MOLECULAR ASPECTS OF ALPORT RENAL DISEASE PROGRESSION

描述（由申请人提供）：Alport综合征由三种IV型胶原基因COL 4A 3、COL 4A 4或COL 4A 5中的任何一种突变引起，其中任何一种突变导致肾小球基底膜中所有三种蛋白质缺失。这是该疾病的根本原因，全世界每5000人中就有1人以上受到影响。在人类和动物模型中，该疾病表现为延迟发作和进行性，允许治疗干预的窗口。我们和其他人已经发表了明确的著作，指出基质金属蛋白酶（MMPs）的失调作为Alport GBM病理学的根本原因的关键作用。然而，MMP基因调控的失调是如何发生的，以及肾小球疾病的发病机制仍不清楚。我们提供了令人信服的证据支持这一提议的中心假设，即疾病的起始是通过1321整合素激活足细胞中异常层粘连蛋白表达的信号转导改变的结果。这些层粘连蛋白（111和211）在GBM中积累并直接激活粘着斑激酶，引起肌动蛋白细胞骨架重排和适应不良的基因调节，这促进了GBM的足突消失和蛋白水解降解。我们在三个具体目标中检验这一假设。在第一个目标中，我们使用细胞培养和体内方法来探索通过整合素连接激酶的改变的1321整合素信号传导在异常层粘连蛋白表达的激活中的作用，以及异常层粘连蛋白在FAK激活中的作用。在诱导型足细胞特异性启动子控制下表达12和21层粘连蛋白的双转基因小鼠将确定异常GBM层粘连蛋白沉积是否足以触发Alport样肾小球病理，从而构成疾病起始的触发因素。在第二个目标中，我们研究FAK激活和细胞骨架重排和下游适应不良基因调控之间的关系，包括大量诱导基质金属蛋白酶10和12使用的组合的小分子抑制剂的研究培养的足细胞和在体内结合条件FAK基因敲除小鼠。在第三个目标中，我们将研究MMP-10和MMP-12在与Alport肾小球病理学相关的GBM破坏中的协同作用。我们将采用遗传方法产生COL 4A 3和MMP-12无效的小鼠，并条件性地产生MMP-10无效的小鼠，以确定阻断这两种MMPs是否可以提供可行的治疗干预，以可能应用于人类Alport群体。这些目标的成功完成将确定Alport肾小球疾病起始的分子机制，这是朝着开发旨在将疾病抑制在临床前状态的治疗迈出的重要一步。 公共卫生相关性： 目前尚不存在针对Alport综合征患者的靶向治疗方法。目前，每20，000人中就有1人患有这种疾病，接受ACE抑制剂，透析和移植治疗。这项工作利用动物模型来探索新的治疗靶点，旨在将疾病控制在早期阶段。

DESCRIPTION (provided by applicant): Alport syndrome results from mutations of any of three type IV collagen genes COL4A3, COL4A4, or COL4A5, any of which result in the absence of all three proteins in the glomerular basement membrane. This is the underlying cause of the disease, which affects more than 1 in 5000 people worldwide. In humans and animal models, the disease presents as delayed onset and progressive, allowing a window for therapeutic intervention. We and others have published definitive works which point to a key role for dysregulation of matrix metalloproteinases (MMPs) as an underlying cause of Alport GBM pathology. How dysregulation of MMP gene regulation occurs, however, has remained unclear, as has the mechanism of glomerular disease initiation. We provide compelling evidence supporting the central hypothesis of this proposal that disease initiation results from altered signaling through 1321 integrin which activates expression of abnormal laminin in podocytes. These laminins (111 and 211) accumulate in the GBM and directly activate focal adhesion kinase, causing actin cytoskeletal rearrangement and maladaptive gene regulation, which promotes foot process effacement and proteolytic degradation of the GBM. We test this hypothesis in three specific aims. In the first aim we use cell culture and in vivo approaches to explore the role of altered 1321 integrin signaling through integrin linked kinase in the activation of abnormal laminin expression, and the role of abnormal laminins in FAK activation. A double transgenic mouse expressing 12 and 21 laminins under control of an inducible podocyte-specific promoter will determine whether abnormal GBM laminin deposition is sufficient to trigger Alport-like glomerular pathology and thus constitute the trigger for disease initiation. In the second aim we examine the relationship between FAK activation and cytoskeletal rearrangement and downstream maladaptive gene regulation including massive induction of matrix metalloproteinase 10 and 12 using a combination of small molecule inhibitor studies on cultured podocytes and in vivo combined with a conditional FAK knockout mouse. In the third aim we will examine the collaborative role of MMP-10 and MMP-12 in GBM destruction associated with Alport glomerular pathology. We will employ a genetic approach to produce mice that are null for COL4A3 and MMP-12, and conditional null for MMP-10 to determine whether blocking both of these MMPs provides a viable therapeutic intervention for possible application in the human Alport population. Successful completion of these aims will define the molecular mechanism of Alport glomerular disease initiation, which is a major step towards development of therapies aiming to arrest the disease in its pre-clinical state. PUBLIC HEALTH RELEVANCE: A targeted therapeutic approach for patients with Alport syndrome does not exist. Currently the 1 in 20,000 people with the disease are treated with ACE inhibitors, dialysis, and transplant. This work utilizes animal models to explore new therapeutic targets aimed at arresting the disease in its early stages.