Partial attenuation of cytotoxicity and apoptosis by SOD1 in ischemic renal epithelial cells.
Reactive oxygen species (ROS) contribute significantly to apoptosis in renal ischemia-reperfusion (IR) injury, however the exact mechanisms are not well understood. We used novel lentiviral vectors to over-express superoxide dismutase 1 (SOD1) in proximal tubular epithelial (LLC-PK(1)) cells and determined effects of SOD1 following ATP depletion-recovery, used as a model to simulate renal IR. SOD1 over-expression partially protected against cytotoxicity (P < 0.001) and decreased superoxide (O(2) (*-)) in ATP depleted cells. The ATP depletion-mediated increase in nuclear fragmentation, an index of apoptosis and activation of caspase-3 was also partially blocked by SOD1 (P < 0.05). However, SOD1 over-expression was insufficient to completely attenuate caspase-3, indicating that ROS other than cytoplasmic O(2) (*-) are involved in ATP depletion mediated injury. To test the contribution of hydrogen peroxide, a subset of enhanced green fluorescent protein (EGFP) and SOD1 (serum free and injured) cells were treated with polyethylene glycol-catalase (PEG-catalase). As expected there was 50% reduction in cytotoxicity and caspase-3 in SOD1 cells compared to EGFP cells; catalase treatment decreased both indices by an additional 28% following ATP depletion. To test the role of mitochondrial derived superoxide, we also treated a subset of LLC-PK(1) cells with the mitochondrial antioxidant, MitoTEMPO. Treatment with MitoTEMPO also decreased ATP depletion induced cytotoxicity in LLC-PK(1) cells in a dose dependant manner. These studies indicate that both SOD1 dependent and independent pathways are integral in protection against ATP depletion-recovery mediated cytotoxicity and apoptosis, however more studies are needed to delineate the signaling mechanisms involved.
Liang
Huan Ling
HL
Division of Transplant Surgery, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Arsenault
Jody
J
Mortensen
Jordan
J
Park
Frank
F
Johnson
Christopher P
CP
Nilakantan
Vani
V
D000255:Adenosine Triphosphate / Q000172:deficiency; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3 / Q000378:metabolism; D002374:Catalase / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology*; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D007511:Ischemia / Q000201:enzymology*; D007687:Kidney Tubules, Proximal / Q000098:blood supply* / Q000166:cytology*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D018374:LLC-PK1 Cells; D016086:Lentivirus / Q000235:genetics; D010880:Piperidines / Q000494:pharmacology; D015203:Reproducibility of Results; D013482:Superoxide Dismutase / Q000378:metabolism*; D000072105:Superoxide Dismutase-1; D013481:Superoxides / Q000378:metabolism; D013552:Swine; D013997:Time Factors
C528764:2,2,6,6-tetramethyl-1-piperidine; D010880:Piperidines; C000606290:SOD1 protein, human; C485184:enhanced green fluorescent protein; D013481:Superoxides; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide; D007770:L-Lactate Dehydrogenase; D002374:Catalase; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D053148:Caspase 3
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