Mitochondrial dysfunction is a primary event in renal cell oxalate toxicity

Abstract

In cultured renal epithelial cells, exposure to oxalate, a constituent of many kidney stones, elicits a cascade of responses that often leads to cell death. Oxalate toxicity is mediated via generation of reactive oxygen species (ROS) in a process that depends at least in part upon lipid signaling molecules that are generated through membrane events that culminate in phospholipase A2 (PLA2) activation. The present studies asked whether mitochondria, a major site of ROS production, were targets of oxalate toxicity, and if so, whether mitochondrial responses to oxalate were mediated by PLA2 activation. Effects of oxalate and various lipids on mitochondrial membrane potential (DeltaPsim) were measured in Madin-Darby canine kidney (MDCK) cell monolayers using 5,5',6,6'-tetrachloro 1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), a DeltaPsim-sensitive dye. Other studies assayed caspases, serine proteases activated during apoptosis, in response to oxalate or lipid signaling molecules. Additional studies asked whether oxalate or lipids produced by PLA2 activation promoted ROS formation in isolated renal mitochondria. Oxalate exposure decreased MDCK cell DeltaPsim within 30 minutes, a response attenuated by arachidonyl trifluoromethyl ketone (AACOCF3), an inhibitor of cytosolic PLA2 (cPLA2). Exposure to arachidonic acid or to lysophosphatidylcholine (lyso-PC), lipid products of PLA2 activation, or to ceramide, another lipid signal generated in MDCK cells following oxalate exposure, also depolarized MDCK cell DeltaPsim and increased the number of caspase-positive cells. Isolated renal mitochondria responded to oxalate, arachidonic acid, lyso-PC, and ceramide by increasing their accumulation of ROS, lipid peroxides, and oxidized thiol proteins. These studies suggest that lipid signaling molecules released after oxalate-induced PLA2 activation trigger marked, rapid changes in mitochondrial function that may mediate toxicity in renal epithelial cells.