Mitochondria-Targeted Antioxidant Peptide SS31 Prevents Hypoxia/Reoxygenation- Induced Apoptosis by Down-Regulating p66Shc in Renal Tubular Epithelial Cells

Background/Aims: Ischemia/reperfusion injury plays a crucial role in renal transplantation and represents a significant risk factor for acute kidney injury and delayed graft function. Mitochondria-targeted antioxidant peptide SS31 has been shown to attenuate ischemia/ reperfusion injury by inhibiting oxidative stress. The present study was carried out to investigate whether the pretreatment of SS31 could reduce hypoxia/reoxygenation (H/R)-induced injury by inhibiting p66Shc. Methods: The cultured rat renal proximal tubular cell line NRK52E cells were exposed to 24 h hypoxia (5% CO2, 1% O-2, 94% N-2) followed by 6 h reoxygenation (5% CO2, 21% O-2, 74% N-2). SS31 was added to the culture medium 4 h prior to the treatment. Then the cell viability, apoptosis, and oxidative stress levels were determined. In addition, western blot analysis was performed to determine the expression of p66Shc, p-p66Shc, cytochrome c, and caspase-3. Results: H/R induced apoptotic cell death, accompanied with activation of total and p-p66Shc in NRK52E cells. Pretreatment with SS31 or overexpression of a dominant-negative Ser36 mutant p66Shc (p66Shc S36A) or p66Shc siRNA prevented cell death, whereas the protection effect of SS31 was completely blocked by overexpression of wild-type p66Shc. Furthermore, SS31 pretreatment reduced H/R-induced intracellular oxidative stress, cytochrome c translocation to the cytoplasm, and caspase-3 activation through inhibiting p66Shc. Conclusion: This study revealed that SS31 pretreatment serves a protective role against H/R-induced apoptosis of human renal tubular epithelial cells, and the mechanism is related to suppression of p66Shc. Copyright (C) 2013 S. Karger AG, Basel