Glomerular Cell-Cell Crosstalk and Injury

描述(申请人提供)：节段性硬化是一种特征性病变，定义为原发性和继发性肾小球硬化。在大多数引起肾小球硬化的疾病中，足细胞中TGFb的表达是与硬化性病变相关的应激反应信号。为了准确定义足细胞中TGFbI型受体(TBRI)信号的激活如何影响体内的肾小球细胞，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)表达具有结构性活性的TBRI突变体(TBRI(AAD))。当足细胞中的TbRI(AAD)信号被激活时，小鼠最终会出现蛋白尿，并随后出现与足细胞耗竭相关的进行性肾小球硬化。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，使用内皮素1型受体抑制剂(Ednra)或线粒体靶向的超氧化物清除剂治疗，可以消除内压，防止蛋白尿和节段性硬化症。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冥想内应力和ED在进行性硬化症中足细胞损伤/丢失的表现中是必不可少的。利用PodTbrI(AAD)足细胞与小鼠肾小球内皮细胞(MGEC)的体外共培养体系，我们证实Dox激活的PodTbrI(AAD)条件培养液或mGEC的Edn1处理足以启动ED，随后的mGEC上清液足以诱导足细胞凋亡。最后，我们对Dox停药进行了计时，发现即使在没有Dox的情况下，尿白蛋白/肌酐比值100倍的增加和足细胞至少40%的丢失是硬化症不可逆转进展的表型标志。假说：足细胞动态平衡的紊乱可能与Edn1的释放有关，以启动Edn1/EdnrA介导的相邻内皮细胞的mtStress和功能障碍，作为响应，释放可溶性信号(S)，介导以进行性节段性硬化症为特征的邻近足细胞的进行性损害和耗竭。具体目的：1)鉴定血管内皮细胞产生的可溶性分子信号(S)，该信号是在体外表现足细胞凋亡所必需的，受到EDN1激活功能障碍的影响。2)改进和精确定位表型标志物(阈值)和精确界定PodTbrI(AAD)小鼠肾小球硬化不可逆进展的潜在分子机制。3)阐明人类原发和继发性足细胞病变中的节段性硬化和足细胞病变是否与人类肾脏活检病例中潜在的足细胞-内皮串扰有关。长期：拟议的研究应阐明调节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，以确定肾小球疾病进展过程中不可逆节段性硬化的特征。这项研究旨在确定新的肾小球病变特异性治疗靶点。

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.