Identifying Novel Therapies to Reduce Injury following Ischemia Reperfusion in Kidney

Renal ischemia reperfusion injury (IRI) is associated with early inflammation, including neutrophil infiltration, that exacerbates the initial ischemic insult. The molecular pathways involved in these responses are poorly characterized and there is no treatment targeting these early events. We first performed a proteomic analysis of porcine kidneys biopsies collected during transplantation to identify early changes in protein expression following IRI. Systems biology analysis identified animportant role of inflammation, specifically pathways converging on NFκB. Normothermic exvivo kidney perfusion of kidneys during transplantation attenuated inflammation. Based on these findings, we then performed an in silico analysis and confirmed marked changes in NFκB-mediated gene expression in early renal IRI. We then evaluated the effect of NFκB-blockade with a BRD4 inhibitor on neutrophil adhesion to endothelial cells in vitro prior to testing the effect of BRD4 inhibition in an in vivo murine IRI model. BRD4 inhibition attenuated TNFα-induced and hypoxia re-oxygenation-induced neutrophil capture and adhesion to endothelial cells in vitro, and improved cell viability following hypoxia re-oxygenation. In vivo, IRI led to increased expression of pro-inflammatory cytokines and adhesion molecules at 6 hours post-IRI with sustained upregulated expression up to 48 hours post-IRI. These effects were attenuated, in part, by pretreatment with the BRD4 inhibitor. Flow cytometry analysis showed absolute neutrophil counts increased significantly in the bone marrow, blood, and kidney 24 hours post-IRI. Activated neutrophils also increased in the blood and kidney as early as 6 hours post-IRI and remained elevated at 24 and 48 hours-post IR. BRD4 inhibition reduced both total and activated neutrophil counts in the kidney. IR-induced tubular injury correlated with the neutrophil infiltration and was reduced by BRD4 inhibition. In summary, NFκB signaling is the primary pathway upregulated following SCS, and targeting NFκB with a BRD4 inhibitor reduces neutrophil infiltration, neutrophil activation, and tubular cell injury in kidneys subjected to IRI. BRD4 inhibition may represent a new therapeutic approach to the management of acute kidney injury.Ph.D