Shp2 a Non-Receptor Tyrosine Phosphatase Regulates Nephrin Phosphorylation and Po

描述（由申请人提供）：蛋白尿肾病，包括糖尿病肾病是导致大多数终末期肾病患者的原因。蛋白尿不仅对肾脏疾病的进展很重要，而且也是一个重要的心血管危险因素。足细胞一直是蛋白尿肾小球疾病研究的主要焦点。最近的研究表明足细胞足突消退是足细胞流动性的一种形式，是板足形成增加的结果。Nephrin能够以磷酸化依赖的方式调节足细胞肌动蛋白动力学、板足形成和局灶粘附动力学，这表明它在足突消退中起作用。假设：抑制增加足细胞流动性或阻止板足形成的信号事件将阻止足细胞足突在损伤后的扩散和损失。理由：足细胞损伤后，观察到肾素磷酸化增加。我们的初步数据表明Src激酶依赖的Nephrin磷酸化是由非受体酪氨酸磷酸酶shp2调控的。此外，抑制Shp2可防止硫酸鱼精蛋白足细胞损伤模型中足细胞足突的消失。具体目标：我们将通过追求以下两个具体目标来检验我们的假设。1)明确Shp2对Nephrin磷酸化的调控。利用生化和细胞生物学技术，我们将扩展我们的初步观察，并定义蛋白质-蛋白质相互作用和Shp2依赖性Nephrin磷酸化的功能后果。2)表征整合素激活调控Nephrin磷酸化的信号机制。我们将研究导致Nephrin磷酸化的分子机制。我们的初步实验表明，Nephrin磷酸化是整合素激活的结果。2)确定Shp2和Nephrin磷酸化在体内足细胞稳态中的作用。我们将生成诱导型和非诱导型足细胞特异性条件shp2敲除小鼠。这只小鼠将使我们能够研究shp2在发育和损伤后的作用。我们还将能够检查在足细胞损伤模型中预防足突消失的益处，例如硫酸鱼精蛋白，特别是在足细胞损失和蛋白尿方面。使用这些小鼠，我们也将能够测试足细胞损伤的其他模型。

DESCRIPTION (provided by applicant): Proteinuric kidney diseases, including diabetic nephropathy are responsible for majority of the patients that develop end stage renal disease. Proteinuria is not only important for progression of kidney disease but is also an important cardiovascular risk factor. Podocytes have been the main focus of investigations in glomerular diseases that present with proteinuria. Recent studies suggest that podocyte foot process effacement is a form of podocyte mobility that is a result of increase in lamellipodia formation. Nephrin ability to regulate podocyte actin dynamics, lamellipodia formation and focal adhesion dynamics in a phosphorylation dependent manner suggests its role in foot process effacement. Hypothesis: Hypothesis: Inhibition of signaling events that increase podocyte mobility or prevent lamellipodia formation will prevent podocyte foot process spreading and loss following injury. Rationale: Increase in Nephrin phosphorylation has been observed following podocyte injury. Our preliminary data suggests that Src kinase dependent Nephrin phosphorylation is regulated by non- receptor tyrosine phosphatase shp2. Furthermore, inhibition of Shp2 prevents podocyte foot process effacement in protamine sulfate model of podocyte injury. Specific Aims: We will test our hypothesis by pursuing the following two specific aims. 1) Define regulation of Nephrin phosphorylation by Shp2. Using biochemical and cell biology techniques we will expand on our preliminary observations and define the protein-protein interaction and functional consequences of Shp2 dependent Nephrin phosphorylation. 2) Characterize signaling mechanism by which integrin activation regulates Nephrin phosphorylation. We will examine the molecular mechanism that result in Nephrin phosphorylation. Our preliminary experiments suggest that Nephrin phosphorylation occurs as a result of integrin activation. 2) Determine the role of Shp2 and Nephrin phosphorylation on podocyte homeostasis in vivo. We will generate an inducible and a non-inducible podocyte specific conditional shp2 knockout mouse. This mouse will enable us to study the role of shp2 during development and following injury. We will also be able to examine the benefit of preventing foot process effacement in podocyte injury models like protamine sulfate specifically in regards to podocyte loss and proteinuria. Using these mice we will also be able to test other models of podocyte injury.