MOLECULAR ASPECTS OF ALPORT RENAL DISEASE PROGRESSION

描述(申请人提供)：Alport综合征是由三种IV型胶原基因Col4A3、Col4A4或Col4A5中的任何一种突变引起的，任何一种突变都会导致肾小球基底膜中所有三种蛋白质的缺失。这是这种疾病的根本原因，全世界每5000人中就有一人受到影响。在人类和动物模型中，这种疾病表现为发病延迟和进展，为治疗干预提供了一个窗口。我们和其他人已经发表了权威性的研究成果，指出基质金属蛋白酶(MMPs)的失调是Alport GBM病理的根本原因之一。然而，目前还不清楚基质金属蛋白酶基因调控的失调是如何发生的，肾小球疾病的发病机制也是如此。我们提供了令人信服的证据支持这一建议的中心假设，即疾病的起始是通过1321整合素的信号改变导致的，它激活了足细胞中异常的层粘连蛋白的表达。这些层粘连蛋白(111和211)聚集在基底膜，直接激活粘着斑激酶，引起肌动蛋白细胞骨架重排和基因调节失调，从而促进基底膜的足突消失和蛋白降解。我们从三个具体目标来检验这一假设。在第一个目的中，我们采用细胞培养和体内实验的方法，探讨通过整合素连接激酶改变的1321整合素信号在层粘连蛋白异常表达的激活中的作用，以及层粘连蛋白异常在FAK激活中的作用。在可诱导的足细胞特异性启动子的控制下，表达12和21层粘连蛋白的双转基因小鼠将确定异常的GBM层粘连蛋白沉积是否足以触发Alport样肾小球病变，从而构成疾病发生的触发因素。在第二个目标中，我们结合小分子抑制剂对培养的足细胞和体内条件FAK基因敲除小鼠的研究，研究了FAK激活与细胞骨架重排和下游不良适应基因调控的关系，包括大量诱导基质金属蛋白酶10和12的表达。在第三个目标中，我们将研究基质金属蛋白酶-10和基质金属蛋白酶-12在Alport肾小球病理相关的基底膜破坏中的协同作用。我们将使用一种遗传方法来产生COL4A3和MMP12缺失以及MMP10条件性缺失的小鼠，以确定阻断这两种MMPs是否为可能应用于人类Alport人群提供了可行的治疗干预。这些目标的成功完成将确定Alport肾小球疾病启动的分子机制，这是朝着旨在阻止疾病处于临床前状态的治疗方法发展的重要一步。 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人接受血管紧张素转换酶抑制剂、透析和移植治疗。这项工作利用动物模型来探索新的治疗目标，旨在阻止疾病的早期阶段。

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.