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

描述(申请人提供)：蛋白尿性肾脏疾病，包括糖尿病肾病，是大多数发展为终末期肾脏疾病的患者的原因。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。足细胞一直是以蛋白尿为主要表现形式的肾小球疾病的研究重点。最近的研究表明，足细胞足突消失是足细胞运动的一种形式，这是板脂形成增加的结果。Nephin以磷酸化依赖的方式调节足细胞肌动蛋白动力学、板脂形成和局部黏附动力学的能力表明它在足突消失中起作用。假设：假设：抑制增加足细胞流动性或防止板脂形成的信号事件将防止足细胞足突的扩散和损伤后的丢失。理论基础：足细胞损伤后观察到Nephin磷酸化增加。我们的初步数据表明，非受体酪氨酸磷酸酶Shp2调控着依赖于Src激酶的Nephin的磷酸化。此外，在硫酸鱼精蛋白损伤的足细胞损伤模型中，抑制Shp2可以防止足细胞足突的消失。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Nephin磷酸化的调节。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Shp2依赖的Nephin磷酸化的蛋白质-蛋白质相互作用和功能后果。2)研究整合素激活调节Nephin磷酸化的信号机制。我们将研究导致Nephin磷酸化的分子机制。我们的初步实验表明，Nephin的磷酸化是整合素激活的结果。2)确定Shp2和Nephin磷酸化在体内足细胞内稳态中的作用。我们将产生一个诱导型和非诱导型足细胞特异性条件性Shp2基因敲除小鼠。这只小鼠将使我们能够研究Shp2在发育和损伤后的作用。我们还将能够研究在足细胞损伤模型(如硫酸鱼精蛋白)中预防足突消失的益处，特别是在足细胞丢失和蛋白尿方面。利用这些小鼠，我们还将能够测试其他足细胞损伤模型。

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.