Elsevier

Journal of Hepatology

Volume 38, Issue 6, June 2003, Pages 717-727
Journal of Hepatology

Adaptive changes in hepatobiliary transporter expression in primary biliary cirrhosis

https://doi.org/10.1016/S0168-8278(03)00096-5Get rights and content

Abstract

Background/Aims: Information about alterations of hepatobiliary transporter expression in primary biliary cirrhosis (PBC) could provide important insights into the pathogenesis of cholestasis. This study aimed to determine the expression of hepatobiliary transport systems for bile salts (Na+/taurocholate cotransporter, NTCP; bile salt export pump, BSEP), organic anions (organic anion transporting protein, OATP2; canalicular conjugate export pump, MRP2; basolateral MRP homologue, MRP3), organic cations (canalicular multidrug export pump, MDR1), and phospholipids (canalicular phospholipid flippase MDR3) in livers from patients with advanced stages of PBC.

Methods: Transporter mRNA and protein levels were assessed by reverse transcription polymerase chain reaction and Western blot analysis. Tissue distribution of transporters was investigated by immunohistochemistry and immunofluorescence microscopy. Hepatic bile acids were measured by gas chromatography-mass spectrometry.

Results: Compared to controls, basolateral uptake systems (NTCP, OATP2) were reduced, canalicular export pumps for bile salts and bilirubin (BSEP, MRP2) were preserved, while canalicular MDR P-glycoproteins (MDR1, MDR3) and the basolateral efflux pump MRP3 were increased in PBC. Double immunofluorescence labeling with a canalicular marker (dipeptidyl peptidase IV) demonstrated proper canalicular localization of BSEP and MRP2 in PBC. OATP2 and MRP2 expression correlated inversely with hepatic levels of hydrophobic bile acids, while positively correlating with hepatic enrichment with ursodeoxycholic acid.

Conclusions: Down-regulation of basolateral uptake systems and maintenance/up-regulation of canalicular and basolateral efflux pumps may represent adaptive mechanisms limiting the accumulation of toxic biliary constituents.

Introduction

Hepatic uptake and excretion of biliary constituents are mediated by transport systems at the basolateral and apical membrane of heptocytes [1], [2] (Fig. 1). Reduced transporter expression may contribute to impaired excretory liver function in animal models of cholestasis [2], [3], [4] and patients with cholestatic liver diseases [5], [6], [7], [8]. However, recent experimental studies suggest, that, particularly with prolonged cholestasis, maintenance or even up-regulation of hepatocellular efflux pumps may reflect adaptive and compensatory mechanisms limiting hepatocellular accumulation of potentially toxic biliary constituents [9], [10], [11], [12], [13], [14]. However, significant differences between rodents and humans in the regulation of bile salt metabolism/transport and the duration of cholestasis necessitate studies to determine whether the adaptive response concepts derived from animal studies [9], [10], [11], [12], [13], [14] are operational in human liver disease.

By studying primary biliary cirrhosis (PBC) as prototypic cholestatic liver disease [15], [16] we aimed to test the hypothesis, that chronic cholestasis in humans induces adaptive changes in transporter expression aimed at protecting the hepatocyte from toxic effects of retained biliary constituents. This information could contribute to our understanding of the pathogenesis of liver injury associated with PBC. Moreover, this could have major clinical implications, since therapeutic strategies aiming to maintain or even up-regulate hepatocellular transporter expression would be expected to ameliorate cholestatic liver injury [17], [18], [19].

Using reverse-transcription polymerase chain reaction, Western blotting, immunohistochemistry and immunofluorescence microscopy, we studied alterations of seven hepatobiliary transport systems in advanced stages of PBC. The results of this study indicate, that down-regulation of basolateral uptake systems and maintenance or even up-regulation of canalicular and basolateral export pumps in PBC may represent adaptive response mechanisms counteracting longstanding cholestasis. These changes are interpreted as an attempt to mitigate tissue damage resulting from accumulating potentially toxic biliary constituents such as bile salts.

Section snippets

Tissue specimens and patients characteristics

Twenty six liver specimens comprising samples from patients with PBC (n=13) and controls without liver disease (n=13) were analyzed. PBC specimens were obtained during liver transplantation (n=11) or liver biopsy (n=2). The diagnosis of PBC was based on standard criteria [15], [16]. Six patients had PBC stage III (PBC III) and seven stage IV (PBC IV) according to Ludwig [20]. Most PBC patients (4/6 PBC III; 5/7 PBC IV) received standard ursodeoxycholic acid (UDCA) treatment [15]. Control liver

Reduced basolateral NTCP and OATP2 expression is accompanied by inverse up-regulation of MRP3 in PBC

NTCP protein levels (95±23% of controls) and immunostaining were preserved in PBC III (Fig. 2, Fig. 3). NTCP protein levels were significantly decreased in PBC IV (48±30% of controls, P<0.01) (Fig. 2). NTCP immunostaining was reduced in PBC IV, which was most evident in the periphery of cirrhotic nodules with ballooned hepatocytes (zone of cholate stasis) (Fig. 3, Fig. 4). NTCP mRNA levels were increased in PBC III (176±49% of controls, P<0.01) and showed a trend for lower expression in PBC IV

Discussion

We investigated seven major hepatobiliary transport systems in advanced stages of PBC to gain insights into transporter regulation in longstanding cholestasis. As suggested by animal studies, chronic alterations of hepatobiliary transporter expression in cholestasis could represent adaptive changes aimed at protecting the hepatocyte from ongoing retention of toxic biliary constituents [9], [10], [11], [12], [13], [14]. Whether this could be relevant in chronic cholestatic liver diseases in

Acknowledgements

This work was supported by grants P15502 (to M. T.) and S7401-MOB (to K. Z.) from the Austrian Science Foundation, grants 7171 and 8522 from the Jubilee Funds of the Austrian National Bank (to M. T.) and the Joseph Skoda Prize from the Austrian Society of Internal Medicine (to M. T.). We gratefully acknowledge Dr James Neuberger (Birmingham, UK) for providing liver specimens from explanted PBC livers and Dr Bruno Stieger (Zurich, Switzerland) for providing the antibodies against NTCP, OATP2 and

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