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: 10253029
• 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/10253029
• 关键词: Address; Advisory Committees; Age; Albuminuria; Anti-Inflammatory Agents; Applications Grants; Attenuated; Basic Science; Binding Sites; Biopsy; Biopsy Specimen; CD46 Antigen; CD55 Antigens; Chronic Kidney Failure; Clinical; Clinical Research; Complement; Complement 3; Complement Activation; Complement Membrane Attack Complex; Congestive; 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 Transplantation; Laboratories; Luciferases; Maintenance; Malignant Hypertension; Measures; Mediating; Mediator of activation protein; Medical center; Medicine; Membrane; Mentors; Mentorship; Modeling; Molecular; Mus; NOS3 gene; Nephrology; Nitric Oxide Synthase; Pathology; Pathway interactions; Pharmacology; Phenotype; Physicians; Plasminogen Activator Inhibitor 1; Postdoctoral Fellow; Predisposition; Prevalence; Promoter Regions; Publications; Regulation; Renal glomerular disease; Reporter; Research; Research Personnel; Research Training; Role; Scientist; Shiga Toxin; Shiga-Like Toxin II; Specimen; Swelling; Testing; Training; Transcript; Transcriptional Regulation; Translational Research; Transplantation; United States Department of Veterans Affairs; Vascular Cell Adhesion Molecule-1; Vascular Endothelial Growth Factors; Wild Type Mouse; Yang; Zinc Fingers; antibody-mediated rejection; base; cadherin 5; career development; cell injury; chromatin immunoprecipitation; 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; prevent; promoter; response to injury; small molecule; success; thrombotic; transcription factor; transcriptome sequencing; vascular bed; 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）是肾内皮细胞疾病最严重的表现 损伤包括内皮细胞 (EC) 肿胀、EC 下扩张、炎症和微血栓， 未经治疗，会因各种病因（包括）而发展为慢性肾病。 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 移植后抗体介导的排斥反应，尽管原因多种多样，但通常与 关键血栓和炎症 EC 转录物和补体激活的失调。 膜结合（DAF、CD59、CD46）补体调节因子的表达在许多 TMA 亚型中发生改变 目前尚不清楚这是否是伤害的驱动因素或后果，以调查其机制。 补体激活和 EC 转录本在 TMA 中失调，我们回顾了 RNA-的表达阵列 使用 TMA 对肾活检进行测序，并观察到 ​​Krüppel 样因子 4 (KLF4)（一种锌指） 转录因子，是差异表达最高的转录因子。 全身血管床中抗血栓和抗炎表型的关键介质，但其作用 TMA 中微血管损伤和补体激活的调节仍有待研究。 这个和我们的初步数据，我的中心假设，需要 EC-KLF4 来预防补体依赖性 和 TMA 中独立的肾微血管 EC 损伤，将通过以下具体目标进行研究：1） 确定 TMA 中 EC 特异性 KLF4 的肾脏保护作用；2) 检验 KLF4-DAF 相互作用的假设； 需要减轻 TMA 中的补体激活；以及 3) 研究 KLF4 的机制 减轻 TMA 中的肾微血管 EC 损伤 在子目标 1A 下，我们将确定小鼠是否患有 使用两种小鼠模型诱导的 EC-Klf4 (iKlf4ΔEC) 损失加速了肾 EC 损伤和补体激活 TMA 模型（抗 VEGFR2 Ab 和志贺毒素） 在 subaim 1B 中，将确定 KLF4 的保护作用。 使用受到 VEGFR2 抑制的 EC 过表达 KLF4 的小鼠来评估损伤程度。 通过组织学、超微结构和功能测量，以及炎症和血栓转录本和 在subaim 2.A.中，我们将评估KLF4和DAF之间的相互作用机制 使用 ChIP 测定和荧光素酶报告基因在 subaim 2.B. 中，我们将测试 EC-Daf 敲低的小鼠。 通过使用 VEGFR2 抑制剂治疗并交叉治疗，增加了 EC 补体激活的易感性 在 subaim 2.C 中，我们将使用 Klf4ΔEC 小鼠对人体活检中的 DAF 和 C3 进行 IF。 TMA 样本证实了我们的发现。最后，在目标 3 中，我们将研究其机制。 KLF4 通过补体依赖性 (subaim 3.A) 和独立 (subaim 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 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) 下进行 Sandeep Mallipattu 博士（石溪医学 (SBM) 肾病科主任）及其工作人员的主要指导 肾病专家（NVAMC），肾小球疾病专家，杨文森博士（主席）的共同指导 SBM 医学）、KLF 病理学专家和补体专家 Berhane Ghebrehiwet 博士。 导师是经验丰富的联邦资助的调查员，他们将指导我的职业发展以及 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：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富有成效的、独立的医师科学家、从事肾脏病学的长期目标 (25%) 并在 NVAMC 进行转化和基础科学 (75%) 除了强大的临床培训之外， 我拥有实现这些目标所需的研究基础，包括：1）博士后肾病学研究 培训 2) 临床研究硕士 3) 发表论文和资助申请成功。