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Partial attenuation of cytotoxicity and apoptosis by SOD1 in ischemic renal epithelial cells

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Abstract

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-PK1) 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 (O2 •−) 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 O2 •− 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-PK1 cells with the mitochondrial antioxidant, MitoTEMPO. Treatment with MitoTEMPO also decreased ATP depletion induced cytotoxicity in LLC-PK1 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.

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Abbreviations

EGFP:

Enhanced green fluorescent protein

IM:

Injury media

IR:

Ischemia-reperfusion

LDH:

Lactate dehydrogenase

PBS:

Phosphate-buffered saline

ROS:

Reactive oxygen species

SOD:

Superoxide dismutase

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Acknowledgments

We greatly appreciate the gift of human SOD1 cDNA from Dr. Charles Epstein (University of California, San Francisco). We are also grateful to Dr. Balaraman Kalyanaraman, Jacek Zielonka and the Free Radical Research Center at the Medical College of Wisconsin for help with measurement of 2-OH-E+. This work was supported in part by a pilot and feasibility project in an NIH P50 (1DK-079306-01), an American Heart Association-Scientist Development Grant (0930326G) and departmental funds to V. Nilakantan.

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  1. Jody Arsenault

    Present address: Eli Lilly, Indianapolis, IN, USA

Authors and Affiliations

  1. Division of Transplant Surgery, Medical College of Wisconsin, Milwaukee, WI, 53226, USA

    Huan Ling Liang, Jordan Mortensen, Christopher P. Johnson & Vani Nilakantan

  2. Kidney Disease Center, H4135, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, 53226, USA

    Huan Ling Liang, Jordan Mortensen, Frank Park, Christopher P. Johnson & Vani Nilakantan

  3. Department of Physiology, Medical College of Wisconsin, Milwaukee, WI, 53226, USA

    Frank Park

  4. VA Medical Center, Medical College of Wisconsin, Milwaukee, WI, 53226, USA

    Christopher P. Johnson

  5. Department of Medicine (Division of Nephrology), Medical College of Wisconsin, Milwaukee, WI, 53226, USA

    Frank Park

  6. Department of Pharmacology, LSU Health Sciences Center, New Orleans, LA, USA

    Jody Arsenault

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  1. Huan Ling Liang
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Correspondence to Vani Nilakantan.

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Liang, H.L., Arsenault, J., Mortensen, J. et al. Partial attenuation of cytotoxicity and apoptosis by SOD1 in ischemic renal epithelial cells. Apoptosis 14, 1176–1189 (2009). https://doi.org/10.1007/s10495-009-0393-z

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