title: De novo demonstration and co-localization of free-radical production and apoptosis formation in rat kidney subjected to ischemia/reperfusion.
abstract: 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.
authors.affiliation: Office for Medical Research Administration, National Taiwan University Hospital, Taipei, Taiwan.
authors.forename: Chiang-Ting
authors.initials: CT
authors.lastname: Chien
authors.affiliation: Office for Medical Research Administration, National Taiwan University Hospital, Taipei, Taiwan.|Department of Surgery, National Taiwan University College of Medicine, Taipei, Taiwan.
authors.forename: Po-Huang
authors.initials: PH
authors.lastname: Lee
authors.affiliation: Department of Physiology, National Taiwan University College of Medicine, Taipei, Taiwan.
authors.forename: Chau-Fong
authors.initials: CF
authors.lastname: Chen
authors.affiliation: Department of Physiology, National Taiwan University College of Medicine, Taipei, Taiwan.
authors.forename: Ming-Chieh
authors.initials: MC
authors.lastname: Ma
authors.affiliation: Department of Urology, National Taiwan University College of Medicine, Taipei, Taiwan.
authors.forename: Ming-Kuen
authors.initials: MK
authors.lastname: Lai
authors.affiliation: Department of Pathology, National Taiwan University College of Medicine, Taipei, Taiwan.
authors.forename: Su-Ming
authors.initials: SM
authors.lastname: Hsu
mesh_terms: 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
chemical_list: 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