AMPK/SIRT1对TGF-beta/Smad信号通路的调控在输尿管梗阻早期肾损伤修复中的机制及作用研究

Obstructive nephropathy is a common disorder in urinary surgery. In children, obstructive nephropathy represents 16.1% of all pediatric kidney transplantation. At present, dialysis or transplantation is then necessary, with loss of quality of life and increase costs to health -care system. We performed a comparative proteomics study of the kidney from the rats with ureteral obstruction. We identified 39 differentially expressed proteins, there were some identified to be involved in the process of mitochondria biogenesis, oxidative stress and TGF-β pathway. Moreover, mitochondria energy sensors AMPK and SIRT1 have crosstalk with TGF-β/Smad signal pathway which is an important pathway leading to apoptosis, EMT and fibrosis in kidney diseases. Therefore, indicated earlier renal injury is due to mitochondria dysfunction and kidney energy loss, which activate TGF-β pathway, leading to renal cell apoptosis and fibrosis. Our previous study also demonstrated the spatio-temporal pathological changes of the kidney in animal model with partial ureteral obstruction and after the obstruction was released. In this study, we will develop a cell strain of rat tubular epithelial cell and podocyte, and TGF-β1 and fluid shear stress were used to induce cell EMT. RNAi and pc-DNA expression vetor will be employed at different points of TGFβ-Smad signal pathway by knocking-down or knocking-up AMPK(according to the in vitro experiment results) and knocking-up SIRT1 in reanl epithelial cell and podocyte. We will study the effects of regulating expression of AMPK and SIRT1 on cell injury in vitro. Then partial ureteral obstruction model was established. In vivo studies will be performed to confirm the in vitro results and to confirm the anti-fibrosis effects of regulating AMPK/SIRT1 expression on TGF-β/Smad pathway. We also want to find a suitable time point for gene theray. Through this study, we aimed to explore the combination therapies that target different points of TGF-β/Smad pathway which involves earlier renal injury and to find a new target for further therapy.

梗阻性肾病是泌尿外科常见疾病，儿童梗阻性肾病导致的终末期肾脏疾病占所有儿童肾移植的16.1%，除终身的血液透析或肾移植尚无好的治疗方法。前期应用蛋白质组学研究首次发现线粒体能量失衡是输尿管梗阻肾脏早期病理改变基础，而和线粒体能量代谢相关的二个能量监控器AMPK和AIRT1与导致肾脏凋亡和纤维化发生的TGF-beta/Smad信号通路密切相关，因此，我们提出输尿管梗阻后肾脏早期病理改变可能是由于线粒体功能障碍，肾脏能量逐渐丧失，并激活TGF-β通路，最后导致细胞凋亡和肾脏纤维化的发生。以此为切入点，建立AMPK及SIRT1干扰或表达载体，在细胞水平进行干扰，观察细胞生物学改变，并以此为基础，在部分输尿管梗阻前后不同时间点，将携带目的基因RNAi或pc-DNA的慢病毒载体转入活体动物体内，观察调控AMPK/SIRT1的表达对肾脏损伤发生的影响，探讨作用机制，为梗阻性肾病肾损伤寻找新的治疗靶点。

先天性梗阻性肾病是小儿泌尿外科常见疾病，儿童梗阻性肾病导致的终末期肾脏疾病占所有儿童肾移植的16.1%，除终身的血液透析或肾移植尚无好的治疗方法。前期应用蛋白质组学研究首次发现线粒体能量失衡是输尿管梗阻肾脏早期病理改变基础，而线粒体能量代谢相关的二个能量监控器AMPK和SIRT1与导致肾脏凋亡和纤维化发生的TGF-beta/Smad信号通路密切相关。我们以此为切入点，本研究中建立AMPK及SIRT1干扰载体，在体内和体外水平进行干扰，观察调控AMPK/SIRT1的表达对肾脏损伤发生的影响，发现通过调控能量和代谢感应关键蛋白AMPK/SIRT1的表达，可以不同角度负性调节与肾脏细胞炎症浸润、凋亡和EMT发生相关的TGF-beta/Smad信号通路。