Lipidomic analysis of MDCK-parental cells and MDCK-MDR1 cells exposed to GF120918 and clarithromycin highlights the role of P-glycoprotein in sphingolipid transport

Abstract

P-glycoprotein (P-gp) is one of the most studied drug transporters, as it enables the cellular efflux of 50 % of marketed drugs and plays a key role in many drug-drug interactions. While a high number of studies has investigated the use of drug probes to phenotype P-gp activity, identifying endogenous markers remain a not fulfilled challenge. A previous untargeted lipidomic study in healthy volunteers highlighted some sphingolipid species as putative endogenous substrates of P-gp. Based on an untargeted lipidomic analysis of Madin-darby canine kidney parental cells (MDCK-parental cells) and MDCK cells transfected with the MDR1 gene (MDCK-MDR1), the present study aimed to investigate the role of P-gp in intracellular sphingolipid transport and, more generally, to demonstrate the interest of metabolomics strategy in the study of in vitro drug transporters. Using an untargeted lipidomic approach based on liquid chromatography high resolution mass spectrometry, allowed us to detect 131 sphingolipid species within MDCK-parental cells and MDCK-MDR1. Among them, modifications of this sphingolipidome were observed because of MDR1 transfection, mainly involving glucosylceramides. Exposure to two P-gp inhibitors, GF120918 and clarithromycin, increased glucosylceramides, particularly in MDCK-parental cells, probably due to expression of endogenous canine P-gp in this cell line. These results support the hypotheses of a role for P-gp as well as glucosylceramide flippase within the Golgi apparatus of cells. It also provides a proof of concept for understanding pharmaco-metabolomic results in the study of drug transporters in humans.