Role of a3a4a5(IV) collagen in podocyte behavior

描述(申请人提供)：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成部分。这个项目的长期目标是了解基底膜中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型胶原在未成熟的基底膜中表达为a1a1a2(IV)网络，但在成熟的基底膜中被a3a4a5(IV)网络所取代。由于A3、A4或A5链中的任何一个突变而未能切换到成熟网络，会导致人类出现Alport综合征，并在该疾病的小鼠和狗模型中导致Alport样表型。A3a4a5(IV)胶原是长期维持肾小球功能所必需的，其机制尚不清楚。我们假设，足细胞合成的GBM的IV型胶原反过来通过整合素或其他细胞受体向足细胞提供信号，这是维持足细胞功能所必需的。为了验证这一假说，我们将追求两个特定的目标：(1)体外表达、分离和鉴定a3a4a5(IV)胶原；(2)确定a3a4a5(IV)胶原对足细胞表型的影响，包括比较足细胞在a3a4a5(IV)和a1a1a2(IV)胶原上的黏附和迁移，鉴定与a3a4a5(IV)胶原相互作用的足细胞整合素，以及鉴定与足细胞相互作用的IV型胶原结构域。该项目的成功完成将有助于提高我们对Alport综合征分子缺陷的了解，同时拓宽申请者的研究专长，并为她过渡到独立研究员身份做好准备。

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.