Improved hepatic MRP2/ABCC2 transport activity in LPS-induced cholestasis by aquaporin-1 gene transfer
Introduction
The Multidrug Resistance-associated Protein 2 (MRP2/ABCC2) transporter is expressed at the canalicular pole of hepatocytes and mediates the biliary transport of glutathione and glutathione, sulfate, and glucuronide conjugates of a large number of endo- and xenobiotics, and is thus important to bile acid-independent bile formation [1]. The administration of lipopolysaccharide (LPS) to rodents is an experimental approach commonly used to explore the pathogenesis of cholestasis associated to sepsis [2,3]. LPS, a bacterial endotoxin, downregulates the expression of key biliary transporters, i.e., MRP2/ABCC2, bile acid export pump BSEP/ABCB11, and aquaporin-8 water channels [2,4,5].
We previously demonstrated that gene delivery of human aquaporin-1 (hAQP1) water channels to hepatocytes improves the cholestasis triggered by LPS in part by stimulating BSEP/ABCB11-mediated biliary bile salt output [6]. We studied now whether hepatocyte-expressed hAQP1 is also able to promote MRP2/ABCC2 transport activity.
Section snippets
Animals and experimental protocol
Adult male Wistar rats were maintained on a standard laboratory diet and water ad libitum and housed in a humidity- and temperature-controlled environment under a constant 12-h light-dark cycle. Laboratory animals received Human care and were treated following the Guide for the Care and Use of Laboratory Animals (National Institutes of Health). Protocols were approved by the local animal welfare committee.
Rats were anesthetized with ketamine/xylazine (100 mg/kg body weight/15 mg/kg body weight,
Biliary glutathione excretion
As we have already reported [6], hAQP1 gene delivery normalized the diminished bile flow in LPS-triggered rats without affecting that of non-cholestatic animals (Fig. 1A). We now found that biliary glutathione output was markedly reduced by 65% in LPS-triggered rats and that hAQP1 transfer increased it significantly (Fig. 1B).
Biliary glutathione transport provides a major driving force for bile acid-independent bile flow, thus its increase in AdhAQP1-treated rats would certainly contribute to
Conclusion
In conclusion, we found that hepatic adenovirus-mediated transfer of hAQP1 increased MRP2/ABCC2 transport activity assessed in situ with endogenous and exogenous substrates, probably by a hAQP1-induced augmentation of canalicular membrane cholesterol amount. Our results suggest that hAQP1-induced MRP2/ABCC2 activation contributes to the improvement of the cholestasis triggered by LPS, a finding that might have therapeutic implications for sepsis-associated cholestatic diseases.
Conflicts of interest
The authors declare no conflict of interest.
Author contributions
Conceived and designed the experiments: JM, ADM, RAM. Performed the experiments: JM, GNT, MD. Analyzed the data: JM, ADM, RAM. Wrote the paper: JM, RAM. All authors read and approved the final manuscript.
Declaration of interests
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests.
Acknowledgments
We would like to thank Dr B. Baum and Dr. RG Goya for donations of AdhAQP1 and control adenovector, respectively. We would like to thank also Prof. María Robson and the staff from the English Department of the Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR) for the language correction of the manuscript. This work was supported by Grants PIP 2015-088 and PUE 0089 from CONICET, PICT 2015-0439 from ANPCyT to RAM and PICT 2017–3393 from ANPCyT to JM. The funders had no role in study design,
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