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

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.

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。

血管钙化是导致糖尿病肾病（DN）继发心血管疾病的重要环节。肾主骨理论认为肾气促进骨骼生长并控制衰老，而血管钙化是衰老的显著标志。研究基于肾主骨理论及DN脾肾亏虚、脉络瘀阻病机，采用db/db小鼠模型，饲以高磷饮食诱导血管钙化。结果显示与db/m组相比，模型小鼠呈现体型肥胖、血糖升高、尿白蛋白肌酐比值（UACR）增加、肾功能异常，病理表现为肾小球基底膜增厚、系膜基质增加、肾小球体积增大等DN特征；胸主动脉弹性纤维断裂、内膜凹凸不平、中膜排列紊乱、α-SMA、OPG表达减弱，钙调蛋白PLCγ、Calcineurin及成骨基因Runx2表达增强，伴钙盐沉积。模型小鼠肾脏Kl表达较正常对照组明显减弱，血清可溶性Kl（sKl）水平降低，而FGF23明显升高；免疫共沉淀显示模型组胸主动脉FGF23与FGFR4结合增强，进一步采用质谱分析，发现各组间存在差异表达的化合物。在不同剂量健脾补肾、祛瘀泄浊中药肾元颗粒干预组，小鼠模型UACR、肾脏病理及肾功能得到显著改善；随着肾脏Kl表达增强，胸主动脉FGF23与FGFR4表达、成骨表型及钙盐沉积明显减轻。体外采用高糖联合高磷诱导大鼠血管平滑肌细胞（VSMCs）钙化模型。检测显示细胞钙含量及ALP活性明显增加，α-SMA、SM22α表达呈下降、成骨因子Runx2表达呈增加趋势。经FGF23处理后，FGFR4、PLCγ表达明显上调。采用BLU9931与U73122分别抑制FGFR4与PLCγ活化，上述蛋白表达得到逆转。经慢病毒转染sKl及肾元颗粒含药血清干预后，VSMCs成骨表型、FGFR4、PLCγ、Runx2表达及钙盐沉积明显减轻。说明肾脏产生的Kl与骨骼分泌的FGF23、血管表达的FGFR4相互应答，参与了DN血管钙化，肾元颗粒通过增强Kl表达，减轻FGF23/FGFR4介导的血管钙化，为临床应用中医药防治DN心血管并发症提供依据。