Role of Focal Adhesion Kinase (FAK) in Nephrosis and Nephritis

描述(申请人提供)：慢性肾脏疾病(CKD)通常导致不可逆转的肾功能恶化，并进展到终末期肾脏疾病(ESKD)。慢性病已经成为一个严重的公共卫生问题，从美国疾病控制和预防中心获得的数据显示，到2010年，美国ESKD的新病例预计将达到650,000例，伴随着280亿美元的医疗保险支出。由于足细胞功能障碍继发的肾小球疾病占所有ESKD的90%，详细的分子和遗传学方法来确定足细胞发育和修复的机制可能为我们开发治疗药物和靶点提供新的见解。目前，可用于治疗肾小球疾病的治疗选择仅限于血管紧张素受体阻滞剂。血管紧张素转换酶抑制剂、类固醇和烷化剂。由于许多接受这些药物治疗的患者仍进展为ESKD，这表明可能涉及其他导致损伤的机制。当足细胞受损时，细胞体收缩，导致细胞消失，随后出现蛋白尿。为了使细胞消失，细胞必须调节肾小球基底膜和细胞外基质之间的粘连接触，细胞外基质由局灶性粘连和整合素组成。本研究的目的是明确粘着斑蛋白、粘着斑激酶(FAK)在损伤后足细胞调节中的作用，FAK是细胞运动的关键调节因子。初步结果表明，足细胞损伤后FAK被高度激活，在肾病和肾病综合征的小鼠模式中，缺乏足细胞FAK表达的条件性基因敲除小鼠似乎对损伤具有抵抗力。本研究的目的是通过在体内诱导足细胞损伤和用shRNA抑制足细胞FAK的表达，并在体外突变关键的FAK调节位点来评估FAK激活的功能相关性(特异性目标1)。由于一种新的特异性FAK抑制剂(诺华)目前已上市，将使用该化合物(特异性目标2)在小鼠足细胞损伤前后测试体内对FAK抑制的功能反应。最后，为了解决FAK在其下游信号分子调节中的作用，如诱导基质金属蛋白酶2(MMP2)活性，将利用小鼠以及前两个特定目标中定义的细胞培养模型来确定诱导损伤的机制(特定目标3)。 公共卫生相关性：肾损伤导致炎症、蛋白丢失和尿液中的血液，通常会导致不可修复的损害，最终需要进行肾移植和/或透析来治疗受影响的患者。在美国，预计到2010年，慢性肾脏疾病的新病例数量将超过65万人。目前，可用的治疗选择数量有限。该提案的目的是确定肾脏损伤过程中涉及的机制，并利用能够对抗导致疾病进展的致病因子(S)的抑制剂。

DESCRIPTION (provided by applicant): Chronic kidney disease (CKD) often leads to irreversible deterioration of renal function and progresses to End Stage Kidney Disease (ESKD). CKD has emerged as a serious public health problem and data obtained from the USRDS reveals that the number of new cases of ESKD in the United States is projected to be 650,000by 2010, with accompanying Medicare expenditures of $28 billion. As glomerular diseases secondary to podocyte dysfunction contribute up to 90% of all ESKD, a detailed molecular and genetic approach to identify mechanisms for podocyte development and repair may give us new insights for developing therapeutic agents and targets. Currently the therapeutic options available to treat glomerular diseases are limited to Angiotensin Receptor Blockers. Angiotensin Converting Enzyme Inhibitor, Steroids, and Alkylating Agents. As many patients treated with these agents still progress to ESKD, this suggests that other mechanisms responsible for injury are likely involved. When podocytes are damaged, the cell body retracts resulting in effacement and subsequently, proteinuria. For effacement to occur, cells must regulate adhesive contacts between the glomerular basement membrane and the extracellular matrix, which is comprised of focal adhesions and integrins. The goal of this research project is to define the role of focal adhesion protein, Focal Adhesion Kinase (FAK), a critical regulator of cell movement, in podocyte regulation following injury. Preliminary results demonstrate that FAK is highly activated following podocyte injury and a conditional knockout mice lacking podocyte FAK expression appear resistant to injury in murine modes of nephrotic and nephritic syndromes. The aim of the current proposal is to assess the functional relevance of FAK activation by inducing podocyte injury in-vivo and by knocking down podocyte FAK expression with shRNA, and mutating critical FAK regulatory sites in-vitro (Specific Aim 1). As a novel specific FAK inhibitor (Novartis) is currently available, the functional response to FAK inhibition in vivo will be tested before and after podocyte injury in mice using this compound (Specific Aim 2). Finally, to address FAK's role in its regulation of downstream signaling molecules such as inducing matrix metalloproteinase 2 (MMP-2) activity, mice as well as cell culture models defined in the first two specific aims will be utilized to determine the mechanism inducing injury (Specific Aim 3). PUBLIC HEALTH RELEVANCE: Kidney injury that results in inflammation, loss of protein, and blood in the urine often leads to irreparable damage that ultimately requires a kidney transplantation and/or dialysis to treat the affected patient. In the United States, the number of new cases of chronic kidney disease is projected to be greater than 650,000 by year 2010. Currently there are a limited number of therapeutic options available. The goal of the proposal is to define the mechanisms that are involved during kidney injury and to utilize inhibitors that antagonize against the offending agent(s) responsible for disease progression.