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

项目摘要 蛋白尿性肾病，包括糖尿病肾病，是大多数患者的原因 发展为终末期肾病。蛋白尿不仅对肾脏疾病的进展很重要，而且 一个重要的心血管危险因素。足细胞一直是肾小球研究的主要焦点。 出现蛋白尿的疾病。最近的研究表明，足细胞足突消失是一种形式 足细胞移动性是片状脂膜形成增加的结果。内弗林调节足细胞的能力 磷酸化依赖的肌动蛋白动力学、片状脂膜形成和局部黏附动力学 提示它在足部突起消退中的作用。假设：假设：抑制信号事件 增加足细胞移动性或防止板脂形成将防止足细胞足突扩散和 受伤后的损失。理论基础：足细胞后观察到Nephin磷酸化增加 受伤。我们的初步数据表明，依赖于Src激酶的Nephin的磷酸化是由非 受体酪氨酸磷酸酶Shp2。此外，抑制Shp2可阻止足细胞足突 硫酸鱼精蛋白致足细胞损伤模型的消退。 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)定义监管 Shp2对Nephin的磷酸化。使用生化和细胞生物学技术，我们将扩展我们的 初步观察并确定Shp2的蛋白质-蛋白质相互作用和功能后果 依赖性肾小球蛋白的磷酸化。2)表征整合素激活的信号机制 调节Nephin的磷酸化。我们将研究导致Nephin的分子机制 磷酸化。我们的初步实验表明，Nephin的磷酸化是由于 整合素激活。2)确定Shp2和Nephin磷酸化在足细胞稳态中的作用 在活体内。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。我们也将成为 能够在鱼精蛋白等足细胞损伤模型中检查预防足突消失的益处 硫酸盐尤其在足细胞丢失和蛋白尿方面。利用这些老鼠，我们还将能够测试 足细胞损伤的其他模型。

Project Abstract 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.