Klotho蛋白对草酸钙肾结石大鼠肾脏氧化应激的影响

Effect of Klotho protein on renal oxidative stress in rat with calcium oxalate nephrolithiasis

目的探讨Klotho蛋白对草酸钙肾结石大鼠肾小管上皮细胞氧化应激的影响及其机制。 方法2016年11—12月选取30只6～8周龄雄性SD大鼠，分为正常组、结石组、药物组，每组10只。正常组每天生理盐水灌胃；结石组使用1%乙二醇和2%氯化铵诱导法建立大鼠肾草酸钙结石模型；药物组自由饮水，并用含1%乙二醇和2%氯化铵溶液灌胃的同时，给予2.5 mg福辛普利＋15.0 mg缬沙坦溶于蒸馏水，每次2 ml灌胃。喂养4周后处死各组大鼠。采用双抗体夹心酶联免疫吸附法（ELISA）检测大鼠肾组织中丙二醛（MDA）、超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和谷胱甘肽（GSH）的含量；RT-PCR法检测Klotho基因和核因子E2相关因子2（Nrf2）基因mRNA表达水平；蛋白质印迹法检测Klotho和Nrf2蛋白表达水平。 结果结石组MDA含量（12.43±0.43）μmol/mg，明显高于正常组（8.67±0.84）μmol/mg和药物组（7.97±0.81）μmol/mg（均P〈0.05），而药物组与正常组比较差异无统计学意义（P〉0.05）。正常组、结石组、药物组的SOD分别为（247.89±2.45）、（109.54±4.21）、（189.74±10.47）U/mg，GSH分别为（38.98±4.55）、（26.87±3.92）、（31.29±2.54）μmol/mg，CAT分别为（138.47±8.74）、（119.87±8.45）、（127.46±7.45）U/mg，结石组SOD、GSH、CAT含量均低于正常组和药物组（P〈0.05），而正常组和药物组比较差异无统计学意义（均P〉0.05）。结石组Klotho mRNA（0.208±0.036）和Nrf2 mRNA（0.499±0.086）表达水平明显低于正常组[分别为（1.011±0.174）和（1.023±0.139），均P〈0.05]和药物组[分别为（1.123±0.248）和（1.148±0.089），均P〈0.05]。结石组肾组织中Klotho蛋白（0.341±0.127）和Nrf2蛋白（0.201±0.030）低于正常组[分别为（0.750±0.140）和（0.558±0.095），均P〈0.05]和药物组[分别为（0.841±0.288）和（0.450±0.153），均P〈0.05]。 结论福辛普利和缬沙坦可通过上调乙二醇诱导的Klotho基因低表达，降低肾小管上皮细胞损伤，抑制草酸钙晶体黏附成石；Klotho的抗氧化损伤作用可能与Keap1-Nrf2-ARE信号通路被激活有关。

ObjectiveTo investigate the protective effect and mechanism of Klotho protein on oxidative stress in renal tubular epithelial cells of experimental rat models of renal calcium oxalate stone. MethodsThe 30 SD rats, 6-8 weeks old, were randomly divided into 3 groups（10 of each）, normal control group（group A）, calcium oxalate model group（group B）, drug plus calcium oxalate model group（group C）. Group A was established with physiological saline by garage each day, group B was established with 1% ethylene glycol in drinking water＋ 2% ammonium chloride by garage（2 ml/d）, group C was established with Fosinopril 2.5mg＋ Valsartan 15mg aqueous solution 2 ml by gavage on the basis of group B（2 ml/d）. 4 weeks later, the level of malondialdehyde （MDA）, superoxide dismutase（SOD）, catalase（CAT） and glutathione peroxidase（GSH） in the kidney homogenate were measured by double antibody sandwich enzyme-linked immunosorbent assay （ELISA）, Polymerase chain reaction （RT-PCR） was used to measure expression of Klotho and Nrf2 mRNA, and Western Blot was used to measure the expression of Klotho and Nrf2 protein. ResultsThe level of MDA in group B [（12.43±0.43）μmol/mg] was significantly increased compared to group A[（8.67±0.84）μmol/mg, P〈0.05] and group C [（7.97±0.81）μmol/mg, P〈0.05], while group A was close to group C （P〉0.05）. In group A, B, and C, the levels of SOD were （247.89±2.45）, （109.54±4.21）, and （189.74±10.47）U/mg, respectively; the levels of GSH were （38.98±4.55）, （26.87±3.92）, and （31.29±2.54）μmol/mg, respectively; CAT were （138.47±8.74）, （119.87±8.45）, and（127.46±7.45）U/mg, respectively. The levels of SOD, GSH, CAT in group B were significantly lower than that in group A and C, while those in group B were close to group A （P〉0.05）. The expression of Klotho and Nrf2 mRNA in group B [（0.208±0.036）and （0.499±0.086）] were significantly lower than group A（1.011±0.174 and 1.023±0.139, P〈0.05）and group C（1.123±0.248 and 1.023±0.139, P〈0.05）. The expression of Klotho and Nrf2 protien were also significantly lower than that in group A and C （P〈0.05）. ConclusionsValsartan and Fosinopril could prevent the formation of renal CaOx stones by upregulating expression of low level Klotho gene induced by ethylene glycol.This effect may be involved with activation of Keapl-Nrf2-ARE signaling pathway.