Rolf Nüsing

Generation of 8-epi-prostaglandin F2α in isolated rat kidney glomeruli by a radical-independent mechanism

Isoprostanes comprise a group of free radical‐catalyzed products of arachidonic acid. However, there is recent evidence pointing towards an enzyme‐dependent formation of isoprostanes. With the use of isolated rat glomeruli we addressed the mechanisms of isoprostane generation. Synthesis of prostanoids and isoprostanes, including 8‐epi‐PGF2α, was studied under conditions favouring radical formation. Cultured glomeruli formed different prostanoids including 8‐epi‐PGF2α. Upon LPS challenge cyclo‐oxygenase (COX)‐2 expression was enhanced, and this was paralleled by a 2–9‐fold increase in prostanoid formation, including isoprostanes. Addition of COX‐isoform unselective inhibitors (diclofenac, indomethacin) or a selective inhibitor (NS‐398) suppressed the synthesis of prostanoids, 8‐epi‐PGF2α and total isoprostane fraction; however, inhibition of the latter was less pronounced. Antioxidants such as butylated hydroxytoluene (BHT), nordihydroguaiaretic acid (NDGA), or dimethylurea exhibited an only minimal inhibitory effect on 8‐epi‐PGF2α synthesis. Moreover, ROS‐generating drugs (menadione, methylviologen) or NADPH‐driven radical formation were unable to cause the generation of significant amounts of 8‐epi‐PGF2α by rat glomeruli. In contrast, the total isoprostane fraction could be increased by menadione addition. These data provide further evidence for a radical‐independent, but COX‐dependent formation of 8‐epi‐PGF2α in renal tissue. Regarding the other isoprostanes, both radicals and COX enzymes contribute to their formation. Based on our data we assume that elevated release of vasoactive 8‐epi‐PGF2α has to be expected under conditions when the prostanoid system in the kidney is stimulated, e.g. under inflammatory conditions. Regarding renal oxidative injuries, the usefulness of 8‐epi‐PGF2α as a representative marker molecule of oxidative stress has to be questioned.