Elsevier

Journal of Hepatology

Volume 44, Issue 4, April 2006, Pages 768-775
Journal of Hepatology

Mice lacking Mrp3 (Abcc3) have normal bile salt transport, but altered hepatic transport of endogenous glucuronides

https://doi.org/10.1016/j.jhep.2005.07.022Get rights and content

Background/Aim

Multidrug Resistance Protein 3 (MRP3) transports bile salts and glucuronide conjugates in vitro and is postulated to protect the liver in cholestasis. Whether the absence of Mrp3 affects these processes in vivo is tested.

Methods

Mrp3-deficient mice were generated and the contribution of Mrp3 to bile salt and glucuronide conjugate transport was tested in (1): an Ussing-chamber set-up with ileal explants (2), the liver during bile-duct ligation (3), liver perfusion experiments, and (4) in vitro vesicular uptake experiments.

Results

The Mrp3(−/−) mice show no overt phenotype. No differences between WT and Mrp3-deficient mice were found in the trans-ileal transport of taurocholate. After bile-duct ligation, there were no differences in histological liver damage and serum bile salt levels between Mrp3(−/−) and WT mice, but Mrp3-deficient mice had lower serum bilirubin glucuronide concentrations. Glucuronide conjugates of hyocholate and hyodeoxycholate are substrates of MRP3 in vitro and in livers that lack Mrp3, there is reduced sinusoidal secretion of hyodeoxycholate-glucuronide after perfusion with hyodeoxycholate.

Conclusions

Mrp3 does not have a major role in bile salt physiology, but is involved in the transport of glucuronidated compounds, which could include glucuronidated bile salts in humans.

Introduction

Members of the ATP-binding cassette (ABC) family of transporters are large membrane proteins that couple the energy released from ATP hydrolysis to active transport of a substrate across the membrane [1]. The ABCC subfamily contains nine transporters, the Multidrug Resistance Proteins 1–9 (MRP1–9) [2], [3], [4]. MRPs transport organic anions with broad substrate specificity but their physiological roles are still not fully defined. An example is MRP3, which in humans localizes to the basolateral membranes of epithelial cells from the gut, liver, pancreas, kidney, and the adrenal gland [5], [6], [7].

Initial studies on MRP3 focused on the liver. The basal hepatic levels of Mrp3 in rats are very low, but high levels are found in the naturally occurring Mrp2-deficient rat strains and in livers of Dubin–Johnson patients [7], [8], [9], [10]. Moreover, treatments that induce cholestasis (e.g. common bile-duct ligation; BDL) also result in a substantial induction of hepatic Mrp3 in rats [9], [10], [11], [12], and to some induction in mice as well [13].

A possible explanation for these findings comes from transport assays with rat Mrp3 [14], which showed that Mrp3 transports glycocholate, taurocholate, and taurolithocholate-3-sulfate, the latter two with high affinity. This has led to the suggestion that Mrp3 may be important in the absorption of bile salts in the terminal ileum [15] and in protecting the liver under conditions of impaired bile flow, by transporting toxic organic anions (e.g. bile salts) across the sinusoidal membrane into the circulation for subsequent excretion in the urine [12], [16]. It is doubtful whether this would also apply to humans, however, as human MRP3 transports glycocholate and taurocholate with low affinity [16], [17], [18]. Mice with a homozygous disruption of Mrp3 were generated by embryonic stem cell technology to study the role of Mrp3 in bile salt physiology. The characterization of these Mrp3(−/−) mice is reported here, extending a preliminary report on these mice [19].

Section snippets

Animals

The generation of Mrp3(−/−) mice is described in online Supplementary material. Mouse stocks were maintained as a cross of FVB/129Ola (50%/50%). Male mice were used in the experiments described. Mouse handling and experimental procedures were conducted in accordance with institutional guidelines for animal care and use.

Generation and characterization of antibodies against mouse Mrp3

A fragment of mouse Mrp3 corresponding to amino acids 818–952 was cloned by RT-PCR from liver RNA into the pMalC expression plasmid. Fusion proteins were isolated as previously

Generation of Mrp3(−/−) mice

Two independent properly targeted embryonic stem cell clones were obtained (see Supplementary Methods) and injected into mouse blastocysts. This resulted in chimeric mice that transmitted the Mrp3 allele through the germ line to F1 offspring (Supplementary Fig. 1b). Offspring from Mrp3(+/−) intercrosses were born at the expected Mendelian ratio indicating that there is no selection against the mutant allele during development. Absence of intact Mrp3 in Mrp3(−/−) mice was verified by

Discussion

Our results with the Mrp3(−/−) mouse indicate that Mrp3 does not contribute substantially to bile salt physiology in agreement with the preliminary analysis of our mice by Zelcer [19] and a very recent report on an independently generated Mrp3(−/−) mouse by Belinsky et al. [36]. The absence of a role for Mrp3 in intestinal bile salt transport is in line with a recent report that Ostα/Ostβ is a basolateral bile salt carrier in transfected epithelial cells and is coexpressed in ileal enterocytes

Acknowledgements

We thank members of the Borst lab for advice and assistance throughout this study and Dr Wim Nooijen of the clinical chemistry lab for analyzing mouse serum. We are grateful for the excellent biotechnical assistance of A.J. Schrauwers. This study was supported in part by grants of the Netherlands Organization for Scientific Research (NWO program 912-02-073) and of the Dutch Cancer Society (NKI 2001-2474).

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