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

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.

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。

糖尿病肾脏病（DKD）因其高发病率、高致残率、高额医疗费已成为危害人类健康的公共卫生问题。钙磷代谢异常所导致的血管钙化是DKD继发心血管疾病的独立危险因素，其发病机制尚未完全阐明，现有干预措施没有收到理想效果。申请人前期研究及相关报道发现DKD钙磷代谢紊乱与肾脏产生的Klotho(Kl)基因和骨骼分泌的成纤维细胞生长因子23（FGF23）异常相关。本研究基于肾主骨理论及DKD脾肾亏虚、浊邪内蕴病机，以Kl为调控基因，以钙磷代谢为靶点，给予健脾补肾、通腑泄浊中药干预。采用STZ诱导的糖尿病肾病小鼠模型及HK-2细胞模型，运用免疫印记、免疫共沉淀、激光共聚焦、KlsiRNA及Kl质粒转染等技术，解析Kl、早期生长反应基因1（Egr1）、FGF23、24羟化酶(CYP24)的交互作用及对钙磷代谢的影响，验证健脾补肾、通腑泄浊中药的作用机制。.通过超高效液相色谱串联质谱技术，测得大鼠肾元颗粒含药血清中淫羊藿苷、黄芪甲苷、大黄素的含量分别为0.87μg/ml、0.76μg/ml、13.6μg/ml，进而采用该含药血清干预高糖环境下的HK-2细胞，发现肾元颗粒含药血清明显诱导Kl表达上调，协同增加FGF23诱导的Kl与FGFR1表达及共表达，抑制Egr1、CYP24、p-ERK1/2表达，增强α羟化酶（CYP27B1）表达；在siRNA沉默Kl表达的条件下，肾元颗粒含药血清仍能增强Kl、Kl与FGFR1表达及共表达，抑制Egr1与CYP24、增强CYP27B1表达。在体内，经STZ及高磷饮食诱导糖尿病肾病小鼠模型。与空白对照组相比，模型组24小时尿白蛋白、血糖、肌酐、磷及FGF23明显升高，血钙及25（OH）VitD3、肾脏Kl与FGFR1表达及共表达明显降低，CYP24、CYP27、p-ERK1/2明显增强；经肾元颗粒干预后，Kl与FGFR1表达及共表达明显增强，Egr-1、CYP24、CYP27B1及p-ERK1/2表达受到抑制，低钙高磷状态得到纠正，在胸主动脉中观察到钙盐沉积减轻，胫骨骨膜下软骨细胞形态得到一定程度改善。通过研究，在HK-2细胞模型及糖尿病小鼠模型中建立了Kl调控的FGFR1/FGF23/Egr-1信号通路与钙磷代谢及血管钙化的关系，揭示了肾元颗粒通过上述靶点改善糖尿病肾病钙磷代谢、减轻血管钙化的新机制，为拓展肾主骨理论的临床应用提供了依据。