Role of a3a4a5(IV) collagen in podocyte behavior

描述（由申请方提供）：足细胞、内皮细胞和肾小球基底膜（GBM）是肾小球滤过屏障的关键组分。本项目的长期目标是了解GBM中IV型胶原的组成如何影响足细胞（肾小球上皮细胞）的行为。IV型胶原在未成熟GBM中表达为a1 a1 a2（IV）网络，但在成熟GBM中被a3 a4 a5（IV）网络取代。由于α 3、α 4或α 5链中的任一个中的突变而未能切换到成熟网络导致人类中的Alport综合征以及该疾病的小鼠和狗模型中的Alport样表型。长期维持肾小球功能所需的a3 a4 a5（IV）胶原的机制尚不清楚。我们假设，足细胞合成的GBM的IV型胶原蛋白，反过来通过整合素或其他细胞受体向足细胞提供信号，这是维持适当的足细胞功能所必需的。为了验证这一假设，我们将追求两个具体目标：（1）体外表达、分离和表征a3 a4 a5（IV）胶原;和（2）确定a3 a4 a5（IV）胶原对足细胞表型的影响，包括比较足细胞在a3 a4 a5（IV）和a1 a1 a2（IV）胶原上的粘附和迁移，与α 3 α 4 α 5（IV）胶原相互作用的足细胞整联蛋白的鉴定，以及与足细胞相互作用的胶原IV结构域的鉴定。该项目的成功完成将有助于提高我们对Alport综合征分子缺陷的理解，同时扩大申请人的研究专业知识，并为她过渡到独立研究者身份做好准备。

DESCRIPTION (provided by applicant): Podocytes, endothelial cells, and the glomerular basement membrane (GBM) are the key components of the glomerular filtration barrier. The long term goal of this project is to understand how the composition of collagen IV in the GBM influences the behavior of podocytes (glomerular epithelial cells). Type IV collagen is expressed as an a1a1a2(IV) network in the immature GBM but is replaced by the a3a4a5(IV) network in the mature GBM. Failure to switch to the mature network due to mutations in any of the a3, a4 or a5 chains leads to Alport syndrome in humans and Alport-like phenotype in mouse and dog models of the disease. The mechanism whereby the a3a4a5(IV) collagen is required for the long term maintenance of the renal glomerular function is not known. We hypothesize that type IV collagen of the GBM, synthesized by podocytes, in turn provides signals to podocytes through integrins or other cellular receptors, which are required to maintain the appropriate podocyte function. To test this hypothesis, we will pursue two specific aims: (1) to express, isolate, and characterize the a3a4a5(IV) collagen in vitro; and (2) To define the influence of the a3a4a5(IV) collagen on the podocytes phenotype, including comparison of podocytes adhesion and migration on a3a4a5(IV) vs. a1a1a2(IV) collagen, identification of the podocyte integrins interacting with a3a4a5(IV) collagen, and identification of collagen IV domains that interact with podocytes. The successful completion of this project would help improve our understanding of the molecular defects in Alport syndrome, while broadening the applicant's research expertise and preparing her for the transition to an independent investigator status.