ToxSci Advance Access originally published online on October 24, 2007
Toxicological Sciences 2008 101(1):171-178; doi:10.1093/toxsci/kfm261
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Ischemia-Reperfusion of Rat Livers Decreases Liver and Increases Kidney Multidrug Resistance–Associated Protein 2 (Mrp2)
Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas 66160-7417, USA
1 To whom correspondence should be addressed at Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160-7417, USA. Fax: (913) 588-7501. E-mail: cklaasse{at}kumc.edu.
Received July 5, 2007; accepted October 7, 2007
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ABSTRACT |
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Hepatic ischemia-reperfusion (IR) injury during liver transplantation can lead to cholestasis and remote organ dysfunction. Multidrug resistance–associated proteins (Mrps) are efflux transporters known to transport a diverse set of substrates, such as amphipathic chemicals, organic anions, and endogenous molecules. The purpose of this study was to determine the effect of hepatic IR injury on the expression of Mrps in rat liver and kidney. Male Sprague-Dawley rats were subjected to 60 min of partial hepatic ischemia. At various times after reperfusion (0, 3, 6, 24, and 48 h), the ischemic lobes were harvested as well as kidneys. RNA and protein expression of Mrps in livers and kidneys were determined by the branched DNA method, Western blot analysis, and tissue immunofluorescence. Mrp2 mRNA and protein expression in livers decreased after IR. Conversely, Mrp2 mRNA and protein expression in kidneys increased after IR. Mrp3 mRNA expression, and Mrp4 mRNA and protein expression in kidneys transiently increased after IR. The intensity of immunofluorescent staining of Mrp2 corresponded to changes in Mrp2 expression in livers and kidneys after IR as detected by Western blot analysis and was localized to the apical membrane domain in both tissues. These results demonstrate that after hepatic IR, downregulation of hepatic Mrp2 and upregulation of renal Mrp2 occur. These decreases in hepatic Mrp2 may contribute to cholestasis, yet increases in kidney may protect from oxidative stress and/or inflammation after hepatic IR.
Key Words: cholestasis; Mrps; remote organ; ischemia-reperfusion; transplantation.
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INTRODUCTION |
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Several membrane proteins mediating the adenosine triphosphate (ATP)–dependent transport of chemicals conjugated to glutathione (GSH), glucuronide, or sulfate have been identified as members of the multidrug resistance–associated protein (Mrp) family (Homolya et al., 2003
). Mrps play a major role in hepatobiliary and renal elimination of many structurally diverse xenobiotics, including organic anions and drug conjugates (Bodo et al., 2003). Mrps also transport endogenous molecules, such as leukotriene C4 (LTC4), prostaglandin, bilirubin glucuronide, and bile acids (Homolya et al., 2003).
MRP1 (ABCC1), the first member of the MRP family, was originally identified on the basis of its elevated expression in multidrug-resistant lung cancer cells (Cole et al., 1992). Using Mrp1 knockout mice, it has been shown that Mrp1 has an important role in the export of LTC4, a mediator of inflammation, and in protecting the body from a number of toxins, including several antitumor drugs (Loe et al., 1996). Mrp2 (Abcc2) is a 190-kDa phosphoglycoprotein that transports a wide range of endogenous and xenobiotic compounds, including conjugated bilirubin. Mrp2 is mainly expressed on the canalicular membrane of hepatocytes but is also present in renal tubular and intestinal epithelial cells (Cherrington et al., 2002). Lack of MRP2 protein in Dubin-Johnson syndrome patients, as well as in transport-deficient (TR–) rats, which lack Mrp2, leads to conjugated hyperbilirubinemia (Paulusma et al., 1996, 1997). Mrp3 (Abcc3) is an ATP-binding cassette transporter that is able to confer resistance to anticancer drugs, such as etoposide, and to transport lipophilic anions such as bile acids and glucuronides (Hirohashi et al., 2000; Zeng et al., 1999). A recent study showed that Mrp3 transports bilirubin glucuronide (Belinsky et al., 2005). Overexpression of Mrp4 is associated with increased cellular efflux of purine analogs, such as 6-mercaptopurine, and nucleoside-based antiviral drugs, such as 9-(2-phosphonylmethoxyethyl) adenine (Wielinga et al., 2002). Mrp4 also transports cyclic nucleotides, such as cyclic adenosine monophosphate and cyclic guanosine monophosphate (van Aubel et al., 2002). Furthermore, several compounds of physiological and pharmacological importance, such as methotrexate, estradiol-17β-glucuronide, bile acids, prostaglandins, and dehydroepiandrosterone-3-sulfate, were recently shown to be transported by Mrp4 (Kruh and Belinsky, 2003). These Mrps play a widespread role in detoxification, drug resistance, and, because of the role in the export of glutathione disulfide (GSSG) by Mrp1 and Mrp2, in the defense against oxidative stress (Keppler et al., 1997).
Ischemia-reperfusion (IR) injury of the liver is an unavoidable process in liver transplantation. During hepatic IR, an acute inflammatory response that may cause significant liver damage or dysfunction occurs (Lemasters and Thurman, 1997). Furthermore, hepatic IR injury is frequently associated with cholestasis (Ben-Ari et al., 2003). Our recent study has shown that hepatic IR–induced downregulation of canalicular transporters may be involved in the development of cholestasis (Tanaka et al., 2006). Hepatic IR also leads to remote organ injury. The most notable example of remote injury following hepatic IR is neutrophilic infiltration, edema, and intra-alveolar hemorrhage in the lung (Colletti et al., 1990). We have recently demonstrated that hepatic IR causes renal dysfunction in rats and the induction of renal heme oxygenase-1 after hepatic IR protects the kidney from this renal injury (Tanaka et al., 2007).
Whereas several studies have shown that renal Mrp2 increases in bile duct–ligated rats (Lee et al., 2001; Tanaka et al., 2002), regulation of Mrps in kidney during hepatic IR is not known. Therefore, the purpose of this study was to investigate the effects of hepatic IR on expression of Mrps in liver and kidney. After hepatic IR surgery, the following were determined: (1) mRNA expression of hepatic and renal Mrp1–4, (2) Western blot analysis of protein levels of hepatic and renal Mrp1–4, and (3) immunofluorescent analysis of subcellular localization of hepatic and renal Mrp2.
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MATERIALS AND METHODS |
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Materials.
All chemicals were purchased from Sigma-Aldrich (St Louis, MO).
Experimental operation procedure of IR model.
Male Sprague-Dawley rats (290–340 g, N = 4 or 5; Sasco, Omaha, NE) were used after 7 days of acclimation to the animal room. The animals were divided into two groups: (1) IR surgical groups (hepatic ischemia 60 min) and (2) sham surgery. The rats were anesthetized with sodium pentobarbital (50 mg/kg, ip) during surgery. A midline incision was made and the liver exposed. Branches of the hepatic artery and portal vein, supplying the left lateral and median lobes of the liver, were occluded with an atraumatic Glover bulldog clamp for 60 min. After 60 min of partial hepatic ischemia, the clamp was removed to initiate hepatic reperfusion. Sham-operated control rats underwent the same surgical procedure without vascular occlusion. The rats were reanesthetized with pentobarbital (50 mg/kg, ip), and the ischemic lobes of liver were harvested as well as kidneys after the indicated time periods (0, 3, 6, 24, and 48 h) of reperfusion. Animals received humane care as outlined in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication 86-23, revised 1985).
RNA isolation.
Total RNA was isolated using RNAzol B reagent (Tel Test Inc., Friendswood, TX) according to the manufacturer's protocol. The concentration of total RNA in each sample was quantified spectrophotometrically at 260 nm. The integrity of each RNA sample was evaluated by formaldehyde-agarose gel electrophoresis before analysis.
Branched DNA signal amplification (bDNA) assay.
Rat Mrp1–4 transcripts were quantified using the branched signal amplification assay as previously described (QuantiGene High Volume bDNA Signal Amplification Kit; Panomics, Fremont, CA) (Cherrington et al., 2002). Briefly, total RNA (1 µg/µl, 10 µl/well) was added to each well of a 96-well plate containing capture hybridization buffer and 50 µl of each diluted probe set. For each gene, total RNA was allowed to hybridize to the probe set overnight at 53°C. Subsequent hybridization steps were carried out as per the manufacturer's protocol, and luminescence was measured with a Quantiplex 320 bDNA Luminometer interfaced with Quantiplex Data Management Software Version 5.02 for analysis of luminescence from 96-well plates. The luminescence for each well was reported as relative light units per 10 µg total RNA. Oligonucleotide probe sets specific to rat Mrp1–4 mRNA transcripts were previously described (Chen and Klaassen, 2004; Cherrington et al., 2002).
Membrane preparations for Western blot.
Crude membrane samples (a mixture of plasma membranes and intracellular organelle membranes) were prepared from hepatic and renal tissues according to a method described previously (Chen et al., 2005). Briefly, 0.2–0.3 g tissue was minced in 10 ml ice-cold buffer A (0.25M sucrose, 10mM Tris-HCl [pH 7.4–7.6], containing 25 µg/ml leupeptin, 50 µg/ml aprotinin, 40 µg/ml phenylmethylsulfonyl fluoride [PMSF], 0.5 µg/ml pepstatin, and 50 µg/ml antipain). The minced tissue was poured into a Dounce homogenizer (Kontes, Vineland, NJ) and homogenized on ice for 20 strokes. The crude homogenate was further homogenized on ice for five strokes with a Teflon homogenizer. Each homogenate was filtered through two layers of cheesecloth and then centrifuged at 100,000 x g for 60 min at 4°C. The resulting pellet was resuspended in buffer B (0.25M sucrose, 10mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid [pH 7.5], and 40 µg/ml PMSF). Protein concentrations were determined with a bicinchoninic acid assay kit (Pierce, Rockford, IL).
Western blot analysis.
Membrane samples mixed with sample loading buffer (50–100 µg protein/lane) were loaded without heating onto a 7.5% sodium dodecyl sulfate-polyacrylamide gel. Following electrophoresis, proteins in the gel were electrotransferred to nitrocellulose membranes overnight at 20 V at 4°C. Membranes were blocked for 1 h at room temperature with 5% nonfat milk in Tris-buffered saline containing 0.05% Tween-20 (TBS-T). Blots were then incubated overnight at 4°C with one of the following antibodies: anti-Mrp1 (MRPr1, 1:1000), anti-Mrp2 (EAG15, 1:2000), anti-Mrp3 (1:5000), or anti-human MRP4 polyclonal antibody that cross-reacts with rat Mrp4 (1:1000) (Chen et al., 2005). Goat monoclonal antibody to Mrp1 (MRPr1) and rabbit polyclonal antibody to MRP4 were purchased from Alexis Biochemicals (San Diego, CA). Rabbit polyclonal anti-rat Mrp2 antibody EAG15 was generated in our laboratory according to Buchler et al. (1996). Rabbit polyclonal anti-rat Mrp3 antibody was also generated in our laboratory (Slitt et al., 2003). After thorough washing (three 10-min washes with excess TBS-T), blots were incubated for 1 h with goat anti-rat IgG horseradish peroxidase–linked secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA; 1:5000) for detection of Mrp1 or donkey anti-rabbit IgG horseradish peroxidase–linked secondary antibody (Amersham Biosciences Inc., Piscataway, NJ; 1:5000) for detection of Mrp2, 3, and 4. Blots were washed again. Immunoreactive bands were detected with enhanced chemical luminescence kit (Amersham Biosciences Inc.). Mrp1–4 proteins were visualized by exposure to Fuji Medical X-Ray film. Immunoreactive intensity of Mrp1–4 protein in the blots was quantified by densitometric analysis using Scion Image (Scion, Frederick, MD).
Immunohistochemistry.
Livers and kidneys were rapidly frozen in isopentane and subsequently immersed in liquid nitrogen and stored at –80°C until use. Sections of frozen liver and kidney were fixed in acetone at –20°C for 10 min and then rehydrated with phosphate-buffered saline (PBS). Sections were blocked at room temperature for 30 min with 5% goat serum/PBS and then incubated with rabbit anti-rat Mrp2 primary antibody EAG15 diluted 1:500 in PBS containing 5% goat serum at room temperature for 2 h. After incubation in primary antibody, the sections were washed three times for 5 min with PBS and incubated for 1 h at room temperature with fluorescein isothiocyanate–labeled secondary antibody to rabbit IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) diluted 1:200 in PBS containing 5% goat serum for liver sections and cyanin 3 (Cy3)–labeled secondary antibody to rabbit IgG (Jackson ImmunoResearch Laboratories) diluted 1:200 in PBS containing 5% goat serum for kidney sections. Slides were washed in PBS three times for 5 min each and then rinsed in distilled deionized water. The sections were air dried and mounted with Vectashield (Vector Laboratories, Burlingame, CA). Fluorescent staining in sections was visualized on an Olympus BX41 microscope (Olympus, Lake Success, NY). Images were captured using an Olympus DP70 camera.
Statistical analysis.
Statistical differences were determined using Student's t-test with significance set at p < 0.05. Bars represent mean ± SEM.
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RESULTS |
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Mrp1–4 mRNA in Liver after Hepatic IR
In ischemic lobes, the mRNA expression of the canalicular export pump Mrp2 was decreased 55% after 24 h of reperfusion (Fig. 1). However, the basolateral export pumps Mrp1, Mrp3, and Mrp4 were not altered by IR at any time point (Fig. 1).
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Mrp1–4 mRNA in Kidney after Hepatic IR
The effects of hepatic IR on Mrp1–4 mRNA expression in kidney are shown in Figure 2. Mrp1 was not altered by hepatic IR at any time point. In kidney, the mRNA expression of Mrp2 was increased 29 and 283% at 3 and 6 h after reperfusion, respectively. Mrp3 was slightly increased at 6 and 24 h after reperfusion by 56 and 39%, respectively. Mrp4 mRNA was transiently increased at 6 h after reperfusion by 107%.
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Western Blot Analysis of Mrp1–4 Protein in Liver after Hepatic IR
Mrp1–4 protein expression after hepatic IR was quantified in liver membrane fractions (Fig. 3). Rat liver Mrp2 protein expression after 24 and 48 h of reperfusion decreased 88 and 80%, respectively. Hepatic IR did not affect Mrp1, 3, and 4 protein levels, corresponding to the mRNA results.
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Western Blot Analysis of Mrp1–4 Protein in Kidney after Hepatic IR
Mrp1–4 protein expression after hepatic IR was determined in kidney membrane fractions (Fig. 4). Rat kidney Mrp2 protein expression increased 188, 381, and 490% after 6, 24, and 48 h of reperfusion, respectively.
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Mrp4 was transiently increased at 24 h after reperfusion by 97%. Renal Mrp1 and Mrp3 were not altered by hepatic IR at any time point.
Immunohistochemistry of Mrp2 in Liver after Hepatic IR
Downregulation of Mrp2 protein expression in liver was observed by Western blotting (Fig. 3). Therefore, Mrp2 subcellular localization was determined in rat livers by tissue immunofluorescence. As shown in Figure 5, Mrp2 expression appeared restricted to the canalicular membranes of hepatocytes. The intensity of staining was decreased 24 h after IR in comparison with sham-operated controls. Thereafter, the Mrp2 signal increased to control values by 48 h, showing that Mrp2 recovers rapidly. These findings are consistent with the immunoblotting results and confirm that hepatic IR leads to a decrease in hepatic Mrp2 expression.
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Immunohistochemistry of Mrp2 in Kidney after Hepatic IR
In contrast to the decrease in Mrp2 in liver after IR, upregulation of Mrp2 protein expression was noted in kidneys by Western blotting (Fig. 4). Therefore, Mrp2 subcellular localization was determined in rat kidneys by tissue immunofluorescence. In sham-operated rats, no significant Mrp2 signal was observed. However, increased signal was detected at the brush border membrane of the proximal tubules 24 h after IR, indicating that Mrp2 was induced 24 h after hepatic IR (Fig. 6).
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DISCUSSION |
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Hepatic IR injury is a common event in hepatic resectional surgery and liver transplantation (Lemasters and Thurman, 1997), and organs undergoing transplantation often suffer a certain degree of injury caused by IR. Cholestasis is a common complication after liver transplantation (Ben-Ari et al., 2003
). In experimental animal studies, reduced levels of hepatobiliary transport proteins are seen in cholestasis (Elferink et al., 2004; Lee et al., 2001; Tanaka et al., 2002; Trauner et al., 1997). Furthermore, hepatic IR leads to remote organ injury as well (Colletti et al., 1990; Tanaka et al., 2007). Clinical studies have indicated that renal dysfunction following liver transplantation is common (Braun et al., 2003).
A decrease in Mrp2 in liver has been determined in numerous animal models of hepatotoxicity, including bile duct ligation (BDL), lipopolysaccharide (LPS), and ethinylestradiol treatments (Elferink et al., 2004; Lee et al., 2001; Tanaka et al., 2002; Trauner et al., 1997). However, less is known about the regulation of Mrps in kidney (Chen et al., 2005; Denk et al., 2004; Lee et al., 2001; Tanaka et al., 2002). In the present study, Mrp1–4 mRNA, protein expression, and Mrp2 tissue localization in liver and kidney were determined after hepatic IR.
This study demonstrates that IR decreases Mrp2 in liver, both at the mRNA and at the protein level. However, downregulation of Mrp2 protein is more profound than that of mRNA expression, suggesting that posttranscriptional regulation also plays a role in hepatic IR–induced downregulation of Mrp2 (Elferink et al., 2004).
During the initial phase of hepatic IR injury, Kupffer cells are activated and release reactive oxygen species and proinflammatory cytokines, such as tumor necrosis factor-alpha, interleukin 1β (IL-1β), and IL-6 (Colletti et al., 1990; Wanner et al., 1996). Inflammation and proinflammatory cytokines suppress the expression of several hepatic transporters and metabolic enzymes, often resulting in cholestatic liver disease (Denson et al., 2002; Elferink et al., 2004). Activated Kupffer cells secrete cytokines in response to LPS that leads to decreased hepatic Mrp2 expression (Nakamura et al., 1999), and these cytokines may be the reason for decreased Mrp2 expression after hepatic IR. Hepatic Mrp2 expression has been shown to be suppressed by IL-1β–dependent downregulation of retinoid X receptor (RXR)/retinoic acid receptor (RAR) nuclear protein levels and binding activity, in both HepG2 cells as well as in bile duct–ligated rats (Denson et al., 2000, 2002). However, a previous study from this laboratory showed that RXR
protein levels in liver remained unaltered after hepatic IR (Tanaka et al., 2006). Therefore, unlike the involvement of RXR/RAR in the BDL model, another transcription factor might be associated with the downregulation of Mrp2 in the hepatic IR model. One such factor could be the pregnane X receptor (PXR), which is involved in Mrp2 regulation (Kast et al., 2002). PXR can downregulate several hepatic proteins, including Mrp2, during inflammation (Teng and Piquette-Miller, 2005). Thus, a number of different signaling pathways could potentially downregulate Mrp2 during IR, but the exact mechanism is unclear.
Rat Mrp2 expression is decreased in various cholestatic models, including BDL-, LPS-, and ethinylestradiol-induced cholestasis (Elferink et al., 2004; Lee et al., 2001; Tanaka et al., 2002; Trauner et al., 1997). A number of studies have demonstrated that Mrp2-deficient rats are protected from xenobiotic-induced oxidative stress and have lower hepatotoxicity. This is primarily because of higher basal levels of GSH in Mrp2-deficient rat liver (Ji et al., 2004b; Silva et al., 2005). Furthermore, overexpression of Mrp2 increased hepatic levels of 4-hydroxynonenal (4-HNE), a final product of lipid peroxidation. Mrp2 normally eliminates intracellular GSH conjugated with 4-HNE, and the reduction of intracellular GSH results in hepatocyte necrosis (Ji et al., 2004a). Therefore, in the present study, marked downregulation of Mrp2 in liver may play a protective role against oxidative stress generated by hepatic IR.
Under normal physiological conditions, basolateral Mrp1, 3, and 4 are not highly expressed in liver (Chen and Klaassen, 2004; Cherrington et al., 2002). However, hepatic expression of these basolateral Mrps is inducible under various pathophysiological conditions, such as cholestasis and endotoxin treatment (Denk et al., 2004; Tanaka et al., 2002). Induction of basolateral Mrp exporters may serve as a compensatory mechanism to protect the liver from injury caused by an overload of bile acids and/or other toxic constituents. However, in this study, none of the basolateral Mrps were induced after hepatic IR, although Mrp2 expression was markedly decreased. These differences might be due to the degree of cholestasis or the different experimental models. Prostaglandins are known to have cytoprotective effects on livers after hepatic IR (Hossain et al., 2006). Prostaglandins are transported by Mrp4 (Kruh and Belinsky, 2003). It appeared that the cytoprotective effects of prostaglandins may not require any changes in the expression of Mrp4.
In kidney, Mrp2 was increased after IR and transient induction of Mrp4 was observed. The upregulation of Mrp2 and Mrp4 remains uncertain at the present time, but potential mechanisms include pathways activated by oxidative stress, elevated systemic levels of cytokines, conjugated bilirubin, or bile acids, all of which occur in this model (Colletti et al., 1990; Tanaka et al., 2006, 2007). Previous studies have shown that rat renal Mrp2 expression increases after BDL (Lee et al., 2001; Tanaka et al., 2002). In previous studies, mRNA expression of Mrp2 was increased in renal proximal tubular epithelial cells after treatment with conjugated bilirubin, sulfate-conjugated bile acids, or human bile (Tanaka et al., 2002). Therefore, either conjugated bilirubin and/or bile acids, which increased in serum after hepatic IR, could induce renal Mrp2 via nuclear receptors, such as constitutive active/androstane receptor (CAR), PXR, or farnesoid X receptor (Kast et al., 2002). Our recent study showed that a transcription factor, nf-e2–related factor 2 (Nrf2), is activated in kidney after hepatic IR (Tanaka et al., 2007). A number of studies have reported that Mrp2 is regulated by Nrf2, and thus Nrf2 may be a likely mechanism for Mrp2 regulation after IR (Maher et al., in press; Vollrath et al., 2006). Mrp4 is also regulated by CAR and Nrf2 (Assem et al., 2004; Maher et al., in press), and similar mechanisms may be applicable.
Renal Mrp2 and Mrp4 are localized to the brush border membrane where substrates are transported into urine (Chen et al., 2005; Schaub et al., 1997). In the present study, immunofluorescent staining of rat kidney sections confirms that Mrp2 is localized to the apical membrane in the proximal tubular cells as demonstrated previously (Schaub et al., 1997). Furthermore, the intensity of Mrp2 in rats after IR is very strong, whereas in sham rats, there is virtually no staining. Although increases in Mrp2 protein expression were detected by Western blotting, a more prominent increase was observed by immunohistochemistry. This may be because crude membrane samples for Western blot are prepared from different cell types other than the proximal tubular cells and because the membrane preparations contain intracellular organelle membranes in addition to plasma membranes. Because Mrp2 and Mrp4 are localized to the brush border membrane, the increase in these two transporters may play a protective role in excreting chemical burdens generated by hepatic IR, including bile acids, GSSG, a urinary metabolite of 4-hydroxynonenal (4-hydroxynonenal mercapturic acid, 1,4-dihydroxynonene mercapturic acid, etc.), and cysteinyl leukotrienes into urine (Alary et al., 1995; Ji et al., 2002; Keppler et al., 1997; Kruh and Belinsky, 2003; Takamatsu et al., 2004). As mentioned previously, downregulation of Mrp2 in liver may protect against oxidative stress generated by hepatic IR. We speculate that kidney is not affected by the hypoxia and subsequent oxidative stress that occurs during reperfusion, so the induction of renal Mrp2 may be signaled by the accumulation of compounds that would normally be excreted into bile. Furthermore, a previous study from this laboratory showed that renal Mrp4 expression is upregulated in TR– rats, suggesting that upregulation of Mrp4 may be to compensate for the loss of Mrp2 function (Chen et al., 2005). Therefore, the induction of Mrp4 in kidney after hepatic IR may aid in the urinary excretion of overlapping substrates between these two transporters.
In summary, this study is the first to report gene regulation of Mrps in liver and kidney after hepatic IR. Downregulation of Mrp2 in liver and upregulation of Mrp2 in kidney were observed in this study after hepatic IR. Interestingly, these results in hepatic IR are similar to those after BDL (Denk et al., 2004; Lee et al., 2001; Tanaka et al., 2002). Downregulation of Mrp2 in liver may be part of the reason for the observation of cholestasis after hepatic IR. In contrast, increased renal Mrp2 and Mrp4 expression may protect kidneys from injury from hepatic IR by excreting toxic substrates/substances.
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FUNDING |
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National Institutes of Health (ES-09649, ES-09716, ES-07079, and RR021940).
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ACKNOWLEDGMENTS |
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The authors would like to thank Drs Tyra Leazer, Hong Lu, and Jay Petrick for their technical assistance.
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REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
|---|
Alary J, Bravais F, Cravedi JP, Debrauwer L, Rao D, Bories G. Mercapturic acid conjugates as urinary end metabolites of the lipid peroxidation product 4-hydroxy-2-nonenal in the rat. Chem. Res. Toxicol. (1995) 8:34–39.[CrossRef][Web of Science][Medline]
Assem M, Schuetz EG, Leggas M, Sun D, Yasuda K, Reid G, Zelcer N, Adachi M, Strom S, Evans RM, et al. Interactions between hepatic Mrp4 and Sult2a as revealed by the constitutive androstane receptor and Mrp4 knockout mice. J. Biol. Chem. (2004) 279:22250–22257.
Belinsky MG, Dawson PA, Shchaveleva I, Bain LJ, Wang R, Ling V, Chen ZS, Grinberg A, Westphal H, Klein-Szanto A, et al. Analysis of the in vivo functions of Mrp3. Mol. Pharmacol. (2005) 68:160–168.
Ben-Ari Z, Pappo O, Mor E. Intrahepatic cholestasis after liver transplantation. Liver Transpl. (2003) 9:1005–1018.[CrossRef][Web of Science][Medline]
Bodo A, Bakos E, Szeri F, Varadi A, Sarkadi B. The role of multidrug transporters in drug availability, metabolism and toxicity. Toxicol. Lett. (2003) 133:140–141.
Braun N, Dette S, Viebahn R. Impairment of renal function following liver transplantation. Transplant. Proc. (2003) 35:1458–1460.[CrossRef][Web of Science][Medline]
Buchler M, Konig J, Brom M, Kartenbeck J, Spring H, Horie T, 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. (1996) 271:15091–15098.
Chen C, Klaassen CD. Rat multidrug resistance protein 4 (Mrp4, Abcc4): Molecular cloning, organ distribution, postnatal renal expression, and chemical inducibility. Biochem. Biophys. Res. Commun. (2004) 317:46–53.[CrossRef][Web of Science][Medline]
Chen C, Slitt AL, Dieter MZ, Tanaka Y, Scheffer GL, Klaassen CD. Up-regulation of Mrp4 expression in kidney of Mrp2-deficient TR– rats. Biochem. Pharmacol. (2005) 70:1088–1095.[CrossRef][Web of Science][Medline]
Cherrington NJ, Hartley DP, Li N, Johnson DR, Klaassen CD. Organ distribution of multidrug resistance proteins 1, 2, and 3 (Mrp1, 2, and 3) mRNA and hepatic induction of Mrp3 by constitutive androstane receptor activators in rats. J. Pharmacol. Exp. Ther. (2002) 300:97–104.
Cole SP, Bhardwaj G, Gerlach JH, Mackie JE, Grant CE, Almquist KC, Stewart AJ, Kurz EU, Duncan AM, Deeley RG. Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line. Science (1992) 258:1650–1654.
Colletti LM, Remick DG, Burtch GD, Kunkel SL, Strieter RM, Campbell DA Jr. Role of tumor necrosis factor-alpha in the pathophysiologic alterations after hepatic ischemia/reperfusion injury in the rat. J. Clin. Invest. (1990) 85:1936–1943.[Web of Science][Medline]
Denk GU, Soroka CJ, Takeyama Y, Chen WS, Schuetz JD, Boyer JL. Multidrug resistance-associated protein 4 is up-regulated in liver but down-regulated in kidney in obstructive cholestasis in the rat. J. Hepatol. (2004) 40:585–591.[CrossRef][Web of Science][Medline]
Denson LA, Auld KL, Schiek DS, McClure MH, Mangelsdorf DJ, Karpen SJ. Interleukin-1 beta suppresses retinoid transactivation of two hepatic transporter genes involved in bile formation. J. Biol. Chem. (2000) 275:8835–8843.
Denson LA, Bohan A, Held MA, Boyer JL. Organ-specific alterations in RAR alpha:RXR alpha abundance regulate rat Mrp2 (Abcc2) expression in obstructive cholestasis. Gastroenterology (2002) 123:599–607.[CrossRef][Web of Science][Medline]
Elferink MG, Olinga P, Draaisma AL, Merema MT, Faber KN, Slooff MJ, Meijer DK, Groothuis GM. LPS-induced downregulation of MRP2 and BSEP in human liver is due to a posttranscriptional process. Am. J. Physiol. Gastrointest Liver. Physiol. (2004) 287:G1008–G1016.
Hirohashi T, Suzuki H, Takikawa H, Sugiyama Y. ATP-dependent transport of bile salts by rat multidrug resistance-associated protein 3 (Mrp3). J. Biol. Chem. (2000) 275:2905–2910.
Homolya L, Varadi A, Sarkadi B. Multidrug resistance-associated proteins: Export pumps for conjugates with glutathione, glucuronate or sulfate. Biofactors (2003) 17:103–114.[Web of Science][Medline]
Hossain MA, Wakabayashi H, Izuishi K, Okano K, Yachida S, Maeta H. The role of prostaglandins in liver ischemia-reperfusion injury. Curr. Pharm. Des. (2006) 12:2935–2951.[CrossRef][Web of Science][Medline]
Ji B, Ito K, Horie T. Multidrug resistance-associated protein 2 (MRP2) enhances 4-hydroxynonenal-induced toxicity in Madin-Darby canine kidney II cells. Chem. Res. Toxicol. (2004a) 17:158–164.[CrossRef][Web of Science][Medline]
Ji B, Ito K, Sekine S, Tajima A, Horie T. Ethacrynic-acid-induced glutathione depletion and oxidative stress in normal and Mrp2-deficient rat liver. Free. Radic. Biol. Med. (2004b) 37:1718–1729.[CrossRef][Web of Science][Medline]
Ji B, Ito K, Suzuki H, Sugiyama Y, Horie T. Multidrug resistance-associated protein2 (MRP2) plays an important role in the biliary excretion of glutathione conjugates of 4-hydroxynonenal. Free. Radic. Biol. Med. (2002) 33:370–378.[CrossRef][Web of Science][Medline]
Kast HR, Goodwin B, Tarr PT, Jones SA, Anisfeld AM, Stoltz CM, Tontonoz P, Kliewer S, Willson TM, Edwards PA. Regulation of multidrug resistance-associated protein 2 (ABCC2) by the nuclear receptors pregnane X receptor, farnesoid X-activated receptor, and constitutive androstane receptor. J. Biol. Chem. (2002) 277:2908–2915.
Keppler D, Leier I, Jedlitschky G. Transport of glutathione conjugates and glucuronides by the multidrug resistance proteins MRP1 and MRP2. Biol. Chem. (1997) 378:787–791.[Web of Science][Medline]
Kruh GD, Belinsky MG. The MRP family of drug efflux pumps. Oncogene (2003) 22:7537–7552.[CrossRef][Web of Science][Medline]
Lee J, Azzaroli F, Wang L, Soroka CJ, Gigliozzi A, Setchell KD, Kramer W, Boyer JL. Adaptive regulation of bile salt transporters in kidney and liver in obstructive cholestasis in the rat. Gastroenterology (2001) 121:1473–1484.[CrossRef][Web of Science][Medline]
Lemasters JJ, Thurman RG. Reperfusion injury after liver preservation for transplantation. Annu. Rev. Pharmacol. Toxicol. (1997) 37:327–338.[CrossRef][Web of Science][Medline]
Loe DW, Almquist KC, Deeley RG, Cole SP. Multidrug resistance protein (MRP)-mediated transport of leukotriene C4 and chemotherapeutic agents in membrane vesicles. Demonstration of glutathione-dependent vincristine transport. J. Biol. Chem. (1996) 271:9675–9682.
Maher JM, Dieter MZ, Aleksunes LM, Slitt AL, Guo GL, Dalton TP, Chan JY, Scheffer GL, Tanaka Y, Manautou JE, et al. Oxidative and electrophilic stress induces Mrp transporters via the Nrf2 transcriptional pathway. Hepatology (2007) 46:1597–1610.[CrossRef][Medline]
Nakamura J, Nishida T, Hayashi K, Kawada N, Ueshima S, Sugiyama Y, Ito T, Sobue K, Matsuda H. Kupffer cell-mediated down regulation of rat hepatic CMOAT/MRP2 gene expression. Biochem. Biophys. Res. Commun. (1999) 255:143–149.[CrossRef][Web of Science][Medline]
Paulusma CC, Bosma PJ, Zaman GJ, Bakker CT, Otter M, Scheffer GL, Scheper RJ, Borst P, Oude Elferink RP. Congenital jaundice in rats with a mutation in a multidrug resistance-associated protein gene. Science (1996) 271:1126–1128.[Abstract]
Paulusma CC, Kool M, Bosma PJ, Scheffer GL, ter Borg F, Scheper RJ, Tytgat GN, Borst P, Baas F, Oude Elferink RP. A mutation in the human canalicular multispecific organic anion transporter gene causes the Dubin-Johnson syndrome. Hepatology (1997) 25:1539–1542.[CrossRef][Web of Science][Medline]
Schaub TP, Kartenbeck J, Konig J, Vogel O, Witzgall R, Kriz W, 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. (1997) 8:1213–1221.[Abstract]
Silva VM, Thibodeau MS, Chen C, Manautou JE. Transport deficient (TR–) hyperbilirubinemic rats are resistant to acetaminophen hepatotoxicity. Biochem. Pharmacol. (2005) 70:1832–1839.[CrossRef][Web of Science][Medline]
Slitt AL, Cherrington NJ, Maher JM, Klaassen CD. Induction of multidrug resistance protein 3 in rat liver is associated with altered vectorial excretion of acetaminophen metabolites. Drug. Metab. Dispos. (2003) 31:1176–1186.
Takamatsu Y, Shimada K, Chijiiwa K, Kuroki S, Yamaguchi K, Tanaka M. Role of leukotrienes on hepatic ischemia/reperfusion injury in rats. J. Surg. Res. (2004) 119:14–20.[CrossRef][Web of Science][Medline]
Tanaka Y, Chen C, Maher JM, Klaassen CD. Kupffer cell-mediated downregulation of hepatic transporter expression in rat hepatic ischemia-reperfusion. Transplantation (2006) 82:258–266.[Web of Science][Medline]
Tanaka Y, Kobayashi Y, Gabazza EC, Higuchi K, Kamisako T, Kuroda M, Takeuchi K, Iwasa M, Kaito M, Adachi Y. Increased renal expression of bilirubin glucuronide transporters in a rat model of obstructive jaundice. Am. J. Physiol. Gastrointest. Liver Physiol. (2002) 282:G656–G662.
Tanaka Y, Maher JM, Chen C, Klaassen CD. Hepatic ischemia-reperfusion induces renal heme oxygenase-1 (HO-1) via nf-e2-related factor 2 (Nrf2) in rats and mice. Mol. Pharmacol. (2007) 71:817–825.
Teng S, Piquette-Miller M. The involvement of the pregnane X receptor in hepatic gene regulation during inflammation in mice. J. Pharmacol. Exp. Ther. (2005) 312:841–848.
Trauner M, Arrese M, Soroka CJ, Ananthanarayanan M, Koeppel TA, Schlosser SF, Suchy FJ, Keppler D, Boyer JL. The rat canalicular conjugate export pump (Mrp2) is down-regulated in intrahepatic and obstructive cholestasis. Gastroenterology (1997) 113:255–264.[CrossRef][Web of Science][Medline]
van Aubel RA, Smeets PH, Peters JG, Bindels RJ, Russel FG. The MRP4/ABCC4 gene encodes a novel apical organic anion transporter in human kidney proximal tubules: Putative efflux pump for urinary cAMP and cGMP. J. Am. Soc. Nephrol. (2002) 13:595–603.
Vollrath V, Wielandt AM, Iruretagoyena M, Chianale J. Role of Nrf2 in the regulation of the Mrp2 (ABCC2) gene. Biochem. J. (2006) 395:599–609.[CrossRef][Web of Science][Medline]
Wanner GA, Ertel W, Müller P, Höfer Y, Leiderer R, Menger MD, Messmer K. Liver ischemia and reperfusion induces a systemic inflammatory response through Kupffer cell activation. Shock (1996) 5:34–40.[Web of Science][Medline]
Wielinga PR, Reid G, Challa EE, van der Heijden I, van Deemter L, de Haas M, Mol C, Kuil AJ, Groeneveld E, Schuetz JD, et al. Thiopurine metabolism and identification of the thiopurine metabolites transported by MRP4 and MRP5 overexpressed in human embryonic kidney cells. Mol. Pharmacol. (2002) 62:1321–1331.
Zeng H, Bain LJ, Belinsky MG, Kruh GD. Expression of multidrug resistance protein-3 (multispecific organic anion transporter-D) in human embryonic kidney 293 cells confers resistance to anticancer agents. Cancer Res. (1999) 59:5964–5967.
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