Inhibition of Stress Kinase-Dependent Pro-Oxidant Signaling as a Novel Approach for the Prevention of Ischemia/reperfusion Injury (IRI).

Background: Excessive production of reactive oxygen species (ROS) during early reperfusion is causal for the development of IRI during organ transplantation. Antioxidants proved ineffective in the clinical setting and we thus propose approaches, which a priori prevent the increase in cellular ROS. Reperfusion is also characterized by the activation of the stress kinases p38 and JNK. We recently obtained evidence that activated p38 leads to elevated mitochondrial ROS levels and cardiomyocyte death in vitro. Under these conditions also p66SHC translocates to the mitochondria to generate ROS. Phosphorylation of serine 36 on p66SHC is required for mitochondrial translocation, ROS production and cell death and p66SHC ablation prevented IRI in the Langendorff-perfused heart. Here we studied both kinases as possible p66SHC activators. Moreover, we tested the benefit of inhibiting p38 in in vivo models of rat kidney clamping and rat kidney transplantation. Methods: We employed in vitro (cardiomyocytes, MEFs) and in vivo (rat kidney clamping and transplantation) models of ischemia/reperfusion (IR). Kinase function was blocked by small molecule inhibitors or siRNAs. Outcomes were monitored in terms of ROS-induced damage, cell death and organ function. Results: Simulated IR (sIR) results in the activation of p38 and JNK, phosphorylation of p66SHC, and increased ROS production. However, only the inhibition of JNK blocked p66SHC phosphorylation and decreased ROS production, suggesting that the pro-oxidant function of p38 occurs through p66SHC-indpendent mechanism. p38 activation also plays an important role in vivo. In the kidney clamping experiments p38 inhibition almost completely prevented severe functional impairment caused by IR. Using p38 inhibitor as a single agent a significant improvement was also observed in the setting of kidney transplantation. Histological and molecular analyses suggested that protection resulted from decreased redox stress and apoptotic cell death. Conclusion: Interference with p38 and JNK/p66SHC signaling may provide a therapeutic approach for decreasing damage to cells and organs in the setting of IR.