利用外源性胰腺功能支持模型研究糖尿病肾病代谢异常机制

Metabolic abnormality is the main pathogenesis of diabetic nephropathy (DN), previous research mainly focused on the abnormal metabolism of glucose, research related to lipid, amino acid and other abnormal metabolism was insufficient. Additionally, there are lacking of specific biological markers for early warning, early diagnosis, and evaluation of the progress of DN. This project uses parabiosis animal model to construct an exogenous pancreatic functional support DN animal model, to verify the role of exogenous pancreatic functional support in reducing DN and improving its progression. Various kinds of technologies such as Non-targeted metabonomics, cytokine quantitative detection antibody chip and bioinformatics analysis were used to screen key metabolic factors and signaling pathways for DN progression. Through construction of cell and animal models modified by the selected metabolic key factors gene or intervention by the metabolic key factors, both in vivo and in vitro experiments would be conducted, to elucidate the molecular mechanisms of metabolic key factors in regulating the proliferation, apoptosis, autophagy, oxidative stress and inflammation of DN mesangial cells/tubular epithelial cells, and to provide basic research evidence for the biological markers of DN and the new targets of intervention. The project not only enriching the theoretical connotation of DN development and progression, but also laying the experimental foundation for developing new targets for DN prevention and treatment, thus, possessing an important scientific value and clinical significance.

代谢异常是糖尿病肾病（diabetic nephropathy，DN）的主要发病机制，但目前研究主要聚焦在糖代谢异常，脂质代谢、氨基酸等异常代谢研究不足；并且临床缺乏DN预警、早期诊断的特异性生物学标志物，延缓DN进展的防治手段匮缺。本项目利用前期研究建立的成熟连体动物模型，构建DN外源性胰腺功能支持模型，验证外源性胰腺功能支持改善DN发生进展的作用；采用非靶向代谢组学、细胞因子定量检测抗体芯片及生物信息学分析等技术，筛选DN发生进展的代谢关键因子和信号通路；通过构建代谢关键因子基因修饰的细胞和动物模型或代谢关键因子的干预，体内外实验解析代谢关键因子调控DN肾小球系膜细胞/肾小管上皮细胞增殖、凋亡、自噬、氧化应激及炎症的分子机制，为寻找DN生物学标志和干预新靶点提供基础研究证据。从而，丰富DN发生进展的理论内涵，奠定研发DN防治新靶标的实验基础，具有重要的科学价值和临床意义。

糖尿病肾病（diabetic kidney disease, DKD）的发病率逐年升高，目前临床缺乏DKD预警和早期诊断的特异性生物学标志物，延缓DKD进展的特异性防治手段匮缺。筛选出DKD特异性代谢异常是解析DKD发生进展以及干预新靶点的关键所在。利用连体动物模型创建外源性胰腺功能支持动物模型，结合高通量组学技术，为筛选DKD发生进展的关键代谢因子提供了技术保障。本课题中将野生小鼠分别与处于DKD不同时期的小鼠构建连体模型，通过连体动物间血液循环，野生小鼠胰腺分泌的胰岛素等代谢因子进入DKD小鼠体内调控物质代谢；DKD小鼠体内物质进入野生小鼠体内，利用野生小鼠的胰腺进行代谢调控；从而实现野生小鼠对DKD小鼠的胰腺功能支持。通过比较连体模型中DKD小鼠血糖、血脂、尿蛋白、肾功能及肾脏组织病理的变化差异，证实了外源性胰腺功能支持通过调控DKD小鼠的血糖、血脂代谢，减少了蛋白尿、改善了肾小球高滤过，减轻了肾组织损伤，具有延缓 DKD发生进展的作用。同时采用细胞因子定量检测抗体芯片技术，结合系统生物信息学分析，筛选出了DKD发生进展的代谢关键因子Fractalkine。Fractalkine在体内与其受体CX3CR1结合发挥作用，体外实验初步证实该通路激活能够促进肾小管上皮细胞自噬、凋亡及炎症反应，具有增加、促进肾小管肾损伤的作用。在此基础上，应用CX3CR1基因敲除（Cx3cr1 KO）小鼠叠加STZ诱发DKD模型进行体内实验，进一步明确了Fractalkine/CX3CR1信号通路具有促进DKD发生进展的作用。综上，本课题通过构建复合动物模型、筛选关键代谢因子以及体内、体外验证实验，证实了项目的科学假说：外源性胰腺功能支持通过调节DKD小鼠的异常代谢，延缓DKD的发生进展；Fractalkine/CX3CR1信号通路通过调控肾组织凋亡、自噬、炎症及纤维化，促进DKD的发生进展。该发现进一步阐明DKD发生进展的代谢性异常机制和关键环节，为寻找DKD生物学标志和干预靶点提供基础研究证据。