A study on the Changes of localization of canalicular transporters in cholestasis.

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Biliary excretion of bile acids and organic anions is mediated by ATP-dependent primary active transporters at the canalicular membrane. Multidrug resistance protein 2 (MRP2) is a transporter for organic anions such as conjugated bilirubin, and is defective in Dubin-Johnson syndrome. Bile salt export pump (BSEP) is a transporter for amidated bile acids, and is defective in progressive familial intrahepatic cholestasis type 2. Down regulation of MRP2 and BSEP has been reported in various cholestatic models. In addition, the impairment of vesicular targeting of transporters to the canalicular membrane has been postulated as an important mechanism of cholestasis. In the present study, in order to clarify the mechanism of cholestasis, localization ofMRP2 and BSEP in hepatocytes was investigated by immunohistochemistry in bile duct-ligated rats and lipopolysaccharide (LPS)-induced rat cholestasis. Polyclonal anti-MRP2 and anti-BSEP antibodies were obtained by the immunization of rabbits by C-terminal peptides coupled with KLH. Microscopic observation of the liver after the treatment with HRP-labeled second antibody revealed the localization ofMRP2 and BSEP at the canalicular membrane of hepatocytes. In bile duct-ligated rats, a decrease of the staining ofMRP2 and BSEP at the canalicular membrane was observed, suggesting a dysfunction of these transporters. In contrast, the staining of these transporters was the same as controls in LPS-induced cholestatic model. Similar results were obtained by using commercially available monoclonal anti-MRP2 antibody. In future, immunofluorescent observations by a confocal laser microscopy, which was insufficient in the present study, and studies on human liver with cholestasis are planed.