MOLECULAR MECHANISMS IN RENAL INTERSTITIAL FIBROSIS

拟议研究的长期目标是阐明 肾间质纤维化的分子机制 导致终末期肾病(ESRD)的途径。两大 肾小管间质中存在的细胞外途径 将研究的隔室：尿激酶-纤溶酶级联和 金属蛋白酶(MMPs)家族。初步数据显示， 间质纤维化的特征是两者都失活。 蛋白水解酶表达上调所致的蛋白分解途径 抑制物：纤溶酶原激活物抑制物(PAI)和组织抑制物 金属蛋白酶(TIMP)。使用体内和体内的组合 体外方法，将调查三个特定的目的。目标1：达到 验证这样一种假设：肾内尿激酶下调- 纤溶酶级联反应在肾间质纤维化中的作用 阐明细胞起源和涉及的机制途径。目标2： 来检验这样一种假设，即肾内信号的下调 金属蛋白酶活性在肾间质纤维化中的作用 并确定细胞起源和涉及的机制途径。 目的#3：生产、鉴定和测试两种重组蛋白， 自激活基质分解素-Ig和PAI-L抑制-Ig的能力 增强肾脏蛋白水解酶活性，使肾脏变弱 间质纤维化。实验性肾脏疾病模型(蛋白质- 霸王蛋白尿和阿霉素肾病)将在 正常小鼠和进行基因工程操作的小鼠 肾基质降解蛋白水解酶活性：PAI-L过表达 小鼠，PAI-L缺陷小鼠，uPA-受体缺陷小鼠。TIMP-1，TIMP-1- 2、TIMP-1-3缺陷小鼠和TIMP-1过表达小鼠。 将产生重组基质分解素和PAI-L抑制肽 融合到免疫球蛋白链上，并测试它们的能力 减轻肾脏纤维化。主要结果将是严重的 基于间质面积定量的间质纤维化 由基质蛋白、小管区和管周毛细血管占据 表面积。肾内蛋白分解活性将通过以下方法进行评估 检测编码蛋白水解酶及其相关基因的mRNA水平 抑制剂。肾胶原蛋白溶解活性的变化，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物的形成。体外研究 将研究uPA受体调节细胞周期的能力 纤维化分子(转化生长因子-β、血管紧张素II、 L)和基质蛋白。这些研究的结果应该是 明确确定这些肾内血管的作用和重要性 肾脏纤维化中的蛋白分解途径。

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.