Mechanisms of glomerular disesase progression

肾小球疾病进展的机制

• 批准号: 8291405
• 负责人: JEFFREY R SCHELLING
• 金额: $ 32.7万
• 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 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8291405
• 关键词: Adhesions; Albuminuria; Antibodies; Azotemia; Binding; Biological Assay; Blood Urea Nitrogen; Brain; Breeding; Cell Adhesion; Cell Culture Techniques; 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; Docking; Dominant-Negative Mutation; ECM receptor; Embryo; Exhibits; Extracellular Matrix; Family; Focal Adhesions; GTP-Binding Proteins; Genetic; Glomerular Capillary; Glomerular Mesangial Cell; Goals; Guanine Nucleotide Dissociation Inhibitors; Health; Histology; Human; In Vitro; Incubated; Injury; Integrins; Kidney; Kidney Diseases; Knockout Mice; Lead; Ligands; Ligation; Lysine; MMP14 gene; Maps; Metalloproteases; Modeling; Morphology; Mus; Myofibroblast; Nephrectomy; Pathologic; Pathway interactions; Peptides; Phenotype; Physiological; Placenta; Plastics; Precipitation; Rattus; Recruitment Activity; Renal glomerular disease; Reporter; Role; Signal Pathway; Signal Transduction; Smooth Muscle Actin Staining Method; Technology; Testing; Tissues; Transmission Electron Microscopy; Vitronectin; angiogenesis; base; cell transformation; disease phenotype; glomerular basement membrane; glomerular capillary endothelium; glomerulosclerosis; in vivo; interstitial; laminin-10; loss of function; mesangial cell; migration; mouse model; mutant; nidogen-1; podocyte; premature; prevent; protein activation; receptor; research study; rho; working group

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(申请人提供)：整合素是细胞外基质受体，调节重要的细胞功能。？8亚基与α-v配对，其表达仅限于肾脏、脑和胎盘。在体内和体外，8的表达定位于系膜细胞。ITG？8-/-小鼠发展为轻度肾脏疾病，但由于过早死亡，表型尚未完全确定。在肾小球疾病的小鼠模型中，肾脏表达减少，来自ITG？8-/-小鼠的MC发展成肌成纤维细胞特征。在已知的8种配体中，只有潜伏期相关肽(LAP)在肾脏中被检测到，它与转化生长因子形成小的潜伏复合体(SLC)，以维持转化生长因子处于不活跃(潜伏)状态。SLC与β8的结合可通过裂解LAP的MT1-MMPs而导致转化生长因子β的激活，尽管MT1-MMPs在肾小球中不表达，但在疾病状态下可以被诱导。我们发现，LAP在MC中表达，并刺激依赖于8的板足和迁移，这与rac1的激活一致。作为MC？8配体的筛选，我们表明足细胞分泌的基质在依赖于MC？8的细胞黏附方面优于系膜基质--这是首次证明任何8种配体支持稳定的黏附。我们已经证明[8]与Rho GDI相互作用。GDI的主要功能是将Rho家族的G蛋白隔离在胞浆中，从而阻止G蛋白的激活或与质膜效应器的结合。我们发现，8簇增强了MCs中8-GDI的相互作用和rac1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rac1的抑制。我们假设潜在的转化生长因子？与MC？8的结扎将GDI结合的rac1招募到？8细胞质的尾部。8-GDI相互作用使rac1和GDI解离，从而促进rac1的激活以抑制MC肌成纤维细胞的分化。在病理状态下，MC？8表达减少，导致LAP刺激丧失，GDI对rac1的胞浆滞留，并损害MC与GBM的结合。最终结果是肾小球毛细血管内皮细胞发生改变，MC转化为肌成纤维细胞。具体目的：(1)利用MC靶向缺失的小鼠和获得性肾小球硬化的大鼠模型，在体内表征？8的功能。(2)对MC？8配体进行表征。(3)检测？8是否作为RacGDI置换因子抑制MC肌成纤维细胞的分化。公共卫生相关性美国有超过2000万人患有慢性肾脏疾病，通常始于肾小球的损伤，肾小球是肾脏的过滤单元。我们的建议旨在确定一种特定的受体，即表达在肾小球系膜细胞上的β-8整合素，在调节肾脏疾病中的作用。这种整合素在大脑、肾脏和胎盘中被发现，并且只在大脑中得到了很好的表征。我们目前是研究肾脏β-8整合素的唯一小组，我们提供了β-8功能丧失导致肾脏疾病的初步数据。为了实现这一建议的目标，我们使用了小鼠模型，在该模型中，β-8整合素已经从系膜细胞中选择性地删除，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，该模型在该项目完成后更容易操作；我们希望提供有关肾脏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。