Drug-induced liver injury (DILI) is the major cause of pharmaceutical withdrawal from the market and cholestasis is one of the most common DILI observed. Cholestasis stem from abnormalities in the activity of bile acid transporters, namely the sodium-dependent taurocholate co-transporting polypeptide, Ntcp (Slc10a1), and the bile salt export pump, Bsep (Abcb11). The nuclear receptors, pregnane X receptor (PXR) and farnesoid X receptor (FXR), have been implicated in the regulation of Ntcp and Bsep. The aim of this study was to establish the relative roles of FXR and PXR in the regulation of Ntcp and Bsep, in the rat liver and sandwich cultured rat hepatocytes (SCRH) to establish the usefulness of SCRH as a good model for bile acid transport studies. For this purpose, male Sprague Dawley rats were treated with FXR and PXR ligands and siRNAs. Intraperitoneal (IP) injection of FXR ligand, CDCA, resulted in induction in FXR mRNA levels whereas siRNA treatment knocked down FXR transcript levels. FXR induction decreased Ntcp transcript levels that were reversed in FXR knockdown animals. CDCA treatment resulted in greater than 2-fold increase in Bsep mRNA levels that was absent in FXR knockdown animals. The PXR ligand, PCN, showed an induction in PXR mRNA levels while siRNA treatment resulted in the knockdown in PXR transcript levels. PXR induction did not cause any change in Ntcp and Bsep transcript levels. Furthermore, sandwich cultured rat hepatocytes were used to ascertain if the above in vivo findings are reproducible in vitro. Treatment of hepatocytes with FXR ligand, CDCA, and PXR ligand, PCN, caused greater than 5-fold increase in the respective mRNA levels whereas respective siRNA treatment caused >79% knocked in their transcript levels. FXR induction decreased Ntcp mRNA levels that were ablated in FXR knockdown cultures. FXR induction resulted in a 6-fold increase in Bsep mRNA levels that disappeared in FXR knockdown cultures. On the contrary, PXR induction did not influence the expression levels of Ntcp and Bsep. Taken together these data demonstrate that both FXR and PXR are inducible at mRNA and protein levels in vivo and their expression can be knocked down in the rat liver. Moreover, FXR negatively regulate Ntcp and positively regulate Bsep while PXR does not influence Ntcp and Bsep expression. Finally, the data shows that these in vivo findings can be successfully reproduced in sandwich cultured rat hepatocytes. To conclude, sandwich cultured rat hepatocytes mimic the in vivo situation and provide a good model for bile acid transport studies.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:589516 |
| Date | January 2013 |
| Creators | Farooq, Muhammad |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=203406 |
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