Oxalate-induced ceramide accumulation in Madin-Darby canine kidney and LLC-PK1 cells

Background Oxalate exposure produces oxidant stress in renal epithelial cells leading to death of some cells and adaptation of others. The pathways involved in these diverse actions remain unclear, but appear to involve activation of phospholipase A(2) (PLA(2)) and redistribution of membrane phospholipids. The present studies examined the possibility that oxalate actions may also involve increased accumulation of ceramide, a lipid-signaling molecule implicated in a variety of pathways, including those leading to apoptotic cell death. Methods. Ceramide accumulation was examined in renal epithelial cells from pig kidney (LLC-PK1 cells) and from dog kidney [Madin-Darby canine kidney (MDCK cells)] using the diacylglycerol kinase assay. Sphingomyelin degradation was assessed by monitoring the disappearance of H-3-sphingomyelin from cells that had been prelabeled with [H-3]-choline. The effects of oxalate were compared with those of other oxidants (peroxide, xanthine/xanthine oxidase), other mate and citrate), and a known activator of sphingomyelinase in these cells [tumor necrosis factor-alpha (TNF-alpha)]. Separate studies determined whether oxalate-induced accumulation of ceramide could be blocked by pretreatment with antioxidants [Mn (III) tetrakis (1-methyl-4-pyridyl) porphyrin (Mn TMPyP, a superoxide dismutase mimetic) or N-acetylcysteine (NAC; an antioxidant)], with an inhibitor of ceramide synthase [fumonisin B1 (FB1)] or with an inhibitor of PLA(2) [arachidonyl trifluoromethylketone (AACOCF(3))] Results. Oxalate exposure produced a significant time- and concentration-dependent increase in cellular ceramide. A reciprocal decrease in H-3-sphingomyelin was observed under these conditions. Increases in cellular ceramide levels were also observed after treatment with other oxidants (hydrogen peroxide, and xanthine/xanthine oxidase), activators of sphingomyelinase (TNF-alpha), exogenous sphingomyelinase, or arachidonic acid. Formate produced similar (albeit smaller) effects, and citrate did not. The oxidant-induced increases in ceramide were attenuated by pretreatment with NAC (a glutathione precursor) and MnTMPyP (a superoxide dismutase mimetic), suggesting a role for cellular redox states. The oxalate-induced increase in ceramide was also attenuated by pretreatment with AACOCF(3), suggesting a role for PLA(2). Pretreatment with FB1 produced a small but statistically insignificant attenuation of the response to oxalate. Conclusions. Oxalate exposure produces a marked accumulation of ceramide in renal epithelial cells by a process that is redox sensitive and mediated in part by activation of PLA(2). Since cellular sphingomyelin decreased as ceramide increased, it seems likely that oxalate actions are mediated, at least in part, by an increase in sphingomyelinase activity, although alterations in ceramide synthase are also possible. Further study is required to define the steps involved in oxalate actions and to determine the extent to which ceramide signaling mediates oxalate actions.