Transcriptional and epigenetic control of angiogenic genes in sepsis-induced acute kidney injury.

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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.