Cross-talk between nitric oxide and superoxide determines ceramide formation and apoptosis in glomerular cells

Abstract

Background. The modulation of cell signaling by nitric oxide (NO) and superoxide (O2-) is associated with apoptotic cell death in inflammatory kidney diseases. Recently, we have shown that NO induces ceramide production in glomerular mesangial and endothelial cells and the ratio of NO and O2- determines whether cells live or die. Methods. Glomerular endothelial and mesangial cells were labeled with [14C]serine, the precursor of all sphingolipids, then stimulated with reactive oxygen species- or reactive nitrogen species-generating substances and subjected to lipid extraction. Radioactive lipids were separated and analyzed by thin-layer chromatography. DNA fragmentation, as a characteristic feature of apoptosis, was measured by a nucleosome/DNA-ELISA, which quantitatively recorded the histone-associated DNA fragments. Results. Exposure of glomerular endothelial and mesangial cells to either NO donors or superoxide-generating substances led to a delayed and sustained ceramide formation that paralleled the induction of apoptosis in both cell types. Coincubation of endothelial cells with NO and superoxide, which led to the generation of peroxynitrite, caused a synergistic enhancement of ceramide generation and apoptosis when compared to either stimulus alone. By contrast, in glomerular mesangial cells co-stimulation with superoxide neutralized not only NO-induced apoptosis but also NO-induced ceramide formation, although O2- alone triggered ceramide formation in mesangial cells and caused cell death. Moreover, SIN-1, a substance that simultaneously releases NO and O2- and thereby generates peroxynitrite, also stimulated a delayed ceramide formation in endothelial cells but not in mesangial cells. Furthermore, exposure of endothelial cells to glucose oxidase, which generates hydrogen peroxide, or to exogenous hydrogen peroxide, also showed a dose-dependent increase in ceramide formation and apoptosis, although to a lesser extent than did superoxide. Conclusions. These data suggest that ceramide represents an important mediator of reactive oxygen and nitrogen species-triggered cell responses, like apoptosis. There seem to be cell type-specific protective mechanisms that critically depend on a fine-tuned redox balance between reactive nitrogen and oxygen species to determine whether a cell undergoes apoptosis or survives when exposed to oxidative and/or nitrosative stress conditions.