莱菔硫烷通过Keap1/Nrf2通路改善糖尿病肾脏足细胞线粒体损伤的机制

High glucose-induced disruption of mitochondrial homeostasis incurs aberrant generation of reactive oxygen species in podocytes, leading to the process of diabetic podocytopathy which is tightly related to the genesis and development of proteinuria. Sulforaphane, a natural inducer of Nrf2, regulates redox balance by upregulating anti-oxidative genes; while it is still unclear whether sulforaphane-induced activation of Keap1/Nrf2 pathway is involved in mitochondria protection in podocytes under high glucose conditions. Preliminary experiments from our group revealed that sulforaphane protected podocyte mitochondrial from high glucose-induced injury, accompanied with elevation of PINK1 level; but the mechanism is undermined. Hereby, we propose hypothesis that sulforaphane regulates the expressions of PINK1 and p62 by activating Keap1/Nrf2 pathway, and then maintaining the mitochondrial homeostasis in podocyte by selectively scavenging the damaged mitochondria via initiating and facilitating mitophagy. Therefore, we intend to choose mouse podocytes and podocyte-specific Nrf2 knockout mice as experimental subjects, following by utilizing the technique of CRISPR/Cas9 system combined with lentiviral vector transfection and chemical inhibitors, as well as in vivo bioluminescence imaging, functional metabolic analysis by Seahorse XF technology, protein mass spectrometry analysis, immuno-electron microscope, CLAMS system for monitoring metabolism in vivo, chromatin immunoprecipitation and other advanced techniques, so as to explore the effect and mechanism of sulforaphane on regulating mitochondrial homeostasis in podocytes through the perspective of molecules, cells and animals, which will illuminate novel strategy of preventing and controlling diabetic kidney disease.

足细胞损伤是引起糖尿病肾病蛋白尿发生发展的重要因素，线粒体稳态失衡引起活性氧过度释放，在高糖所致足细胞损伤过程中扮演关键角色。莱菔硫烷可激活Keap1/Nrf2通路调控抗氧化基因表达，但其能否调节足细胞线粒体稳态尚不清楚。我们前期研究证实，莱菔硫烷可改善高糖引起的足细胞线粒体损伤，伴随PINK1水平上调，但机制不明。因此我们提出假设：莱菔硫烷可能通过Keap1/Nrf2通路激活PINK1/p62介导的线粒体自噬，选择性清除受损线粒体，维持足细胞线粒体稳态。为此，本研究拟选取小鼠足细胞、足细胞特异性Nrf2敲除小鼠为研究对象，结合CRISPR/Cas9系统、慢病毒载体、化学抑制剂应用，借助荧光素酶活体成像、Seahorse代谢检测、蛋白质谱分析、免疫电镜、CLAMS系统代谢检测、CHIP技术，从分子、细胞及动物整体水平探讨莱菔硫烷调控足细胞线粒体稳态的详细机制，为糖尿病肾病防治提供新思路。

糖尿病肾病（Diabetes kidney disease, DKD）是我国慢性肾脏疾病的首要发病原因。足细胞损伤是引起糖尿病肾病蛋白尿发生发展的重要因素，线粒体稳态失衡引起活性氧过度释放，在高糖所致足细胞损伤过程中扮演关键角色。莱菔硫烷可激活Nrf2通路调控抗氧化基因表达，但其能否调节足细胞线粒体稳态尚不清楚。我们前期研究证实，莱菔硫烷（Sulforaphane，SFN）可改善高糖引起的足细胞线粒体损伤，伴随Pink1水平上调，但机制不明。在本研究中，我们首先分别在体内、外构建小鼠糖尿病模型及高糖诱导的足细胞损伤模型，给予SFN应用后，检测肾脏和足细胞功能相关指标的变化，初步明确SFN在体外和体内对糖尿病肾损伤具有改善作用，并初步了解了Nrf2在SFN作用过程中的变化规律；其次我们构建了Nrf2稳定高表达和Nrf2敲低的足细胞株，应用SFN作用后，发现SFN可通过调控细胞核内Nrf2的表达和激活Nrf2通路，改善高糖诱导的足细胞损伤。随后我们通过构建Pink1敲减慢病毒进行细胞回复实验，发现SFN可以通过Nrf2/Pink1信号通路调节线粒体自噬，改善高糖足细胞线粒体损伤。最后，我们构建了足细胞Nrf2条件性敲除小鼠模型，应用SFN后发现莱菔硫烷在体内可通过调控Nrf2表达和激活Nrf2/Pink1通路，改善糖尿病小鼠足细胞线粒体损伤，延缓DKD进展。本项目的研究成果为糖尿病肾病足细胞线粒体稳态失衡的分子机制提供理论支撑，同时为糖尿病肾病的诊治挖掘新的分子靶点。