Mechanisms of glomerular disesase progression

肾小球疾病进展的机制

• 批准号: 7526528
• 负责人: JEFFREY R SCHELLING
• 金额: $ 32.84万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7526528
• 关键词: Adhesions; Albuminuria; Antibodies; Azotemia; Binding; Biological Assay; Blood Urea Nitrogen; Brain; Breeding; Cell Adhesion; Cell Differentiation process; Cell Line; Cell membrane; Cell physiology; Cells; Characteristics; Chimeric Proteins; Chinese Hamster Ovary Cell; Chronic Kidney Failure; Cicatrix; Coculture Techniques; Collagen; Collagen Type IV; Complex; Confocal Microscopy; Cytoplasmic Tail; Cytosol; Data; Defect; Disease; Disruption; Docking; Dominant-Negative Mutation; ECM receptor; Embryo; Exhibits; Extracellular Matrix; Family; Focal Adhesions; GTP-Binding Proteins; Genetic; Glomerular Capillary; Glomerular Mesangial Cell; Glutathione S-Transferase; Goals; Guanine Nucleotide Dissociation Inhibitors; Histology; Human; In Vitro; Incubated; Injury; Integrins; Kidney; Kidney Diseases; Knockout Mice; Lead; Ligands; Ligation; Localized; Lysine; MMP14 gene; Maps; Medical Surveillance; Metalloproteases; Modeling; Morphology; Mus; Myofibroblast; Nephrectomy; Pathologic; Pathway interactions; Peptides; Personal Satisfaction; Phenotype; Physiological; Placenta; Plastics; Precipitation; Public Health; Rattus; Recruitment Activity; Renal glomerular disease; Reporter; Role; Signal Pathway; Signal Transduction; Smooth Muscle Actin Staining Method; Technology; Testing; Tissues; Transmission Electron Microscopy; Vitronectin; Work; angiogenesis; base; cell transformation; disease phenotype; glomerular basement membrane; glomerular capillary endothelium; glomerulosclerosis; human MMP14 protein; in vivo; interstitial; laminin-10; loss of function; mesangial cell; migration; mouse model; mutant; nidogen-1; podocyte; prevent; protein activation; receptor; research study; rho

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.

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合作，表达仅限于肾脏，大脑和胎盘。在体内和体外，8的表达定位于系膜细胞（MC）。Itg <$8-/-小鼠发生轻度肾病，但由于过早致死，表型不完全确定。在肾小球疾病小鼠模型中，肾<$8表达降低，来自Itg <$8-/-小鼠的MC发育成肌成纤维细胞特征。已知的？在8种配体中，只有潜伏相关肽（Lactency-associated peptide，LTP）与TGF？结合形成小的潜伏复合物（small latent complex，SLC），以维持TGF？处于无活性（潜伏）状态，在肾脏中被检测到。SLC绑定到8可以导致TGF？通过MT 1-MMP切割肾小球的活化，尽管MT 1-MMP在肾小球中不表达，但可在疾病状态中诱导。我们发现，Rac 1在MC中表达，并刺激8依赖性板足和迁移，与Rac 1激活一致。作为MC的屏幕8配体，我们表明足细胞分泌的基质上级系膜基质的<$8依赖性细胞粘附-任何<$8配体支持稳定的粘附的第一个证明。我们已经证明，<$8与Rho GDI相互作用。GDI的主要功能是将Rho家族G蛋白隔离在胞质溶胶中，从而防止G蛋白活化或与质膜效应物结合。我们表明，8集群增强功能？8-GDI相互作用和Rac 1在MC中的激活，而Itg-8-/-和Gdi-/-MC表现出肌成纤维细胞的特征，如α-平滑肌肌动蛋白（SMA）组装和RhoA激活，以及Rac 1抑制。我们假设潜在的TGF β与MC β 8的连接将GDI结合的Rac 1募集到MC β 8的胞质尾区。8-GDI相互作用使Rac 1与GDI解离，这促进Rac 1活化以抑制MC肌成纤维细胞分化。在病理状态下，MC 8的表达降低，从而导致Rac 1刺激的丧失、GDI对Rac 1的细胞质滞留以及MC与GBM的结合受损。净效应是MCs转化为肌成纤维细胞，改变了肾小球毛细血管内皮的监视。具体目的：（1）使用MC靶向<$8缺失小鼠和获得性肾小球硬化大鼠模型体内表征<$8功能。(2)表征MC 8配体。(3)测试Δ 8是否作为RacGDI置换因子发挥功能以抑制MC肌成纤维细胞分化。公共卫生相关性美国有超过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）损伤。我们的建议旨在确定一个特定的受体，β-8整合素，这是肾小球系膜细胞上表达，在调节肾脏疾病的作用。这种整联蛋白存在于脑、肾和胎盘中，并且仅在脑中得到充分表征。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整合素已被选择性地从系膜细胞中删除，以避免大脑的混淆效应。此外，我们正在采用肾小球系膜细胞培养模型，在完成该项目后，可以更容易地操作;我们希望提供有关肾脏β-8整合素的新信息，这可能导致慢性肾脏疾病的新疗法。