P38MAPK Inhibition Prevents Impairment of Kidney Function Resulting from Ischemia/Reperfusion Injury (IRI)

Abstract

Background: In the course of solid organ transplantation excessive production of reactive oxygen species (ROS) is a major contributor to the development of ischemia/reperfusion injury (IRI). Preventing ROS actions through the use of antioxidants so far proved unsatisfactory in the clinical setting, most likely due to the difficulty to timely and efficiently target these substances to the site of ROS production and action. In various in vivo (IR) and in vitro (hypoxia/reoxygenation, HR) models we observed a consistent pattern in the activation of key intracellular signaling pathways. We recently also obtained evidence, which linked the activation of p38MAPK to mitochondrial ROS accumulation and cell death during hypoxia/reoxygenation (HR). These data suggest that mitochondrial changes preceding the onset of cell death are subject to regulation by intracellular signaling pathways. This insight could be therapeutically exploited for the prevention of IR-induced organ damage. Here we further dissected the contribution of p38 to IR- and HR-induced damage and provide first evidence for a therapeutic benefit of p38 inhibition in vivo. Methods: Kidney transplantation and kidney clamping in the rat were used for the induction of IRI. HR was predominantly analyzed in HL-1 cardiomyocytes and primary MEFs. Intracellular signaling was monitored by using phosphorylation-specific antibodies. Mitochondrial ROS levels were determined by imaging of cells pre-loaded with Mitotracker Red CM-H2XROS. ROS/NOS-induced tissue damage was visualized by 3-nitrotyrosine specific antibodies. To assess acute kidney injury HSP70 expression was monitored by immunoblotting, serum creatinine and urea were measured in the clinical routine lab, and serum cystatin c and NGAL concentrations were determined by ELISA. Expression of cytokines and oxidative stress markers was measured by real time qPCR. Apoptosis of cells during IRI was assessed by TUNEL assay. Results: In the work presented here we provide further evidence for the involvement of p38 in causing redox stress and suggest MAPKAP-kinase 2 (MK2) as a downstream effector. Reperfusion following kidney clamping or transplantation was marked by a profound increase in the activity of p38 and the putative effector MK2 which was significantly prevented by the p38 inhibitor BIRB-796. p38 inhibition almost completely prevented functional impairment caused by IR during kidney clamping as measured by reduced serum creatinine, urea, cystatin c and NGAL levels. P38 inhibition also protected from oxidative damage and significantly reduced the percentage of apoptotic cells and the expression of TNFα and HO-1 during IR. Most importantly, a significant reduction of functional impairment was also achieved in the kidney transplant model. Conclusion: Inhibiting p38 signaling during IR may provide a potent strategy for limiting IRI. Supported by funds from the FWF and the Jubiliäumsfond OeNB.