Hydrogen peroxide induces 21-aminosteroid-inhibitable F₂-isoprostane production and cytolysis in renal tubular epithelial cells

F₂-isoprostanes are the newly identified reactive oxygen species-catalyzed peroxidation products of arachidonate. The infusion of these prostaglandin F₂-like prostanaoids into the rat kidney induces profound parallel reductions in RBF and GFR, suggesting that these metabolites may be partly responsible for the hemodynamic alterations seen in free radical-linked acute renal injury models. The present study examined directly in renal proximal tubular (LLC-PK₁) cells whether hydrogen peroxide, a reactive oxygen species implicated in many models of acute renal injury, induces F₂-isoprostane production and whether its production can be inhibited by the recently synthesized lipid peroxidation inhibitor 21-aminosteroid (lazaroid U-74389G). The incubation of LLC-PK₁ cell layers with hydrogen peroxide for 3 h resulted in a dose-related six-fold increase in F₂-isoprostane production, measured by the gas chromatographic-mass spectroscopic method. The preincubation of cells with 21-aminosteroid prevented hydrogen peroxide-induced F₂-isoprostane production, a finding also demonstrable with other lipid peroxidation inhibitors, e.g., 2-methyl aminochroman (U-83836E) and diphenyl-p-phenylenediamine. Besides inhibiting isoprostane production, 21-aminosteroid reduced hydrogen peroxide-induced lipid degradation and peroxidation, and protected the cells against hydrogen peroxide-induced cytolysis. The novel finding that hydrogen peroxide induces 21-aminosteroid-inhibitable F₂-isoprostane production in renal epithelial cells supports the In vivo report that its levels are elevated in reactive oxygen species-linked renal injury models such as ischemia-reperfusion. Besides direct cell injury, lipid peroxidation by generating F₂-isoprostanes may further contribute to renal dysfunction through a vasoconstrictive mechanism. Thus, the inhibition of excess F₂-isoprostane production may be one of the additional mechanisms, besides cytoprotection, by which antioxidants ameliorate renal dysfunction in experimental models of acute renal injury.