基于klotho基因/FGF23/Egr1信号通路研究温补脾肾通腑泄浊法对糖尿病肾病钙磷代谢的影响

钙磷代谢异常所致的血管钙化及肾性骨病是糖尿病肾病的严重并发症，其发生与肾脏分泌的klotho（kl）基因和骨骼产生的纤维原细胞生长因子23（FGF23）表达异常相关。本研究基于肾主骨理论及糖尿病肾病脾肾亏虚、浊毒内蕴病机，采用STZ腹腔注射复制糖尿病肾病小鼠模型，给以温补脾肾、通腑泄浊中药，观察其对模型钙磷代谢、血管钙化和肾性骨病的影响；运用生物质谱技术、适时定量PCR、免疫印记等检测肾组织Kl与FGF23结合，介导早期生长反应基因1（Egr1）及24羟化酶(CYP24)的表达；采用比色法测定胸主动脉、胫骨碱性磷酸酶活性及胸主动脉钙含量。在体外构建Kl的siRNA及高表达质粒转染肾小管上皮细胞，运用免疫共沉淀、免疫细胞化学法等检测Kl与FGF受体1、FGF23共表达，诱导Egr1、CYP24基因表达。阐明Kl基因对钙磷代谢的影响，为肾主骨理论的深入研究及温补脾肾、通腑泄浊法的应用提供依据。

Vascular calcification and renal osteopathy caused by abnormal calcium and phosphate metabolism is a serious complication of diabetic nephropathy. It is associated with the abnormal expression of Klotho secreted by kidney and FGF23 released by bones. Based on the TCM theory of kidney governing bones and kidney deficiency results in weak bones, this research focuses on Klotho-regulated calcium and phosphate metabolism, diabetic nephropathy will be replicated in mouse models via high calcium and phosphate diet and STZ intraperitoneal injection. Combined with TCM treatment such as the pyretic tonification of spleen and kidney and the purgative elimination of turbidity,we will observe the effects on calcium and phosphate metabolism, vascular calcification and renal osteopathy. We seek to examine the binding of Klotho and FGF23 involved in the expression of Egr1 and CYP24 in the early stage. Alkaline phosphatase activities in the thoracic aorta and the shin bone as well as calcium level in the thoracic aorta will be measured using colorimetric method. Through in vitro study, high expression plasmid and siRNA of Klotho will be transfected to renal tubular epithelial cell. The co-expression of Klotho and FGFR1 as well as FGF23 which induce the gene expression of Egr1 and 24hydroxylase(CYP24) will be measured by co-immunoprecipitation and immunocytochemical methods.The expected results from this study could further decipher the theory of kidney governing bones and provide additional insights into diabetic nephropathy. It may also shed light on the therapeutic mechanism of the pyretic tonification of spleen and kidney and the purgative elimination of turbidity on diabetic nephropathy.