Transcriptional Regulation of Thrombotic Microangiopathy in the Renal Microvasculature

肾脏特异性血栓性微血管病变(TMA)是肾内皮细胞最严重的表现 损伤包括内皮细胞(EC)肿胀、亚EC扩张、炎症和微血栓以及 未经治疗，均可发展为慢性肾脏疾病。TMA的发生有多种原因，包括 溶血性尿毒症综合征、抗血管内皮生长因子(VEGF)治疗、恶性高血压和 尽管原因多种多样，但移植后抗体介导的排斥反应通常与 关键的血栓和炎症性EC转录本的失调和补体激活。此外， 膜结合(DAF、CD59、CD46)补体调节因子在许多亚型TMA中的表达发生改变 目前还不清楚这是司机还是受伤的后果。探讨其作用机制(S)。 补体激活和EC转录本在TMA中异常调节，我们综述了RNA-RNA表达阵列。 用TMA对肾活检组织进行测序，并观察到Krüppel样因子4(KLF4)，一个锌指 转录因子，是差异表达最高的转录本。以往的研究表明，KLF4是 全身血管床抗血栓和抗炎表型的关键介质及其作用 TMA对肾微血管损伤和补体激活的调节作用仍有待研究。基于 这和我们的初步数据，也就是我的中心假设，EC-KLF4是防止补体依赖所必需的 以及TMA中独立的肾微血管内皮细胞损伤，将通过以下特定目的进行调查：1) 确定EC特异性KLF4在TMA中的肾脏保护作用；2)检验KLF4-DAF相互作用的假说 以减轻TMA的补体活化；3)研究KLF4的机制(S 减轻TMA大鼠肾微血管内皮细胞损伤。在次级目标1A下，我们将确定小鼠是否具有 两只小鼠肾内皮细胞诱导性缺失KLF4(iKLF4ΔEC)加速肾内皮细胞损伤和补体活化 TMA模型(抗VEGFR2抗体和志贺毒素)。在次级目标1B中，将确定KLF4的保护作用 使用内皮细胞高表达KLF4的小鼠接受VEGFR2抑制。将对受伤程度进行评估 通过组织学、超微结构和功能测量以及炎症和血栓形成转录和 补体激活。在次级目标2.A.中，我们将评估KLF4和DAF之间的相互作用机制 使用芯片分析和荧光素酶报告。在SubAim 2.B.中，我们将测试EC-Daf基因敲除的小鼠 通过使用VEGFR2抑制剂和交叉处理来增加对EC补体激活的易感性 他们与KLF4ΔEC小鼠进行了两次实验。在SubAim 2.C中，我们将对人类活检组织中的DAF和C3执行IF TMA样本来证实我们的发现。最后，在目标3中，我们将探讨(S)通过 KLF4通过补体依赖(亚目标3.A)和非依赖(亚目标3.B)方式减轻EC损伤 在原代肾小球内皮细胞和人微血管内皮细胞中使用小分子C5aR和PAI-1抑制剂。 该项目将在诺斯波特退伍军人事务医疗中心(NVAMC)根据 桑迪普·马利帕图医生(石溪医学(SBM)肾病科主任)和工作人员的主要指导 肾病学家NVAMC)，肾小球疾病专家和文森特·杨博士的共同指导(主席 KLF病理生物学专家)和补体专家Berhane Ghebrehiweet博士。全 导师是经验丰富的联邦政府资助的调查人员，他们将指导我的职业发展 外部咨询委员会。拟议项目和职业发展计划的完成将使 实现我的短期目标，即揭示肾小球EC对补体激活的敏感性机制 和伤害，以及成为一名多产的、独立的内科科学家、从事肾脏病治疗的长期目标 (25%)和从事翻译和基础科学(75%)，在NVAMC。除了强大的临床培训外， 我有必要的研究基础来完成这些目标，包括：1)博士后肾脏病研究 培训2)临床研究硕士3)展示发表和资助申请的成功。

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.