MOLECULAR MECHANISMS IN RENAL INTERSTITIAL FIBROSIS

肾间质纤维化的分子机制

• 批准号: 6635114
• 负责人: ALLISON AUDREY EDDY
• 金额: $ 28.03万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6635114
• 关键词: chronic renal failure; collagen; confocal scanning microscopy; disease /disorder model; extracellular matrix proteins; fibrin; fibrosis; genetically modified animals; immunocytochemistry; laboratory mouse; molecular pathology; nephrectomy; pathologic process; plasmin; plasminogen activator inhibitors; proteolysis; recombinant proteins; tissue /cell culture; tissue inhibitor of metalloproteinases; urokinase

The long-term goal of the proposed research is to elucidate the molecular mechanisms of renal interstitial fibrosis, the final common pathway leading to end-stage renal disease (ESRD). The two major extracellular pathways present in the tubulointerstitial renal compartment will be investigated: the urokinase-plasmin cascade and the metalloproteinase (MMP) family. Preliminary data suggests that interstitial fibrosis is characterized by inactivation of both proteolytic pathways due to up-regulated expression of protease inhibitors: plasminogen activator inhibitors (PAI) and tissue inhibitors of metalloproteinases (TIMP). Using a combination of in vivo and in vitro approaches, three specific aims will be investigated. Aim #1: To test the hypothesis that down-regulation of the intrarenal urokinase- plasmin cascade plays a role in renal interstitial fibrosis and to elucidate the cellular origin and mechanistic pathways involved. Aim #2: To test the hypothesis that down-regulation of intrarenal metalloproteinase activity plays a role in renal interstitial fibrosis and to determine the cellular origin and mechanistic pathways involved. Aim #3: To produce, characterize and test two recombinant proteins, auto-activating stromelysin-Ig and PAI-l-inhibitor-Ig, for their ability to increase renal proteolytic activity and to attenuate renal interstitial fibrosis. Models of experimental renal disease (protein- overlord proteinuria and adriamycin nephropathy) will be studied in normal mice and in mice that have genetically-engineered manipulations in renal matrix-degrading proteolytic activity: PAI-l over-expressing mice, PAI-l deficient mice, uPA-receptor deficient mice. TIMP-1, TIMP-1- 2 and TIMP-1-3 deficient mice and TIMP-1 over-expressing mice. Recombinant stromelysin and a PAI-l inhibiting peptide will be generated fused to an immunoglobulin chain and tested for their ability to attenuate renal fibrosis. The primary outcome will be the severity of interstitial fibrosis based on quantification of the interstitial area occupied by matrix proteins, tubular area and peritubular capillary surface area. Intrarenal proteolytic activity will be evaluated by measuring mRNA levels for genes encoding the proteases and their inhibitors. Changes in renal collagenolytic activity, transforming growth factor-beta (TGF-Beta) activation, fibrin deposition, leukocyte migration and collagen fibril cross-link formation. In vitro studies will investigate the ability of the uPA-receptor to modulate the production of the fibrogenic molecules (TGF-beta, angiotensin II, endothelin-l) and matrix proteins. The results of these studies should clearly establish the role and importance of these intrarenal proteolytic pathways in renal fibrosis.

拟议研究的长期目标是阐明 肾间质纤维化的分子机制，最后共同 导致终末期肾病（ESRD）的途径。两大 肾小管间质细胞外通路 室将进行研究：尿激酶-纤溶酶级联反应和 金属蛋白酶（MMP）家族。初步数据提示 间质性纤维化的特征在于两者的失活。 蛋白酶表达上调导致的蛋白水解途径 抑制剂：纤溶酶原激活物抑制剂（派）和组织抑制剂 金属蛋白酶（TIMP）。使用体内和体内 体外方法，将研究三个具体目标。目标1： 测试肾内尿激酶下调的假设- 纤溶酶级联在肾间质纤维化中起作用， 阐明细胞起源和机制途径。目标二： 为了验证肾内表达下调的假设， 金属蛋白酶活性在肾间质纤维化中的作用 并确定所涉及的细胞起源和机制途径。 目的#3：生产、表征和测试两种重组蛋白， 自激活溶基质素-Ig和PAI-1-通道-Ig，因为它们的能力 以增加肾蛋白水解活性并减弱肾 间质纤维化实验性肾病模型（蛋白质- 霸王蛋白尿和阿霉素肾病）将在 正常小鼠和基因工程改造的小鼠 在肾基质降解蛋白水解活性中：PAI-1过表达 小鼠、PAI-1缺陷小鼠、uPA受体缺陷小鼠。TIMP-1，TIMP-1- 2和TIMP-1-3缺陷小鼠和TIMP-1过表达小鼠。 将产生重组基质溶解素和PAI-1抑制肽 与免疫球蛋白链融合，并测试它们的能力， 减轻肾纤维化。主要结局将是 基于间质面积定量的间质纤维化 被基质蛋白、肾小管区和管周毛细血管占据 表面积肾内蛋白水解活性将通过以下方式进行评价 测量编码蛋白酶的基因的mRNA水平及其 抑制剂的肾脏胶原溶解活性的变化，转化 生长因子-β（TGF-β）活化，纤维蛋白沉积，白细胞 迁移和胶原纤维交联形成。体外研究 将研究uPA受体调节 纤维化分子（TGF-β，血管紧张素II， 内皮素-L）和基质蛋白。这些研究的结果应该 明确这些肾内的作用和重要性， 蛋白水解途径在肾纤维化中的作用