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INTRODUCTION |
Renal organic anion transport plays a vital role in the
elimination of a wide variety of potentially toxic, negatively charged waste products of metabolism, drugs, environmental pollutants, and
their metabolites from the body. The transport mechanisms responsible
for this elimination have been studied extensively (1-3). Based on
these studies, it has been suggested that the transport of organic
anions is a complex process involving distinctly different proteins at
the apical and basolateral membranes of the proximal tubule cells.
Organic anions are transported across the basolateral membrane into the
cell in exchange for intracellular dicarboxylates, which are
subsequently returned into the cell via a sodium-dependent
dicarboxylate transporter. The luminal exit of organic anions is
thought to occur by anion exchange and/or facilitated diffusion
(1-3).
We previously reported the cloning of a mouse cDNA (termed NKT)
that encodes a 546-amino acid membrane protein specifically expressed
in the kidney (7). Subsequently, its counterparts from rat and winter
flounder were isolated independently by other groups (4-6). Although
expression studies in Xenopus oocytes revealed that this
membrane protein had the characteristics of renal organic anion
transporter (4-6), little is known concerning the structural basis for
the function of this transporter. The availability of the cDNA that
encodes the organic anion transporter protein enables us to begin to
unravel the structure-function relationship of this elimination system
at the molecular level.
We predicted in our early study (7) that the extracellular loop between
putative transmembrane regions 1 and 2 contains four potential
N-linked glycosylation consensus sites at positions Asn-56,
Asn-86, Asn-91, and Asn-107. The same profile was also observed in its
isoforms from rat and winter flounder (4-6). The presence of conserved
N-glycosylation sites in all the organic anion transporters
cloned so far suggests that glycosylation may play an important role in
the function of these proteins.
A study using brush-border membrane vesicles from dog kidney (8)
implied that histidine residues may be important for organic anion
transport. To elucidate the diversity of the substrate recognition by
the organic anion transporters, a signal feature of the organic anion
transport system, it is important to clarify the mechanisms involved in
the interaction of the substrates with the essential residues such as
histidine residues of
mOAT.1
Because it is highly desirable to express mammalian transport proteins
in a mammalian expression system to ensure the appropriate post-translational processing, in the present study we have pursued an
alternative expression system in a mammalian cell line, COS-7 (9). We
showed that, when transfected with mOAT cDNA, the COS-7 cell system
is functionally active as an organic anion transporter, and it exhibits
similar general characteristics as described previously in other
systems (4-6, 10). We now show that PAH transport by mOAT can be
inhibited by vitamins and a variety of anionic drugs. Here we provide
the first evidence that glycosylation of mOAT is necessary for the
proper trafficking of the transporter onto the plasma membrane. We also
suggest that histidine residues may be important for the transport
function. To evaluate the role of mOAT during kidney development, we
have studied the expression of mOAT in the developing mouse kidney.
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EXPERIMENTAL PROCEDURES |
DNA Constructs--
Full-length cDNAs encoding mOAT with and
without epitope tags, were subcloned into the mammalian expression
vector pcDNA3.1(
) (Invitrogen). The epitope-tagged constructs
encoded a fusion protein consisting of full-length mOAT with 10 amino
acids of the human c-Myc epitope (EQKLISEEDL, nucleotide
GAACAAAAGCTGATTTCTGAAGAAGACCTG) at the carboxyl terminus. Tagged mOAT
cDNA was synthesized by the polymerase chain reaction
amplification. The polymerase chain reaction product was subcloned into
plasmid pcDNA3.1(
) at XbaI and HindIII
sites. The sequence was confirmed by the dideoxy chain termination method.
Expression in COS-7 Cells--
COS-7 cells were grown in 24-well
plates (50-80% confluency) at 37 °C and 5% CO2 in
Dulbecco's modified Eagle's medium (BioWhittaker) supplemented with
10% fetal bovine serum (Sigma), 100 units/ml penicillin, and 100 mg/ml
streptomycin. Cells were transfected with either 0.25 µg/well of
pcDNA3.1(
)-mOAT, pcDNA3.1(
)-mOAT-myc, or pcDNA3.1(
)
using LipofectAmineTM reagent (Life Technologies, Inc.)
following the manufacturer's instruction. The transfection efficiency
was ~30% in all experiments as estimated by visualization by
fluorescence microscopy after staining of mOAT-myc-transfected cells
using anti-myc antibody in conjunction with fluorescein
isothiocyanate-conjugated secondary antibody. The cells were used for
transport measurements or immunostaining 48-72 h after transfection.
Transport Measurements--
For each well, uptake solution (140 mM NaCl, 2 mM KCl, 1 mM
CaCl2, 1 mM MgCl2, 10 mM Hepes, 5 mM Tris, pH 7, and 20 µM 14C-labeled PAH) was added. At times
indicated in the figure legends, the uptake was stopped by aspirating
off the uptake solution and rapidly washing the plate with ice-cold
washing solution (140 mM choline chloride, 2 mM
KCl, 1 mM CaCl2, 1 mM
MgCl2, 10 mM Hepes, and 5 mM Tris,
pH 7). The cells were solubilized in 0.2 N NaOH and
aliquoted for liquid scintillation counting. The protein concentration was determined using the Bradford dye-binding procedure (11). For the
inhibition studies, PAH uptake was measured in the presence of 0.3 mM to ~1 mM substrates as indicated.
In Vitro Transcription and Translation--
cRNA was synthesized
from various pcDNA3.1(
) subclones using an in vitro
transcription kit (Stratagene). The cRNAs were then translated in a
rabbit reticulocyte lysate system (Promega) with L-[35S]methionine (NEN Life Science Products)
as described earlier (12). Canine pancreatic microsomes (Promega) were
added to some of the translation reactions, which were then centrifuged
at 4° C for 30 min in buffer containing 2.5% glycerol. SDS sample
buffer (125 mM Tris, pH 6.8, 2% SDS, 5%
2-mercaptoethanol, 8 M urea, 20% sucrose, 0.5 mg/ml
bromphenol blue) was added to all of the translation reactions, which
were then boiled for 5 min before SDS-polyacrylamide gel electrophoresis.
Immunofluorescence of Transfected Cells--
72 h after
transfection, COS-7 cells were washed three times in phosphate-buffered
saline (PBS), fixed for 15 min at room temperature in 4%
paraformaldehyde in PBS, and rewashed in PBS. The fixed cells were then
permeabilized with 0.1% Triton X-100 for 10 min. After that, the cells
were incubated for 15 min at room temperature in PBS containing 5%
goat serum and then incubated for 1 h in the same medium
containing anti-myc antibody (1:100) at room temperature. The cells
were washed, and bound primary antibodies were detected by reaction
with fluorescein isothiocyanate-coupled goat anti-mouse IgG (Chemicon)
diluted 1:100 for 1 h. Cells were thoroughly washed, and the cover
glasses were mounted in 90% glycerol plus Citifluor. Samples were
visualized on a fluorescence microscope.
In Situ Hybridization--
Developing mouse kidneys were
collected, rinsed in PBS, and then fixed in ice-cold freshly prepared
4% paraformaldehyde/PBS for 1 h. They were then rinsed in 0.9%
NaCl and dehydrated through a graded series of ethanol and embedded in
paraffin. 7-µm sections were cut, mounted on slides, dewaxed,
pretreated, and prehybridized as described previously (7). Antisense
RNA probes labeled with [
-35S]UTP (Amersham) were produced.
Hybridization was done overnight at 50 °C. Post-hybridization
treatments were as follows: (i) two washes in 50% formamide, 2× SSC,
20 mM 2-mercaptoethanol (FSM) at 60 °C for 30 min, (ii) digestion with 10 µg/ml RNase A in 4× SSC, 20 mM
Tris-HCl (pH 7.6), 1 mM EDTA at 37 °C for 30 min, and
(iii) two washes in FSM at 60 °C for 45 min. Slides were dipped in
Kodak NTB-2 emulsion and exposed for 10 days, and photographs were
taken using a Leica DMRB microscope.
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RESULTS |
Kinetics of PAH Transport--
Our preliminary experiment showed
that pcDNA3.1(
)-mOAT-transfected cells gave a linear uptake of
14C-labeled PAH for 1 h (data not shown). The kinetics
of PAH transport were studied using 10-min uptake points. The initial
rate of PAH uptake over a wide range of PAH concentrations was
determined (Fig. 1a).
Consistent with the previously reported data (4-6, 10), the transport
of PAH across the cell membrane was saturable (Fig. 1b).
Based on Eadie-Hofstee plot analysis (Fig. 1b,
inset), the Km value for PAH was 37.3 µM and Vmax was 210 pmol/mg/10 min.

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Fig. 1.
PAH uptake kinetics into cells
transfected with pcDNA3.1( )-mOAT. a, COS-7 cells
were transfected with pcDNA3.1( )-mOAT (solid
circle) and pcDNA3.1( ) alone (control, open
circle), and the initial rate of uptake was assayed at
25-600 µM PAH. b, in one representative
experiment, the Km is 37.3 µM and
Vmax is 210 pmol/mg/10 min (inset).
The data represent uptake into pcDNA3.1( )-mOATtransfected
cells minus uptake into pcDNA3.1( )-transfected cells. Values are
mean ± S.E. (n = 3).
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Substrate Specificity--
We examined the substrate specificity
of the carrier in competition experiments. A representative experiment
of the relative inhibition of PAH (20 µM) uptake by 0.3 mM to ~1 mM substrates is shown in Fig.
2. Because kidney is typically more
efficient in the elimination of water-soluble compounds than liver,
which is more efficient in the biliary disposal of liposoluble
compounds (13), we tested the inhibition effect of water-soluble
vitamins on mOAT-induced PAH transport. Among the vitamins examined,
riboflavin inhibited the mOAT-mediated PAH transport by 70%. Folic
acid and pantothenic acid showed a moderate inhibition (about 50%).
Ascorbic acid had no significant effect on the transport process (Fig. 2a). We also examined the inhibition effect of a series of
anionic drugs that may have potential nephrotoxicity (Fig.
2b): nonsteroidal anti-inflammatory drugs, indomethacin,
sulindac, diclofenac, and carprofen almost completely blocked PAH
transport. Anti-hypertensive drugs captopril and enalapril and
anti-tumor drugs methotrexate and semustine showed moderate inhibition.
However, lisinopril (anti-hypertensive drug) and cisplatin (anti-tumor
drug) had little effect under our experimental condition.

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Fig. 2.
Substrate specificity of mOAT. The
substrate selectivity was obtained in competition experiments. The
solid columns represent uptake into
pcDNA3.1( )-mOAT-transfected cells, and the open
columns represent uptake into pcDNA3.1( )-transfected
cells. The data are presented as percent of control uptake.
a, inhibition by probenecid and vitamins. b,
inhibition by anionic drugs (1 mM, except methotrexate,
semustine, and cisplatin, which were 0.3 mM).
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The Role of Glycosylation in the Transport Function--
In our
previous study (7), we reported the presence of four potential
glycosylation sites located in the first extracellular loop between
transmembrane domains 1 and 2 of mOAT. To determine whether
carbohydrate moieties play a role in the function of the protein, we
carried out initial experiments using the general inhibitor of
glycosylation, tunicamycin. When COS-7 cells transfected with
pcDNA3.1(
)-mOAT were pretreated with 10 µg/ml tunicamycin for
24 h, an almost complete inhibition in the transport activity was
observed (Fig. 3a). The
potential use of these glycosylation sites was supported by our
in vitro translation data of mOAT cRNA using a rabbit
reticulocyte lysate system followed by 7.5% SDS-polyacrylamide gel
electrophoresis (Fig. 3b). The major mOAT translation
product synthesized in the presence of microsomes is larger than the
~58-kDa product synthesized in the absence of microsomes. The
microsome-dependent shift in the size of the mOAT
translation product was reversed by endoglycosidase H treatment,
indicating that the decreased mobility observed after translation in
the presence of microsomes is due to glycosylation.

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Fig. 3.
The effect of glycosylation on the transport
function. a, the effect of tunicamycin on mOAT-mediated
PAH transport (20 µM). b, in vitro
translation of mOAT cRNA. The figure
shows an autoradiograph of an SDS-polyacrylamide gel electrophoresis
(7.5%) used to analyze the in vitro translation products of
mOAT cRNA obtained in the absence (first lane) and in the
presence (second lane) of pancreatic microsomes after
centrifugation. The third lane shows the product obtained in
the presence of microsomes after deglycosylation with endoglycosidase H
(Endo H).
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Next we investigated the reasons for a decrease in the transport
activity with deglycosylation. Our strategy was to engineer an epitope
tag (c-myc) to the carboxyl terminus of the protein so that the
expressed protein could be detected using an anti-myc monoclonal
antibody. To ensure that the epitope tag had little effect on the
transport function, we measured the [14C]PAH uptake into
both mOAT and mOAT-myc-transfected cells. As shown in Fig.
4, both mOAT and mOAT-myc showed similar
transport activity and were equally sensitive to the inhibitor,
probenecid. The transport activities of both constructs were blocked to
the same extent by treatment of 10 µg/ml tunicamycin, suggesting that the myc-tagged construct retains the functional properties of the
native (unmodified) structure.

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Fig. 4.
Functional comparison of mOAT and its
myc-tagged product. a, PAH uptake into
pcDNA3.1( )-mOAT-transfected cells (solid
columns). b, PAH uptake into
pcDNA3.1( )-mOAT-myc-transfected cells (solid
columns). Open columns represent
pcDNA3.1( ) alone-transfected cells.
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Experiments were next carried out to examine the protein expression
pattern of mOAT-myc before and after the treatment with tunicamycin
using immunofluorescence. We showed that the plasma membrane was
clearly labeled in the mOAT-transfected cells (Fig. 5a) as compared with control
cells (Fig. 5e). In contrast, fluorescence remained mainly
in the intracellular compartment after tunicamycin treatment (Fig.
5c). The right panel (Fig. 5,
b, d, and f) shows that cells were
fully attached to the culture dishes under all conditions, suggesting
that the level of tunicamycin used was not toxic to the cells.

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Fig. 5.
Immunofluorescence study of the effect of
tunicamycin on mOAT expression. Cells were stained with anti-myc
antibody and fluorescein isothiocyanate-coupled goat anti-mouse IgG.
Specific immunostaining appears as green fluorescence. a,
c, and e represent the immnofluorescent
microscopy, and b, d, and f represent
the corresponding phase contrast microscopy of a,
c, and e.
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The Role of Histidine Residues in the Transport Function--
A
previous study using brush-border membrane vesicles from dog kidney (8)
implied that histidine residues may be important for organic anion
transport. Therefore, the effect of the histidyl modifier, DEPC, on PAH
transport was examined. Fig.
6a shows that 1 mM
DEPC completely blocked mOAT-mediated PAH transport, and the presence
of 100 µM unlabeled PAH is capable of providing
significant protection (90%) against the inhibitory effect by
DEPC.

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Fig. 6.
The effect of DEPC treatment on
pcDNA3.1( )-mOAT-mediated PAH transport. a,
transfected cells were treated with 1 mM DEPC in the
presence or absence of 100 µM unlabeled PAH, or
transfected cells were treated with 1 mM DEPC and then
washed with 10 mM DTT, and 14C-labeled PAH
transport activity was measured. b, transfected cells were
treated with N-acetylimidazole, and 14C-labeled
PAH transport activity was measured. The solid
columns represent uptake into
pcDNA3.1( )-mOAT-transfected cells, and the open
columns represent uptake into pcDNA3.1( )-transfected
cells (control).
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The specificity of DEPC was further investigated by examining transport
after washing with a sulfhydryl restoring agent, dithiothreitol (DTT).
If the sulfhydryl groups were modified by DEPC, then washing with DTT
would result in restoration (14). As shown in Fig. 6a,
treatment with DTT (10 mM) did not restore PAH transport
after modification by DEPC.
It is conceivable that tyrosine residues could have been modified by
DEPC under the conditions employed (15). Therefore, another approach to
identify the group modified by DEPC was to examine what effect the
tyrosine-directed reagent N-acetylimidazole had on PAH
transport. As shown in Fig. 6b, N-acetylimidazole
(1 mM) had no effect on PAH transport.
In Situ mRNA Localization of mOAT in Developing Mouse
Kidneys--
To study the maturation of the organic anion transport
system in the developing kidney, mRNA localization in the kidneys
of 1-day- and 1-week-old mice were studied using sense and antisense cRNA, and the data were compared with that of adult kidney. Fig. 7 showed that the most intense signal was
present in kidney cortex, following a pattern characteristic of
proximal tubular localization. There was no detectable signal in the
glomeruli (arrows), distal tubules, or medulla
(G). This mRNA expression pattern was obvious at the
time of birth and continued throughout development.

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Fig. 7.
In situ hybridization of developing
mouse kidneys. Parasagittal sections of developing mouse kidneys
were used. Kidney cortex was from 1-day-old (A), 1-week-old
(C), and adult (E). Kidney medulla was from adult
(G). Hybridization signals were shown as bright color. The
dark fields are labeled as A, C, E,
and G, and the bright fields for the dark fields
A, C, E, and G are labeled
as B, D, F, and H,
respectively.
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DISCUSSION |
The goal of this study was to extend our previous study on the
functional characterization of mOAT and identification of elements important for its function. In this study, mOAT-transfected COS-7 cells
were shown to be a valid model system for the functional analyses. The
affinity (Km) for the protosubstrate PAH in COS-7
cells (~37.3 µM) was comparable with the
Km obtained from other systems (14~80
µM) (4-6, 10). It has been suggested that nephrotoxicity
may be related to the elimination of certain drugs by organic anion
transport system (16, 17). Therefore we examined a series of anionic
drugs that may cause renal dysfunction (18) by examining their
inhibitory effect on mOAT-mediated PAH transport. Our result showed
that nonsteroidal anti-inflammatory drugs, indomethacin, sulindac,
diclofenac, and carprofen almost completely blocked PAH transport.
Anti-hypertensive drugs captopril and enalapril and anti-tumor drugs
methotrexate and semustine showed moderate inhibition suggesting that
renal clearance of these drugs may be through the organic anion
transport system.
Our previous study predicted that four potential N-linked
glycosylation sites were located in an extracellular loop between transmembrane domains 1 and 2. Glycosylation of one or more of these
sites was suggested by our in vitro translation study (Fig. 3b). Furthermore, as demonstrated by the effect of
tunicamycin on PAH uptake (Fig. 3a), N-linked
asparagine glycosylation appeared necessary for functional expression
of the transporter. It has been suggested that the addition of
oligosaccharides to proteins is an integral step in the proper sorting,
translocation, and insertion of polypeptides into membranes (19).
Glycosylation may also promote resistance to proteolytic attack (20).
Therefore, tunicamycin, which is known to prevent the initial step in
the glycosylation pathway, could potentially affect transport proteins by several mechanisms. Among the various possibilities, our
immunofluorescence study suggested that glycosylation was necessary for
proper targeting of mOAT to the plasma membrane. Modifications of the
oligosaccharide structure of glycoproteins related to changes in the
biological activity of transporter proteins have been described. The
GLUT1 glucose transporter (21, 22), the organic cation transporter of
renal brush-border membrane (23), are examples of such a correlation.
However, glycosylation is not required for the transport activity of
the serotonin transporter (24). Here we provide the first evidence
functionally, biochemically, and morphologically that glycosylation of
mOAT is essential for the transport function.
In our present study, we also examined the possible involvement of the
cationic amino acid, histidine, in the normal function of the PAH
transport carrier. Other studies have shown that histidine groups are
involved in the normal function of a number of membrane transporters
(8, 25). The results from our studies with the histidine-specific
reagent DEPC showed that this reagent caused a significant inhibition
in the initial rate of PAH transport (Fig. 6a). DEPC is
considered to be a specific histidine reagent when used at a pH range
of 5.5 to 7.5. At higher pH values, DEPC specificity, however,
decreases, and the compound begins to interact with other groups such
as thiol groups and tyrosine residues. Because our experiment was
conducted at pH 7.0, it was reasonable to assume that DEPC was
interacting with histidine residues in the PAH transporter. This
conclusion was further supported by the finding that DTT was unable to
restore DEPC-induced inactivation, and N-acetylimidazole, a
highly specific tyrosine-modifying reagent, was incapable of inhibiting
PAH transport. This argues against the possibility that DEPC was
interacting with thiol groups and tyrosine residues (Fig. 6). Our
result showing that the presence of substrate protected the transporter
from DEPC inactivation suggested that histidine residue(s) may be close
to the PAH binding sites. Further studies using site-directed
mutagenesis of mOAT will provide additional information regarding the
roles of histidine residues in substrate recognition by the organic
anion transporters.
Finally, early investigations suggested that the ability of the newborn
kidney to eliminate organic anions is limited in comparison with the
ability of an adult kidney (27, 28). Our in situ hybridization study shows that mOAT mRNA was expressed in proximal tubules at the time of birth, and the same pattern was observed throughout development. The correlation of the transport activity and
the mRNA expression needs to be examined further.
In summary, our present study suggests that
pcDNA3.1(
)-mOAT-transfected COS-7 cells can serve as an in
vitro model system for studying the pharmacology and molecular
biology of the cloned mOAT. Many drugs that can cause renal
dysfunction, such as anti-tumor drugs, antibiotics, and nonsteroidal
anti-inflammatory drugs, are transported by the organic anion transport
system. Therefore, our in vitro model system for screening
drugs will facilitate the elucidation of the molecular basis of
drug-related nephrotoxicity. Our study also presents data
characterizing the impact of N-glycosylation and histidine
residue(s) on the function of the cloned mOAT. The results contribute
to our understanding of the relationship between structure and function
of the organic anion transporters.