Sepsis-induced acute kidney injury (AKI) is the most common and life-threatening cause of renal injury in critically ill patients. And yet, there have been no improvements in the treatment of septic AKI in decades. Septic AKI is distinct from non-septic AKI; notably - microcirculatory dysfunction manifested by low blood flow, endothelial cell (EC) activation and vascular leak, play a prominent pathologic roles. The microvasculature consists of luminal EC and pericytes, which encircle the abluminal endothelial wall. The EC receptor tyrosine kinase, Tie-2 (TEK), and its two ligands, angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2), (i.e., Ang-Tie-2 axis) regulate microvasculature. Pericytes are the primary source of Ang-1, which maintains EC quiescence via Tie-2 signaling. Tie-2 expression/signaling is, part, regulated by blood flow, a process that involves transcription factor, Klf2. Tie-2 and Ang-1 gene expression is downregulated in septic kidney, contributing to EC dysfunction. Changes in Tie-2/ Ang-1 expression are associated with epigenetic alterations and loss of Klf2 at these loci. We will test the hypothesis that sepsis-induced Klf2 disengagement from Ang-1 and Tie-2 genes alters dynamic network(s) of transcription and epigenetic factors interacting along Ang-1 and Tie-2 loci which down-regulates their transcription and contributes to endothelial leak. Aim #1. To define kinetics of transcription/epigenetic network changes associated with disengagement of Klf2 from renal Ang-1 and Tie-2 genes in mouse models of sepsis. Correlating kinetics of sepsis-induced transcription/epigenetic alterations at the renal Ang-1 and Tie-2 genes with progression to endothelial leak will identify Klf2-dependent and -independent interactions that will be tested in vitro (Aims #2-3) for their role inTie-2 and Ang-1 expression. Aim #2. To use EC and pericyte cultures to define which interactions of chromatin-bound proteins act (additively, synergistically or antagonistically) to regulate Tie-2 and Ang-1 transcription. Mechanism of Tie-2 and Ang-1 repression will be studied by knocking down/inhibiting candidate factors (e.g., HDACs) tethered to these loci. Aim #3. To characterize which of transcription/epigenetic factor interactions at Tie-2 and Ang-1 loci are responsive to flow/inflammatory mediators and regulate microvascular barrier in in vitro 3D- flow microvessels. We will take advantage of our synthetic human kidney microvessels that model endothelial leak to identify flow-responsive transcription/epigenetic interactions that regulate Ang-1 and Tie-2 genes. We have recently demonstrated, previously unanticipated, epigenetic heterogeneity and uniqueness of gene responses during AKI. Thus, defining key transcription/epigenetic network components engaged at Ang-1 and Tie-2 genes as potential drug targets will provide translational basis for future testing combinatorial rationally- designed pharmacologic interventions to mitigate microvascular leak and kidney injury during sepsis.

Sepsis-induced acute kidney injury (AKI) is the most common cause of renal injury in critically ill patients. Septic AKI is distinct from non-septic AKI, notably, microcirculatory dysfunction manifested by low blood flow, endothelial cell activation and vascular leak, play uniquely prominent pathologic roles. Septic AKI is associated with epigenetic changes and repression of the angiopoietin Ang-Tie-2 axis genes. We propose to test the hypothesis that sepsis alters network(s) of synergistic and antagonistic transcription factors/epigenetic interaction along renal Ang-1 and Tie-2 loci down-regulating their transcription which results in vascular leak.