Gene Delivery of OP-1 in Rodent Model of Renal Fibrosis

肾纤维化啮齿动物模型中 OP-1 的基因传递

• 批准号: 6460312
• 负责人: GLENN A MCDONALD
• 金额: $ 7.21万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6460312
• 关键词: bone morphogenetic proteins; collagen; confocal scanning microscopy; extracellular matrix proteins; fibronectins; fibrosis; kidney; protein degradation; protein localization; tissue /cell culture; ubiquitin

This application for Ro3 award is submitted as a supplement for the K08 award DK02546-02. Osteogenic Protein-1 (OP- 1) or Bone Morphogenetic Protein-7) (BMP-7) is a member of the transforming growth factor beta (TGF-beta) superfamily of secreted growth factors. The kidney is the primary site of OP-1 synthesis. Mice genetically deficient in OP-1 expression have markedly abnormal renal development, suggesting that this protein may be important in regulation of cell division and morphogenesis in the kidney. Furthermore, systematic administration of OP-1 has been shown to delay or halt progress to end stage renal failure in the 5/6 nephrectomy of renal fibrosis. To further explore this phenomenon, we evaluated the effects of OP-1 on the extracellular matrix in an in vitro model of renal fibrosis. Increasing concentrations of OP-1 0-300 ng/ml) resulted in a dose dependent increase in fibronectin levels in mouse messangial and proximal tubular cells. In duplicate samples processed for RNA revealed no significant changes in fibronectin RNA levels, suggesting a non- transcriptional mechanism. Concurrently, our laboratory has demonstrated that fibronectin is a target protein for ubiquitin dependent degradation. The research goals for the final two years of the proposal will focus on the observation that OP-1 decreases ubiquitin dependent degradation of fibronectin. These goals will be realized in the following ways. First we will determine id the effects of OP-1 are specific for fibronectin or if it has similar effects on other extracellular matrix proteins such as collagen I, III and IV. Second we will investigate the potential involvement of OP-1 on ubiquitination of fibronectin. Finally, utilizing confocal microscopy we will evaluate the effects of OP-1 on the subcellular distribution of fibronectin. This finding presented in this presented in this proposal represents a new paradigm in ECM homeostasis. Understanding the normal mechanism of action of this pathway will not only elucidate a novel mechanism of extracellular matrix homeostasis but may have major therapeutic implications.

本Ro 3裁决申请是作为K 08裁决DK 02546 -02的补充提交的。 成骨蛋白-1（OP- 1）或骨形态发生蛋白-7（BMP-7）是分泌生长因子的转化生长因子β（TGF-β）超家族的成员。 肾脏是OP-1合成的主要部位。 OP-1表达基因缺陷的小鼠具有明显异常的肾脏发育，表明该蛋白可能在肾脏细胞分裂和形态发生的调节中起重要作用。 此外，在肾纤维化的5/6肾切除术中，OP-1的系统给药已显示延迟或停止进展至终末期肾衰竭。 为了进一步探索这一现象，我们在肾纤维化的体外模型中评估了OP-1对细胞外基质的影响。OP-1浓度增加（0-300 ng/ml）导致小鼠系膜细胞和近端肾小管细胞中纤连蛋白水平呈剂量依赖性增加。 在一式两份样品中，处理RNA显示纤连蛋白RNA水平无显著变化，表明非转录机制。 同时，我们的实验室已经证明纤连蛋白是泛素依赖性降解的靶蛋白。 该提案最后两年的研究目标将集中在观察OP-1降低纤连蛋白的泛素依赖性降解。 这些目标将通过以下方式实现。 首先，我们将确定OP-1的作用是否对纤连蛋白具有特异性，或者它是否对其他细胞外基质蛋白如胶原蛋白I、III和IV具有类似的作用。 其次，我们将研究OP-1对纤连蛋白泛素化的潜在参与。 最后，利用共聚焦显微镜，我们将评估OP-1对纤连蛋白亚细胞分布的影响。 这一发现提出了在这方面提出的建议代表了一个新的范例ECM稳态。 了解这一途径的正常作用机制不仅将阐明细胞外基质稳态的新机制，而且可能具有重要的治疗意义。