Glomerular Cell-Cell Crosstalk and Injury

肾小球细胞间的串扰和损伤

• 批准号: 8506338
• 负责人: Erwin P. Bottinger
• 金额: $ 36.87万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-15 至 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8506338
• 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; Process; Public Health; Recovery; Renal glomerular disease; Research; Sclerosis; Signal Transduction; Small Interfering RNA; Stress Response Signaling; Superoxides; System; Time; Transgenic Mice; Up-Regulation; Urine; WT1 gene; Withdrawal; Work; base; burden of illness; cell injury; cell type; glomerulosclerosis; in vivo; inhibitor/antagonist; mitochondrial dysfunction; mouse model; mutant; novel; podocyte; prevent; public health relevance; receptor; research study; response; therapeutic target

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）：节段性硬化是原发性和继发性肾小球硬化的特征性病变。在大多数引起肾小球硬化的疾病中，足细胞中Tgfb的表达是与硬化病变相关的应激反应信号。为了精确定义足细胞中Tgfb I型受体（TbrI）信号的激活如何影响体内肾小球细胞，我们建立了一个转基因小鼠模型（PodTbrI(AAD)），该模型能够有条件地（多西环素诱导）表达组成型活性TbrI突变体（TbrI(AAD)）。当足细胞中的TbrI（AAD）信号被激活时，小鼠最终发展为蛋白尿，随后进行性肾小球硬化与足细胞耗竭相关。然而，足细胞形态保持正常，但足细胞最初释放内皮素1 （Edn1）引起内皮细胞线粒体氧化应激（ENDO mtStress）和内皮功能障碍（ED）。此外，使用内皮素1 A型受体（Ednra）抑制剂或线粒体靶向超氧化物清除剂治疗，可消除ENDO mtStress并预防蛋白尿和节段性硬化。这些结果提示了一种新的足细胞-内皮-足细胞串扰，其中Edn1/Ednra介导的ENDO mtStress和ED对进行性硬化症中足细胞损伤/丢失的表现至关重要。通过体外共培养PodTbrI（AAD）足细胞和小鼠肾小球内皮细胞（mGECs），我们证实了dox激活的PodTbrI（AAD）条件培养基或Edn1处理mGEC足以引发ED，随后的mGEC上清足以诱导足细胞凋亡。最后，我们定时停用阿霉素，发现即使在没有阿霉素的情况下，尿白蛋白/肌酐比值增加100倍和足细胞减少至少40%是硬化不可逆进展的表型标志。假设：足细胞内稳态的扰动可能与Edn1的释放有关，从而启动Edn1/ ednra介导的相邻内皮细胞的mtStress和功能障碍，从而释放可溶性信号，介导进行性节段性硬化特征的相邻足细胞的进行性损伤和耗竭。特异性目的：1)鉴定受edn1激活功能障碍影响的内皮细胞产生的可溶性分子信号，这是体外足细胞凋亡表现所必需的。2)完善和确定表型标记（阈值）和潜在的分子机制，这些标记（阈值）精确界定了PodTbrI（AAD）小鼠肾小球硬化不可逆进展的发病。3)确定人原发性和继发性足细胞病变的节段性硬化和足细胞病变是否与人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特征的不可逆节段性硬化。本研究旨在确定新的肾小球病变特异性治疗靶点。