Glomerular Cell-Cell Crosstalk and Injury

描述（由申请人提供）：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，足细胞中的Tgfb表达是与硬化病变相关的应激反应信号。为了精确地定义足细胞中Tgfb I型受体（TbrI）信号传导的激活如何影响体内肾小球细胞，我们产生了转基因小鼠模型（PodTbrI（AAD）），其能够有条件地（强力霉素诱导的）表达组成型活性TbrI突变体（TbrI（AAD））。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，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（Edn 1），导致内皮细胞线粒体氧化应激（ENDO mtStress）和内皮功能障碍（艾德）。此外，用内皮素1型A受体（Ednra）抑制剂或脑内靶向超氧化物清除剂治疗，消除了ENDO mtStress并预防了白蛋白尿和节段性硬化。这些结果表明一种新的足细胞-内皮细胞-足细胞串扰，其中Edn 1/Ednra介导ENDO mtStress和艾德对于进行性硬化中足细胞损伤/损失的表现是必不可少的。利用PodTbrI（AAD）足细胞和小鼠肾小球内皮细胞（mGECs）的体外共培养系统，我们证实了Dox活化的PodTbrI（AAD）条件培养基或Edn 1处理mGEC足以引发艾德，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/肌酐比值增加100倍和足细胞损失至少40%是硬化不可逆进展的表型标志物。假设：足细胞稳态的扰动可与Edn 1释放相关，以启动Edn 1/Ednra介导的线粒体应激和邻近内皮细胞的功能障碍，其作为响应释放可溶性信号，介导进行性节段性硬化症特征性的邻近足细胞的进行性损伤和耗竭。具体目标：1）鉴定由经历Edn 1激活的功能障碍的内皮细胞产生的可溶性分子信号，其是体外足细胞凋亡表现所需的。2)细化并查明表型标记物（阈值）和潜在的分子机制，这些标记物和机制可以精确区分PodTbrI（AAD）小鼠肾小球硬化症不可逆转进展的发生。3)描述人类原发性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传导串扰的要求和机制，这些信号传导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。

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.