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 perfusion technique, and a DNA fragmentation/apoptosis-related protein assay were adapted for demonstration de novo and co-localization of reactive oxygen species (ROS) production and apoptosis formation in rat kidneys subjected to ischemia/reperfusion injury. The results showed that prolonged ischemia potentiated proapoptotic mechanisms, including increases in the Bax/Bcl-2 ratio, CPP32 expression, and poly-(ADP-ribose)-polymerase fragments, and subsequently resulted in severe apoptosis, including increases in DNA fragmentation and apoptotic cell number in renal proximal tubules (PT) and distal tubules (DT) in a time-dependent manner. The increased level of ROS detected on the renal surface was correlated with that in blood and was intensified by a prolonged interval of ischemia. The main source of ROS synthesis was the PT epithelial cells. The ROS and apoptotic nuclei detected in the PT cells can be ameliorated by superoxide dismutase (SOD) treatment before reperfusion. However, the apoptotic nuclei remained in DT in the SOD-treated rats, indicating that formation of apoptosis in DT was not influenced by the small amounts of ROS produced. In PT and DT cell cultures, significant increases in apoptotic cells and ROS were evident in PT cells after hypoxia/reoxygenation insult. Furthermore, the oxidative damage in PT, but not in DT, can be alleviated by ROS scavengers SOD and hexa(sulfobutyl)fullerene, confirming that PT are vulnerable to ROS. These results lead us to conclude that ROS produced in significant amounts in PT epithelium under ischemia/reperfusion or hypoxia/reoxygenation conditions may be responsible for the apoptotic death of these cells.
Chien
Chiang-Ting
CT
Office for Medical Research Administration, National Taiwan University Hospital, Taipei, Taiwan.
Lee
Po-Huang
PH
Office for Medical Research Administration, National Taiwan University Hospital, Taipei, Taiwan.|Department of Surgery, National Taiwan University College of Medicine, Taipei, Taiwan.
Chen
Chau-Fong
CF
Department of Physiology, National Taiwan University College of Medicine, Taipei, Taiwan.
Ma
Ming-Chieh
MC
Department of Physiology, National Taiwan University College of Medicine, Taipei, Taiwan.
Lai
Ming-Kuen
MK
Department of Urology, National Taiwan University College of Medicine, Taipei, Taiwan.
Hsu
Su-Ming
SM
Department of Pathology, National Taiwan University College of Medicine, Taipei, Taiwan.
D000818:Animals; D017209:Apoptosis; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D053938:DNA Fragmentation; D005260:Female; D005609:Free Radicals / Q000378:metabolism; D005786:Gene Expression Regulation; D007668:Kidney / Q000098:blood supply* / Q000293:injuries / Q000378:metabolism / Q000473:pathology; D008163:Luminescent Measurements; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D015427:Reperfusion Injury / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D051028:bcl-2-Associated X Protein
C494828:Bax protein, rat; D005609:Free Radicals; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D011065:Poly(ADP-ribose) Polymerases; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases