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Vol. 63, Issue 3, 590-596, March 2003
Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina (A.G.A., Y.-H.H., X.-P.Y., J.B.P.); and Division of Basic Medical Sciences, Mercer University, School of Medicine, Macon, Georgia (R.K.Z.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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Mercuric ions are highly reactive and form a variety of organic
complexes or conjugates in vivo. The renal proximal tubule is a primary
target for mercury uptake and toxicity, and circumstantial evidence
implicates organic anion transporters in these processes. To test this
hypothesis directly, the transport and toxicity of mercuric-thiol
conjugates were characterized in a Madin-Darby canine kidney cell line
stably transfected with the human organic anion transporter 1 (hOAT1).
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-terazolium bromide assays
(for mitochondrial dehydrogenase) confirmed that mercuric conjugates of
the thiols N-acetylcysteine (NAC), cysteine, or
glutathione were more toxic in hOAT1-transfected cells than in the
nontransfected cells. The NAC-Hg2+ conjugate was most
cytotoxic, inducing greater than 50% cellular death over 18 h at
a concentration of 100 µM. The cytotoxic effects were fully reversed
by probenecid (an OAT1 inhibitor) and partially reversed by
p-aminohippurate (an OAT1 substrate). Toxicity of this
conjugate was reduced by the OAT1-exchangeable dicarboxylates 
-ketoglutarate, glutarate, and adipate, but not by succinate, a
nonexchangeable dicarboxylate. 203Hg-uptake studies showed
probenecid-sensitive uptake of mercury-thiol conjugates in the
hOAT1-transfected cells. The apparent Km for the NAC-Hg2+ conjugate was 44 ± 9 µM. Uptake of the
NAC-Hg2+ conjugate was cis-inhibited by
glutarate, but not by methylsuccinate, paralleling their effects on
toxicity. Probenecid-sensitive transport of the NAC-Hg2+
conjugate was also shown to occur in Xenopus laevis
oocytes expressing the hOAT1 or the rOAT3 transporters, suggesting that
OAT3 may also transport thiol-Hg2+ conjugates. Thus, renal
accumulation and toxicity of thiol-Hg2+ conjugates may
depend in part on the activity of the organic transport system.
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Introduction |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The
renal organic anion transport system is an elimination route for many
xenobiotics, as well as for endogenous organic anions (Burckhardt et
al., 2001
). The basolateral uphill step for organic anion transport is
mediated by transporters of the OAT family. OAT1 is a
dicarboxylate/organic anion exchanger that takes up organic anions in
exchange for intracellular -ketoglutarate (Pritchard, 1995), and its
gene was cloned recently for the rat (Sekine et al., 1997; Sweet et
al., 1997), winter flounder (Wolff et al., 1997), and human (Reid et
al., 1998; Cihlar et al., 1999; Hosoyamada et al., 1999; Lu et al.,
1999). This transporter is multispecific and mediates the uptake of
structurally dissimilar organic acids and some neutral compounds,
including p-aminohippurate (PAH), nonsteroidal
anti-inflammatory drugs (Apiwattanakul et al., 1999), 
-lactam
antibiotics (Jariyawat et al., 1999), cyclic nucleotides (Sekine et
al., 1997), and nucleoside analogs (Cihlar et al., 1999). The other
basolateral organic anion transporter, OAT3, also handles a diverse
number organic anion substrates by a yet undescribed mechanism, and its
gene has been cloned in the rat (Kusuhara et al., 1999), human (Cha et
al., 2001), and the mouse (Brady et al., 1999), in which its
role was characterized recently with the use of knockout mice (Sweet et
al., 2002). Apical efflux of organic anions into the lumen occurs by
ATP-binding cassette transport efflux pumps as well as by a yet
uncharacterized potential-sensitive transporter (Krick et al., 2000;
Burckhardt et al., 2001).
It is well established that inorganic mercury
(Hg2+) is nephrotoxic (Zalups, 2000).
Accumulation and uptake of Hg2+ is high in all
three segments of the proximal tubule (Zalups and Barfuss, 1990;
Zalups, 1991a,b). All of the epithelial cells in these segments contain
the OAT1 and OAT3 transporters in their basolateral membrane
(Hosoyamada et al., 1999; Sweet et al., 1999; Tojo et al., 1999; Cha et
al., 2001; Hasegawa et al., 2002). Recent in vivo data from rats
indicates that OAT1 and/or OAT3 provides a route for basolateral uptake
of mercuric species. For example, intravenous coadministration of
HgCl2 with the organic anion transporter substrate PAH inhibits Hg2+ uptake when
Hg2+ was administered as
HgCl2 or as thiol-Hg2+
conjugates (Zalups, 1998a,b). In another study using the same technique, it was found that pretreatment with small aliphatic dicarboxylic acids (i.e., glutarate and adipate) before
Hg2+ administration inhibited renal
Hg2+ uptake in a dose-dependent manner (Zalups
and Barfuss, 1998b). Because these acids are all exchangeable
substrates of OAT1, it was concluded that there was a basolateral
uptake pathway for mercuric ion species involving the organic anion
transporter (Zalups, 2000; Zalups and Koropatnick, 2000). Recently, PAH
and glutarate-sensitive basolateral uptake of
thiol-Hg2+ conjugates was shown to occur in
rabbit isolated perfused proximal (pars recta) tubules (Zalups and
Barfuss, 2002), which further supports the potential role of
basolateral organic anion transporters.
Because OAT1 and OAT3 are located in the basolateral membrane, they can
act upon mercuric species present in the blood. More than 98% of the
mercuric ions in the serum are bound to albumin. A smaller fraction is
presumed to be bound to endogenous, low-molecular-weight thiols (Lau
and Sarkar, 1979). However, the mercuric-albumin complex seems to be
labile because the in vivo concentration of mercuric species in plasma
decreases rapidly over time (Zalups, 1998b), suggesting that ligand
exchange occurs between a mercuric-albumin complex and
low-molecular-weight thiols (Zalups and Koropatnick, 2000). The
low-molecular-weight thiol conjugates seem to be much less labile in an
aqueous environment (Rabenstein, 1989; Farrell et al., 1990; Oram et
al., 1996). In fact, administration of such thiols alters both the
toxicity and disposition of Hg2+ in the kidney
(Zalups and Barfuss, 1996, 1998a; Zalups, 1998b). For example,
intravenous coadministration of endogenous low-molecular-weight thiols
(including Cys, homocysteine, NAC) with Hg2+ to
rats has been demonstrated to increase both the level of renal accumulation and intoxication, indicating that these
low-molecular-weight thiol conjugates are taken up by specific
molecular mechanisms (Zalups and Barfuss, 1996, 19998a; Zalups, 1998b),
potentially including the organic anion transport system.
The goal of the present investigation was to determine whether the organic anion transporters play a mechanistic role in the uptake, accumulation, and toxicity of inorganic mercury in renal cells, thereby supporting prior in vivo studies. To this end, we used a cell-line transfected with hOAT1 to assess both uptake and cytotoxicity of Hg2+ and thiol- Hg2+ complexes. In addition, we characterized the uptake of the NAC-Hg2+ conjugate in both hOAT1- and rOAT3-expressing oocytes.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Tissue Culture. A mycoplasma-free type II MDCK subclone with low transepithelial resistance was supplied by Dr. Daniel Balkovetz (University of Alabama at Birmingham, Birmingham, AL). This line was originally developed in the laboratory of Dr. Kai Simmons (European Molecular Biology Laboratory, Heidelberg, Germany). These cells were grown in a humidified atmosphere of 5% CO2/95% O2 at 37°C in culture media composed of EMEM (Invitrogen, Carlsbad, CA). supplemented with 1 mM sodium pyruvate and 10% fetal bovine serum (Invitrogen). This EMEM formulation will be referred to as "supplemented EMEM" throughout this article. Cells were split every 3 to 7 days, and 5 to 10% of the culture was inoculated into new flasks.
Cell Transfection. MDCK cells were transfected with hOAT1 cDNA ligated to pcDNA3.1 (Invitrogen) using SuperFect Reagent (QIAGEN, Valencia, CA) according to the manufacturer's protocol (5 µl of SuperFect/µg of DNA). Surviving cell clones were maintained in culture media with 200 µg/ml G418 (Invitrogen) and screened for organic anion transport activity by assaying the uptake of [3H]PAH, as described below. The clone displaying the greatest level of [3H]PAH uptake (greater than 20 times the level in nontransfected MDCK cells) was used in the present study.
Production of Isotopic Hg2+. Mercuric oxide (3 mg) containing the stable isotope 200Hg2+ and enriched 202Hg2+ (target) were weighed and doubly sealed in quartz tubing (actual mercuric oxide isotopic composition, <0.05% 196Hg, 1.5% 198Hg, 2.82% 199Hg, 4.24% 200Hg, 3.11% 201, 86.99% 202Hg, and 1.34% 204Hg). The double-encapsulated target was sent to the Missouri University Research Reactor facility to be irradiated (by neutron activation) for 4 weeks. The irradiated target was placed in protected storage for 10 days to allow for the isotopic decay of the newly formed 197Hg2+. The target was removed from the quartz tubing with four 50-µl rinses of 1N HCl. All four rinses were placed and sealed in a single 1.7-ml polypropylene vial. A sample of the solution was then used to determine the precise solid content of Hg using plasma-coupled elemental mass spectrometry. The radioactivity of the solution was determined by use of a PerkinElmer Wallac (Gaithersburg, MD) Wizard 3" 1480 Automatic Gamma Counter (203Hg counting efficiency, ~50%). The specific activities of the 203Hg2+ used in the present study ranged between 8 and 12 mCi/mg Hg.
Hg2+ Tracer Uptake in hOAT1 Transfected and Nontransfected MDCK Cells. In the MDCK cell clone selected for these experiments, hOAT1 is expressed in both apical and basolateral faces (J.B. Pritchard et al., unpublished results). Apical expression of hOAT1 allows for the uptake to be studied in cells grown on solid support. Thus, for these experiments, cells were plated in 24-well (2.0 cm2) cell-culture cluster plates (Corning Glassworks, Corning, NY) in supplemented EMEM at a density of 0.5 × 106 cells/well (added as 2 ml). They were then grown in a humidified atmosphere of 5% CO2/95% air at 37°C for 2 days. Media were changed after the first 24 h.
For transport assays, media were aspirated from wells, and cells were rinsed three times with three volumes of 3 ml each of Hank's buffered saline solution (HBSS) supplemented with 10 mM HEPES, pH 7.4. Transport buffer (333 µl; specific to each experiment) containing radioactive 203Hg2+ was added to each well. Thiols were always added in a 4:1 M ratio to the mercuric cation concentration to ensure the formation of linear II coordinate complexes (Rabenstein, 1989). At defined times, wells were rinsed with cold
(4°C) stop buffer [HBSS supplemented with 10 mM HEPES, pH 7.4, with
1 mM 2,3-dimercapto-1-propane-sulfonic acid (DMPS) and 200 µM
probenecid]. Because DMPS oxidizes rapidly in aqueous solutions, it
was added less than 1 h before use. Cellular Hg2+ uptake was determined after adding 1 ml of 1 N NaOH to each well and shaking (in an orbital shaker at 500 rpm) for
24 h. Cell lysate (700 µl) from each well was neutralized with
700 µl of 1 N HCl and added to 15 ml of EcoLume (ICN Biomedicals
Inc., Costa Mesa, CA) scintillation fluid. The radioactivity of each
sample was determined using a Tri-Carb 2900TR Liquid Scintillation
Analyzer (PerkinElmer Life Sciences, Boston, MA;
203Hg counting efficiency,
~80-90%). Of the remaining cell lysate in each well, 50 µl was processed for Bradford protein assay (Bradford, 1976). Data
for each well were normalized to the corresponding protein concentration.
Cell Viability/Toxicity Testing. Cell viability was measured with an MTT-based in vitro toxicology assay (TOX1; Sigma, St. Louis, MO). This assay measures mitochondrial dehydrogenase activity by the conversion of the yellow tetrazolium dye MTT to purple formazan crystals. Cells were plated in supplemented EMEM at a density of 5.0 × 104 cells/well (added as 200 µl/well) in sterile 96-well microtiter plates (Corning Glassworks) and allowed to grow for 48 h in a humidified atmosphere of 5% CO2/95% air at 37°C. Supplemented EMEM was changed after the first 24 h by inversion. Excess media adhering to the plate was blotted off with sterile gauze (Johnson & Johnson Medical, Arlington, TX). After 48 h, wells were again washed with two washes of 200 µl/well of HBSS. After washing, test compounds were added to individual wells (200 µl/well) in unsupplemented EMEM, and cells were grown for 18 h (unless otherwise specified) in a humidified atmosphere of 5% CO2/95% O2 at 37°C. At the conclusion of the exposure period, media were removed by inversion and blotting, wells were washed with 200 µl of HBSS, and 100 µl of 0.5 mg/ml (1.2 mM) MTT in HBSS was added to each well. Cells were incubated for 2 h, and 100 µl of solubilization buffer (10% Triton X-100, 0.1 N HCl in isopropyl alcohol) were added to each well. This buffer both lysed the cells (releasing the formazan) and dissolved the water-insoluble formazan crystals. After overnight incubation at room temperature, full solubilization had occurred, and plates were read at 570 nm with a SpectraMax 340 microtiter plate reader (Molecular Devices Corporation, Sunnyvale, CA) running SoftMax Pro.
Oocyte Preparation and Hg2+ Tracer Uptake in hOAT1-
and rOAT3-Expressing Oocytes.
Female Xenopus laevis
liver-fed frogs were obtained from Xenopus I (Ann Arbor, MI). The
ovaries were removed from tricaine-anesthetized frogs, and oocytes were
isolated and defolliculated as described previously (Sweet et al.,
1997). Briefly, this procedure uses collagenase A digestion followed by
an incubation in a K2HPO4 buffer. Oocytes were then stored in an incubator at 18°C and allowed to recover overnight in oocyte Ringer's 2 (OR-2; 82.5 mM NaCl, 2.5 mM
KCl, 1 mM Na2HPO4, 3 mM
NaOH, 1 mM CaCl2, 1 mM
MgCl2, 1 mM sodium pyruvate, and 5 mM HEPES, pH
7.6) supplemented with 5% horse serum and 50 µg/ml gentamicin. After
the recovery period, stage IV and V oocytes were microinjected with
16.1 nl of either high-performance liquid chromatography water or
capped RNA (hOAT1 or rOAT3 clones at 1.93 µg/µl).
Statistical Analysis. Results are presented as representative data from at least two experiments. Data are expressed as the mean ± standard error. For cytotoxicity studies, a sample size of n = 4 was used, and for uptake studies, a sample size of n = 3 was used. Assuming that each sample was mutually independent, statistical analysis was performed using two-way analysis of variance on log-transformed data followed by either Tukey's or Dunnett's post hoc test. Data were analyzed with the use of SAS version 8.0 (SAS Institute, Cary, NC). Differences among means were considered statistically significant at P < 0.05.
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cellular Viability.
As shown in Fig.
1, sensitivity of control and
hOAT1-transfected MDCK cells to 18 h of exposure to
HgCl2 was similar through the range of
concentrations tested. The LD50 for mercuric
chloride was approximately 16 µM. Mercuric conjugates of Cys, NAC, or
GSH were less toxic than HgCl2 in both cell
types. However, sensitivity of the nontransfected cells was markedly
lower than that in the cells expressing hOAT1. As seen in Fig. 1A,
there was no significant toxicity of the
thiol-Hg2+ conjugates in the control cells, even
at Hg2+ concentrations four times greater than
the concentration of HgCl2 that yielded complete
loss of viability. In contrast, exposure to the
thiol-Hg2+ conjugates caused marked toxic effects
in hOAT1-transfected cells over the same concentration range (Fig. 1B).
Cellular viability in the cells transfected with hOAT1 was reduced by
81% during the 18 h of exposure to 100 µM of the
NAC-Hg2+ conjugate. The
Cys-Hg2+ conjugate (100 µM) was intermediate in
toxicity, with a 54% decrease in survival. The
GSH-Hg2+ conjugate was the least toxic, with only
13% mortality at the same concentration and time of exposure.
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-ketoglutarate, adipate, and suberate (Pritchard and
Miller, 1993). Other dicarboxylates, such as succinate, fumarate, and
malate, are not effective inhibitors of OAT1. As shown in Fig.
4, the level of cellular death induced by
mercuric conjugates of NAC was significantly reduced by the
exchangeable dicarboxylates glutarate, -ketoglutarate, and adipate.
In contrast, succinate, a nonexchangeable dicarboxylate (Chatsudthipong
and Dantzler, 1992), did not provide significant protection.
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Isotopic Hg2+ Uptake.
The data presented above
suggest that the presence of hOAT1 provides a specific pathway for
thiol-Hg2+ conjugates into cells and that
increased cellular uptake leads to a corresponding increase in the
level of toxicity. To test this directly, we measured the uptake of
Hg2+ in control and hOAT1-expressing cells. As
seen in Fig. 5, there was 3- to 4-fold
greater uptake of the NAC-Hg2+ and
Cys-Hg2+ conjugates in hOAT1-expressing cells
than in control cells. However, uptake of the
GSH-Hg2+ conjugate (the least toxic mercuric
thiol species) was similar in both cell types. The hOAT1-dependent
uptake (difference between uptake in hOAT1 transfected and
nontransfected cells) of the NAC-Hg2+ and
Cys-Hg2+ conjugates was almost completely
abolished by probenecid (200 µM).
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1·min1 for the
nontransfected cells and 4.2 pmol·mg · protein1·min1 for the
hOAT1-transfected cells. By 2 h, uptake in the hOAT1-transfected cells had not reached steady state and was 6-fold greater than that in
nontransfected cells. Because uptake was linear at 40 min (Fig. 6),
this time was used for kinetic studies. As shown in Fig.
7, the apparent
Km for hOAT1-dependent uptake of
mercuric conjugates of NAC was 44 ± 9 µM, and the
Vmax was 9 ± 1 pmol·(mg · protein)1·min 1.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The kidney is the primary target organ for inorganic mercury
toxicity (Zalups, 2000). Within the kidney, the primary sites of
mercury accumulation and toxicity are the S2 and S3 segments of the
proximal tubule (Zalups and Barfuss, 1990; Zalups, 1991a,b). These are
also the primary sites of OAT1 and OAT3 expression (Hosoyamada et al.,
1999; Sweet et al., 1999; Tojo et al., 1999; Cha et al., 2001; Hasegawa
et al., 2002). Moreover, in vivo studies suggest that OAT1 and/or OAT3
may participate in uptake of toxic mercuric species, because a variety
of OAT inhibitors decrease the severity of the nephropathy induced by
the mercuric species (Zalups, 1998a,b; Zalups and Barfuss, 1998b). In
agreement with in vivo studies, the data reported here provide
molecular evidence that hOAT1 and rOAT3 can mediate the uptake of
thiol-Hg2+ conjugates (particularly the
NAC-Hg2+ and Cys-Hg2+
conjugates). Both uptake and toxicity of the
thiol-Hg2+ conjugates were much greater in MDCK
cells expressing hOAT1 than in control cells lacking this transporter
(Figs. 1 and 5). Furthermore, both uptake and toxicity were reduced by
inhibitors of OAT-mediated transport, indicating that
thiol-Hg2+ conjugates are substrates of hOAT1
(Figs. 3-5). This is also the case for uptake of the
NAC-Hg2+ conjugate by rOAT3 in the X. laevis oocyte expression system (Fig. 9). In contrast, uptake of
inorganic mercuric ions seems to occur by an hOAT1-independent
mechanism, because this form of mercury was equally toxic to
hOAT1-transfected and -nontransfected cells. It is important to note
that inorganic mercury was far more toxic than any of the conjugated
species. Indeed, not only was the overall toxicity greater, but
toxicity was observed at much lower concentrations. Thus, formation of
mercury complexes or conjugates was generally protective. However, as
shown in the present study, some of these conjugates are OAT
substrates. Therefore, OAT1 and OAT3 seem to provide specific paths for
the entry of certain thiol-Hg2+ conjugates into
renal cells, potentially leading to proximal tubular damage. Likewise,
competition for this transport leads to the prevention of toxic effects
of Hg2+ upon administration of OAT inhibitors
such as PAH and probenecid in vivo (Zalups, 1998a,b) and in vitro (Fig.
3).
The basis for transport of mercuric-thiol conjugates by hOAT1 and rOAT3
seems to rest in the structure of the conjugates (Burckhardt et al.,
2001). For instance, the NAC-Hg2+ conjugate is
similar in structure to many mercapturic acids
(N-acetylcysteine S conjugates of various organic
molecules). In fact, mercapturic acids have been shown previously to be
secreted by the renal proximal tubule (Stevens and Jones, 1989). In
addition, it was recently shown that several mercapturic acids [i.e.,
S-(2,4-dinitrophenyl)-N-cysteine] are specific
hOAT1 substrates (Pombrio et al., 2001).
Among the thiol conjugates studied, both the hOAT1-dependent uptake and
toxicity of the NAC-Hg2+ conjugate were
particularly striking. This conjugate was also taken up by the
rOAT3-expressing oocytes. These results are entirely consistent with
the in vivo demonstration by Zalups and Barfuss (1998a) that uptake of
this conjugate is localized entirely to the basolateral membrane, the
site of OAT1 and OAT3 expression (Hosoyamada et al., 1999; Sweet et
al., 1999; Tojo et al., 1999; Cha et al., 2001; Hasegawa et al., 2002).
However, the relative contributions of the various
thiol-Hg2+ conjugates to mercury toxicity in vivo
remain an open question. Certainly, the plasma concentrations of
cysteine- and GSH-mercuric conjugates far exceed that of NAC in vivo
(Mansoor et al., 1992; Chassaing et al., 1999). On the other hand,
clinical administration of thiols, such as NAC for various medical
conditions (i.e., acetaminophen poisoning, etc.) (Schiodt et al.,
2002), may greatly alter the potential for formation and renal
accumulation of specific thiol-Hg2+ conjugates.
Finally, although this study clearly links the uptake and toxicity of
mercuric compounds with OAT1 and potentially OAT3, this does not mean
that organic anion transporters are the only potential paths for
proximal tubular uptake of mercuric conjugates. Evidence already exists
for the involvement of certain luminal amino acid transporters as well
(Cannon et al., 2001). Thus, the story is not yet complete, but the
ability of OAT1 and OAT3 to transport the mercuric conjugates, coupled
with in vivo evidence that OAT inhibitors reduce renal toxicity of
mercury, indicates that this transport plays an important role in the
development of renal damage induced by Hg2+.
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Acknowledgments |
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We thank Dr. Safaraz Ahmed, Ramsey Walden, and Porché D. Kirkland for their invaluable assistance in the radioisotopic uptake portion of this project. We also thank Laura Hall for her assistance with the X. laevis oocyte preparation and injection. Furthermore, we thank Dr. Shyamal D. Peddeda for his assistance in the statistical analysis. We also thank Dr. Delon Barfuss at Georgia State University for his assistance in supplying radioactive mercury for the experiments discussed in this manuscript.
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Footnotes |
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Received May 9, 2002; Accepted November 18, 2002
R.K.Z. is supported, in part, by National Institute of Environmental Health Science grants ES05157, ES05980, and ES11288.
Address correspondence to: Dr. John B. Pritchard, National Institute of Environmental Health Sciences, Laboratory of Pharmacology and Chemistry, P.O. Box 12233, F1-03, Research Triangle Park, NC 27709. E-mail: pritcha3{at}niehs.nih.gov
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Abbreviations |
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OAT, organic anion transporter; DMPS, 2,3-dimercapto-1-propane-sulfonic acid; EMEM, Eagle's modified essential medium; Cys, cysteine; GSH, glutathione (reduced); HBSS, Hanks' buffered saline solution; hOAT1, human organic anion transporter 1; MDCK, Madin-Darby canine kidney cells; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide; NAC, N-acetylcysteine; PAH, p-aminohippurate; ANOVA, analysis of variance; rOAT3, rat organic anion transporter 3; OR-2, oocyte Ringer's 2.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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-ketoglutarate controls the efficacy of renal organic anion transport.
J Pharmacol Exp Ther
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), NAC (
), and Cys (
). Points reflect means of
replicates of four ± S.E. Two-way ANOVAs (Tukey's test) of
logarithmically transformed data were used to compare the presence of
hOAT1 at each of the different chemical doses. *, P < 0.05.
) was fitted to a
hyperbolic equation with the use of KaleidaGraph 3.0 (Synergy Software,
Reading, PA). Points reflect means of replicates of three ± S.E.
Two-way ANOVAs (Tukey's test) of logarithmically transformed data were
used to compare the presence of hOAT1 at each of the treatments. *,
P < 0.05.
