Inhibition of transport across the hepatocyte canalicular membrane by the antibiotic fusidate

Abstract

Hyperbilirubinemia is a frequent side effect induced by long-term therapy with the antibiotic fusidate. The aim of this study was to elucidate the molecular mechanisms of fusidate-induced hyperbilirubinemia by investigating its influence on hepatic transport systems in the canalicular membrane. Using canalicular membrane vesicles from rat liver, we determined the effect of fusidate on the adenosine 5′-triphosphate (ATP)-dependent transport of substrates of the apical conjugate export pump, multi-drug resistance protein 2 (Mrp2, symbol Abcc2) and the bile salt export pump (Bsep, symbol Abcb11). Fusidate inhibited the ATP-dependent transport of the Mrp2 substrates 17β-glucuronosyl estradiol and leukotriene C₄, and the transport of cholyltaurine by Bsep with K_i values of 2.2±0.3, 7.6±1.3, and 5.5±0.8 μM, respectively. To elucidate the in vivo implication of these findings, the effect of fusidate treatment on the elimination of intravenously administered tracer doses of 17β-glucuronosyl estradiol and cholyltaurine into bile was studied in rats. Treatment with fusidate (100 μmol/kg body weight) reduced the biliary excretion rate of 17β-glucuronosyl [${}^3\text{H}$]estradiol and [${}^3\text{H}$]cholyltaurine by 75 and 80%, respectively. Extended treatment of rats with fusidate (100 μmol/kg body weight, three times daily i.p. for 3 days) reduced hepatic Mrp2 protein levels by 61% (P<0.001). Our data suggest that there are at least two different mechanisms involved in the impairment of transport processes and hepatobiliary elimination by fusidate, direct inhibition of transport of Mrp2 and Bsep substrates by competitive interaction and impairment by a decreased level of hepatic Mrp2.

Introduction

Fusidate is a mono-anionic steroidal antibiotic widely used for the treatment of infectious diseases with methicillin-resistant or, more recently, multi-resistant Staphylococcus aureus strains [1], [2], [3]. One of the major side effects of fusidate treatment is the induction of conjugated hyperbilirubinemia which may occur as early as 2 days after starting the treatment [4], [5]. The incidence of fusidate-induced hyperbilirubinemia ranged from 17 to 48% when given intravenously [6], [7]. When treatment is discontinued, elevated serum bilirubin levels may return to normal within 4–6 days [7], [8]. So far the molecular mechanisms responsible for fusidate-induced hyperbilirubinemia have not been elucidated.

Cholestasis may be a side effect of drug treatment provoked by a variety of substances such as macrolides [9], penicillins [9], ethinylestradiol [10] and cyclosporin A [11]. Interference with the ATP-dependent transport of amphiphilic anions into bile is one of the mechanisms considered to play a key role [12], [13]. Membrane proteins mediating the ATP-dependent transport of amphiphilic anions across the canalicular membrane include the apical multi-drug resistance protein 2 (MRP2) [14], [15] and the bile salt export pump (BSEP), a member of the ABCB subfamily of the ATP-binding cassette transporters [16].

MRP2 mediates the ATP-dependent transport of conjugated bilirubin from the hepatocyte into bile with high affinity [17]. The absence of functional MRP2 in the human hepatocyte canalicular membrane is the molecular basis of the Dubin–Johnson syndrome [18], which is characterized by conjugated hyperbilirubinemia. Inhibition of MRP2 has so far been demonstrated for cyclosporin A [17], [19] and 17β-D-glucuronosyl estradiol [13].

BSEP is predominantly expressed in the liver and localized to the canalicular membrane [16], [20]. In contrast to MRP2 with its broad substrate specificity, BSEP has been defined as the specific bile salt transporter with high affinity for cholyltaurine [13], [16]. Progressive familial intrahepatic cholestasis type 2, an inherited liver disease of childhood characterized by cholestasis and elevated serum γ-glutamyltransferase activity, was shown to be associated with mutations in the BSEP gene [20]. Cyclosporin A, rifamycin SV, rifampicin, and glibenclamide were found to inhibit BSEP transport function [12], [13], [19]. It is of interest to note that fusidate [3] is both, a structural analog of the prototypic Bsep substrate cholyltaurine [16] and of the prototypic Mrp2 substrate 17β-D-glucuronosyl estradiol [15].

The aim of the present study was to investigate whether fusidate affects the transport across the hepatocyte canalicular membrane. Although fusidate-induced hyperbilirubinemia was described in humans, we chose the rat model for our investigations as the human and the rat orthologs of MRP2 exhibit very similar transport characteristics for conjugated bilirubin [17]. Further, the rat represents a suitable model for in vivo experiments on hepatobiliary elimination of many substances. Evidence derived from both, in vitro and in vivo experiments supports the assumption that fusidate acts as an inhibitor of the ATP-dependent transport by Mrp2 and Bsep into bile. Furthermore, fusidate administration to rats leads to a decrease in Mrp2 protein levels upon long-term treatment.