MRP-1 and BCRP Promote the Externalization of Phosphatidylserine in Oxalate-treated Renal Epithelial Cells: Implications for Calcium Oxalate Urolithiasis

Abstract

To investigate the possible involvement of multidrug resistance-associated protein 1 (MRP-1) and breast cancer resistance protein (BCRP) in the oxalate-induced redistribution of phosphatidylserine (PS) in renal epithelial cell membranes. A western blot analysis was used to examine the MRP-1 and BCRP expression levels. Surface-expressed PS was detected by the annexin V-binding assay. The cell-permeable fluorogenic probe 2,7-dichlorofluorescein diacetate was used to measure the intracellular reactive oxygen species (ROS) level. A rat model of hyperoxaluria was obtained using 0.5% ethylene glycol and 1.0% ammonium chloride. In addition, certain animals received verapamil (50 mg/kg body weight), which is a common inhibitor of MRP-1 and BCRP. The degree of nephrolithiasis was assessed histomorphometrically using sections stained by Pizzolato method and by measuring the calcium oxalate crystal content in the renal tissue. Oxalate produced a concentration-dependent increase in the synthesis of MRP-1 and BCRP. Treatment with MK571 and Ko143 (MRP-1- and BCRP-specific inhibitors, respectively) significantly attenuated the oxalate-induced PS externalization. Adding the antioxidant N-acetyl-l-cysteine significantly reduced MRP-1 and BCRP expression. In vivo, markedly decreased nephrocalcinosis was observed compared with that in the rat model of hyperoxaluria without verapamil treatment. Oxalate induces the upregulation of MRP-1 and BCRP, which act as phospholipid floppases causing PS externalization in the renal epithelial cell membrane. The process is mediated by intracellular ROS production. The ROS-mediated increase in the synthesis of MRP-1 and BCRP can play an important role in hyperoxaluria-promoted calcium oxalate urolithiasis by facilitating phosphatidylserine redistribution in renal epithelial cells.