Multidrug resistance-associated protein 4 is up-regulated in liver but down-regulated in kidney in obstructive cholestasis in the rat

Abstract

Background/Aims

Multidrug resistance-associated protein 4 (Mrp4, ABCC4) transports cyclic nucleotides, anti-retroviral compounds, and sulfated bile acids. Mrp4 expression is increased in farnesyl/bile acid receptor knockout mice. Our aim was to investigate Mrp4 expression and function in rat liver and kidney in obstructive cholestasis.

Methods

Male Sprague–Dawley rats were subjected to bile duct ligation (BDL) or sham-surgery. Animals were sacrificed after 3, 7, and 14 days and tissues were harvested for Western blot analysis, real-time reverse transcriptase-mediated polymerase chain reaction (RT-PCR), and immunohistochemistry.

Results

Western blot analysis revealed a progressive, more than seven-fold increase $\text{(P}\text{<0.05)}$ of Mrp4 expression in cholestatic livers, 14 days after BDL. In contrast, Mrp4 in 14-day BDL kidneys decreased to 26±4% of controls $\text{(P}\text{<0.005).}$ Immunohistochemistry localized Mrp4 to the basolateral hepatocyte membrane and corroborated its hepatic up-regulation after BDL. Real-time RT-PCR demonstrated no major changes of Mrp4 mRNA levels in liver and kidney after BDL. Cyclic adenosine monophosphate, an MRP4 substrate, was increased in plasma and urine, consistent with these findings.

Conclusions

Obstructive cholestasis in rats results in progressive up-regulation of Mrp4 protein in liver but down-regulation in kidney. The absence of corresponding changes in Mrp4 mRNA suggests posttranscriptional mechanisms as predominant regulators of Mrp4 expression in BDL rats.

Introduction

The multidrug resistance-associated proteins (MRP/Mrp) function as adenosine triphosphate (ATP)-dependent conjugate export pumps for a wide range of organic anionic substrates [1]. The hepatic isoform of Mrp2 (Abcc 2) transports bilirubin diglucuronide, sulfates, glutathione conjugates as well as a variety of other organic anionic conjugates from the liver into the bile canaliculus and is the main driving force for bile salt-independent bile flow [2], [3]. In the kidney Mrp2 is localized to the apical membrane of the proximal tubule and may be involved in renal secretion of bile salt conjugates in cholestasis [4]. Mutations of the MRP2 gene result in the Dubin-Johnson syndrome which is characterized by conjugated hyperbilirubinemia and impaired hepatobiliary secretion of a wide range of amphipathic compounds [1]. In addition, different forms of cholestasis result in down-regulation of hepatic Mrp2 [5], [6]. Other members of the Mrp family in the liver function as efflux pumps but are localized to the basolateral membrane including Mrp1 (Abcc 1) [7] and Mrp3 (Abcc 3) [8]. Although the expression of the Mrp1 and Mrp3 isoforms is low under physiological conditions, these transporters, particularly Mrp3, are substantially induced during cholestatic liver injury, reflecting physiologically important adaptive responses [6], [9], [10].

MRP4 (ABCC4) was recently found to be the first transporter that supported efflux of nucleoside monophosphate analogs like 9-(2-phosphonylmethoxyethyl)adenine (PMEA) and azidothymidine monophosphate from mammalian cells [11]. Studies, carried out in membrane vesicles with recombinant MRP4, also demonstrated transport of cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP) [12], [13], estradiol-17β-d-glucuronide [12], methotrexate [14], and dehydroepiandrosterone-3-sulfate (DHEAS) [15]. Recently, MRP4/Mrp4 has been localized to the basolateral hepatocyte membrane [16]. In contrast to Mrp1 [17] and Mrp3 [18] immunofluorescence studies have localized MRP4/Mrp4 to the apical membrane of the proximal tubule [13]. Mrp4 expression is substantially increased in livers of mice with disruption of the farnesyl/bile acid nuclear receptor (FXR/BAR), which have increased levels of serum and hepatocellular bile acids, and Mrp4 can be further up-regulated by cholic acid-feeding, consistent with a role in bile acid homeostasis [19]. A recent study has identified conjugated bile acids, especially sulfated derivatives, and steroids as substrates of MRP4 when expressed in membrane vesicles from HEK-293 and Sf9 cells [15].

Based on these findings our aim was to investigate Mrp4 expression and function in obstructive cholestasis in the rat. In addition to liver, Mrp4 expression was also determined in kidney because urinary excretion represents an alternative route for the elimination of bile acids in cholestasis.