Transcriptional Regulation of Thrombotic Microangiopathy in the Renal Microvasculature

肾微血管中血栓性微血管病的转录调控

• 批准号: 10512050
• 负责人: Chelsea Estrada
• 金额: --
• 依托单位: NORTHPORT VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-10-01 至 2026-09-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10512050
• 关键词: Acceleration; Advisory Committees; Age; Albuminuria; Anti-Inflammatory Agents; Applications Grants; Attenuated; Basic Science; Binding; Binding Sites; Biopsy; Biopsy Specimen; Blood Vessels; CD46 Antigen; CD55 Antigens; Chronic Kidney Failure; Clinical Research; Complement; Complement Activation; Complement Membrane Attack Complex; Cre lox recombination system; Data; Development Plans; Disease; End stage renal failure; Endothelial Cells; Endothelium; Etiology; Exhibits; Foundations; Funding; GKLF protein; Gene Chips; Genes; Genetic Transcription; Glomerular Capillary; Goals; Growth Factor Inhibition; Hemolytic-Uremic Syndrome; Histologic; Histology; Homeostasis; Human; Inflammation; Inflammatory; Injury; KDR gene; Kidney; Kidney Diseases; Kidney Transplantation; Laboratories; Luciferases; Maintenance; Malignant Hypertension; Measures; Mediating; Mediator; Medical center; Medicine; Membrane; Mentors; Mentorship; Modeling; Molecular; Mus; NOS3 gene; Nephrology; Nitric Oxide Synthase; Pathology; Pathway interactions; Phenotype; Physicians; Plasminogen Activator Inhibitor 1; Postdoctoral Fellow; Predisposition; Prevalence; Productivity; Promoter Regions; Publications; Regulation; Renal glomerular disease; Reporter; Research; Research Personnel; Research Training; Role; Scientist; Shiga Toxin; Shiga-Like Toxin II; Specimen; Swelling; Testing; Thrombus; Transcript; Transcriptional Regulation; Translational Research; Transplantation; United States Department of Veterans Affairs; Vascular Cell Adhesion Molecule-1; Vascular Endothelial Cell; Vascular Endothelial Growth Factors; Vascular Endothelium; Wild Type Mouse; Zinc Fingers; antibody-mediated rejection; cadherin 5; career development; cell injury; chromatin immunoprecipitation; clinical training; differential expression; experience; experimental study; genetic regulatory protein; glomerular endothelium; human RNA sequencing; in silico; inhibitor; insight; kidney biopsy; knock-down; military veteran; mouse model; new therapeutic target; novel; overexpression; pharmacologic; prevent; promoter; response to injury; small molecule; success; thrombotic; transcription factor; transcriptome sequencing; vascular bed; vascular injury; von Willebrand Factor

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.

肾特异性血栓性微血管病（TMA）是肾内皮细胞增生最严重的表现， 损伤包括内皮细胞（EC）肿胀、亚EC扩张、炎症和微血栓， 未经治疗的患者均会发展为慢性肾病。TMA发生于各种病因之后，包括 溶血性尿毒综合征、抗血管内皮生长因子（VEGF）治疗、恶性高血压和 移植后抗体介导的排斥反应，尽管原因多种多样，但通常与 关键血栓形成和炎性EC转录物的失调和补体激活。此外，委员会认为， 在许多TMA亚型中，膜结合补体调节因子（CD 46、CD 59、CD 46）的表达改变 目前还不清楚这是司机还是受伤的后果。研究以下机制： 补体激活和EC转录在TMA中失调，我们综述了RNA- 从肾活检组织中用TMA进行测序，并观察到Krüppel样因子4（KLF 4），一种锌指蛋白， 转录因子，是差异表达最高的转录本。以前的研究表明，KLF 4是 是全身血管床抗血栓形成和抗炎表型的关键介质，但其作用 在TMA中调节肾微血管损伤和补体激活仍有待研究。基于 这和我们的初步数据，我的中心假设，EC-KLF 4是必要的，以防止补体依赖性 和独立的肾微血管EC损伤的TMA，将通过以下具体目的进行研究：1） 确定EC特异性KLF 4在TMA中的肾保护作用; 2）检验KLF 4-KLF 4相互作用的假设， 是缓解TMA中补体激活所必需的;和3）研究KLF 4 减轻TMA中的肾微血管EC损伤。在子目标1A下，我们将确定是否具有 EC-Klf 4（iKlf 4 ΔEC）诱导性缺失加速了肾EC损伤和补体激活，使用两种小鼠 TMA模型（抗VEGFR 2 Ab和滋贺毒素）。在子目标1B中，将确定KLF 4的保护作用 使用EC过表达KLF 4的小鼠进行VEGFR 2抑制。将评估损伤程度 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物， 补体激活在分目标2.A.中，我们将评估KLF 4和KLF之间的相互作用机制， 使用ChIP测定和荧光素酶报告子。在分目标2.B.中，我们将测试EC-Daf基因敲低的小鼠 通过用VEGFR 2抑制剂治疗增加了对EC补体激活的敏感性， 在两个实验中，Klf 4 ΔEC小鼠。在子目标2.C中，我们将对人类活检组织中的C3和C4进行IF TMA样本来证实我们的发现。最后，在目标3中，我们将研究 KLF 4通过补体依赖性（子目的3.A）和非依赖性（子目的3.B）方式减轻EC损伤 在原代肾小球EC和人微血管EC中使用小分子C5 aR和派-1抑制剂。 该项目将在北港退伍军人事务医疗中心（NVAMC）进行， Sandeep Mallipattu博士（斯托尼布鲁克医学部（SBM）肾脏科主任和工作人员）的主要指导 肾脏病学家NVAMC），肾小球疾病专家，并与Vincent Yang博士（ KLF病理生物学专家Berhane Ghebrehiwet博士和补体专家Berhane Ghebrehiwet博士。所有 导师是经验丰富的，联邦资助的调查人员，将指导我的职业发展沿着 外部咨询委员会。完成拟议的项目和职业发展计划将使 实现了我的短期目标，即揭示肾小球EC对补体激活的敏感性机制 和伤害和长期目标，成为一个富有成效的，独立的医生，科学家，从事肾脏病学 (25%）和执行转化和基础科学（75%），在NVAMC。除了强大的临床培训， 我有必要的研究基础，以实现这些目标，包括：1）博士后肾脏病学研究 培训2）临床研究硕士3）证明出版和资助申请成功。