分泌型Klotho蛋白抑制肾间质纤维化的机制研究

目的 研究分泌型Klotho蛋白抑制肾间质纤维化的机制,为治疗慢性肾脏病提供理论基础和新思路。方法 通过单侧输尿管梗阻（UUO）诱导C57BL/6小鼠肾纤维化,并分5组处理：假手术Sham+磷酸盐缓冲液（PBS）]组、模型（UUO+PBS）组、UUO+Klotho组、UUO+Klotho+胰岛素组、UUO+Klotho+胰岛素样生长因子-1（IGF-1）组。检测小鼠左肾组织活性氧水平,并用HE及Masson染色评估肾脏病理改变及纤维化程度。实时荧光定量PCR、蛋白免疫印迹法以及免疫荧光化学检测肾组织中超氧化物歧化酶2 （SOD2）、纤连蛋白和α-平滑肌肌动蛋白（α-SMA）表达水平,此外用蛋白免疫印迹法检测磷酸化蛋白激酶B（p-AKT）、AKT、磷酸化磷脂酰肌醇（-3）激酶（p-PI3K）、PI3K、胰岛素受体底物-1（IRS-1）蛋白表达水平。结果 成功建立了UUO小鼠模型。与UUO+PBS组比较,UUO+Klotho组小鼠肾组织的纤维化缓解（P <0.001）,且肾纤维化标志物纤连蛋白和α-SMA mRNA及蛋白的表达降低（P均 < 0.001）,保护因子SOD2 mRNA及蛋白表达升高（P均 < 0.001）。胰岛素及IGF-1可激活肾组织IRS-1/PI3K/AKT通路并破坏Klotho对肾脏的保护作用（P均 < 0.001）。结论 Klotho通过抑制胰岛素下游IRS-1/PI3K/AKT信号通路从而缓解肾脏的纤维化。

Soluble Klotho attenuates unilateral ureteral obstruction-induced renal fibrosis in mouse models

Objective To investigate the mechanism of soluble Klotho in inhibiting renal interstitial fibrosis, and provide theoretical evidence and novel ideas for the treatment of chronic kidney disease. Methods C57BL/6 mouse models with renal fibrosis were established by unilateral ureteral obstruction （UUO） and divided into five groups： Sham+PBS, UUO+PBS, UUO+Klotho, UUO+Klotho+insulin, and UUO+Klotho+insulin-like growth factor （IGF）-1 groups. The reactive oxygen species （ROS） level in the left kidney was detected. The renal pathological changes and fibrosis were assessed by HE and Masson staining. The expression levels of superoxide dismutase 2 （SOD2）, fibronectin and α-smooth muscle actin （α-SMA） in the renal tissues were measured by qRT-PCR, Western blot and immunofluorescence assay, respectively. In addition, the expression levels of p-AKT, AKT, p-PI3K, PI3K and IRS proteins were also assessed by Western blot. Results The UUO mouse models were successfully established. Compared with the UUO+PBS group, the renal fibrosis was significantly alleviated, the expression levels of fibronectin and α-SMA mRNA and proteins were significantly down-regulated （all P < 0.001）, whereas those of SOD2 mRNA and protein were considerably up-regulated （both P < 0.001） in the UUO+Klotho group. Insulin and IGF-1 treatment activated the IRS-1/PI3K/AKT signaling pathway in the renal tissues and impaired the protective role of Klotho upon the UUO-induced renal fibrosis （both P < 0.001）. Conclusion Klotho can alleviate renal fibrosis by inhibiting the IRS-1/PI3K/AKT signaling pathway in the UUO-induced mouse models.