p53/miR-34a/SIRT1信号通路介导足细胞损伤参与糖尿病肾病发病的机制研究

Diabetic nephropathy (DN) is the most common cause of end-stage renal disease (ESRD) and its incidence has been increasing worldwide over the past decade, which is a worldwide public health problem that threatens human health seriously. We demonstrated previously that the role of transcription factor (TF) acetylation by Sirtuin 1 (SIRT1) in podocyte to mediate hyperglycemia-induced pathologic changes in diabetic nephropathy (DN). And we also showed that advanced glycation end products (AGEs) induced p53 acetylation and miR-34a expression, down-regulated the expression of SIRT1 in cultured human podocytes.Recent evidence suggests the activation of p53/miR-34a/SIRT1 pathway in the pathogenesis of DN, however, the mechanisms are not well characterized.Locked nucleic acid (LNA) miR-34a inhibitor was administered to diabetic db/db mice to down regulate miR-34a in vivo. Histology and morphological analysis are determined by periodic acid Schiff–stained sections of kidney.Hyperglycemia induced mitochondrial damage and autophagy response in DN are observed using isolated glomeruli and primary podocyte culture. In vitro conditionally immortalized human podocytes are transfected with miR-34a mimic or inhibitors, or infected with lentiviral plasmids. To confirm the mechanistic role of p53/miR-34a/SIRT1 pathway underlying podocyte injury in DN, podocytes are harvested at 24h posttransfection and processed for cell viability, autophagy and genes-encoding essential mitochondrial proteins.And the expression of TF acetylation and the induction of p53/miR-34a/SIRT1 positive feedback loop in AGEs-BSA treated podocytes are also examined. Targeting podocyte p53/miR-34a/SIRT1 pathway could be a potential new therapy for DN.

糖尿病肾病（DN）是严重威胁人类健康的世界性公共卫生问题。我们前期研究证实：晚期糖基化终末产物（AGEs）诱导人足细胞p53转录上调miR-34a表达，靶向抑制组蛋白脱乙酰酶SIRT1，SIRT1去乙酰化是减轻DN足细胞损伤的重要调控机制，因此，高糖可以激活p53/miR-34a/SIRT1信号通路，但具体机制尚未阐明。本研究运用锁核苷酸修饰技术构建miR-34a敲减糖尿病肾病（db/db)小鼠模型，评估病理改变，提取原代足细胞了解该信号通路表达和功能；体外人足细胞系运用miR-34a 模拟物、抑制物和慢病毒质粒干预 p53/miR-34a/SIRT1信号通路表达，深入解析该通路三者调控关系，了解SIRT1下游蛋白乙酰化后参与调控的足细胞自噬、凋亡和线粒体生物合成等改变，明确该通路介导足细胞损伤参与DN发病的具体机制。本研究将为临床DN足细胞损伤和修复提供新的理论依据和干预策略。

该课题聚焦p53/miR-34a/SIRT1通路在足细胞病发生发展的中的作用及其关键分子机制。根据研究计划，我们证实：（1）体内研究：以STZ诱导糖尿病肾病（DN）小鼠模型和阿霉素小鼠（AN）模型为研究对象，运用锁核苷酸（LNA）修饰技术敲减miR-34a从而体内干预 p53/miR-34a/SIRT1信号通路传导，发现敲除miR-34a可以下调DN和AN小鼠模型肾小球乙酰化P53水平，抑制miR-34a表达，上调SIRT1和足细胞自噬水平，显著缓解DN和AN蛋白尿水平，改善病理损伤。（2）体外研究：a.体外人足细胞阿霉素和AGE刺激均可以上调miR-34a和乙酰化P53表达，下调SIRT1，减少足细胞自噬水平，线粒体合成减少，足细胞凋亡增加；b. 敲除足细胞miR-34a可以上调SIRT1表达，去乙酰化p53增加，从而缓解阿霉素和AGE诱导的足细胞损伤；miR-34a下游靶基因NOX4参与高糖诱导的足细胞氧化应激ROS，丹参酚酸B可以抑制NOX4生成缓解AGE诱导足细胞损伤。c.敲除SIRT1可以增加乙酰化p53水平，加重足细胞损伤，因而miR-34a介导DN足细胞损伤依赖于SIRT1表达；d. 抑制p53可以下调乙酰化p53水平进而下调miR-34a，SIRT1表达增加缓解足细胞损伤，但无法抵消miR-34a高表达靶向抑制SIRT1造成的足细胞损伤，因而 P53介导高糖造成的足细胞损伤依赖miR-34a。（3）临床患者：选取了从2018.1-2019.12我科肾活检明确诊断DN和FSGS的患者各40例，肾组织行组化检测，临床患者外周血检测miR-34a表达，发现以微小病变患者为对照，SIRT1/乙酰化p53在DN患者肾小球中表达增强伴随外周血中循环miR-34a升高。因而，p53/miR-34a/SIRT1通路是介导足细胞参与DN发生发展的重要分子机制。目前该项目资助申请人第一作者已发表SCI 1篇，国内权威期刊论著1篇 ，培养博士研究生1名，2019年美国肾脏病年会（ASN）壁报1篇，资助期间另有2篇相关SCI论著发表。