Genetic Programming in Progressive Renal Disease

足细胞耗竭是进行性肾小球硬化和糖尿病肾病的标志。CD2- 相关蛋白(CD2AP)是一种广泛表达的细胞质接头蛋白。 与足细胞裂隙膜蛋白复合体的形成有关。CD2AP基因缺失小鼠发展为进行性 生命早期肾小球硬化和CD2AP单倍体功能不全与肾小球疾病相关 人类的易感性。然而，目前尚不清楚CD2AP功能丧失是如何导致的 肾小球硬化。在这个续订应用程序中，我们将扩展我们的工作，以测试CD2AP的假设 通过作为抑制转化生长因子-β的分子开关促进肾上皮细胞存活 I型受体(TGFBR1)/Smad3依赖的细胞凋亡途径并激活Tgfbr1/磷脂酰肌醇-3- 依赖激酶(PI3K)的生存通路。如果得到证实，我们的研究将提供一种分子机制 解释CD2AP的缺失如何促进足细胞的凋亡和耗竭，从而促进肾小球疾病 在小鼠和人中，基于CD2AP作为TBRI中多途径调节因子的新的、必要的功能作用。 诱导信号转导。最近的观察进一步强调了这项工作的相关性 提示在没有直接遗传缺陷的情况下，CD2AP表达的功能性下调是一种 在小鼠模型和人类肾小球疾病中常见的发现。 具体目标：1.确定转化生长因子-β诱导因子的分子决定因素和调控特征 CD2AP与胞质Tgfbrl的相互作用我们将a)确定序列基序和结构模型 与CD2AP相互作用的Tgfbrl的基础，以及b)将其与Smad结合模型进行比较 由Tgfbr1的GS区和L45环介导。2.研究CD2AP如何介导负调控 TGFBR1/SMAD3信号转导通路。我们将检查a)CD2AP是否与SMADS竞争绑定到 活化的Tgfbrl；和b)CD2AP靶向内化的Tgfbrl复合体，用于降解和终止 发信号。3.明确足细胞特异性转化生长因子-b受体信号在足细胞凋亡中的作用 体内的耗竭，并确定这种足细胞特异性的机制是否支持了 CD2AP-/-小鼠肾小球损伤与肾小球硬化我们将在体内确定a)有条件的 使用cre/lox技术缺失足细胞中的转化生长因子-b受体II型(TGFBR2)可防止足细胞 CD2AP-/-小鼠的细胞凋亡、衰竭和/或肾小球硬化；以及b)足细胞特异性， 受控激活转化生长因子-(3)受体信号足以诱导足细胞凋亡 利用多西环素诱导的结构性活性表达在体内耗竭和肾小球硬化 Tgfbrl(Tgfbrl(AAD))。

Podocyte depletion is a hallmark of progressive glomerulosclerosis and diabetic nephropathy. CD2- associated protein (CD2AP) is a widely-expressed cytoplasmic adaptor protein that has previously been implicated in podocyte slit diaphragm protein complex formation. CD2AP null mice develop progressive glomerulosclerosis early in life and CD2AP haploinsufficiency is associated with glomerular disease susceptibility in humans. However, it remains unclear how loss of CD2AP function may cause glomerulosclerosis. In this renewal application, we will extend our work to test the hypothesis that CD2AP promotes renal epithelial cell survival by functioning as a molecular switch that suppresses the TGF-beta receptor type I (Tgfbr1)/Smad3-dependent apoptosis pathway and activates a Tgfbrl/phosphoinositide-3- kinase (PI3K) -dependent survival pathway. If confirmed, our studies will provide a molecular mechanism to explain how loss of CD2AP may enhance podocyte apoptosis and depletion to promote glomerular disease in mice and men based on a novel, essential functional role for CD2AP as multi-pathway regulator in TbRI- induced signal transduction. The relevance of this work is further underscored by recent observations suggesting that in the absence of direct genetic defects, functional downregulation of CD2AP expression is a common finding in mouse models and human glomerular disease. Specific Aims: 1. Identify the molecular determinants and characterize the regulation of the TGF-p-inducible interaction of CD2AP and cytoplasmic Tgfbrl. We will a) determine the sequence motif and structural model of Tgfbrl that underlies the interaction with CD2AP, and b) compare it with the Smad-binding model mediated by the GS region and L45 loop of Tgfbrl. 2. Investigate how CD2AP mediates negative regulation of Tgfbr1/Smad3 signaling. We will examine whether a) CD2AP competes with SmadS for binding to activated Tgfbrl; and b) CD2AP targets internalized Tgfbrl complexes for degradation and termination of signaling. 3. Define the role of podocyte-specific TGF-b receptor signaling in podocyte apoptosis and depletion in vivo, and determine whether this podocyte-specific mechanism underlies the progression of glomerular injury to glomerulosclerosis in CD2AP-/- mice? We will determine in vivo whether a) conditional deletion of TGF-b receptor type II (Tgfbr2) in podocytes using cre/lox technology prevents podocyte apoptosis, depletion, and/or glomerulosclerosis in CD2AP-/- mice; and b) whether podocyte-specific, controlled activation of TGF-(3 receptor signaling is sufficient to induce podocyte apoptosis, podocyte depletion and glomerulosclerosis in vivo using doxycycline-inducible expression of constitutively active Tgfbrl (Tgfbrl (AAD)).