Mechanisms of glomerular disesase progression

描述（由申请人提供）：整合素是细胞外基质受体，调节重要细胞功能。¿8亚基与α -v结合，表达仅限于肾脏、大脑和胎盘。体内和体外表达均局限于系膜细胞（MCs）。Itg¿8-/-小鼠发生轻度肾病，但由于过早死亡，表型不完全确定。肾小球疾病小鼠模型中肾¿8表达降低，来源于Itg¿8-/-小鼠的MCs发育成肌成纤维细胞特征。在已知的¿8配体中，肾脏中仅检测到与TGF¿结合形成小潜伏复合物（SLC）的潜伏期相关肽（LAP），使TGF¿保持无活性（潜伏）状态。SLC与¿8结合可通过MT1-MMP切割LAP导致TGF -激活，尽管MT1-MMP在肾小球中不表达，但在疾病状态下可被诱导。我们发现LAP在MCs中表达，并刺激¿8依赖性板足和迁移，与Rac1激活一致。作为MC¿8配体的筛选，我们发现足细胞分泌的基质在依赖¿8的细胞粘附方面优于系膜基质，这是首次证明任何¿8配体支持稳定的粘附。我们已经证明了¿8与Rho GDI相互作用。GDI的主要功能是将Rho家族g蛋白隔离在细胞质溶胶中，阻止g蛋白活化或与质膜效应物结合。我们发现，在MCs中，¿8聚类增强了¿8- Gdi相互作用和Rac1激活，而Itg¿8-/-和Gdi-/- MCs表现出肌成纤维细胞特征，如α -平滑肌肌动蛋白（SMA）组装和RhoA激活，以及Rac1抑制。我们假设TGF与MC¿8的潜在连接将gdi结合的Rac1招募到胞质尾部。8-GDI相互作用使Rac1与GDI分离，促进了Rac1的激活，抑制了MC肌成纤维细胞的分化。在病理状态下，MC¿8的表达减少，导致LAP刺激丧失，胞质中Rac1被GDI保留，MC与GBM的结合受损。净效应是MCs转化为肌成纤维细胞，并改变肾小球毛细血管内皮的监测。具体目的：(1)利用mc -靶向¿8缺失小鼠和获得性肾小球硬化大鼠模型来研究¿8的体内功能。(2)表征MC¿8配体。(3)检测¿8是否作为RacGDI位移因子抑制MC肌成纤维细胞分化。美国有超过2000万人患有慢性肾脏疾病，通常始于肾小球（肾脏的过滤单位）的损伤。我们的建议旨在确定在肾小球系膜细胞上表达的特异性受体β -8整合素在调节肾脏疾病中的作用。这种整合素存在于大脑、肾脏和胎盘中，并且只在大脑中有很好的特征。我们是目前唯一一个研究肾脏β -8整合素的小组，我们提供了β -8功能丧失导致肾脏疾病的初步数据。为了实现这一提议的目标，我们正在使用小鼠模型，其中β -8整合素被选择性地从系膜细胞中删除，以避免来自大脑的混淆效应。此外，我们正在使用系膜细胞培养模型，在这个项目完成后，可以更容易地操作；我们希望提供关于肾脏β -8整合素的新信息，这可能会导致慢性肾脏疾病的新疗法。

DESCRIPTION (provided by applicant): Integrins are extracellular matrix receptors, which regulate vital cell functions. The ¿8 subunit partners with alpha-v, and expression is restricted to kidney, brain, and placenta. ¿8 expression is localized to mesangial cells (MCs) in vivo and in vitro. Itg¿8-/- mice develop mild renal disease, but the phenotype is incompletely defined due to premature lethality. Kidney ¿8 expression is decreased in mouse models of glomerular disease, and MCs derived from Itg¿8-/- mice develop myofibroblast features. Of known ¿8 ligands, only latency-associated peptide (LAP), which combines with TGF¿ to form the small latent complex (SLC), to maintain TGF¿ in an inactive (latent) state, is detected in kidney. SLC binding to ¿8 can lead to TGF¿ activation through MT1-MMP cleavage of LAP, though MT1-MMP is not expressed in glomeruli, but can be induced in disease states. We show that LAP is expressed in MCs, and stimulates ¿8-dependent lamellopodia and migration, consistent with Rac1 activation. As a screen for MC ¿8 ligands, we show that matrix secreted by podocytes is superior to mesangial matrix for ¿8-dependent cell adhesion - the first demonstration of any ¿8 ligand supporting stable adhesions. We have shown that ¿8 interacts with Rho GDI. The major function of GDI is to sequester Rho family G-proteins in cytosol, which prevents G-protein activation or association with plasma membrane effectors. We show that ¿8 clustering enhances ¿8-GDI interaction and Rac1 activation in MCs, whereas Itg¿8-/- and Gdi-/- MCs exhibit myofibroblast features, such as alpha-smooth muscle actin (SMA) assembly and RhoA activation, as well as Rac1 suppression. We hypothesize that latent TGF¿ ligation with MC ¿8 recruits GDI-bound Rac1 to the ¿8 cytoplasmic tail. The ¿8-GDI interaction dissociates Rac1 from GDI, which facilitates Rac1 activation to suppress MC myofibroblast differentiation. In pathologic states, MC ¿8 expression is decreased, resulting in loss of LAP stimulation, cytoplasmic retention of Rac1 by GDI, and impaired MC binding to GBM. The net effect is that MCs transform into myofibroblasts with altered surveillance of the glomerular capillary endothelium. Specific aims: (1) In vivo characterization of ¿8 function using mice with MC-targeted ¿8 deletion and a rat model of acquired glomerulosclerosis. (2) Characterize the MC ¿8 ligand. (3) Test whether ¿8 functions as a RacGDI displacement factor to suppress MC myofibroblast differentiation. PUBLIC HEALTH RELEVANCE Over 20 million people in the U.S. suffer from chronic kidney disease, which usually starts with injury to the glomerulus, the filtering unit of the kidney. Our proposal aims to define the role of a specific receptor, the beta-8 integrin, which is expressed on the glomerular mesangial cell, in regulating kidney disease. This integrin is found in the brain, kidney and placenta and has been well-characterized only in brain. We are currently the only group working on kidney beta-8 integrin, and we provide preliminary data that beta-8 loss of function leads to kidney disease. To achieve the goals of this proposal, we are using mouse models, in which the beta-8 integrin has been selectively deleted from mesangial cells, to avoid confounding effects from brain. In addition, we are employing mesangial cell culture models, which can be more easily manipulated, upon completion of this project; we hope to provide new information about the kidney beta-8 integrin, which could lead to new therapies for chronic kidney diseases.