APE1在高草酸诱导肾小管上皮细胞线粒体氧化应激损伤中的作用

Oxalate-induced mitochondrial oxidative stress injury in renal tubular epithelial cells is the key step in calcium oxalate calculus formation. Apurinic/apyrimidinic endonuclease-1(APE1) plays a central role in the development of mitochondrial DNA damage repair process and is crucial to apoptosis. However, whether APE1 is involved in stone formation is still unknown. It was shown in our studies that, mitochondria-mediated apoptotic pathway was in the highest flight in renal tubular epithelial cells’ apoptosis in patients with nephrolithiasis and the expression of APE1 was notable positive compared to those expressed in the control group. Therefore, we propose that APE1 is crucial to the development of oxalate-induced mitochondrial oxidative stress injury and apoptosis in renal tubular epithelial cells. In this study, we will investigate the cellular localization of APE1 in renal tubular epithelial cells induced by oxalate, explore the relationship between APE1 gene’s mitochondrial-localized expression and the function of mitochondria as well as cell proliferation. Also, we will map the post transcriptional modification patterns and analyze the impact of APE1 expression to certain downstream transcription factors’ expression activity and expression level. Through these efforts, we hope to reveal the mechanism and role of APE1 in the development of oxalate-induced mitochondrial oxidative stress injury in renal tubular epithelial cell so as to provide new ideas for the research of pathogenesis of nephrolithiasis.

肾小管上皮细胞的线粒体氧化应激损伤是草酸钙结石形成的关键环节，脱嘌呤/脱嘧啶核酸内切酶（APE1）对线粒体DNA损伤修复起关键作用，但APE1是否参与草酸钙结石的形成尚不明确。我们前期研究发现，草酸钙结石患者肾脏组织APE1表达与正常人相比显著增高，且电镜观察凋亡的肾小管上皮细胞内存在特异性的线粒体氧化应激损伤。据此我们推测，在高草酸诱导的肾小管上皮细胞氧化应激损伤中，APE1对线粒体DNA修复功能改变及其介导的细胞凋亡起到重要作用。本课题将在此基础上进一步明确高草酸暴露下肾小管上皮细胞内APE1的定位，研究其对线粒体功能和细胞存活、增殖能力的影响；分析APE1在线粒体氧化应激损伤下的转录后修饰改变及其对下游特定转录因子表达水平和活性的影响，阐明APE1在高草酸诱导的肾小管上皮细胞线粒体氧化应激损伤中的作用和分子机制，为肾结石发病机制研究提供新的理论依据。

肾小管上皮细胞的线粒体氧化应激损伤是草酸钙结石形成的关键环节，脱嘌呤/脱嘧啶核酸内切酶（APE1）对线粒体DNA损伤修复起关键作用，但APE1是否参与草酸钙结石的形成尚不明确。我们前期研究发现，草酸钙结石患者肾脏组织APE1表达与正常人相比显著增高，且电镜观察到凋亡的肾小管上皮细胞内存在特异性的线粒体氧化应激损伤。据此我们推测，在高草酸诱导的肾小管上皮细胞氧化应激损伤中，APE1对线粒体DNA修复功能改变及其介导的细胞凋亡起到重要作用。本研究主要针对肾小管上皮细胞在结石形成的氧化应激损伤中发生显著变化的APE1，开展其在结石形成过程中的作用及其具体作用机制研究，进一步明确高草酸暴露下肾小管上皮细胞内APE1的定位以及分析APE1在线粒体氧化应激损伤下的转录后修饰改变。研究发现，高浓度草酸和一水草酸钙刺激肾脏上皮细胞HK-2细胞后，APE1蛋白表达水平上调。同时应用MTT方法检测了细胞的增殖活力。高草酸刺激HK-2细胞使其细胞内活性氧成分增加，诱导凋亡，而应用抗氧化剂N-乙酰半胱氨酸后，能够逆转这一进程。凋亡发生时，细胞内定位发现APE1蛋白发生了核转移，以及APE1的泛素化水平升高；同时, 通过研究APE1氧化还原剂抑制剂E3330对高草酸造成APE1核转移及细胞死亡的保护，发现E3330能够抑制高草酸诱导的APE1的核转移和细胞凋亡。因此，高草酸诱导的肾小管上皮损伤这一进程与APE1蛋白异常表达、修饰和重分布密切相关。本课题揭示了APE1在高草酸诱导的肾小管上皮细胞线粒体氧化应激损伤中的作用和分子机制，为肾结石发病机制研究提供新的理论依据，也为治疗该疾病提供了新的干预靶标与候选药物分子。