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

本RO3奖申请书是对K08奖DK02546-02的补充。成骨蛋白-1(OP-1)或骨形态发生蛋白-7(BMP-7)是分泌型生长因子超家族中的一员。肾脏是OP-1合成的主要部位。OP-1基因表达缺陷的小鼠肾脏发育明显异常，提示该蛋白可能在调节肾脏细胞分裂和形态发生中起重要作用。此外，在肾纤维化的5/6肾切除术中，系统地应用OP-1已被证明可以延缓或阻止终末期肾功能衰竭的进展。为了进一步探讨这一现象，我们在体外肾纤维化模型中评估了OP-1对细胞外基质的影响。OP-1浓度增加(0-300 ng/ml)可引起小鼠肾小管上皮细胞和近端肾小管上皮细胞纤维连接蛋白水平呈剂量依赖性增加。在为RNA处理的重复样本中，纤维连接蛋白RNA水平没有显著变化，这表明一种非转录机制。同时，我们的实验室已经证明，纤维连接蛋白是泛素依赖的降解的靶蛋白。该提案最后两年的研究目标将集中于观察OP-1减少泛素依赖的纤维连接蛋白的降解。这些目标将通过以下方式实现。首先，我们将确定OP-1对纤维连接蛋白的作用是特异的，还是对其他细胞外基质蛋白如I型、III型和IV型胶原具有类似的作用。其次，我们将研究OP-1对纤维连接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对纤维连接蛋白亚细胞分布的影响。本提案中提出的这一发现代表了ECM稳态的一个新范式。了解该通路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。

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.