate (DHEAS). These test compounds are known to be
substrates of the organic anion transport system, and/or mainly excreted in
the urine via tubular secretion (Bergeron
et al., 1975; Patel et al.,
1989; Burnette and de Miranda,
1994; Griffin et al.,
1997; Dresser et al.,
2001).
The kinetic parameters for the uptake by rOat1- and rOat3-expressing cells were compared with those determined for the uptake by kidney slices. In addition, to estimate the contribution of rOat1 and rOat3 to the total uptake by kidney slices, the uptake clearance was also examined using rOat1- and rOat3-expressing cells, and the uptake clearance by cDNA-transfected cells was compared with the corresponding uptake clearance by kidney slices.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). The
radiochemical purity of [14C]temocaprilat was checked by thin-layer
chromatography (n-butanol/acetic acid/distilled water; 4:1:1) and was
found to exceed 95%. Unlabeled PAH, AZT, ACV, salicylate, estrone sulfate,
DHEAS, and 2,4-D were purchased from Sigma-Aldrich (St. Louis, MO), and
unlabeled PCG was purchased from Wako Pure Chemicals (Osaka, Japan). All other
chemicals were of analytical grade and commercially available.
Cell Culture. rOat1- and rOat3-expressing LLC-PK1 cells were
established as described previously
(Sugiyama et al., 2001).
Transfectants were grown in M199 (Invitrogen, Carlsbad, CA) supplemented with
10% fetal bovine serum, penicillin (100 U/ml), streptomycin (100 µg/ml),
and G418 (400 µg/ml) (Invitrogen) at 37°C with 5% CO2 and
95% humidity. Cells were seeded in 12-well plates at a density of 1.2 x
105 cells/well. Cell culture medium was replaced with culture
medium supplemented with sodium butyrate (5 mM) 24 h before the transport
studies to induce the expression of rOat1 and rOat3.
Transport Studies. Transport studies were carried out as described
previously (Hasegawa et al.,
2002). Uptake was initiated by adding medium containing
radiolabeled ligands after cells were washed twice and preincubated with
Krebs-Henseleit buffer at 37°C for 15 min. The Krebs-Henseleit buffer
consists of 118 mM NaCl, 23.8 mM NaHCO3, 4.83 mM KCl, 0.96 mM
KH2PO4, 1.20 mM MgSO4, 12.5 mM HEPES, 5 mM
glucose, and 1.53 mM CaCl2 adjusted to pH 7.4. The final
concentration of [3H]AZT, [3H]ACV, and
[3H]PCG was 1.0 µM, that of [3H]2,4-D was 0.5 µM,
that of [3H]estrone sulfate and [3H]DHEAS was 0.1 µM,
and that of [14C]salicylate and [14C]temocaprilat was
5.0 and 3.4 µM, respectively. The uptake was terminated at a designed time
by adding ice-cold Krebs-Henseleit buffer after removal of the incubation
buffer. Then, cells were washed twice with 1 ml of ice-cold Krebs-Henseleit
buffer, dissolved in 500 µl of 0.2 N NaOH and kept overnight. Aliquots (450
µl) were transferred to scintillation vials after adding 100 µl of 1 N
HCl. The radioactivity associated with the cells and medium was determined by
liquid scintillation counting. The remaining 50 µl of the aliquots of cell
lysate was used to determine the protein concentration by the method of Lowry
with bovine serum albumin as a standard. Ligand uptake is given by the
cell-to-medium concentration ratio determined as the amount of ligand
associated with the cells divided by the medium concentration. Specific uptake
was obtained by subtracting the uptake into vector-transfected cells from that
into cDNA-transfected cells.
Uptake by Kidney Slices. Uptake studies were carried out as
described in a previous report (Hasegawa
et al., 2002). Slices (0.3 mm in thickness) of whole kidneys from
male Sprague-Dawley rats (7–8 weeks old; SLC Co., Ltd, Shizuoka, Japan)
were placed in ice-cold oxygenated incubation buffer. The incubation buffer
consists of 120 mM NaCl, 16.2 mM KCl, 1 mM CaCl2, 1.2 mM
MgSO4, and 10 mM
NaH2PO4/Na2HPO4 adjusted to pH
7.5. Two slices, each weighing 10 to 20 mg, were randomly selected and then
incubated in a 12-well plate with 1 ml of oxygenated incubation buffer in each
well after preincubation for 5 min. The uptake study was carried out at
37°C. After incubating for an appropriate time, each slice was rapidly
removed from the incubation buffer, washed in ice-cold saline, blotted on
filter paper, weighed, and dissolved in 1 ml of soluene-350 (PerkinElmer Life
Sciences) at 50°C for 3 h. The radioactivity was determined in a liquid
scintillation counter after adding 10 ml of scintillation fluid (Hionic Flour;
PerkinElmer Life Sciences).
Kinetic Analyses. Kinetic parameters were obtained using the following equation.
One-saturable (two-saturable) component,
 |
One-saturable and one nonsaturable component,
 |
).
Inhibition constants (Ki values) of a series of compounds
were obtained by examining their inhibitory effects on rOat1- and
rOat3-mediated uptake assuming competitive inhibition using the following
equation:
 |
Fitting was performed by the nonlinear least-squares method using a MULTI
program (Yamaoka et al.,
1981), and the Damping Gauss Newton Method algorithm was used for
fitting.
Estimation of Uptake Clearance in Kidney Slices from cDNA
Transfectants. Recently, several prediction approaches for assessing the
contribution of multiple cytochrome P-450 (P-450) enzymes to certain metabolic
reactions in human liver microsomes have been reported
(Nakajima et al., 2002).
Crespi (1995) proposed the
relative activity factor (RAF), as the ratio of human liver microsomal
activity divided by P-450 activity for an isoform-specific reaction. Based on
this concept, we estimated the contribution of rOat1 and rOat3 to the total
uptake by kidney slices. Because the renal uptake of PAH and pravastatin was
predominantly accounted for by rOat1 and rOat3, respectively
(Hasegawa et al., 2002), they
were used as reference compounds for rOat1 and rOat3. The uptake clearances by
cDNA-transfected cells (CLrOat1 and CLrOat3) were
multiplied by RrOat1 and RrOat3, respectively, as
described in the following equation.
 |
Statistical Analysis. Statistical differences were determined using one-way analysis of variance followed by Fisher's least significant difference method. Differences were considered significant at P < 0.05.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
|
Kinetic Study of the Uptake of Anionic Compounds and Nucleoside Derivatives by rOat1-Expressing LLC-PK1 Cells. Eadie-Hofstee plots of [14C]temocaprilat, [3H]2,4-D, and [3H]AZT are shown in Fig. 2. A nonsaturable component was observed in the Eadie-Hofstee plot, even for the specific uptake of [14C]temocaprilat and [3H]AZT by rOat1 (Fig. 2, A and C), which was determined by subtraction of the uptake by vector-transfected cells from that by rOat1-expressing cells. The Ki values of estrone sulfate, ACV, DHEAS, PCG, and salicylate on PAH uptake by rOat1-expressing LLC-PK1 cells were examined in the rOat1-expressing LLC-PK1 cells (Fig. 3), and kinetic parameters are summarized in Table 1. Furthermore, inhibition studies were carried out to examine the mode of inhibition of the ligands, whose uptake in rOat1-expressing cells was detected, but which was not high enough to allow for further characterization. rOat1-mediated uptake of [3H]PAH was measured at several substrate concentrations in the presence or absence of estrone sulfate, ACV, and salicylate (Fig. 4). All of these compounds increased the apparent Km values but not affect the Vmax values (data not shown), suggesting that these weak substrates of rOat1 inhibit the uptake of [3H]PAH in a competitive manner.
|
|
|
|
|
|
|
Kinetic Study of the Uptake of Anionic Compounds and Nucleoside Derivatives by rOat3-Expressing LLC-PK1 Cells. Eadie-Hofstee plots of [14C]temocaprilat, [3H]PCG, [3H]estrone sulfate, and [3H]DHEAS are shown in Fig. 5. The affinity of 2,4-D, AZT, ACV, and salicylate for rOat3 was examined as the Ki value for [3H]pravastatin uptake by rOat3-expressing cells (Table 1; Fig. 6). Furthermore, inhibition studies were carried out to test the mode of inhibition of the 2,4-D and AZT, whose uptake in rOat3-expressing cells was minimally detected. Eadie-Hofstee plot analyses demonstrated that these two compounds inhibited rOat3-mediated uptake of [3H]pravastatin in a competitive manner (Fig. 7).
|
Uptake of Anionic Compounds and Nucleoside Derivatives by Kidney Slices. The time profiles for the uptake of anionic compounds and nucleoside derivatives by kidney slices are shown in Fig. 8. Eadie-Hofstee plots of the uptake of [3H]2,4-D, [3H]DHEAS, [3H]PCG, [14C]temocaprilat, [14C]salicylate, and [3H]estrone sulfate are shown in Fig. 9. One saturable and one nonsaturable component were observed in the uptake of [3H]2,4-D, [3H]PCG, and [3H]DHEAS (Fig. 9, A–C). Two saturable components were observed in the uptake of [14C]temocaprilat (Table 1; Fig. 9D). Two saturable and one nonsaturable components were observed in the uptake of [14C]salicylate (Fig. 9E). Kinetic parameters were calculated with the Km value of the low-affinity component (Km2) fixed as 550 µM, which was the Ki value of salicylate for rOat1. In the uptake of [3H]estrone sulfate, two saturable and one nonsaturable component were also observed (Fig. 9F). Km and Vmax values of the uptake of anionic compounds are shown in Table 1. The concentration dependence of the uptake of [3H]AZT and [3H]ACV is shown in Fig. 9G. Even in the presence of 3 mM unlabeled ligands, the uptake of [3H]AZT and [3H]ACV was reduced to only 80 and 60% of the controls, respectively (Fig. 9G).
|
Estimation of Uptake Clearance in Kidney Slices from cDNA Transfectants. Comparison of the observed and estimated renal uptake clearance is shown in Fig. 10. The absolute values showing the predicted rOat1-mediated uptake (CLtest, rOat1 · RrOat1), rOat3-mediated uptake (CLtest, rOat3 · RrOat3), the sum of the rOat1- and rOat3-mediated uptake (CLtest, slice), and observed uptake clearance are summarized in Table 2. The observed and predicted uptake of PCG, temocaprilat, estrone sulfate, and DHEAS were similar to each other. The observed values of salicylate and ACV were 7- and 18- fold higher than the predicted values, whereas that of 2,4-D was smaller than the predicted value.
|
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Transfection of rOat1 resulted in a significant increase in the uptake of
temocaprilat, 2,4-D, AZT, ACV, and salicylate, and a slight increase in the
uptake of estrone sulfate was observed in rOat1-expressing cells compared with
the uptake by vector-transfected cells
(Fig. 1). In rOat3-expressing
cells, a significant increase in the uptake of temocaprilat, estrone sulfate,
DHEAS, and PCG was observed, and the uptake of 2,4-D and AZT was slightly
greater than that by vector-transfected cells
(Fig. 1). The difference in the
spectrum of substrate specificity of rOat1 and rOat3 was clearly observed
except for temocaprilat, a common substrate of rOat1 and rOat3 with similar
transport activity and Km values
(Fig. 1;
Table 1). The
Ki values of 2,4-D, ACV, and salicylate, determined for
the uptake of pravastatin by rOat3, were similar to those for rOat1, and the
Ki value of AZT for rOat3 was approximately 3-fold greater
than that for rOat1 (Table 1).
Although their uptake by rOat3-expressing cells was quite small or zero, their
affinity was very close to that of rOat1. Because mutual inhibition study
suggested that 2,4-D, and AZT share the substrate-binding site with
pravastatin in rOat3 molecules (Fig.
7), the Ki values can be used to estimate
Km values for rOat3. It is possible that the relatively
low uptake of 2,4-D and AZT by rOat3 is ascribed to the efficacy of the
translocation process. However, the Ki values of estrone
sulfate, DHEAS, and PCG for rOat1 were 
7- to 10-fold greater than their
Km values for rOat3
(Table 1), suggesting that
their lower affinity for rOat1 is one explanation of the low uptake by
rOat1-expressing cells. Although significant uptake of PCG was not detected in
rOat1-expressing LLC-PK1 cells (Fig.
1), the uptake of PCG was greater in rOat1-expressing Xenopus
laevis oocytes than in control oocytes
(Jariyawat et al., 1999).
Thus, PCG will be a substrate of rOat1 with low transport activity, like
estrone sulfate.
To determine the importance of the transporter(s) in the renal uptake of
xenobiotics, including drugs and endogenous substrates, the contribution of
rOat1 and rOat3 to the total renal uptake needs to be investigated. The renal
uptake of test compounds was investigated using kidney slices. All test
compounds significantly accumulated in kidney slices compared with mannitol
used for the correction of adherent water volume. The uptake of 2,4-D, estrone
sulfate and DHEAS was relatively high followed by temocaprilat, salicylate,
and PCG (Fig. 8). The uptake of
nucleoside derivatives by kidney slices was relatively low compared with the
uptake of other test compounds (Fig.
8). The concentration dependence of the uptake of test compounds
was investigated (Fig. 9).
Kinetic analyses revealed that a single saturable component was involved in
the uptake of 2,4-D, DHEAS, and PCG, whereas two were involved in the uptake
of temocaprilat, salicylate, and estrone sulfate. The Km
value of 2,4-D for the uptake by kidney slices was comparable with that for
rOat1 (Table 1), whereas those
of DHEAS and PCG were comparable with those of rOat3
(Table 1). Taking the results
of the transport studies using cDNA-transfected cells into consideration, it
is possible that the renal uptake of 2,4-D is mainly mediated by rOat1 and
that of DHEAS and PCG is mediated by rOat3. The uptake of temocaprilat and
estrone sulfate consisted of two saturable components and/or one nonsaturable
component (Fig. 9). The
high-affinity components of temocaprilat and estrone sulfate account for 60
and 50% of the total uptake by kidney slices, respectively
(Table 1). The
Km values for the high-affinity components of the uptake
of temocaprilat and estrone sulfate were not necessarily comparable with those
of rOat1 and/or rOat3 (Table
1). The kinetic parameters of the high-affinity components, which
are determined by the nonlinear least-squares method, tends to be low when
multiple saturable components are involved. Taking this into consideration,
the difference in the Km values will be in the range of
experimental deviation. It may be that rOat1 and rOat3 mediate the
high-affinity component of temocaprilat, whereas the high-affinity component
of estrone sulfate uptake by kidney slices is mediated by rOat3. The uptake of
estrone sulfate was significantly reduced in kidney slices from mOat3 knockout
mice, but still greater than the uptake in the presence of inhibitors such as
probenecid and bromosulfophthalein (Sweet
et al., 2002). This observation was consistent with our kinetic
analysis, and the remaining uptake by kidney slices in mOat3 knockout mice may
be partly accounted for by rOat1 because the Km value of
the low-affinity component in kidney slices in this study was similar to the
Ki value for rOat1
(Table 1).
The Km value of salicylate for the uptake by kidney
slices was much smaller than the Ki value for rOat1 or
Oat3 (Table 1). Even though we
assumed the presence of a low-affinity component using the Ki value
of low-affinity component fixed as that for rOat1 and rOat3, the contribution
of the low-affinity component was approximately 10% of the total saturable
component, suggesting involvement of another transporter. Among the members of
the rOat family, rOat2 has been shown to accept salicylate as a substrate
(Morita et al., 2001).
However, it is not localized on the basolateral membrane of the proximal
tubules (Kojima et al., 2002),
and the Km value for rOat2 (82 µM) is much greater than
the Km value determined in kidney slices
(Morita et al., 2001).
Member(s) of the monocarboxylate transporter family are alternative candidate
transporters (Takanaga et al.,
1995; Eladari et al.,
1999).
The uptake of nucleoside derivatives was saturated at high substrate concentrations, but the saturable components can account for the limited fraction of the total uptake, approximately 20 and 40% of the total uptake of AZT and ACV, respectively, as far as the concentrations examined are concerned (Fig. 9). The uptake of AZT did not exhibit any saturation at a concentration sufficient to saturate rOat1- and rOat3-mediated uptake, whereas the uptake of ACV by kidney slices was significantly reduced at a concentration similar to the Km and Ki values for rOat1 and rOat3, respectively. These results suggest that another transporter with lower affinity and/or passive diffusion mediates the uptake of AZT, whereas the uptake of ACV is mediated by rOat1, and possibly, by rOat3, at least in part.
In addition to the comparison of the Km values, the
contribution was evaluated in terms of the relative transport activity. Crespi
(1995) proposed the RAF as the
ratio of the human liver microsomal metabolic activity divided by the P-450
activity for an isoform-specific reaction. According to the RAF concept, the
uptake clearance of test compounds by kidney slices is compared with the
values obtained from the transport activity by cDNA-transfected cells
(Fig. 10;
Table 2). Also, PAH and
pravastatin were used as reference compounds for rOat1 and rOat3,
respectively, assuming the kinetics of transport determined in
cDNA-transfected cells, in particular, that the relative transport activities
are comparable with those in the kidney slices. A nice correlation was
observed between the observed and predicted uptake clearance except for 2,4-D,
where the uptake clearance by kidney slices was smaller than the expected
value (Fig. 10). Based on this
result, we propose the contribution of rOat1 and rOat3 to the total renal
uptake of the test compounds is as follows; 1) rOat1 plays a major role in the
renal uptake of 2,4-D; 2) rOat3 accounts for the uptake of PCG and DHEAS, and
for the high-affinity component of estrone sulfate uptake by kidney slices; 3)
both rOat1 and rOat3 can account for the high-affinity component of
temocaprilat uptake by kidney slices; however, the uptake clearance corrected
by the RAF value for rOat3 was greater than that for rOat1, suggesting that
the contribution of rOat3 will be greater than rOat1; and 4) the uptake of ACV
by rOat1 was too low to account for the uptake of ACV by kidney slices.
The uptake of 2,4-D by kidney slices was lower than the predicted value (Fig. 10; Table 2). There are two possibilities to account for this. First, diffusion limited the uptake when the ligand is extensively taken up by kidney slices. Second, rOat1-expressing LLC-PK1 cells are not an appropriate model to investigate the renal uptake process in a quantitative manner in terms of the driving force and local membrane environment that affects the transport activity and substrate specificity of the transporter. Because the intrinsic transport activity of 2,4-D by rOat1 was 6-fold greater than that of PAH, it is possible that the uptake of 2,4-D by kidney slices is diffusion-limited.
Although the Ki value of estrone sulfate for rOat1 was comparable with the Km value for the low-affinity component of the uptake by kidney slices (Table 1), the uptake corrected by the RAF value was too low. Therefore, the contribution of rOat1 to the low-affinity component of estrone sulfate uptake by kidney slices will be minor.
Finally, the number of rOat1 substrates for which renal uptake is supposed to be mediated by rOat1 was limited in this study. Further studies are required to examine whether the RAF concept can be applied to rOat1-mediated uptake or not by increasing the number of rOat1 substrates the renal uptake of which is mainly mediated by rOat1.
The present study addressed the importance of investigating the contribution made by each transporter to the total uptake process in addition to the transport studies using cDNA-transfected cells. Even although a compound is a good substrate, it is possible that the contribution of the transporter, supposed to be involved, is not the major mechanism. A good correlation between the observed and predicted values suggests that the RAF concept can be applied; at least, as far as rOat3 substrates are concerned. This approach is useful for predicting the contribution by transport studies using cDNA-transfected cells once the absolute value of the uptake of the transporter-specific ligand by the target organ has been determined. Further studies are necessary to establish this approach by investigating the rationale of the in vitro model and by examining the effect of selective inhibitors of each transporter.
In conclusion, we have demonstrated that the kidney is equipped with an uptake system for amphipathic organic anions, which is mainly mediated by rOat3. Although rOat1 transports certain hydrophobic compounds, its contribution to the total renal uptake is minor. It is also suggested that there is an additional uptake system(s) involved in the renal uptake of salicylate, temocaprilat, estrone sulfate, and ACV.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: OAT, organic anion transporter; PAH, p-aminohippurate; PCG, benzylpenicillin; 2,4-D, 2,4-dichlorophenoxyacetate; ACV, acyclovir; AZT, 3'-azido-3'-deoxythymidine; DHEAS, dehydroepiandrosterone sulfate; P-450, cytochrome P-450; RAF, relative activity factor.
Address correspondence to: Dr. Yuichi Sugiyama, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail: sugiyama{at}mol.f.u-tokyo.ac.jp
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Bergeron MG, Gennari FJ, Barza M, Weinstein L, and Cortell S (1975) Renal tubular transport of penicillin G and carbenicillin in the rat. J Infect Dis 132: 374-383.[Medline]
Buist SC, Cherrington NJ, Choudhuri S, Hartley DP, and Klaassen CD
(2002) Gender-specific and developmental influences on the
expression of rat organic anion transporters. J Pharmacol Exp
Ther 301:
145-151.
Burnette TC and de Miranda P (1994) Metabolic disposition of the acyclovir prodrug valaciclovir in the rat. Drug Metab Dispos 22: 60-64.[Abstract]
Cha SH, Sekine T, Fukushima JI, Kanai Y, Kobayashi Y, Goya T, and
Endou H (2001) Identification and characterization of human
organic anion transporter 3 expressing predominantly in the kidney.
Mol Pharmacol 59:
1277-1286.
Cha SH, Sekine T, Kusuhara H, Yu E, Kim JY, Kim DK, Sugiyama Y,
Kanai Y, and Endou H (2000) Molecular cloning and
characterization of multispecific organic anion transporter 4 expressed in the
placenta. J Biol Chem
275:
4507-4512.
Crespi CL (1995) Xenobiotic-metabolizing human cells as tools for pharmacological and toxicological research. Adv Drug Res 26: 179-235.
Dresser M, Leabman M, and Giacomini K (2001) Transporters involved in the elimination of drugs in the kidney: organic anion transporters and organic cation transporters. J Pharm Sci 90: 397-421.[CrossRef][Medline]
Eladari D, Chambrey R, Irinopoulou T, Leviel F, Pezy F, Bruneval P,
Paillard M, and Podevin RA (1999) Polarized expression of
different monocarboxylate transporters in rat medullary thick limbs of Henle.
J Biol Chem 274:
28420-28426.
Enomoto A, Takeda M, Shimoda M, Narikawa S, Kobayashi Y, Yamamoto
T, Sekine T, Cha SH, Niwa T, and Endou H (2002) Interaction of
human organic anion transporters 2 and 4 with organic anion transport
inhibitors. J Pharmacol Exp Ther
301:
797-802.
Griffin RJ, Godfrey VB, Kim YC, and Burka LT (1997)
Sex-dependent differences in the disposition of 2,4-dichlorophenoxyacetic acid
in Sprague-Dawley rats, B6C3F1 mice and Syrian hamsters. Drug Metab
Dispos 25:
1065-1071.
Hasegawa M, Kusuhara H, Sugiyama D, Ito K, Ueda S, Endou H, and
Sugiyama Y (2002) Functional involvement of rat organic anion
transporter 3 (rOat3; Slc22a8) in the renal uptake of organic anions.
J Pharmacol Exp Ther
300:
746-753.
Hosoyamada M, Sekine T, Kanai Y, and Endou H (1999) Molecular cloning and functional expression of a multispecific organic anion transporter from human kidney. Am J Physiol 276: F122-F128.
Inui KI, Masuda S, and Saito H (2000) Cellular and molecular aspects of drug transport in the kidney. Kidney Int 58: 944-958.[CrossRef][Medline]
Jariyawat S, Sekine T, Takeda M, Apiwattanakul N, Kanai Y, Sophasan
S, and Endou H (1999) The interaction and transport of
-lactam antibiotics with the cloned rat renal organic anion transporter
1. J Pharmacol Exp Ther
290:
672-677.
Kobayashi Y, Hirokawa N, Ohshiro N, Sekine T, Sasaki T, Tokuyama S, Endou H, and Yamamoto T (2002) Differential gene expression of organic anion transporters in male and female rats. Biochem Biophys Res Commun 290: 482-487.[CrossRef][Medline]
Kojima R, Sekine T, Kawachi M, Cha SH, Suzuki Y, and Endou H
(2002) Immunolocalization of multispecific organic anion
transporters, OAT1, OAT2 and OAT3, in rat kidney. J Am Soc
Nephrol 13:
848-857.
Kusuhara H, Sekine T, Utsunomiya-Tate N, Tsuda M, Kojima R, Cha SH,
Sugiyama Y, Kanai Y, and Endou H (1999) Molecular cloning and
characterization of a new multispecific organic anion transporter from rat
brain. J Biol Chem 274:
13675-13680.
Kusuhara H and Sugiyama Y (2002) Role of transporters in the tissue-selective distribution and elimination of drugs: transporters in the liver, small intestine, brain and kidney. J Control Release 78: 43-54.[CrossRef][Medline]
Lu R, Chan BS, and Schuster VL (1999) Cloning of the human kidney PAH transporter: narrow substrate specificity and regulation by protein kinase C. Am J Physiol 276: F295-F303.
Morita N, Kusuhara H, Sekine T, Endou H, and Sugiyama Y
(2001) Functional characterization of rat organic anion
transporter 2 in LLC-PK1 cells. J Pharmacol Exp Ther
298:
1179-1184.
Nakajima M, Tane K, Nakamura S, Shimada N, Yamazaki H, and Yokoi T (2002) Evaluation of approach to predict the contribution of multiple cytochrome P450s in drug metabolism using relative activity factor: effects of the differences in expression levels of NADPH-cytochrome P450 reductase and cytochrome b5 in the expression system and the differences in the marker activities. J Pharm Sci 91: 952-963.[CrossRef][Medline]
Patel BA, Chu CK, and Boudinot FD (1989) Pharmacokinetics and saturable renal tubular secretion of zidovudine in rats. J Pharm Sci 78: 530-534.[CrossRef][Medline]
Pritchard JB and Miller DS (1993) Mechanisms mediating
renal secretion of organic anions and cations. Physiol
Rev 73:
765-796.
Russel FG, Masereeuw R, and van Aubel RA (2002) Molecular aspects of renal anionic drug transport. Annu Rev Physiol 64: 563-594.[CrossRef][Medline]
Schwab AJ, de Lannoy IA, Goresky CA, Poon K, and Pang KS (1992) Enalaprilat handling by the kidney: barrier-limited cell entry. Am J Physiol 263: F858-F869.
Sekine T, Cha SH, and Endou H (2000) The multispecific organic anion transporter (OAT) family. Pfluegers Arch 440: 337-350.[CrossRef][Medline]
Sekine T, Cha SH, Tsuda M, Apiwattanakul N, Nakajima N, Kanai Y, and Endou H (1998) Identification of multispecific organic anion transporter 2 expressed predominantly in the liver. FEBS Lett 429: 179-182.[CrossRef][Medline]
Sekine T, Watanabe N, Hosoyamada M, Kanai Y, and Endou H
(1997) Expression cloning and characterization of a novel
multispecific organic anion transporter. J Biol Chem
272:
18526-18529.
Sugiyama D, Kusuhara H, Abe T, Meier PJ, Sekine T, Endou H, Suzuki
H, and Sugiyama Y (2001) Characterization of the efflux transport
of 17-estradiol-D-17-glucuronide from the brain across
the blood-brain barrier. J Pharmacol Exp Ther
298: 1-6.
Sweet DH, Chan LM, Walden R, Yang XP, Miller DS, and Pritchard JB (2003) Organic anion transporter 3 [Slc22a8] is a dicarboxylate exchanger indirectly coupled to the Na+ gradient. Am J Physiol 284: F763-F769.
Sweet DH, Miller DS, Pritchard JB, Fujiwara Y, Beier DR, and Nigam SK (2002) Impaired organic anion transport in kidney and choroid plexus of organic anion transporter 3 (Oat3 [Slc22a8]) knockout mice. J Biol Chem 13:13.
Sweet DH, Wolff NA, and Pritchard JB (1997) Expression
cloning and characterization of ROAT1. The basolateral organic anion
transporter in rat kidney. J Biol Chem
272:
30088-30095.
Takanaga H, Tamai I, Inaba S, Sai Y, Higashida H, Yamamoto H, and Tsuji A (1995) cDNA cloning and functional characterizaton of rat intestinal monocarboxylate transporter. Biochem Biophys Res Commun 217: 370-377.[CrossRef][Medline]
Yamaoka K, Tanigawara Y, Nakagawa T, and Uno T (1981)
A pharmacokinetic analysis program (multi) for microcomputer. J
Pharmacobiodyn 4:
879-885.[Medline]
This article has been cited by other articles:
|
|
 |
|
  T. Watanabe, K. Maeda, T. Kondo, H. Nakayama, S. Horita, H. Kusuhara, and Y. Sugiyama Prediction of the Hepatic and Renal Clearance of Transporter Substrates in Rats Using in Vitro Uptake Experiments Drug Metab. Dispos., July 1, 2009; 37(7): 1471 - 1479. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Nakakariya, Y. Shima, Y. Shirasaka, K. Mitsuoka, T. Nakanishi, and I. Tamai Organic anion transporter OAT1 is involved in renal handling of citrulline Am J Physiol Renal Physiol, July 1, 2009; 297(1): F71 - F79. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Kwon, W.-W. Wang, and S. Miller Renal organic anion transporter 1 is maldistributed and diminishes in proximal tubule cells but increases in vasculature after ischemia and reperfusion Am J Physiol Renal Physiol, December 1, 2008; 295(6): F1807 - F1816. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Nishimura, Y. Seki, K. Sato, T. Chishu, N. Kose, T. Terasaki, Y.-S. Kang, Y. Sai, and E. Nakashima Enhancement of Zidovudine Uptake by Dehydroepiandrosterone Sulfate in Rat Syncytiotrophoblast Cell Line TR-TBT 18d-1 Drug Metab. Dispos., October 1, 2008; 36(10): 2080 - 2085. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Yamada, K. Maeda, E. Kamiyama, D. Sugiyama, T. Kondo, Y. Shiroyanagi, H. Nakazawa, T. Okano, M. Adachi, J. D. Schuetz, et al. Multiple Human Isoforms of Drug Transporters Contribute to the Hepatic and Renal Transport of Olmesartan, a Selective Antagonist of the Angiotensin II AT1-Receptor Drug Metab. Dispos., December 1, 2007; 35(12): 2166 - 2176. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Mizuno, T. Takahashi, Y. Iwase, H. Kusuhara, T. Niwa, and Y. Sugiyama Human Organic Anion Transporters 1 (hOAT1/SLC22A6) and 3 (hOAT3/SLC22A8) Transport Edaravone (MCI-186; 3-methyl-1-phenyl-2-pyrazolin-5-one) and Its Sulfate Conjugate Drug Metab. Dispos., August 1, 2007; 35(8): 1429 - 1434. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Nozaki, H. Kusuhara, T. Kondo, M. Hasegawa, Y. Shiroyanagi, H. Nakazawa, T. Okano, and Y. Sugiyama Characterization of the Uptake of Organic Anion Transporter (OAT) 1 and OAT3 Substrates by Human Kidney Slices J. Pharmacol. Exp. Ther., April 1, 2007; 321(1): 362 - 369. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Hasegawa, H. Kusuhara, M. Adachi, J. D. Schuetz, K. Takeuchi, and Y. Sugiyama Multidrug Resistance-Associated Protein 4 Is Involved in the Urinary Excretion of Hydrochlorothiazide and Furosemide J. Am. Soc. Nephrol., January 1, 2007; 18(1): 37 - 45. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Kikuchi, H. Kusuhara, N. Hattori, K. Shiota, I. Kim, F. J. Gonzalez, and Y. Sugiyama Regulation of the Expression of Human Organic Anion Transporter 3 by Hepatocyte Nuclear Factor 1{alpha}/beta and DNA Methylation Mol. Pharmacol., September 1, 2006; 70(3): 887 - 896. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Tahara, H. Kusuhara, K. Maeda, H. Koepsell, E. Fuse, and Y. Sugiyama INHIBITION OF OAT3-MEDIATED RENAL UPTAKE AS A MECHANISM FOR DRUG-DRUG INTERACTION BETWEEN FEXOFENADINE AND PROBENECID Drug Metab. Dispos., May 1, 2006; 34(5): 743 - 747. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Tahara, H. Kusuhara, M. Chida, E. Fuse, and Y. Sugiyama Is the Monkey an Appropriate Animal Model to Examine Drug-Drug Interactions Involving Renal Clearance? Effect of Probenecid on the Renal Elimination of H2 Receptor Antagonists J. Pharmacol. Exp. Ther., March 1, 2006; 316(3): 1187 - 1194. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Lowes, D. Sykes, C. M. Breen, L. J. Ragone, and D. S. Miller Multiple Components of 2,4-Dichlorophenoxyacetic Acid Uptake by Rat Choroid Plexus J. Pharmacol. Exp. Ther., October 1, 2005; 315(1): 136 - 143. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Morita, H. Kusuhara, Y. Nozaki, H. Endou, and Y. Sugiyama FUNCTIONAL INVOLVEMENT OF RAT ORGANIC ANION TRANSPORTER 2 (SLC22A7) IN THE HEPATIC UPTAKE OF THE NONSTEROIDAL ANTI-INFLAMMATORY DRUG KETOPROFEN Drug Metab. Dispos., August 1, 2005; 33(8): 1151 - 1157. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Deguchi, M. Takemoto, N. Uehara, W. E. Lindup, A. Suenaga, and M. Otagiri Renal Clearance of Endogenous Hippurate Correlates with Expression Levels of Renal Organic Anion Transporters in Uremic Rats J. Pharmacol. Exp. Ther., August 1, 2005; 314(2): 932 - 938. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-J. Lee, H. Kusuhara, J. W. Jonker, A. H. Schinkel, and Y. Sugiyama Investigation of Efflux Transport of Dehydroepiandrosterone Sulfate and Mitoxantrone at the Mouse Blood-Brain Barrier: A Minor Role of Breast Cancer Resistance Protein J. Pharmacol. Exp. Ther., January 1, 2005; 312(1): 44 - 52. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Hirano, K. Maeda, Y. Shitara, and Y. Sugiyama Contribution of OATP2 (OATP1B1) and OATP8 (OATP1B3) to the Hepatic Uptake of Pitavastatin in Humans J. Pharmacol. Exp. Ther., October 1, 2004; 311(1): 139 - 146. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Nagata, H. Kusuhara, S. Hirono, H. Endou, and Y. Sugiyama CARRIER-MEDIATED UPTAKE OF H2-RECEPTOR ANTAGONISTS BY THE RAT CHOROID PLEXUS: INVOLVEMENT OF RAT ORGANIC ANION TRANSPORTER 3 Drug Metab. Dispos., September 1, 2004; 32(9): 1040 - 1047. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
|
|
S. Ohtsuki, T. Kikkawa, S. Mori, S. Hori, H. Takanaga, M. Otagiri, and T. Terasaki Mouse Reduced in Osteosclerosis Transporter Functions as an Organic Anion Transporter 3 and Is Localized at Abluminal Membrane of Blood-Brain Barrier J. Pharmacol. Exp. Ther., June 1, 2004; 309(3): 1273 - 1281. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 N. Mizuno, T. Niwa, Y. Yotsumoto, and Y. Sugiyama Impact of Drug Transporter Studies on Drug Discovery and Development Pharmacol. Rev., September 1, 2003; 55(3): 425 - 461. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
