De novo demonstration and co-localization of free-radical production and apoptosis formation in rat kidney subjected to ischemia/reperfusion

Ischemia-induced oxidative damage to the reperfused kidney was examined. A modified chemiluminescence method, an in situ nitro blue tetrazolium perfus ion technique, and a DNA fragmentation/apoptosis-related protein assay were adapted for demonstration de novo and co-localization of reactive oxygen s pecies (ROS) production and apoptosis formation in rat kidneys subjected to ischemia/reperfusion injury. The results showed that prolonged ischemia po tentiated proapoptotic mechanisms, including increases in the Bax/Bcl-2 rat io, CPP32 expression, and poly-(ADP-ribose)-polymerase fragments, and subse quently resulted in severe apoptosis, including increases in DNA fragmentat ion and apoptotic cell number in renal proximal tubules (PT) and distal tub ules (DT) in a time-dependent manner. The increased level of ROS detected o n the renal surface was correlated with that in blood and was intensified b y a prolonged interval of ischemia. The main source of ROS synthesis was th e PT epithelial cells. The ROS and apoptotic nuclei detected in the PT cell s can be ameliorated by superoxide dismutase (SOD) treatment before reperfu sion. However, the apoptotic nuclei remained in DT in the SOD-treated rats, indicating that formation of apoptosis in DT was not influenced by the sma ll amounts of ROS produced. in PT and DT cell cultures, significant increas es in apoptotic cells and ROS were evident in PT cells after hypoxia/reoxyg enation insult. Furthermore, the oxidative damage in PT, bur not in DT, can be alleviated by ROS scavengers SOD and hexa(sulfobutyl)fullerene, confirm ing that PT are vulnerable to ROS. These results lead us to conclude that R OS produced in significant amounts in PT epithelium under ischemia/reperfus ion or hypoxia/reoxygenation conditions may be responsible for the apoptoti c death of these cells.