The rat canalicular conjugate export pump (Mrp2) is down-regulated in intrahepatic and obstructive cholestasis

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Abstract

Background & Aims: The excretion of various organic isoform of the multidrug resistance protein (Mrp2). 5,6 anions into bile is mediated by an adenosine triphos-Mrp2 is characterized functionally by the absence of canaphate-dependent conjugate export pump, which has licular transport activity for various organic anions (e.g., been identified as the canalicular isoform of the multiconjugated bilirubin, glutathione (GSH) conjugates, oxidrug resistance protein (Mrp2).Mrp2 function is imdized glutathione, cysteinyl-leukotrienes, bile acid sulpaired in various experimental models of intrahepatic fates, and glucuronides) in Groningen yellow/transportand obstructive cholestasis, but the underlying molecudeficient (GY/TR 0 ) rats and Eisai hyperbilirubinemic lar mechanisms are unclear.The aim of this study was (EHBR) rats. 7 The molecular basis for this hereditary to investigate these molecular mechanisms.Methods:defect is a single-nucleotide deletion in the mrp2 gene, 5,8 The effects of endotoxin, ethinylestradiol, and common resulting in a stop codon and absence of the Mrp2 protein bile duct ligation (CBDL) on Mrp2 protein, messenger from the livers of GY/TR 0 and EHBR rats. 5,6 The human RNA (mRNA) expression, and Mrp2 tissue localization homologue of the canalicular conjugate pump has also were determined in rat livers by Northern blotting, Western analysis, and tissue immunofluorescence.To been cloned 9,10 and is absent in the Dubin-Johnson synassess whether changes were specific for Mrp2, we drome. 11This syndrome is characterized by conjugated also examined the expression of canalicular ecto-adenhyperbilirubinemia and a selective abnormality in the osine triphosphatase (ecto-ATPase) and mdr P-glycoexcretion of conjugated anions, 12,13 similar to the GY/ proteins (P-gp).Results: All three cholestatic models TR 0 and EHBR mutant rat strains. 7resulted in a marked decrease in Mrp2 protein (P õThe canalicular excretion of various organic anions is 0.01) and its tissue localization at the canalicular memimpaired in different experimental models of cholestasis, brane.Mrp2 mRNA levels diminished profoundly after including endotoxin (lipopolysaccharide [LPS])-treated endotoxin (P õ 0.0005) and CBDL (P õ 0.05), but did rats, 14-17 ethinylestradiol (EE)-treated rats, 18-20 and after not change after ethinylestradiol.In contrast to Mrp2, common bile duct ligation (CBDL), 21 indicating funcprotein expression of ecto-ATPase and P-gp remained tional impairment of Mrp2 in these forms of cholestasis.unchanged in endotoxin-and ethinylestradiol-treated However, the underlying molecular mechanisms for these animals, whereas P-gp levels increased after CBDL (P õ 0.05).Conclusions: Down-regulation of Mrp2 expresfunctional changes and their relationship to Mrp2 expression may explain impaired biliary excretion of amphiphision remain unknown.Basolateral bile acid and organic lic anionic conjugates in these models of cholestasis.3][24][25][26][27] On the other hand, expression of the canalicular enzyme and putative T bile acid transporter Ca 2/ ,Mg 2/ -ecto-adenosine triphos-he liver plays a major role in the elimination of various endo-and xenobiotic, lipophilic compounds, many of which are excreted into bile after con-Abbreviations used in this paper: CBDL, common bile duct ligation; version into amphiphilic anionic conjugates with gluta-Mrp2, canalicular multidrug resistance protein; ecto-ATPase, Ca 2/ , thione, glucuronate, or sulfate. 1,2Hepatobiliary excretion Mg 2/ -ecto-adenosine triphosphatase; EE, ethinylestradiol; EHBR, Eiof these conjugates is mediated by a primary-active, adensai hyperbilirubinemic; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; P-gp, P-glycoprotein; GSH, reduced glutathione; GY/TR 0 , osine triphosphate (ATP)-dependent conjugate export Groningen yellow/transport-deficient; kb, kilobase; LPS, lipopolysacpump, functionally known as the canalicular multispecharide; mdr, multidrug resistance; SDS, sodium