We have studied regulation of the multidrug resistance protein 2 (mrp2) during bile duct ligation (BDL) in the rat. In hepatocytes isolated after 16, 48, and 72 hours of BDL, mrp2-mediated dinitrophenyl-glutathione (DNP-GS) transport was decreased to 65%, 33%, and 33% of control values, respectively. The impaired mrp2-mediated transport coincided with strongly decreased mrp2 protein levels, without any significant changes in mrp2 RNA levels. Restoration of bile flow after a 48-hour BDL period resulted in a slow recovery of mrp2-mediated transport and protein levels. Immunohistochemical detection of the protein in livers of rats undergoing BDL showed strongly reduced mrp2 staining after 48 hours, which was initiated in the periportal areas of the liver lobule and progressed toward the pericentral areas after 96 hours. Immunofluorescent detection of mrp2 in livers of rats undergoing 48 hours of BDL revealed decreased staining accompanied by intracellular localization of the protein in pericanalicular vesicular structures. Within this intracellular compartment, mrp2 colocalized with the bile salt transporter (bsep) and was still active as shown by vesicular accumulation of the fluorescent organic anion glutathione-bimane (GS-B). We conclude that downregulation of mrp2 during BDL-induced obstructive cholestasis is mainly posttranscriptionally regulated. We propose that this down-regulation is caused by endocytosis of apical transporters followed up by increased breakdown of mrp2, probably in lysosomes. This breakdown of mrp2 is more severe in the periportal areas of the liver lobule. (HEPATOL-OGY 2000;31:684-693.)
The multidrug resistance protein 2 (mrp2), previously called canalicular multispecific organic anion transporter, is an adenosine triphosphate (ATP)-dependent transporter that mediates the biliary excretion of a wide variety of endogenous and xenobiotic compounds. 1 The mrp2 protein has been extensively characterized in transport-mutant rats (the TR ฯช rat, derived from the Wistar strain), 1 and the Eisai hyperbilirubinemic rat (EHBR rat, derived from the Sprague-Dawley rat strain), 2 which lack this protein owing to mutations in the mrp2 gene. [3][4][5] In humans, mutations in the orthologous MRP2 gene cause the Dubin-Johnson syndrome, 6-8 an autosomal recessive defect in the hepatobiliary excretion of a broad range of organic anions. 1,9 The human MRP2 and rat mrp2 are members of the ATP-binding cassette transporter family, and are abundantly and specifically expressed in the canalicular membrane of the hepatocyte. 3,4 Substrates transported by this protein include conjugates of glucuronide, sulphate, and glutathione, and complexes (or cotransport) of heavy metals and oxyanions with glutathione. 1 Recently, we have shown an important role for mrp2 in the transport of reduced glutathione (GSH), 10 which probably serves a physiological function in maintenance of the bile acid-independent flow.
Extrahepatic or obstructive cholestasis is a pathological condition caused by biliary obstruction leading to impaired bile flow. [11][12][13] As a result, bile salt and non-bile salt organic anions (including taurocholate and bilirubin-glucuronides) accumulate in hepatocytes and regurgitate into the circulation. Morphological changes associated with obstructive cholestasis include dilation of the canaliculus, loss of microvilli, and rearrangement of tight-junctional structures. [11][12][13] Kawaguchi et al. 14 have recently shown that alterations in tight-junctional structures are heterogenously distributed throughout the liver lobule during bile duct ligation (BDL) in the rat.
Several studies have been published that describe an association between activity and expression levels of canalicular transporters and obstructive cholestasis using the BDL model in the rat. P-glycoprotein (P-gp) expression levels and activity are increased during BDL, 15 with a concomitant increase in both mdr1a and mdr1b RNA levels. 15 In addition, P-gp (partly) redistributes to the pericanalicular vesicles of the hepatocyte. 15,16 Trauner et al. 17 have shown that BDL in the rat is associated with decreased mrp2 protein levels and mrp2 RNA levels, indicating that down-regulation of mrp2 is