氧化应激诱导MUTYH异常活化在肾间质纤维化中的作用及其线粒体机制研究

Tubulointerstitial Fibrosis (TIF) is the common final pathway of diverse forms of chronic renal disease that contributes to both organ insufficiency and failure. Although a number of experimental studies have attempted to clarify the pathogenesis of TIF, its evaluation and management remain poorly defined. 8-Oxoguanine (8-oxoG), a common DNA lesion caused by reactive oxygen species, is associated with TIF. But the mechanism by which 8-oxoG causes TIF is unknown. The human mutY homologue (MUTYH) gene is involved in the base excision repair system and removes the adenine inserted opposite 8-oxoG in template DNA during DNA replication. Previously, we reported a novel variation in MUTYH (AluYb8MUTYH), which was showed to increase the risk for renal insufficiency and failure. However, MUTYH deficiency would improve the TIF in unilateral urethral obstruction (UUO) mice, and DNA mutations could not been detected in the cells with AluYb8MUTYH variation. More evidences show that TIF is a complex process caused by 8-oxoG accumulation in mitochondrial of renal tubular epithelial cells (RTECs). Different signaling pathways were triggered by the accumulation of single-strand breaks (SSBs) in each type of DNA generated during base excision repair initiated by MUTYH, suggesting that MUTYH may have the effect on TIF under conditions of oxidative stress. We then focused on the mitochondrial pathway involving SSBs, which is related with Calpain activation, and then cell apoptosis and caspase-1 dependent inflammation action. Firstly, we will study the effect of MUTYH on TIF in vivo, using mutant or transfer mice lacking or over-expressing MUTYH under the condition of UUO, and patients with chronic renal disease. Secondly, we will study the effect of MUTYH on apoptosis and inflammation in vivo, using mutant or transfer RTECs lacking or over-expressing MUTYH under the condition of H2O2. Thirdly, we will demonstrate the mechanism regarding oxidative stress, oxidative DNA lesions, SSBs, mitochondrial Calpain signal pathway, and its role on apoptosis and caspase-1 dependent inflammation. In a word, this project is planned to provide a new insight into the involvement of base excision repair as a possible mitochondrial mechanism for oxidative stress induced TIF.

肾间质纤维化（TIF）是肾脏病领域的难点问题，氧化应激被证实参与TIF的发生、发展；而MUTYH是DNA氧化损伤修复系统的重要组分。我们前期首次证实MUTYH与TIF的关系，认识到氧化应激下MUTYH的异常活化，可能通过Calpain介导的线粒体途径调控细胞凋亡和炎症，参与TIF的过程。研究拟利用MUTYH差异表达小鼠，构建UUO模型以及临床病例，明确该基因在TIF中的作用；结合MUTYH差异表达的肾小管上皮细胞(RTECs)，探讨MUTYH与H2O2诱导RTECs凋亡及炎症的关系。在此基础上，体内、外阻断Calpain活性，阐明MUTYH通过Calpain介导的线粒体途径，参与细胞凋亡和Caspase-1依赖炎症级联反应的作用机制。项目从体内到体外，从基础到临床，深入探讨氧化应激、MUTYH和TIF的关系及其线粒体机制，从而丰富氧化损伤修复与疾病发生的学说，并为TIF的预治提供新的理论。

目前，在世界范围内，慢性肾脏病(CKD)俨然成为了严重的公共卫生问题之一。如何减轻肾脏小管间质的纤维化对于有效地延缓 CKD的进展有着十分重要的意义。近年来，关于线粒体功能障碍在CKD疾病进展中的作用得到越来越多的揭示。MUTYH基因位于一号常染色体，大小约11.2Kb，其包括16个外显子，主要编码两种MUTYH蛋白：Ⅰ型蛋白质作用于线粒体DNA，Ⅱ型蛋白作用于细胞核DNA，通过切除与8氧鸟嘌呤错配的腺嘌呤从而减少氧化应激导致的DNA G:C向A:T的突变，维持基因的稳定性。.1. 观察MUTYH基因敲除对于肾间质纤维化及线粒体功能的影响。发现：⑴ 在野生型小鼠UUO模型中，MUTYH基因的表达随着梗阻时间的延长而增高。⑵ ]与野生型UUO模型相比，MUTYH基因敲除使得细胞外基质沉积增多，α-SMA、TGF-β、collagenⅠ和collagen Ⅲ的表达增高及炎症因子的释放更为明显。⑶ 相较于对照组，UUO模型中mtDNA的拷贝数、MMP下降，ROS的产生增多，提示UUO模型中存在线粒体功能障碍，这一现象在MUTYH基因敲除的UUO模型中更为严重。可见，MUTYH基因的敲除加重了UUO模型的肾间质纤维化及线粒体功能障碍。.2. 过表达MUTYH对于TGF-β1诱导的HK-2细胞纤维化及线粒体功能障碍的作用。发现：⑴ 加入TGF-β1刺激后，qRT-PCR检测发现MUTYH基因的表达随着刺激时间的延长而增多，编码的Ⅰ型线粒体蛋白(～57kDa)表达减少，而Ⅱ型核蛋白(～53kDa)的表达增多。⑵ 给予TGF-β1刺激后细胞纤维化指标α-SMA、collagenⅠ和collagen Ⅲ的表达较对照组增多，8-oxoG的水平增高，过表达h-mutyh可以抑制TGF-β1诱导产生的上述现象。⑶ 与对照组相比，TGF-β1刺激的HK-2细胞mtDNA拷贝数、MMP下降、ROS产生增多，提示TGF-β1刺激可导致HK-2细胞线粒体功能受损，而过表达h-mutyh可使得mtDNA拷贝数、MMP下降的程度减轻，ROS的产生减少。可见：过表达MUTYH减轻TGF-β1诱导的HK-2细胞纤维化及线粒体功能障碍。