Transcriptional Regulation of Thrombotic Microangiopathy in the Renal Microvasculature

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Renal-specific thrombotic microangiopathy (TMA) represents the most severe manifestation of renal endothelial injury consisting of endothelial cell (EC) swelling, subEC expansion, inflammation and microthrombi and untreated progresses uniformly to chronic kidney disease. TMA occurs subsequent to various etiologies including hemolytic uremic syndrome, anti-vascular endothelial growth factor (VEGF) therapy, malignant hypertension and antibody mediated rejection after transplant, and despite diverse causes, is typically associated with dysregulation of key thrombotic and inflammatory EC transcripts and complement activation. Furthermore, expression of membrane bound (DAF, CD59, CD46) complement regulators, is altered in many subtypes of TMA and it is unclear whether this is a driver or consequence of injury. To investigate the mechanism(s) by which complement activation and EC transcripts are dysregulated in TMA, we reviewed expression arrays of RNA- sequencing from kidney biopsies with TMA, and observed that Krüppel-Like Factor 4 (KLF4), a zinc finger transcription factor, is the highest differentially expressed transcript. Previous studies demonstrate that KLF4 is a critical mediator of anti-thrombotic and anti-inflammatory phenotype in systemic vascular beds, but its role modulating renal microvascular injury and complement activation in TMA remains to be investigated. Based on this and our preliminary data, my central hypothesis, that EC-KLF4 is required to prevent complement-dependent and independent renal microvascular EC injury in TMA, will be investigated by the following specific aims: 1) Determine the renoprotective role of EC-specific KLF4 in TMA; 2) Test the hypothesis that KLF4-DAF interaction is required to mitigate complement activation in TMA; and 3) Investigate the mechanism(s) by which KLF4 attenuates renal microvascular EC injury in TMA. Under subaim 1A, we will determine whether mice with the inducible loss of EC-Klf4 (iKlf4ΔEC) have accelerated renal EC injury and complement activation using two murine TMA models (anti-VEGFR2 Ab and Shiga toxin). In subaim 1B, the protective role of KLF4 will be determined using mice with EC-overexpression of KLF4 subjected to VEGFR2 inhibition. Extent of injury will be evaluated by histology, ultrastructure and functional measures, as well as inflammatory and thrombotic transcripts and complement activation. In subaim 2.A., we will evaluate the mechanism of interaction between KLF4 and DAF using ChIP assay and luciferase reporter. In subaim 2.B., we will test whether mice with EC-Daf knockdown have increased susceptibility to EC complement activation by treating them with a VEGFR2 inhibitor and crossing them with Klf4ΔEC mice in two experiments. In subaim 2.C, we will perform IF for DAF and C3 in human biopsies TMA specimens to corroborate our findings. Finally, in aim 3, we will investigate the mechanism(s) by which KLF4 attenuates EC injury via complement dependent (subaim 3.A), and independent (subaim 3.B) manner using small-molecule C5aR and PAI-1 inhibitors in primary glomerular ECs and human microvascular ECs. This project will be carried out at the Northport Veterans Affairs Medical Center (NVAMC) under the primary mentorship of Dr. Sandeep Mallipattu (Chief of Nephrology, Stony Brook Medicine (SBM) and Staff Nephrologist NVAMC), an expert in glomerular diseases and co-mentorship of Dr. Vincent Yang (Chair of Medicine at SBM), an expert in KLF pathobiology, and Dr. Berhane Ghebrehiwet, an expert in complement. All mentors are experienced, federally funded investigators and will guide my career development along with an external advisory committee. The completion of proposed project and career development plan will allow the fulfillment of my short-term goal of uncovering mechanisms of glomerular EC sensitivity to complement activation and injury and long-term goals of becoming a productive, independent physician-scientist, practicing nephrology (25%) and performing translational and basic science (75%), at the NVAMC. In addition to robust clinical training, I have the necessary research foundation to fulfill these goals including: 1) Post-doctoral nephrology research training 2) Masters in Clinical Research 3) Demonstrated publication and grant application success.