REACTIVE OXYGEN SPECIES IN PUROMYCIN AMINONUCLEOSIDE NEPHROSIS - IN-VITRO STUDIES

We examined the role of reactive oxygen species (ROS) in puromycin ami nonucleoside (PAN)-induced changes to glomerular epithelial cells (GEC s) in vitro. Levels of superoxide anion (O-2(.-)), hydrogen peroxide ( H2O2) and hydroxyl radical (HO.) were measured in rat kidney-slice cul tures containing PAN with or without antioxidants (allopurinol, probuc ol and alpha-tocopherol/ascorbic acid). GEC morphology was assessed af ter three days of culture using transmission (TEM) and scanning (SEM) electron microscopy. The effects of hypoxanthine on GEC ultrastructure was also assessed. O-2(.-), H2O2 and HO. were generated when PAN was added to kidney-slice cultures in Medium 199. TEM morphometry revealed that incubation with PAN (100 mu g/ml) significantly (P < 0.05 at lea st) retarded the loss of GEC foot processes normally seen in vitro. Wh en the hydrophobic antioxidants probucol or alpha-tocopherol/ascorbic acid, which scavenged/inhibited generation of O-2(.-), H2O2 and HO., w ere added to cultures containing PAN, the effect of PAN on foot proces ses was abolished. The TEM appearance of GECs now resembled that seen in control cultures. On the other hand, SEM revealed that probucol and alpha-tocopherol/ascorbic acid provided no protection against the cha nges induced by PAN in GEC cell bodies or major processes. Allopurinol provided no protection against the changes induced by PAN in GEC cell bodies, major processes or foot processes. The addition of hypoxanthi ne to kidney-slice cultures did not result in the generation of O-2(.- ), H2O2 or HO., or alter GEC ultrastructure. These findings indicate t hat ROS play a role in PAN-induced alterations to GEC foot process arc hitecture in vitro. However, the xanthine oxidase pathway does not app ear to play a major role in generating ROS from PAN in vitro.