Glomerular Cell-Cell Crosstalk and Injury

肾小球细胞间的串扰和损伤

基本信息

批准号：
9234528
负责人：
Ilse Sofia Daehn
金额：
$ 36.87万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-04-15 至 2018-09-19
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9234528
关键词：
Affect Albumins Albuminuria American Animal Model Apoptosis Biopsy Cell Culture Techniques Cell Line Cells Characteristics Chronic Kidney Failure Coculture Techniques Conditioned Culture Media Creatinine Development Dicer Enzyme Disease Disease Progression Doxycycline Endothelial Cells Endothelin-1 Focal Segmental Glomerulosclerosis Foot Process Functional disorder Health Expenditures Homeostasis Human In Vitro Injury Kidney Knock-out Lead Lesion Ligands Mediating MicroRNAs Mitochondria Modeling Molecular Morphology Mus Oxidative Stress Phenotype Phosphotransferases Public Health Receptor Signaling Recovery Renal glomerular disease Research Sclerosis Signal Transduction Small Interfering RNA Stress Response Signaling Study models Superoxides System Transgenic Mice Up-Regulation Urine WT1 gene Withdrawal base burden of illness cell injury cell type endothelial dysfunction experimental study glomerulosclerosis in vivo inducible gene expression inhibitor/antagonist knock-down mitochondrial dysfunction mouse model mutant novel phenotypic biomarker podocyte prevent public health relevance receptor response therapeutic target

项目摘要

DESCRIPTION (provided by applicant): Segmental sclerosis is a characteristic lesion defining both primary and secondary glomerulosclerosis. In most diseases causing glomerulosclerosis, Tgfb expression in podocytes is a stress response signal associated with sclerosis lesions. To define precisely how activation of Tgfb type I receptor (TbrI) signaling in podocytes affects glomerular cells in vivo, we generated a transgenic mouse model (PodTbrI(AAD) that enables conditional (Doxycycline-inducible) expression of a constitutively-active TbrI mutant (TbrI(AAD). When TbrI(AAD) signaling is activated in podocytes, mice eventually develop albuminuria and subsequently progressive glomerulosclerosis associated with podocyte depletion. However, podocyte morphology remains normal while podocytes initially release endothelin 1 (Edn1) to cause endothelial cell mitochondrial oxidative stress (ENDO mtStress) and endothelial dysfunction (ED). Furthermore, treatment with an inhibitor of endothelin 1 type A receptor (Ednra) or mitochondrial-targeted superoxide scavenger, eliminated ENDO mtStress and prevented albuminuria and segmental sclerosis. These results suggest a novel podocyte-to-endothelial-to- podocyte crosstalk, where Edn1/Ednra meditated ENDO mtStress and ED is essential for manifestation of podocyte damage/loss in progressive sclerosis. Using in vitro co-culture system of PodTbrI(AAD) podocytes and mouse glomerular endothelial cells (mGECs), we confirmed that Dox-activated PodTbrI(AAD) conditioned medium or Edn1 treatment of mGEC was sufficient to initiate ED, and subsequent mGEC supernatant was sufficient to induce podocyte apoptosis. Finally, we timed Dox withdrawal and found that 100-fold increase of urine albumin/creatinine ratio and at least 40% loss of podocytes were phenotypic markers for irreversible progression of sclerosis even in the absence of Dox. HYPOTHESIS: Perturbation of podocyte homeostasis can be associated with Edn1 release to initiate Edn1/Ednra-mediated mtStress and dysfunction of adjacent endothelial cells, which in response release soluble signal(s) that mediate progressive damage and depletion of adjacent podocytes characteristic of progressive segmental sclerosis. SPECIFIC AIMS: 1) Identify soluble molecular signal(s) produced by endothelial cells, subject to Edn1-activated dysfunction, that is required for manifestation of podocyte apoptosis in vitro. 2) Refine and pinpoint the phenotypic markers (thresholds) and the underlying molecular mechanisms that demarcate precisely the onset of irreversible progression of glomerulosclerosis in PodTbrI(AAD) mice. 3) Delineate whether segmental sclerosis and podocyte lesions in human primary and secondary podocytopathies are correlated with underlying podocyte-endothelial crosstalk in human kidney biopsy cases. LONGTERM: the proposed studies should elucidate the requirements and mechanisms for interdependent signaling crosstalk between dysregulated podocytes and endocapillary cells that determine irreversible segmental sclerosis characteristic of glomerular disease progression. This research aims to identify novel glomerular lesion-specific therapeutic targets.

描述（由申请人提供）：节段性硬化症是定义原发性和继发性肾小球硬化症的特征性病变。在大多数引起肾小球硬化的疾病中，足细胞中的Tgfb表达是与硬化病变相关的应激反应信号。为了准确定义足细胞中 Tgfb I 型受体 (TbrI) 信号传导的激活如何影响体内肾小球细胞，我们生成了转基因小鼠模型 (PodTbrI(AAD)，该模型能够条件性（多西环素诱导型）表达组成型活性 TbrI 突变体（当 TbrI(AAD) 信号在足细胞中被激活时，小鼠最终会出现白蛋白尿，并随后出现进展。然而，足细胞形态保持正常，而足细胞最初释放内皮素 1 (Edn1)，导致内皮细胞线粒体氧化应激 (ENDO mtStress) 和内皮功能障碍 (ED)。此外，用 A 型内皮素 1 抑制剂治疗。受体 (Ednra) 或线粒体靶向超氧化物清除剂，消除 ENDO mtStress 并预防白蛋白尿和这些结果表明一种新的足细胞-内皮-足细胞串扰，其中 Edn1/Ednra 介导的 ENDO mtStress 和 ED 对于进行性硬化症中足细胞损伤/损失的表现至关重要。使用PodTbrI（AAD）足细胞和小鼠肾小球内皮细胞（mGEC）的体外共培养系统，我们证实Dox激活的PodTbrI（AAD）条件培养基或mGEC的Edn1处理足以引发ED，随后的mGEC上清液是足以诱导足细胞凋亡。最后，我们确定了 Dox 停药的时间，发现即使在没有 Dox 的情况下，尿白蛋白/肌酐比值增加 100 倍，足细胞损失至少 40%，也是硬化不可逆进展的表型标志。假设：足细胞稳态的扰动可能与 Edn1 释放有关，从而启动 Edn1/Ednra 介导的 mt 应激和邻近内皮细胞功能障碍，从而释放可溶性信号，介导进行性节段性硬化症特征的邻近足细胞的进行性损伤和耗竭。具体目标：1) 鉴定内皮细胞产生的可溶性分子信号，这些信号受到 Edn1 激活功能障碍的影响，这是体外足细胞凋亡表现所必需的。 2) 细化和查明表型标记（阈值）和潜在的分子机制，精确划分 PodTbrI(AAD) 小鼠肾小球硬化不可逆进展的开始。 3) 描绘人类原发性和继发性足细胞病中的节段性硬化和足细胞病变是否与人类肾活检病例中潜在的足细胞-内皮串扰相关。长期：拟议的研究应阐明失调的足细胞和毛细血管内细胞之间相互依赖的信号串扰的要求和机制，这些信号串扰决定了肾小球疾病进展的不可逆节段性硬化特征。本研究旨在确定新的肾小球病变特异性治疗靶点。