基于Klotho调控的FGF23/FGFR4/PLCγ信号通路研究糖尿病肾病血管钙化机制及中药的干预作用

血管钙化是导致糖尿病肾病（DN）继发心血管疾病的重要因素。前期工作发现温补脾肾、祛瘀泄浊中药-肾元颗粒明显增强肾脏Klotho（Kl）基因与骨骼分泌的成纤维细胞生长因子（FGF）23结合而改善DN钙磷代谢。本研究基于肾主骨理论及DN脾肾亏虚、脉络瘀阻病机，以血管钙化为靶点，以Kl为调控基因，以db/db小鼠为DN模型，以肾元颗粒进行干预；运用RT-PCR、Western-blot等技术检测肾脏Kl、FGF23、胸主动脉FGF23、FGF受体4(FGFR4)、磷脂酶Cγ（PLCγ）及血管钙化标志物的表达；采用生物质谱检测胸主动脉FGF23/FGFR4复合物；在体外构建Kl高表达质粒转染大鼠主动脉平滑肌细胞，运用免疫共沉淀检测Kl与FGF23、FGFR4共表达，诱导PLCγ表达；阐明Kl调控的FGF23/FGFR4/PLCγ信号通路对血管钙化的影响及中药的干预作用，为中医药防治DN提供依据。

The vascular calcification acts as a leading cause to make diabetic nephropathy (DN) patients combine with cardiovascular disease (CVD).We have found that Klotho gene（Kl）secreted by kidney regulates calcium-phosphorus metabolism through fibroblast growth factor (FGF)23 secreted by bones. According to the theory of “kidneys govern bones” and mechanism of “the spleen and kidney deficiency, stasis of blood vessel”, targeting on the vascular calcification, we will conduct a study on the effects of the mediation of Kl to FGF23/FGFR4 PLCγ Signaling and TCM on the vascular calcification using spontaneous DN (db/db)models. It will be detected that protein and mRNA of Kl and FGF23 expresse in kidney, FGF23, FGF receptor 4(FGFR4), phospholipase C-γ(PLCγ) and biomarkers of vascular calcification in thoracic aortic by real-time quantitative fluorescence PCR and Western blot. The FGF23/FGFR4 complex will be detected in thoracic aortic by MALDI/TOF MS. In vitro, high expression plasmid of Kl will be constructed and transfected to aortic smooth muscle cells of rats. The co-expression of Kl, FGF23、FGFR4 which can induce PLCγ/calcineurin signaling will be examined by co-immunoprecipitation method. The study will reveal the mechanism of vascular calcification modulated by Kl and the effects of “tonifying spleen and kidney, dispel blood stasis and discharge turbid”on the vascular calcification of DN to facilitate the development of TCM.