BASIC SCIENCE INVESTIGATIONSTransport of 99mTc-MAG3 via Rat Renal OrganicAnion Transporter 1
Naoto Shikano, MS1; Yoshikatsu Kanai, MD, PhD2; Keiichi Kawai, PhD3; Nobuyoshi Ishikawa, MD, PhD1;and Hitoshi Endou, MD, PhD2
1Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences, Ibaraki, Japan; 2Department ofPharmacology and Toxicology, Kyorin University School of Medicine, Tokyo, Japan; and 3School of Health Sciences, Faculty of
Medicine, Kanazawa University, Kanazawa, Japan

Recently, complementary DNA (cDNA) encoding a p-aminohippurate (PAH) transporter designated rat organic anion transporter 1 (OAT1) was isolated. OAT1, a multispecific organic
anion transporter at the basolateral membrane, is exclusively
expressed in the middle segment of the proximal tubule in the
rat kidney. It has been proposed that OAT1 is indirectly involved
in PAH uptake via the Na⫹ dicarboxylate cotransporter. In thisstudy, in molecular biologic experiments using OAT1-expressing Xenopus laevis oocytes, we obtained evidence that 99mTcmercaptoacetylglycylglycylglycine (MAG3) is transported via
OAT1. Methods: Capped OAT1 complementary RNA (cRNA)was synthesized from library plasmid cDNA linearized with
BamHI using in vitro transcription. Defolliculated oocytes were
injected with 10 ng of OAT1 cRNA. Two to 3 d after injection,
uptake of 99mTc-MAG3 was measured using ND96 solution containing 18.5 kBq of 99mTc-MAG3. Before the uptake experiments, OAT1-expressing oocytes were preincubated for 2 hwith 1 mmol/L glutarate (a dicarboxylate), to generate an outwardly directed glutarate gradient. Then, after incubation for 60
min at room temperature, radioactivity of oocytes was determined. For the inhibition experiments, uptake was assessed in
the absence or presence of inhibitor: 2 mmol/L of PAH, oiodohippurate (OIH), probenecid, 3,5-diiodo-4-pyridone-N-acetate (iodopyracet), furosemide, ethacrynic acid, glucoheptonate, maleic acid, L-Tyr, or tetraethylammonium (TEA) or 0.1
mmol/L of 2,4-dinitrophenol (DNP). Results: Na⫹ had a significant effect on 99mTc-MAG3 uptake (P ⬍ 0.05). Accumulated
glutarate stimulated simultaneous 99mTc-MAG3 uptake and glutarate excretion (P ⬍ 0.001). The following compounds significantly inhibited 99mTc-MAG3 uptake: PAH, 8.5% ⫾ 16.2% of
99mTc-MAG3 uptake in the absence of an inhibitor; OIH,
26.4% ⫾ 21.7%; probenecid, 29.1% ⫾ 12.4%; iodopyracet,
15.8% ⫾ 7.9%; furosemide, 30.5% ⫾ 15.7%; ethacrynic acid,
21.6% ⫾ 10.6%; glucoheptonate, 35.6% ⫾ 22.6%; and maleic
acid, 60.1% ⫾ 18.7%. 99mTc-MAG3 accumulation in Xenopus
laevis oocytes was not significantly inhibited by TEA, L-Tyr, or
DNP. Conclusion: The following substances had a cis-inhibitory effect on 99mTc-MAG3 transport: PAH, OIH, probenecid,
iodopyracet, furosemide, ethacrynic acid, and glucoheptonate.

Received Mar. 10, 2003; revision accepted Jul. 10, 2003.
For correspondence or reprints contact: Naoto Shikano, MS, Department ofRadiological Sciences, Ibaraki Prefectural University of Health Sciences,4669-2 Ami, Ami-machi, Inashiki-gun, Ibaraki 300-0394, Japan.
E-mail: sikano@ipu.ac.jp

80

Glutarate had a trans-stimulative effect on 99mTc-MAG3 transport. 99mTc-MAG3 acts as a substrate of OAT1, an organic
anion/dicarboxylate exchanger.
Key Words: 99mTc-MAG3; organic anion transporter 1; Xenopus
laevis oocyte; membrane transport; renal function
J Nucl Med 2004; 45:80 – 85T

he organic anion 99mTc-mercaptoacetylglycylglycylglycine (MAG3) was first used to replace 131I-o-iodohippurate (OIH) in renal function studies in 1986 (1). Comparedwith 99mTc-MAG3, 131I-OIH gives poor spatial resolution
because the permissible injection dose is limited and its
photon energy requires the use of coarse-resolution collimators (2). Also, 99mTc-MAG3 has been shown to havebiologic properties similar to those of 131I-OIH in animals
and humans and to be a superior single-photon emitter inscintigraphy (1,3). Renal scintigraphy with 99mTc-MAG3
can provide excellent image quality, even in patients withseverely decreased renal function (4).
Organic anion transporters have been defined as transporters of organic compounds that are inhibited by anexcess of p-aminohippurate (PAH) (5). They are importanttransporters, because many clinically important drugs andtoxic compounds are organic anions, and these compounds
are eliminated via the organic anion transport system (5).
Generally, transepithelial secretion of organic anions occurs
via accumulative transport from blood into proximal tubule
cells across the basolateral membrane (6). Organic anions
such as 99mTc-MAG3 are excreted through the proximal tubular cells of the nephron in a PAH-competitive manner (4).
Recently, Sweet et al. and Sekine et al. independently
isolated complementary DNA (cDNA) encoding a PAHtransporter designated rat organic anion transporter 1(OAT1; Fig. 1) (6,7). OAT1, a multispecific organic aniontransporter at the basolateral membrane, is exclusively expressed in the middle segment of the proximal tubule in the
rat kidney (6). To date, 4 isoforms of mammalian OAT havebeen identified (6 –10). It is unclear which transporter is

THE JOURNAL OF NUCLEAR MEDICINE • Vol. 45 • No. 1 • January 2004

FIGURE 1. Putative membrane topology of OAT1. OAT1 comprises 551 amino acids and has 12 putative membrane-spanning domains. Four N-glycosylation sites and 4 protein kinase
C– dependent phosphorylation sites (PKC sites) have been predicted in first and sixth hydrophilic loops, respectively.

involved in transport of 99mTc-MAG3, which is widely usedas the imaging agent of choice for renal scintigraphy (4). In
the present study, molecular biologic experiments showedthat 99mTc-MAG3 is transported via OAT1 expressed by
Xenopus laevis oocytes.
MATERIALS AND METHODS
Isolation of Xenopus laevis Oocytes
The ethical committees of all relevant universities approved theanimal experiments. All chemicals used were reagent grade (Aldrich Chemical Co. and Kanto Chemical Co., Inc.). Collagenase
was purchased from Boehringer Mannheim. Adult female Xenopus
laevis (Japan SLC Inc.) were anesthetized by hypothermia anddecapitated. Ovaries were then removed and stored in OR2 solution. Stage V and VI oocytes were manually dissected from theovary. Xenopus laevis oocyte follicles were removed by digestionin OR2 solution (pH 7.5) containing 82.5 mmol/L NaCl, 2 mmol/L
KCl, 1 mmol/L MgCl2, 5 mmol/L 2-hydroxyethylpiperazine-N⬘2-ethanesulfonic acid (HEPES), and 1.5 mg/mL collagenase, for
30 – 40 min at room temperature. In oocyte experiments, 6 –22
oocytes were used.
Complementary RNA (cRNA) Synthesis and Oocyte
Injection
Capped OAT1 cRNA (GenBank/EBI Data Bank accession no.
AB004559) was synthesized in vitro using T7 RNA polymerase(Stratagene) and plasmid DNA linearized with BamHI (Invitrogen), as described elsewhere (6). Defolliculated oocytes were
injected with 10 ng of capped cRNA and then incubated in modified Bath’s solution (pH 7.4) containing 88 mmol/L NaCl, 1
mmol/L KCl, 0.33 mmol/L Ca(NO3)2.4H2O, 0.4 mmol/L
CaCl2.2H2O, 0.8 mmol/L MgSO4.7H2O, 2.4 mmol/L NaHCO3, 10
mmol/L HEPES, and 150 mg/mL gentamicin, at 18°C for 2–3 d.
Uptake Assays
99mTc-MAG3 was obtained from Nihon Medi-Physics Co. andDaiichi Radioisotope Laboratories Ltd. and was purified by highperformance liquid chromatography under the conditions described by Shattuck et al. (11).
The uptake experiments were performed using ND96 solution(pH 7.4) containing 96 mmol/L NaCl, 2 mmol/L KCl, 1.8 mmol/L

CaCl2.2H2O, 1 mmol/L MgCl2.6H2O, and 5 mmol/L HEPES.
Before the uptake experiments, oocytes were preincubated in
ND96 containing 1 mmol/L glutarate for 2 h, to generate an
intracellular-to-extracellular gradient of glutarate concentration. In
all uptake experiments, incubation was performed in ND96 solution containing 18.5 kBq of 99mTc-MAG3, at 25°C for 60 min,unless otherwise indicated.
In the time course experiments, uptake of 99mTc-MAG3 wasmeasured with incubation for 10, 30, 60, or 120 min. To assess
dependence of 99mTc-MAG3 transport on extracellular Na⫹, uptake of 99mTc-MAG3 into Xenopus laevis oocytes expressing
OAT1 was measured in the presence or absence of Na⫹ in ND96
(in the absence of NaCl, an equal concentration of choline-Cl wasused). To assess dependence of 99mTc-MAG3 transport on intracellular glutarate, OAT1-expressing Xenopus laevis oocytes werepreincubated in ND96 with or without 1 mmol/L glutarate for 2 h
before being used in uptake experiments. The control oocytes were
injected with a volume of water equal to that of the OAT1 cRNAand vehicle. For inhibition experiments, the following inhibitors
were used: PAH, OIH, probenecid, 3,5-diiodo-4-pyridone-N-acetate (iodopyracet), furosemide, ethacrynic acid, glucoheptonate,
L-Tyr, maleic acid, tetraethylammonium (TEA), and 2,4-dinitrophenol (DNP) (5,12–17). DNP was used at a concentration of 0.1
mmol/L (16); the other inhibitors were used at a concentration of
2 mmol/L. The control oocytes were incubated in the absence of
inhibitor.
In all experiments, incubation was terminated by washing with
ice-cold ND96 solution 5 times. The radioactivity of each oocyte
was measured using a well-scintillation counter (ARC-380;
Aloka). Each experiment was repeated 3 times using different
batches of Xenopus laevis oocytes.
Statistical Analysis
The values obtained in each experiment were expressed as themean ⫾ SD. Statistical comparisons between groups were performed using the Student unpaired t test.
RESULTSIn the preparation of the radiolabeled compound 99mTcMAG3, labeling efficiency was greater than 95.0%. After
purification, the radiochemical purity was greater than
99.9%. The specific activity of the 99m Tc-MAG3 was 6.6 ⫻1014 Bq/mol.
As shown in Figure 2, the 99mTc-MAG3 concentration in
OAT1-expressing Xenopus laevis oocytes reached a steadystate at 60 min after addition of the tracer. Consequently,
in the uptake experiments, the oocytes were incubated for
60 min.
Figures 3 and 4 summarize the characterization of 99mTcMAG3 transport in OAT1-expressing Xenopus laevis oocytes under various incubation conditions. 99mTc-MAG3
uptake was significantly higher in OAT1-expressing oocytes than in water-injected control oocytes (P ⬍ 0.001)(Figs. 3 and 4). In general, results obtained in the presenceof Na⫹ were similar to those obtained in the absence of Na⫹(Fig. 3). In OAT1-expressing oocytes, Na⫹ had a slight but
significant effect (P ⬍ 0.05) on 99mTc-MAG3 uptake (Fig.
3). 99mTc-MAG3 uptake was higher in oocytes preincubatedwith glutarate than in oocytes that were not preincubated

TRANSPORT OF MAG3 VIA OAT1 • Shikano et al.

81

FIGURE 2. Accumulation of 99mTc-MAG3 in OAT1-expressing
Xenopus laevis oocytes. Values represent the mean ⫾ SD (n ⫽6 – 8 oocytes).

with glutarate (Fig. 4). Accumulation of glutarate in Xenopus laevis oocytes trans-stimulated uptake of 99mTc-MAG3
via OAT1.
Figure 5 shows the results of the inhibition experiments.
99mTc-MAG3 uptake was significantly inhibited (P ⬍ 0.001)
by the following compounds: PAH, 8.5% ⫾ 16.2% of
99mTc-MAG3 uptake in the absence of an inhibitor; OIH,
26.4% ⫾ 21.7%; probenecid, 29.1% ⫾ 12.4%; iodopyracet,
15.8% ⫾ 7.9%; furosemide, 30.5% ⫾ 15.7%; ethacrynic
acid, 21.6% ⫾ 10.6%; and glucoheptonate, 35.6% ⫾22.6%. TEA, an inhibitor of organic cation transport (6), did
not inhibit 99mTc-MAG3 uptake. Other compounds that did
not significantly inhibit 99mTc-MAG3 uptake were DNPFIGURE 3. 99mTc-MAG3 transport dependence on extracellular Na⫹ in OAT1-expressing Xenopus laevis oocytes. Uptake of
99mTc-MAG3 was measured in the presence of Na⫹ (Na⫹) orabsence of Na⫹ (choline⫹). The control oocytes were injectedwith same volume of water, instead of OAT1 cRNA. Values
represent mean ⫾ SD (n ⫽ 15–20 oocytes). *P ⬍ 0.05.

82

FIGURE 4. 99mTc-MAG3 transport dependence on intracellular glutarate in OAT1-expressing Xenopus laevis oocytes. Control oocytes were injected with same volume of water, instead of
OAT1 cRNA. Values represent mean ⫾ SD (n ⫽ 15–20 oocytes).
*P ⬍ 0.001.

(16) and L-Tyr. Maleic acid, which causes experimental
Fanconi syndrome (17), significantly inhibited 99mTcMAG3 transport (60.1% ⫾ 18.7%) (P ⬍ 0.01).
DISCUSSION

Organic anion transport is thought to be an importantsystem for diagnosis of kidney function, because manyanions, whether of endogenous or environmental origin, areeliminated from the body by the organic anion secretory
system of the renal proximal tubule (5), and it has been
proposed that this system is involved in drug metabolism

FIGURE 5. Inhibition of OAT-mediated 99mTc-MAG3 uptake
by various drugs. Control value is 99mTc-MAG3 uptake in absence of inhibitor. 99mTc-MAG3 uptake in presence of inhibitor isexpressed as percentage of control value (mean ⫾ SD; n ⫽16 –22 oocytes). *P ⬍ 0.001. †P ⬍ 0.01.

THE JOURNAL OF NUCLEAR MEDICINE • Vol. 45 • No. 1 • January 2004

and renal toxicity (6). Transepithelial excretion of organic
anions occurs via accumulative transport of these ions fromblood into proximal tubule cells across the basolateral membrane, and subsequent secretion into the urine through theapical membrane (6). Based on experiments using isolated
segments of proximal tubules, it has been estimated that, inthe course of secretion, the intracellular concentration of theorganic anion PAH rises to 100 –300 times the concentration in either the peritubular or luminal fluid (18). Accumulative transport of radiolabeled organic anions such as
99mTc-MAG3 enables scintigraphic imaging of renal function.
The molecular structure and properties of organic aniontransporters have gradually been clarified. In the last decade, there has been great interest in the specificity of thebasolateral organic anion transport system (which transports
a remarkably broad spectrum of agents) and in the energetics of transport (which is driven by complex tertiary coupling to metabolic energy) (5). Recently, OAT1 cDNA wascloned. Figure 1 shows the putative membrane topology of
OAT1. OAT1 cDNA consists of 2,294 nucleotides andcontains an open reading frame encoding a 551-amino-acidprotein with 12 putative membrane-spanning domains (6).
Human (19,20) and mouse (21,22) homologues of OAT1
have been cloned and characterized. The characteristics of
OAT1 are the same as those of the organic anion/dicarboxylate exchanger predicted by physiologic studies to be located at the basolateral membrane of proximal tubules.
Northern blotting and in situ hybridization have revealed
that OAT1 is exclusively expressed in particular segments
of proximal tubules, presumably the middle segment (6).
Using an immunohistochemical assay, Tojo et al. detected
expression of OAT1 in the basolateral membrane of rat
proximal tubule cells (23). It is generally assumed that thefirst step in 99mTc-MAG3 secretion by proximal tubule cells
is extraction of 99mTc-MAG3 from peritubular plasma byproximal tubule cells through the basolateral membrane.
OAT1 transcript has not been detected in rat heart, brain,
spleen, lung, liver, skeletal muscle, or testis, but it has been
detected (as a strong Northern blot signal) in rat kidney (7).
Shimada et al. have proposed that, in the basolateral
membrane, 3 transport systems cooperate to accumulate
PAH in cells (Fig. 6). The primary active transport systemat the basolateral cell membrane is Na⫹/K⫹-adenosine
triphosphatase, which maintains an electrochemical potential difference by transporting Na⫹ across the basolateralcell membrane. Intracellular accumulation of certain dicarboxylates far above plasma concentrations occurs as a resultof cotransport of dicarboxylates with 3 Na⫹ (24). In addition, an Na⫹-coupled system mediates uptake of Krebs
cycle intermediates across the luminal membrane (25). Thishypothesis is supported by findings indicating that metabolic fuels stimulate accumulation of PAH in renal cortex(26 –28). Dicarboxylates that accumulate intracellularly(most likely 2-oxoglutarete) are subsequently exchanged
with extracellular PAH, providing a possible mechanism for

FIGURE 6. Proposed scheme for membrane transport of
99mTc-MAG3 in proximal tubule cell. According to anion exchange model of renal organic anion transport model, organic
anions are transported into cell in exchange for intracellular
dicarboxylates, which subsequently return to cell interior via
rNaDC-1. In this model, OAT1 is indirect Na⫹-coupled organic
anion transporter driven by complex tertiary coupling to metabolic energy. ATPase ⫽ adenosine triphosphatase.

accumulation of PAH at high levels within cells. PAH may
leave the cell via the brush-border membrane, by following
its electrochemical gradient. This proposed model is very
consistent with the present results.
In the uptake experiment with OAT1-expressing Xenopus
laevis oocytes, intracellular concentration of 99mTc-MAG3
was 200.8 ⫾ 75.6 times its extracellular concentration (n ⫽22). This indicates that uptake of 99mTc-MAG3 is due toactive transport (tertiary active transport in Fig. 6) against
an outwardly directed 99mTc-MAG3 concentration gradient,rather than facilitated diffusion. In a study using isolated
segments, a similar level of accumulation of PAH wasobserved (18).
Figures 2 and 3 show that 99mTc-MAG3 acts as a substrate of OAT1 in OAT1-expressing Xenopus laevis oocytes. 99mTc-MAG3 transport via OAT1 is slightly but significantly dependent on extracellular Na⫹ concentration(P ⬍ 0.05) (Fig. 3). This suggests that Na⫹ indirectly
contributes to 99mTc-MAG3 transport.
Figure 4 shows the effect of preincubation with glutarate
on transport of 99mTc-MAG3 by OAT1-expressing Xenopus
laevis oocytes. Accumulated glutarate stimulates 99mTcMAG3 uptake via OAT1. The trans-stimulative effect of
glutarate indicates that 99mTc-MAG3 acts as a substrate of
OAT1, which is an organic anion/dicarboxylate exchanger.
It has been reported that coexpression of OAT1 and rat Na⫹
dicarboxylate transporter (rNaDC-1) in Xenopus laevis oocytes results in greater PAH uptake than does expression of
OAT1 only, because of the outwardly directed dicarboxylate gradient created by rNaDC-1 (6). The anion exchange
model suggests that, at the basolateral membrane, 99mTcMAG3 is also exchanged with anions generated duringcellular metabolic processes including the Krebs cycle; forexample, lactate or dicarboxylates.

TRANSPORT OF MAG3 VIA OAT1 • Shikano et al.

83

On the other hand, it is important to know which drugs
affect 99mTc-MAG3 accumulation in kidneys. We tested
several drugs in the present study. In human studies, PAH
loading with infusion caused a significant decrease in 99mTcMAG3 clearance (3). Figure 5 shows that both PAH andOIH (induced hippurates) inhibited 99mTc-MAG3 OAT1mediated transport (P ⬍ 0.001). Probenecid, which is arenal secretion blocker and is also used as a uricosuric drug(13), inhibited renal clearance of 99mTc-MAG3 (P ⬍ 0.001)(29). OAT1-mediated 99mTc-MAG3 uptake was also inhibited (P ⬍ 0.001) by iodopyracet, which has long been used
as a radiographic contrast medium. In general, loop diuretics (such as ethacrynic acid and furosemide) are bound toplasma protein (18). Also, results of micropuncture studies
indicate that most diuretics are not filtered at the glomerulus(18). Thus, a possible mechanism of appearance of diuretics
in proximal tubules is secretion into proximal tubules via
active transport (18). Furosemide and ethacrynic acid had
cis-inhibitory effects on 99mTc-MAG3 uptake in the present
study (P ⬍ 0.001). These compounds had a similar effect on
14C-PAH uptake in a study by Sekine et al. (6). It appears
that secretion of potent loop diuretics into the urine of thepars recta may be an important feature of their mode ofaction (18). Figure 5 shows that both glucoheptonate and99mTc-MAG3 are transported by OAT1. Glucoheptonate, anorganic anion, is a ligand of 99mTc-glucoheptonate (30).
Maleic acid, which causes experimental Fanconi syndrome(17), had a significant inhibitory effect (P ⬍ 0.01).
TEA, which has previously been found to inhibit organic
cation transport, did not inhibit 99mTc-MAG3 transport (5).
L-Tyr (a neutral amino acid) and 99mTc-MAG3 are not bothtransported by OAT1. Unexpectedly, 99mTc-MAG3 transport was not significantly inhibited by DNP, which inhibits
generation of high-energy phosphates (16); organic aniontransport is thought to involve a tertiary active transportsystem, as described above. Other important clinically relevant drugs that inhibit transport via OAT1 include penicillin and thiazide diuretics. Further investigation of theeffects of these compounds on 99mTc-MAG3 transport isneeded.
The most conspicuous feature of this transport system is
its extremely wide substrate selectivity, which includes not
only endogenous anions but also several clinically important drugs (6). For example, it has been reported that OAT1transports the following compounds: diclofenac, diflunisal,
flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamate, naproxen, oxyphenbutazone, phenacetin, phenylbutazone, piroxicam, tolmetin (hydrophobic nonsteroidal
antiinflammatory drugs [NSAIDs]), and salicylate (hydrophilic NSAIDs) (31). Transport of 14C-PAH via OAT1 isstrongly inhibited by cephaloridine (␤-lactam antibiotic),
nalidixic acid (old quinolone), valproic acid (antiepilepticdrug), ␣-ketoglutarate, and urate (6). There have been reports of the substrate structure of organic anion transporters.
Binding of organic anions with the renal organic aniontransporter depends mainly on hydrophobic interactions,

84hydrogen bonding, and electrostatic interactions between
the substrate and the carrier (32). The organic anion transporter interacts with substrates that contain a hydrophobic
core with negative charges or negative partial charges (33).
The organic anion transport system requires only a hydrophobic backbone and negative or partial negative charges
optimally separated by 60 –70 nm (34). The substrates of
OAT1 are apparently structurally unrelated to each other.
These previous findings provide a possible explanation for
the extremely wide substrate selectivity of OAT1.
To date, 4 isoforms of OAT have been identified: OAT1,OAT2 (8), OAT3 (9,10), and OAT4 (35). Transcellulartransport of organic anions has been demonstrated in various tissues, including kidney, brain, liver, and placenta.
Incidentally, sporadic liver and gallbladder visualization has
been observed during studies of renal transport of 99mTcMAG3, and fasting increases hepatobiliary excretion (11).
Because 99mTc-MAG3 is dianionic at physiologic pH, it islikely that it is transported by hepatocytes using the anionictransport system at physiologic pH (11). OAT2 is expressedstrongly in the liver and weakly in the kidney; OAT3 isexpressed strongly in the liver, kidney, and brain andweakly in the eye. Indocyanine green has been found to
inhibit transport of substrates such as PAH via both OAT2
and OAT3 (8,9). Furthermore, blocking the anionic transport system of the liver by infusing rats with indocyanine
green before injecting 99mTc-MAG3 has been found to significantly decrease the amount of 99mTc-MAG3 excreted
into the bile (11). This suggests a connection between
99mTc-MAG3 excretion and PAH excretion via the isoforms
of OAT in the hepatobiliary system.
Numerous organic anions, including 99mTc-MAG3, areefficiently excreted from renal proximal tubules via carriermediated pathways. In the basolateral membrane, thepresent results indicate that 99mTc-MAG3 is a substrate of
OAT1, whereas 99mTc-MAG3 transport in the brush border
membrane is less clearly understood (Fig. 6). We believethat transport experiments using Xenopus laevis oocytes canhelp to lay the foundation for development of improved
pharmaceuticals for evaluation of kidney function. We anticipate future studies of 99mTc-MAG3 transport via otherisoforms of OAT, including analysis of relative kinetics.
CONCLUSION

PAH, OIH, probenecid, iodopyracet, furosemide,
ethacrynic acid, and glucoheptonate had cis-inhibitory effects on 99mTc-MAG3 transport. Glutarate had a transstimulative effect. The present results are consistent with theproposed model of organic anion transport into cells in
exchange for intracellular dicarboxylates, which subsequently return to the cell interior via a rNaDC-1. Thus,
99mTc-MAG3 acts as a substrate of OAT1, an organic anion/
dicarboxylate exchanger.

THE JOURNAL OF NUCLEAR MEDICINE • Vol. 45 • No. 1 • January 2004

ACKNOWLEDGMENTS

We thank Kohichi Hamazaki, Tomoaki Murakami, and
Makoto Hira (Ibaraki Prefectural University) for their excellent technical assistance. We are grateful to Jun Inatomi
and Do Kyung Kim (Kyorin University School of Medicine) for providing cDNA. This work was supported
by Grants-in-Aid for Scientific Research (10770451,
14770498, and 13557075) from the Ministry of Education,
Science, Sports, and Culture of Japan and the Japan Society
for Promotion of Science. Financial support was also provided by Ibaraki Prefectural University Project Research(grants 9808-3, 0118-1, and 0220-1) and Ibaraki Prefectural
University Grants-in-Aid for the Encouragement of Young
Scientists (2001 and 2002).
REFERENCES
1. Fritzberg AR, Kasina S, Eshima D, Johnson DL. Synthesis and biologicalevaluation of technetium-99m MAG3 as a hippuran replacement. J Nucl Med.
1986;27:111–116.
2. Taylor A, Eshima J, Alazraki N. 99mTc-MAG3, a new renal imaging agent:
preliminary results in patients. Eur J Nucl Med. 1987;12:510 –514.
3. Bubek B, Brandau W, Weber E, Kalble T, Parekh N, Georgi P. Pharmacokineticsof technetium-99m-MAG3 in humans. J Nucl Med. 1990;31:1285–1293.
4. Itoh K. 99mTc-MAG3: review of pharmacokinetics, clinical application to renal
diseases and quantification of renal function. Ann Nucl Med. 2001;15:179 –190.
5. Pritchard JB, Miller DS. Mechanisms mediating renal secretion of organic anions
and cations. Physiol Rev. 1993;73:765–796.
6. Sekine T, Watanabe N, Hosoyamada M, Kanai Y, Endou H. Expression cloning
and characterization of a novel multispecific organic anion transporter. J Biol
Chem. 1997;272:18526 –18529.
7. Sweet DH, Wolff NA, Pritchard JB. Expression cloning and characterization of
ROAT1. J Biol Chem. 1997;272:30088 –30095.
8. Sekine T, Cha SH, Tuda M, Apiwattanakul N, Kanai Y, Endou H. Identificationof multispecific organic anion transporter 2 expressed predominantly in the liver.
FEBS Lett. 1998;492:179 –182.
9. Kusuhara H, Sekine T, Utunomiya-Tate N, Kanai Y, Sugiyama Y, Endou H.
Molecular cloning and characterization of a new multispecific organic aniontransporter from rat brain. J Biol Chem. 1999;274:13675–13680.
10. Race JE, Grassl SM, Williams WJ, Holtzman EJ. Molecular cloning and characterization of two novel human renal organic anion transporters (hOAT1 andhOAT3). Biochem Biophys Res Commun. 1999;255:508 –514.
11. Shattuck LA, Eshima D, Taylor AT, et al. Evaluation of the hepatobiliary
excretion of technetium-99m-MAG3 and reconstitution factors affecting radiochemical purity. J Nucl Med. 1994;35:349 –355.
12. Heathcote R St A, Gardner RA. Per-Abrodil (Pelviren D): an experimental
investigation. Br J Radiol. 1933;6:304 –312.
13. Brazeau P. Inhibitors of tubular transport of organic compounds. In: Goodman L,
Gilman A, eds. The Pharmacologic Basis of Therapeutics. New York, NY:
Macmillan Publishing Co., Inc.; 1975:862– 863.
14. Culter RE, Blair AD. Clinical pharmacokinetics of frusemide. Clin Pharmacokinet. 1979;4:279 –296.

15. Byer KH, Baer JE, Michaelson JK, Russo HF. Renotropic characteristics of
ethacrynic acid. J Pharmacol Exp Ther. 1965;147:1–22.
16. Rosengerg LE, Blair A, Segal S. Transport of amino acid by slices of rat-kidneycortex. Biochem Biophys Acta. 1961;54:479 – 488.
17. Dimopoulou CS, Sigalas I, Margaritis L. Induction of experimental Fanconi
syndrome in mice: its effect on the glomerular filtration function studied by
99mTc-DTPA. Nucl Med Biol. 1996;23:807– 812.
18. Grantham JJ, Irish JM III. Organic acid transport and fluid secretion in the pars
recta (PST) of the proximal tubule. Int Congr Ser. 1977;422:83– 87.
19. Hosoyamada M, Sekine T, Kanai Y, Endou H. Molecular cloning and functional
expression of a multispecific organic anion transporter from human kidney. Am J
Physiol. 1999;276:F122–F128.
20. Lu R, Chan BS, Schuster VL. Cloning of the human kidney PAH transporter:
narrow substrate specificity and regulation by protein kinase C. Am J Physiol.
1999;276:F295–F303.
21. Lopez-Nieto CE, You G, Bush KT, Barros EJG, Beier DR, Nigam SK. Molecularcloning and characterization of NKT, a gene product related to the organic cationtransporter family that is almost exclusively expressed in the kidney. J Biol
Chem. 1997;272:6471– 6478.
22. Kuze K, Graves P, Leahy A, Wilson P, Stuhlmann H, You G. Heterologous
expression and functional characterization of a mouse renal organic anion transporter in mammalian cells. J Biol Chem. 1999;274:1519 –1524.
23. Tojo A, Sekine T, Nakajima N, et al. Immunohistochemical localization ofmultispecific renal organic transporter (OAT1) in rat kidney. J Am Soc Nephrol.
1999;10:464 – 471.
24. Bureckhardt G. Sodium-dependent dicarboxylate transport in rat renal basolateral
membrane vesicles. Pflugers Arch. 1984;401:254 –261.
25. Wright EM, Wright SH, Hirayama B, Kippen I. Interactions between lithium andrenal transport of Krebs cycle intermediates. Proc Natl Acad Sci USA. 1982;79:7514 –7517.
26. Cross RJ, Taggart JV. Renal tubular transport: accumulation of p-aminohippurateby rabbit kidney slices. Am J Physiol. 1950;161:181–190.
27. Kippen I, Klinenberg JR. Effects of renal fuels on uptake of PAH and uric acidby separated tubules of rabbit. Am J Physiol. 1978;235:F137–F141.
28. Maxild J, Moller JV. Metabolic studies on renal transport of p-aminohippurate invitro. Biochem Biophys Acta. 1969;184:614 – 624.
29. Eshima D, Taylor A Jr, Fritzberg AR, Kasina S, Hansen L, Sorenson JF. Animalevaluation of technetium-99m triamide mercaptide complexes as potential renal
imaging agents. J Nucl Med. 1987;28:1180 –1186.
30. Lee HB, Blaufox MD. Mechanism of renal concentration of technetium-99m
glucoheptonate. J Nucl Med. 1985;26:1308 –1313.
31. Apiwattanakul N, Sekine T, Chairoungdua A, et al. Transport properties ofnonsteroidal anti-inflammatory drugs by organic anion transporter 1 expressed in
Xenopus laevis oocytes. Mol Pharmacol. 1999;55:847– 854.
32. Moller JV, Sheikh MI. Renal organic anion transport system: pharmacological,
physiological and biochemical aspects. Pharmacol Rev. 1983;34:315–358.
33. Ullrich KJ, Rumrich G. Contraluminal transport systems in the proximal renal
tubule involved in secretion of organic anions. Am J Physiol. 1988;254:F453–
F463.
34. Fritzsch G, Rumrich G, Ullrich KJ. Anion transport through the contraluminal
cell membrane of renal proximal tubule: the influence of hydrophobicity andmolecular charge distribution on the inhibitory activity of organic anions. Biochim Biophys Acta. 1989;978:249 –256.
35. Cha SH, Sekine T, Kusuhara H, et al. Molecular cloning and characterization ofmultispecific organic anion transporter 4 expressed in the placenta. J Biol Chem.
2000;275:4507– 4512.

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