1 Third Department of Internal Medicine, Mie University School of Medicine, 2 - 174 Edobashi, Tsu City, 514 - 8507; and the 2 Department of Hygiene, Kinki University School of Medicine, Osakasayama, 589 - 8511, Japan
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ABSTRACT |
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Regulation of bilirubin glucuronide transporters during hyperbilirubinemia in hepatic and extrahepatic tissues is not completely clear. In the present study, we evaluated the regulation of the bilirubin glucuronide transporters, multidrug resistance-associated proteins (MRP)2 and 3, in rats with obstructive jaundice. Bile duct ligation (BDL) or sham operation was performed in Wistar rats. Liver and kidneys were removed 1, 3, and 5 days after BDL (n = 4, in each group). Serum and urine were collected to measure bilirubin levels just before animal killing. MRP2 And MRP3 mRNA expressions were determined by real-time RT-PCR. Protein expression of MRP2 and MRP3 was determined by Western blotting. Renal MRP2 function was evaluated by para-aminohippurate (PAH) clearance. The effect of conjugated bilirubin, unconjugated bilirubin, human bile, and sulfate-conjugated bile acid on MRP2 gene expression was also evaluated in renal and hepatocyte cell lines. Serum bilirubin and urinary bilirubin excretion increased significantly after BDL. In the liver, the mRNA expression of MRP2 decreased 59, 86, and 82%, and its protein expression decreased 25, 74, and 93% compared with sham-operated animals after 24, 72, and 120 h of BDL, respectively. In contrast, the liver expression of MRP3 mRNA increased 138, 2,137, and 3,295%, and its protein expression increased 560, 634, and 612% compared with sham-operated animals after 24, 72, and 120 h of BDL, respectively. On the other hand, in the kidneys, the mRNA expression of MRP2 increased 162, 73, and 21%, and its protein expression increased 387, 558, and 472% compared with sham-operated animals after 24, 72, and 120 h of BDL, respectively. PAH clearance was significantly increased after BDL. The mRNA expression of MRP2 increased in renal proximal tubular epithelial cells after treatment with conjugated bilirubin, sulfate-conjugated bile acid or human bile. Upregulation of MRP2 in the kidneys and MRP3 in the liver may be a compensatory mechanism to improve bilirubin clearance during obstructive jaundice.
bile duct ligation; multidrug resistance-associated protein 2; multidrug resistance-associated protein 3.
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INTRODUCTION |
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MULTIDRUG RESISTANCE PROTEIN (MRP)2 ABCC2 is a 190-kDa phosphoglycoprotein (3, 30, 34, 42, 45) that transports a wide range of glutathione, glucuronate, and sulfate conjugates, including conjugated bilirubin, out of cells by an ATP-dependent mechanism (10, 27, 28, 35). MRP2 is mainly expressed on the canalicular membrane of hepatocytes but is also present in renal tubular and intestinal epithelial cells (23, 32, 37, 39).
The regulation of MRP2 expression is not well understood. It has been reported that hepatocyte expression of MRP2 is downregulated in the rat model with cholestasis and that it is upregulated in cultured rat hepatocytes exposed to 2-acetylaminofluorene or cyclohexamide (4, 5, 20, 31, 41). Another member of the multidrug resistance-associated transporters, MRP3 (ABCC3), has been recently identified. MRP3 is located on the basolateral membrane of hepatocytes (16). It mediates basolateral efflux of organic anions, bile salts, anticancer drugs, methotrexate, etoposide, and bilirubin monoglucuronide (7, 17, 22). MRP3, which is normally expressed at low levels in hepatocytes, is upregulated during obstructive cholestasis (11, 19, 21). This contrasts with MRP2, which is known to be downregulated progressively during obstructive jaundice (20).
The function of MRP2 in renal cells under physiological and pathological conditions has not been, as yet, fully elucidated. It has been reported that paraaminohippurate (PAH), one of the substrates for renal organic anion transport, is transported by human apical MRP2 (1). Octreotide is also transported across the brush border membrane of renal proximal tubules by both P-glycoprotein and MRP2 (8). A recent study demonstrated that cisplatin induces renal expression of both human P-glycoprotein and MRP2 (26). In the present study, we hypothesized that expression of MRP2 in the kidneys may compensate downregulation of this transporter in the liver during obstructive jaundice. To demonstrate this hypothesis we measured the renal expression of MRP2 in an experimental model of obstructive jaundice caused by bile duct ligation (BDL) in rats.
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MATERIALS AND METHODS |
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Reagents. Bilirubin ditaurate disodium salt was purchased from Porphyrin Products (Logan, Utah). Bilirubin was purchased from Nacalai Tesque (Kyoto, Japan). Taurolithocholic acid 3-sulfate disodium salt (TLCA sulfate) and taurine were purchased from Sigma (St. Louis, MO).
Bile. Bile was sampled through a bile drainage tube from an 86-yr-old woman with obstructive jaundice caused by common bile duct carcinoma. The bile sample was sterilized through a 0.22-µm filter (Millipore, Bedford, MA) before use. The concentration of conjugated bilirubin in bile was 238 µM. Informed consent was obtained from the patient before sampling.
Animal model. Male Wistar rats weighing 200-250 g were subjected to BDL under general anesthesia (pentobarbital sodium 50 mg/kg body wt ip). All rats received care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals, recommended by the National Academy of Science and published by the National Institutes of Health (NIH publication 86-23, revised 1985). Liver and kidneys were removed after 24, 72, and 120 h of BDL (n = 4 each). The organs were perfused with phosphate-buffered saline, cut into small pieces, and snap-frozen in liquid nitrogen until use. Control animals underwent sham surgery that consisted in exposure of bile duct but without ligating it. Liver and kidneys were removed 24, 72, and 120 h after sham surgery (n = 4 each).
Blood samples were collected during organ removal. For urine sampling, the rats were placed in metabolic cages and urine was collected for 24 h. The concentrations of bilirubin and bilirubin glucuronide were determined separately using a test kit (Wako Pure Chemical, Tokyo, Japan) by the alkaline azobilirubin method.Cell culture. The human hepatoma cell line HepG2 (Riken Cell Bank, Tsukuba, Japan) was grown in DMEM supplemented with 10% (vol/vol) fetal bovine serum, 50 µg/ml penicillin, and 50 µg/ml streptomycin. Human renal proximal tubular epithelial cells (RPTEC), obtained from Bio- Whittaker (Walkersville, MD), were cultured in renal epithelial cell basal medium supplemented with 10% (vol/vol) fetal bovine serum and 0.5 mg/ml hydrocortisone, 10 µg/ml human epidermal growth factor, 0.5 mg/ml epinephrine, 10 mg/ml transferrin, 5 mg/ml insulin, 6.51 µg/ml triiodothyronine, gentamicin sulfate, and amphotericin-B. Cells were passaged using standard trypsinization procedures. All cell lines were incubated at 37°C in a humidified atmosphere consisting of 95% air and 5% CO2.
RNA isolation from cultured cells and PCR. Total RNA was isolated from HepG2 and RPTEC cells using TRIzol reagent (n = 3, each; Invitrogen, Carlsbad, CA) after treatment for 24 h with 100 µM bilirubin ditaurate, 10 µM unconjugated bilirubin, 100 µM TLCA sulfate, or 20% (vol/vol) human bile. Reverse transcription and PCR were carried out as described previously with minor modifications (36, 43). The primers used for amplification of human MRP2 were 5'-TGTCTTCACCATCATCGTCATT-3' (nucleotides 3400~3420) and 5'-TCCTGCCCACCACACCAATCTT-3' (nucleotides 4023~4043). The primers for GAPDH amplification were 5'-CCACCCATGGCAAATTCCATGGCA-3' (nucleotides 150~169) and 5'-TCTAGACGGCAGGTCAGGTCCACC-3' (nucleotides 720~743). PCR was performed under the following conditions: 94°C for 60 s, annealing for 60 s, and elongation at 72°C for 60 s for a total of 30 cycles for MRP2 and 25 cycles for GAPDH. A final elongation at 72°C for 10 min was performed. Annealing temperature for both MRP2 and GAPDH was 55°C. The PCR products were separated on 1% agarose gel and visualized by ethidium bromide staining. The bands were scanned and semiquantified by densitometric analysis using the public domain NIH Image program (Wayne Rasband, Research Service Branch of the National Institute of Mental Health, Bethesda, MD).
Real-time PCR.
Total RNA for real-time PCR was extracted from liver and kidney
specimens using the Total SV RNA Isolation System Kit (Promega, Madison, WI) according to the manufacturer's instructions. Isolated RNA was purified by ethanol precipitation and stored at 80°C until
use. Primers and probes for MRP2 and MRP3 were prepared using Primer
Express version 1 (PE Biosystems, Foster city, CA). For the rat MRP2,
the sense primer was 5'-TTCTGTTCCGCCTTGCT-3' (nucleotides 3658~3674),
the antisense primer was 5'-CTTCTGCCGTCATCCTCAC-3' (nucleotides
3784~3766), and the Taqman probe was
5'-TGTCCAACGCCCTCAATATCACA-3' (nucleotides 3725~3747)
(29). For rat MRP3, the sense primer was
5'-GAAGACACACTCAGCACCC-3' (nucleotides 2694~2712), the antisense primer was 5'-GCTTGCGGACCTCGTAT-3' (nucleotides 2760~2743), and the Taqman probe was 5'-CACAGACCTGACAGACACCGAGC-3' (nucleotides 2714~2736). The rat GAPDH was used as a control. For amplification of
the rat GAPDH, the sense primer was 5'-TCTCCACCACTATCGCAGAA-3' (nucleotides 1758~1777), and the antisense primer was
5'-TTGGCAGCTTGGACTATGCT-3' (nucleotides 1833~1814) were used. For the
rat GAPDH, the Taqman probe 3'-TCCGTTTTGGCAGAGAAGATGCAA-3'
(nucleotides 1782~1805) was used.
Western blotting analysis. The rabbit polyclonal antibody against rat MRP2, EAG15 (30), was kindly provided by Professor D. Keppler (Deutsches Krebsforschungszentrum, Heidelberg, Germany). The rabbit polyclonal antibody against rat MRP3 (17) was a kind gift from Professor Y. Sugiyama (Graduate School of Pharmaceutical Science, University of Tokyo, Tokyo, Japan). Crude plasma membrane (CPM) was prepared from rat liver and kidney homogenates as described previously (33, 41). Fifty micrograms of liver CPM or 80 µg of kidney membrane proteins were separated on a 7.5% polyacrylamide gel. After the membrane was transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA), it was blocked for 1 h at room temperature with 5% nonfat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TBST). The membrane was then incubated with anti-MRP2 (1:5,000) or anti-MRP3 (1:1,000) antibody, and after it was washed appropriately, it was incubated for 1 h with a peroxidase-conjugated goat anti-rabbit IgG antibody (1:3,000). After the membrane was washed four times with TBST buffer, chemiluminescence development was performed using an Immune-Star chemiluminescent protein-detection system (Bio-Rad). The immunoreactive bands on the autoradiography films were scanned and semiquantified by densitometric analysis using the public domain NIH Image program. Control blot was prepared in parallel following the same protocol but using nonimmune rabbit serum.
Evaluation of renal function by PAH clearance.
PAH clearance was determined after 24, 72, and 120 h of BDL
(n = 4 each) after previously reported methods with
minor modifications (38, 44). In brief, the right jugular
vein and the left carotid artery were cannulated with heparinized
polyethylene tubes. Rats were given an intravenous bolus infusion (0.4 ml/100 g body wt) of 1% PAH in saline solution via the jugular vein
catheter followed by infusion (50 µl/min) of PAH solution through a
pump for 1 h. Bladder catheter was placed to collect urine for 30 min after the infusion. Arterial blood samples (300 µl) were drawn 15 min after the infusion. Serum and urine PAH concentration were
determined by an automated chemistry analyzer. PAH clearance
(ml · min1 · 100 g
1) was
calculated using the following formula: [urine volume (ml/min)/body wt
(100 g)]×[urine PAH (mg/dl)/plasma PAH (mg/dl)].
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RESULTS |
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Serum and urinary bilirubin levels after BDL.
BDL was associated with an increase in the serum bilirubin level and in
the daily urinary conjugated bilirubin excretion. Total serum bilirubin
increased at 24 and 72 h after BDL and slightly recovered after
120 h of operation (Fig.
1A). Most of elevated serum
bilirubin was of direct-reacting type. Daily urinary conjugated bilirubin excretion markedly increased at 24 and 72 h after BDL and recovered slightly 120 h after operation (Fig. 1B).
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Effect of BDL on MRP2 and MRP3 mRNA expression.
MRP2, MRP3 and GAPDH mRNA expressions were determined by real-time
RT-PCR (Fig. 2). The standard curve
performed with serial dilutions of the samples showed a constant slope
between experiments, indicating the constant efficiency of the PCR. The
liver MRP2 mRNA significantly decreased 59, 86, and 82% compared with
sham-operated animals after 24, 72, and 120 h of BDL,
respectively.
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Effect of BDL on MRP2 and MRP3 protein levels.
MRP2 protein levels on the apical membrane were determined in liver and
kidneys after 24, 72, and 120 h of BDL (Fig.
3, A and B). Rat
liver MRP2 protein expression significantly decreased in 25, 74, and
93% of sham-operated animals after 24, 72, and 120 h of BDL,
respectively. On the other hand, liver MRP3 protein level significantly
increased in 560, 634, and 612% of sham-operated animals after 24, 72, and 120 h of BDL, respectively. In contrast to liver MRP2, renal
MRP2 protein expression increased significantly in 387, 558, and 472%
of sham-operated animals after 24, 72, and 120 h of BDL,
respectively.
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Effect of BDL on PAH clearance.
Renal function was evaluated by PAH clearance. BDL induced a remarkable
increment of PAH clearance of 0.42 ± 0.16, 0.76 ± 0.23, and
0.53 ± 0.27 ml · min1 · 100 g body
wt
1 compared with sham-operated animals after 24 and
72 h of BDL, respectively (Fig. 4).
PAH clearance changed in parallel with daily urinary bilirubin
excretion.
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Effect of bilirubin ditaurate, TLCA sulfate, unconjugated
bilirubin, and human bile on the mRNA expression of MRP2 in HepG2 and
RPTEC cells.
The mRNA expression of MRP2 was evaluated by RT-PCR, and it was
normalized by the GAPDH content. The mRNA expression of MRP2 in HepG2
cells did not change significantly compared with controls in the
presence of bilirubin ditaurate, TLCA sulfate, and so on. (data not
shown). However, bilirubin ditaurate, TLCA sulfate, or human bile
increased the mRNA expression of MRP2 in RPTEC cells (Fig.
5).
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DISCUSSION |
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In this study, we evaluated the expression of MRP2 and MRP3 in liver and kidneys from BDL rats. The serum levels of the conjugated bilirubin, which mainly increased in our experimental model, decreased after 120 h, suggesting the occurence of extrahepatic excretion of bilirubin during BDL. In the present study, we hypothesized that the kidneys excrete the excess circulating levels of bilirubin during obstructive jaundice.
It has been reported that liver MRP2 protein is downregulated after BDL; however, the precise mechanism of this MRP2 downregulation in the liver is still controversial (20, 31). In the present study, we carried out real-time RT-PCR to quantify precisely the level of mRNA expression of MRP2 during BDL-associated obstructive jaundice. The results showed that liver MRP2 mRNA expression is decreased after BDL in rats. This decrease in mRNA expression was also associated with a concomitant decrease in protein expression of MRP2, as demonstrated by Western blotting analysis. This result agrees with findings of previous studies (20).
Under normal physiological conditions, the expression of MRP2 is high and that of MRP3 is low in hepatocytes; therefore, in the normal liver, most of conjugated organic anions are transported to bile canaliculi via MRP2. Low expression of MRP2 and concomitant increased expresssion of MRP3, which is located on basolateral membrane of hepatocytes (16, 21), may explain the elevation of serum bilirubin during BDL. MRP3 in the liver is upregulated by bilirubin and bilirubin glucuronides (11, 12, 19). This compensatory effect of MRP3 may play an important role in reducing injury to hepatocytes from cytotoxic materials that increase during obstructive jaundice.
In addition to liver excretion, conjugated bilirubin has been reported to be excreted also through the kidneys. Previous studies suggested that urinary excretion occurs mainly by glomerular filtration (13, 25). However, in conditions associated with cholestasis, renal tubular excretion of sulfate-conjugated bile acids may also takes place (9). In our present experimental BDL model, in contrast to the liver, the protein and mRNA expressions of MRP2 were significantly increased in the kidneys after 24 h. This upregulation of MRP2 was associated with a concomitant elevation of renal PAH clearance up to 72 h after BDL. These results suggest that increased renal MRP2 is functionally active during obstructive jaundice and that bilirubin is transported into urine, at least in part, via renal MRP2. The increased MRP2 expression in the kidneys may provide an alternative pathway for accelerating excretion of bilirubin conjugates during obstructive jaundice. However, to what degree this tubular secretion contributes to renal excretion of conjugated bilirubin needs to be clarified.
An important finding that needs clarification is the mechanism by which BDL affects the expression of MRP2 in the liver and kidneys. To gain some insights into this mechanism, in the present study, we evaluated the effects of synthetic conjugated bilirubin, sulfate-conjugated bile acid, human bile, and unconjugated bilirubin on human MRP2 expression in an in vitro system using hepatocyte and renal cell lines. In our in vitro study, we found that bilirubin ditaurate, TLCA sulfate, and human bile upregulate the expression of MRP2 in renal tubular cells but not in hepatocytes. These findings are consistent with changes in renal MRP2 expression observed in our animal BDL model. These results suggest that conjugated bilirubin, sulfate-conjugated bile acid, and some components of bile may, by themselves, regulate the expression of renal MRP2 and that this regulation varies according to the type of cells. Both conjugated bilirubin and sulfate-conjugated bile acid are well-characterized substrates of MRP2. The fact that conjugated bilirubin (bilirubin ditaurate), TLCA sulfate, and human bile but not unconjugated bilirubin upregulated the expression of MRP2 in renal tubular cells suggests that stimulation of MRP2 expression in these cells is substrate specific (14, 15, 40). Overall, findings in our in vitro experiments carried out using liver and renal tubular cells may explain the contrasting effect of BDL in our animal model. Further studies should be carried out to identify the cellular mechanisms involved in the opposing effects of glucuronide conjugates in different cell types.
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ACKNOWLEDGEMENTS |
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We are thankful to D. Keppler (Deutsches Krebsforschungszentrum, Heidelberg, Germany) for providing the EAG15 antibody and to Y. Sugiyama (Graduate School of Pharmaceutical Science, Tokyo University, Tokyo, Japan) for providing the anti-rat MRP3 antibody.
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FOOTNOTES |
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This study was supported by a grant-in-aid (no. 10470133, 1998-2000) from the Ministry of Education, Science and Culture of Japan.
Address for reprint requests and other correspondence: Y. Adachi, Third Dept. of Internal Medicine, Mie Univ. School of Medicine, 2-174 Edobashi, Tsu City, Mie, 514-8507 Japan (E-mail: adachi-y{at}clin.medic.mie-u.ac.jp).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpgi.00383.2001
Received 30 August 2001; accepted in final form 20 December 2001.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Büchler, M,
König J,
Brom M,
Kartenbeck J,
Spring H,
Horie T,
and
Keppler D.
cDNA cloning of the hepatocyte canalicular isoform of the multidrug resistance protein, cMRP, reveals a novel conjugate export pump deficient in hyperbilirubinemic mutant rats.
J Biol Chem
271:
15091-15098,
1996
2.
Budiman, T,
Bamberg E,
Koepsell H,
and
Nagel G.
Mechanism of electrogenic cation transport by the cloned organic cation transporter 2 from rat.
J Biol Chem
275:
29413-29420,
2000
3.
Cantz, T,
Nies AT,
Brom M,
Hofmann AF,
and
Keppler D.
MRP2, a human conjugate export pump, is present and transports fluo 3 into apical vacuoles of Hep G2 cells.
Am J Physiol Gastrointest Liver Physiol
278:
G522-G531,
2000
4.
Chiang, PW,
Song WJ,
Wu KY,
Korenberg JR,
Fogel EJ,
van Keuren ML,
Lashkari D,
and
Kurnit DM.
Use of a fluorescent-PCR reaction to detect genomic sequence copy number and transcriptional abundance.
Genome Res
6:
1013-1026,
1996[Abstract].
5.
Corbett, CL,
Bartholomew TC,
Billing BH,
and
Summerfield JA.
Urinary excretion of bile acids in cholestasis: evidence for renal tubular secretion in man.
Clin Sci (Colch)
61:
773-780,
1981[ISI][Medline].
6.
Demeule, M,
Brossard M,
and
Beliveau R.
Cisplatin induces renal expression of P-glycoprotein and canalicular multispecific organic anion transporter.
Renal Physiol
46:
832-840,
1999.
7.
Evers, R,
Kool M,
van Deemter L,
Janssen H,
Calafat J,
Oomen LC,
Paulusma CC,
Elferink RP,
Bass F,
Schinkel AH,
and
Borst P.
Drug export activity of the human canalicular multispecific organic anion transporter in polarized kidney MDCK cells expressing cMOAT (MRP2) cDNA.
J Clin Invest
101:
1310-1319,
1998
8.
Gibson, UEM,
Held CA,
and
Willaims PM.
A novel method for real time quantitative RT-PCR.
Genome Res
6:
995-1000,
1996[Abstract].
9.
Gutmann, H,
Miller DS,
Droulle A,
Drewe J,
Fahr A,
and
Fricker G.
P-glycoprotein- and MRP2-mediated octreotide transport in renal proximal tubule.
Br J Pharmacol
129:
251-256,
2000
10.
Harvey, RB,
and
Brothers AJ.
Renal extraction of p-aminohippurate and creatinine measured by conscious in vivo sampling of arterial and renal vein blood.
Ann NY Acad Sci
102:
46-54,
1962.
11.
Heid, CA,
Stevens J,
Livak KJ,
and
Williams PM.
Real time quantitative PCR.
Genome Res
6:
986-994,
1996[Abstract].
12.
Hirohashi, T,
Suzuki H,
Ito K,
Ogawa K,
Kume K,
Shimizu T,
and
Sugiyama Y.
Hepatic expression of multidrug resistance-associated protein- like proteins maintained in Eisai hyperbilirubinemic rats.
Mol Pharmacol
53:
1068-1075,
1998
13.
Hirohashi, T,
Suzuki H,
and
Sugiyama Y.
Characterization of the transport properties of cloned rat multidrug resistance-associated protein 3 (MRP3).
J Biol Chem
274:
15181-15185,
1999
14.
Hirohashi, T,
Suzuki H,
Takikawa H,
and
Sugiyama Y.
ATP-dependent transport of bile salts by rat multidrug resistance-associated protein 3 (MRP3).
J Biol Chem
275:
2905-2910,
2000
15.
Hosokawa, S,
Tagaya O,
Mikami T,
Nozaki Y,
Kawaguchi A,
Yamatsu K,
and
Shamoto M.
A new rat mutant with chronic conjugated hyperbilirubinemia and renal glomerular lesions.
Lab Anim Sci
42:
27-34,
1992[Medline].
16.
Ishikawa, T,
Allikimets R,
Dean M,
Higgins C,
Ling V,
and
Wain HM.
New nomenclature of human ABC transporter genes.
Xenobio Metabol and Dispos
15:
8-19,
2000.
17.
Ito, K,
Suzuki H,
Hirohashi T,
Kume K,
Shimizu T,
and
Sugiyama Y.
Functional analysis of a canalicular multispecific organic anion transporter cloned from rat liver.
J Biol Chem
273:
1684-1688,
1998
18.
Ito, K,
Suzuki H,
Hirohashi T,
Kume K,
Shimizu T,
and
Sugiyama Y.
Molecular cloning of canalicular multispecific organic anion transporter defective in EHBR.
Am J Physiol Gastrointest Liver Physiol
272:
G16-G22,
1997
19.
Kauffmann, HM,
Keppler D,
Kartenbeck J,
and
Schrenk D.
Induction of cMRP/cMoat gene expression by cisplatin, 2-acetylaminofluorene, or cycloheximide in rat hepatocytes.
Hepatology
26:
980-985,
1997[ISI][Medline].
20.
Kawaguchi, T,
Sakisaka S,
Mitsuyama K,
Harada M,
Koga H,
Taniguchi E,
Sasatomi K,
Kimura R,
Ueno T,
Sawada N,
Mori M,
and
Sata M.
Cholestasis with altered structure and function of hepatocyte tight junction and decreased expression of canalicular multispecific organic anion transporter in rat model of colitis.
Hepatology
31:
1285-1295,
2000[ISI][Medline].
21.
Keppler, D,
Kamisako T,
Leier I,
Cui Y,
Nies AT,
Tsujii H,
and
König J.
Localization, substrate specificity, and drug resistance conferred by conjugate export pumps of the MRP family.
Adv Enzyme Regul
40:
339-349,
2000[ISI][Medline].
22.
Keppler, D,
and
König J.
Hepatic canalucular membrane 5: expression and localization of the conjugate export pump encoded by MRP2 (cMRP/cMOAT) gene in liver.
FASEB J
11:
509-516,
1997
23.
König, J,
Rost D,
Cui Y,
and
Keppler D.
Characterization of the human multidrug resistance protein isoform MRP3 localized to the basolateral hepatocyte membrane.
Hepatology
29:
1156-1163,
1999[ISI][Medline].
24.
Kool, M,
de Haas M,
Scheffer GL,
Scheper RJ,
van Eijk MJ,
Juijn JA,
Baas F,
and
Borst P.
Analysis of expression of cMOAT(MRP2), MRP3, MRP4, and MRP5, homologues of the multidrug resistance-associated protein gene (MRP1), in human cancer cell lines.
Cancer Res
57:
3537-3547,
1997[Abstract].
25.
Kool, M,
van der Linden M,
de Haas M,
Scheffer GL,
de Vree JM,
Smith AJ,
Jansen G,
Peters GJ,
Ponne N,
Scheper RJ,
Elferink RP,
Bass F,
and
Borst P.
MRP3, an organic anion transporter able to transport anticancer drugs.
Proc Natl Acad Sci USA
96:
6914-6919,
1999
26.
Kullak-Ublick, GA,
Beuers U,
and
Paumgartner G.
Hepatobiliary transport.
J Hepatol
32, Suppl1:
3-18,
2000[Medline].
27.
Kusuhara, H,
Suzuki H,
and
Sugiyama Y.
The role of P-glycoprotein and canalicular multispecific organic anion transporter (cMOAT) in the hepatobiliary excretion of drugs.
J Pharm Sci
87:
1025-1040,
1998[ISI][Medline].
28.
Leier, I,
Hummel-Eisenbeiss J,
Cui Y,
and
Keppler D.
ATP-dependent para-aminohippurate transport by apical multidrug resistance protein MRP2.
Kidney Int
57:
1636-1642,
2000[ISI][Medline].
29.
Meyer, PJ,
and
Boyer JL.
Preparation of basolateral (sinusoidal) and canalicular plasma membrane vesicles for the study of hepatic transport processes.
Methods Enzymol
192:
534-545,
1990[Medline].
30.
Munoz, ME,
Esteller A,
and
Gonzalez J.
Substrate induction of bilirubin conjugation and biliary excretion in the rat.
Clin Sci (Colch)
73:
371-375,
1987[ISI][Medline].
31.
Nies, AT,
Cantz T,
Brom M,
Leier I,
and
Keppler D.
Expression of the apical conjugate export pump, MRP2, in the polarized hepatoma cell line, WIF-B.
Hepatology
28:
1332-1340,
1998[ISI][Medline].
32.
Ogawa, K,
Suzuki H,
Hirohashi T,
Ishikawa T,
Meier PJ,
Hirose K,
Akizawa T,
Yoshioka M,
and
Sugiyama Y.
Characterization of inducible nature of MRP3 in rat liver.
Am J Physiol Gastrointest Liver Physiol
278:
G438-G446,
2000
33.
Oude Elfelink, RP,
Meijer DK,
Kuipers F,
Jansen PL,
Groen AK,
and
Groothuis GM.
Hepatobiliary secretion of organic compounds; molecular mechanisms of membrane transport.
Biochem Biophys Acta
1241:
215-268,
1995[ISI][Medline].
34.
Paulusma, CC,
Bosma PJ,
Zaman GJ,
Bakker CT,
Otter M,
Scheffer GL,
Scheper RJ,
Borst P,
and
Oude Elferink RP.
Congenital jaundice in rats with a mutation in a multidrug resistance-associated protein gene.
Science
271:
1126-1128,
1996[Abstract].
35.
Paulusma, CC,
Kothe MJ,
Bakker CT,
Bosma PJ,
Van Bokhoven I,
van Marle J,
Bolder U,
Tytgat GN,
and
Oude Elferink RP.
Zonal down-regulation and redistribution of the multidrug resistance protein 2 during bile duct ligation in rat liver.
Hepatology
31:
684-693,
2000[ISI][Medline].
36.
Rigler, R,
Foldes-Papp Z,
Meyer-Almes FJ,
Sammet C,
Volcker M,
and
Schnetz A.
Fluorescence cross-correlation: a new concept for polymerase chain reaction.
J Biotechnol
63:
97-109,
1998[ISI][Medline].
37.
Robert, AG,
Robert AR,
and
Salah DK.
A new method of measuring renal function in conscious rats without the use of radioisotopes.
J Pharmacol Toxicol Methods
36:
189-197,
1996[ISI][Medline].
38.
Sauna, ZE,
Smith MM,
Müller M,
and
Ambudkar SV.
Evidence for the vectorial nature of drug (substrate)-stimulated ATP hydrolysis by human P-glycoprotein.
J Biol Chem
276:
33301-33304,
1999[Medline].
39.
Schaub, TP,
Kartenbeck J,
König J,
Spring H,
Dörsam J,
Staehler G,
Storkel S,
Thon WF,
and
Keppler D.
Expression of the MRP2 gene-encoded conjugate export pump in human kidney proximal tubules and in renal cell carcinoma.
J Am Soc Nephrol
10:
1159-1169,
1999
40.
Schaub, TP,
Kartenbeck J,
König J,
Vogel O,
Witzgall R,
Kriz W,
and
Keppler D.
Expression of the conjugate export pump encoded by the MRP2 gene in the apical membrane of kidney proximal tubules.
J Am Soc Nephrol
8:
1213-1221,
1997[Abstract].
41.
Soroka, CJ,
Lee JM,
Azzaroli F,
and
Boyer JL.
Cellular localization and up-regulation of multidrug resistance-associated protein 3 in hepatocytes and cholangiocytes during obstructive cholestasis in rat liver.
Hepatology
33:
783-791,
2001[ISI][Medline].
42.
Suzuki, H,
and
Sugiyama Y.
Excretion of GSSG and glutathione conjugates mediated by MRP1 and cMOAT/MRP2.
Semin Liver Dis
18:
359-376,
1998[ISI][Medline].
43.
Trauner, M,
Arrese M,
Soroka CJ,
Ananthanarayanan M,
Koeppel TA,
Schlosser SF,
Suchy FJ,
Keppler D,
and
Boyer JL.
The rat canalicular conjugate export pump (MRP2) is down-regulated in intrahepatic and obstructive cholestasis.
Gastroenterology
113:
255-264,
1997[ISI][Medline].
44.
Vos, TA,
Hooiveld GJE,
Koning H,
Childs S,
Meijer DKF,
Moshage H,
Jansen PLM,
and
Müller M.
Up-regulation of the multidrug resistance genes, Mrp1 and Mdr1b, and down-regulation of the organic anion transporter, Mrp2, and the bile salt transporter, spgp, in endotoxemic rat liver.
Hepatology
28:
1637-1644,
1998[ISI][Medline].
45.
Yamazaki, K,
Mikami T,
Hosokawa S,
Tagaya O,
Nozaki Y,
Kawaguchi A,
Funami H,
Katoh H,
Yamamoto N,
and
Wakabayashi T.
A new mutant rat with hyperbilirubinuria (hyb).
J Hered
86:
314-317,
1995[ISI][Medline].