基于醛固酮/MR/SGK1通路探讨UUO诱导巨噬细胞表型转化介导对侧肾脏损伤的机制及中药的保护作用

The mechanism of unilateral renal injury lead to chronic renal failure is not clear, but infiltration of macrophage and translate to myofibroblasts induce by aldosterone which plays an important role in it. Increased aldosterone with UUO in experimental animals when binds to the mineralocorticoid receptor of renal natural cell in contralateral kidney through blood circulation. The macrophage phenotypic transformation and cell pyroptosis induced with inflammasome component which leading to chronic renal failure because of renal interstitial fibrosis through MR/SGK-1 pathway. In this study, the macrophage in contralateral kidney as the target, the nuclear translocation of NR3C2, co-expression of F4 / 80 and α-SMA will observe with immunofluorescence and confocal microscopy for verify the activation of MR and the phenotypic transformation of macrophages. Expression of NF-κB and NLRP3 were detect with Real time-PCR, Western blot and immunofluorescence method. The mechanism of macrophage phenotypic transformation, cell pyroptosis, cell proliferation and renal blood vessels injure induced by aldosterone should be investigated. Protection of Buxujieduhuoxuehuayu herbs and MR blockade also observe for mechanism of chronic renal failure induced with unilateral kidney injury partly. Expand the application of the ‘both disease prevention and change prevention’ in the treatment of chronic kidney disease and provide a theoretical basis for clinical treatment.

单侧肾脏损伤进展为慢性肾衰竭的机制尚不明了，醛固酮刺激巨噬细胞浸润并转化为肌成纤维细胞在其中发挥着重要作用。采用UUO动物模型诱导醛固酮分泌增多，经血液循环作用于对侧肾脏盐皮质激素受体（MR)，通过MR/SGK1信号通路招募巨噬细胞集聚并刺激其表型转化；或分泌炎性小体诱导细胞焦亡，形成间质纤维化导致慢性肾衰竭。本研究以“健侧”肾脏巨噬细胞为靶点，采用免疫荧光及共聚焦显微镜观察NR3C2的核转位、F4/80与α-SMA的共表达验证MR的活化及巨噬细胞表型转化，实时荧光定量PCR、蛋白印迹、免疫荧光等检测SGK1、NF-κB、炎性小体等表达，探讨醛固酮刺激巨噬细胞表型转化、炎性坏死、血管损伤的机制，同时观察补虚解毒活血化瘀中药及醛固酮受体阻断剂的干预作用，揭示单侧肾脏损伤诱导慢性肾衰竭的部分机制，拓展中医“既病防变”理论在慢性肾脏病治疗中的应用，为临床治疗提供理论依据。

单侧肾脏损伤诱导慢性肾衰竭的机制尚不明了，对侧肾脏纤维化是关键的病理改变，巨噬细胞浸润及表型转化参与这一过程。本研究观察益气活血解毒中药抑制盐皮质激素受体（MR）活化诱导巨噬细胞表型转化参与脏器纤维化的机制，从炎症诱导纤维化途径阐明“浊毒致瘀”病机的分子生物学机制。首先采用UUO大鼠复制肾间质纤维化实验动物模型，观察对侧肾脏巨噬细胞浸润及表型转化在其中的作用，给以盐皮质受体阻断剂和益气解毒化瘀中药干预，证实MR的活化在其中发挥着关键作用，中药抑制其活化可以减轻肾脏损伤；在此基础上采用醛固酮灌注方式观察对小鼠肾脏巨噬细胞浸润及表型转化的作用，实验结果显示皮下持续输注醛固酮可以诱导小鼠血压升高、肾功能损伤，大量炎性细胞浸润，巨噬细胞的集聚和表型转化参与肾脏纤维化的形成，盐皮质激素受体抑制剂依普利酮、艾沙利酮可以减轻病理损伤，中药的作用与抑制MR活化有关。体外分别观察醛固酮诱导RAW264.7以及骨髓来源巨噬细胞的增殖、迁移、管形成及表型转化，证实中药血清和盐皮质激素受体阻断剂可以抑制醛固酮诱导的细胞表型转化。此外，益气解毒化瘀中药还可以减轻UUO大鼠诱导的心脏、肺脏、血管的病理损伤，验证了中医“整体观念”及“治未病”理论的指导意义。本课题从炎症损伤角度探讨单侧肾脏损伤诱导慢性肾衰竭的机制，阐明肾素-血管紧张素-醛固酮系统的激活诱导盐皮质激素受体活化在其中发挥着重要作用，为临床治疗提供了实验依据。