线粒体自噬在急性高血糖导致的“肾毒性”损伤中的作用机制及小檗碱干预疗效研究

Traditional view believes that the major dangers of acute hyperglycemia are acute metabolic disorders induced by acute hyperglycemia like ketosis and acute hyperglycemic toxicity to islet β-cells. Our previous work show hyperglycemia keeping around 16.7mmol/L for 6 hours can damage renal tubular epithelial cell morphology and reabsorption function, with mitochondria injured and oxidative stress activated, but the mechanism is still not clear. Mitophagy plays a very important role in both clearing away reactive oxygen species and keeping a stable state in cells. Berberine has specific anti-oxidative stress and renal protective effect. So we hypothesize that acute hyperglycemia suppress mitophagy in tubular epithelial cells through AMPK-mTOR pathway, reactive oxygen species accumulate and finally lead to renal tubular epithelial cell injury. Based on the hypothesis, we set up acute hyperglycemic rat model using hyperglycemic clamp technique, combined with in vitro experiment, to observe the impact of acute hyperglycemia on AMPK-mTOR pathway and indicators related to mitophagy using RT-PCR, Western blot and morphological observation, etc. Discuss further the damage is reversible or not, and whether the damage is reliable on glucose concentration. We will treat both rats and renal tubular epithelial cells using berberine to see the renal protective effect from the mitophagy perspective. In conclusion, we will further reveal the possible mechanism of acute hyperglycemia induced renal injury, and enrich the theory of danger brought from acute hyperglycemia.

传统观点认为，急性高血糖危害主要是引起酮症等急性代谢紊乱及对胰岛β细胞的“高糖毒性”。课题组前期发现，16.7mmol/L高糖持续作用6小时可导致大鼠肾小管结构和功能损伤，伴线粒体受损和氧化应激激活，具体机制不清。线粒体自噬在清除活性氧物质、维持细胞稳态中起重要作用。小檗碱具有明确抗氧化应激及肾保护作用。据此提出假说：急性高血糖通过AMPK-mTOR通路抑制肾小管上皮细胞线粒体自噬，活性氧堆积，参与急性高糖导致的“肾毒性”损伤发生。课题组通过高糖钳夹技术建立大鼠急性高血糖模型，结合体外实验，运用RT-PCR、Western blot、组织形态学等方法观察急性高糖对AMPK-mTOR自噬调控通路及线粒体自噬的影响，探讨急性高糖“肾毒性”损伤的可逆性及浓度梯度关系，并观察小檗碱对急性高糖“肾毒性”损伤的干预效果及机制。研究结果将进一步揭示急性高血糖导致肾脏损伤的机制，丰富急性高血糖危害的理论。

研究背景. 急性高血糖可导致大鼠肾小管结构和功能损伤伴线粒体受损和氧化应激激活，线粒体自噬在清除活性氧物质、维持细胞稳态中起重要作用。小檗碱具有明确抗氧化应激及肾保护作用。据此提出假说：急性高血糖通过AMPK-mTOR通路抑制肾小管上皮细胞线粒体自噬，活性氧堆积，参与急性高糖导致的“肾毒性”损伤发生。.研究内容. 通过高糖钳夹技术建立大鼠急性高血糖模型，结合体外实验，运用RT-PCR、Western blot、组织形态学等方法观察急性高糖对AMPK-mTOR自噬调控通路及线粒体自噬的影响，探讨急性高糖“肾毒性”损伤的可逆性及浓度梯度关系，并观察小檗碱对肾小管上皮细胞损伤的干预效果及机制。.研究结果. 1、急性高糖可导致肾脏结构和功能损伤，这种损伤在血糖浓度达到11.1mmol/L时即已出现，且以肾小管损伤为主；急性高糖导致的肾脏损伤呈浓度依赖性，即血糖浓度越高损伤越重；2、急性高糖导致的肾小管损伤一定程度上可逆，随着解除高糖作用时间的延长，肾小管损伤逐渐恢复；3、在急性高糖导致的肾小管损伤中，肾小管上皮细胞线粒体自噬受到抑制，且与血糖浓度相关，血糖浓度越高，线粒体自噬抑制程度越重；AMPK-mTOR信号通路参与急性高糖导致的肾小管上皮细胞线粒体自噬的抑制调节；4、小檗碱可在一定程度上改善白蛋白负荷导致肾小管上皮细胞自噬抑制，减轻细胞损伤。.科学意义. 1、进一步验证了急性高血糖导致“肾毒性”损伤的浓度依赖关系，并在一定程度上可逆，AMPK-mTOR信号通路起关键作用，丰富了急性高血糖危害的理论；2、验证了小檗碱可通过AMPK通路改善自噬，减轻细胞损伤。