Klotho与自噬在脓毒症急性肾损伤小鼠中的表达趋势

The trend of changes in expression of Klotho and autophagy in sepsis-induced acute kidney injury mice model

目的研究脓毒症急性肾损伤小鼠中Klotho与自噬的关系。方法选用清洁级健康雄性Balb／c小鼠，用盲肠结扎穿刺术（cecalligationandpuncture，CLP）建立小鼠脓毒症急性肾损伤模型，分别在CLP造模后3h、6h、12h、1d、2d、3d、5d处死小鼠（n=12只）；假手术组（Sham）（n=6）1d处死，正常组（Normal）（n=6）同时处死。留取血清检测血肌酐（Scr）、尿素氮（BUN），肾组织行HE和PAS染色，观察光镜病理结果，电镜超微结构观察自噬体的形成，Westernblot和免疫组化检测肾组织Klotho蛋白、LC3蛋白和P62蛋白的表达以及分布。SPSS23．0软件采用独立样本资料的t检验进行统计学分析。结果CLP术后Scr和BUN明显升高，1d时达高峰，分别为（165．64±20．56）~mol／L和（45．51±d．05）mmol／L；HE和PAS染色显示肾小管刷状缘上皮细胞脱落，基底膜暴露，间质炎症细胞浸润，电镜显示肾脏可见自噬小体和吞噬泡。与Sham组相比，Klotho蛋白浓度从3h开始逐渐变淡，至1d时达最低值（t=51．851，P〈0．01），2d时有所恢复，然后逐渐转浓；LC3-Ⅱ呈相反的变化趋势，从3h开始逐渐增浓，至1d时达峰值（t=-22．676，P〈0．01），2d时有所下降，然后逐渐变淡，而P62（t=6．43，P=0．002）作为自噬的相关蛋白与LC3-Ⅱ反应相反，表明自噬的激活作用，在1d这个时间点最显著。免疫组化显示Klotho蛋白和LC3-II蛋白主要分布于肾小管胞浆内。CLP术后1d，Klohto蛋白明显减弱（t=-8．371，P〈0．01），LC3-Ⅱ蛋白明显增浓（t=4．995，P=0．001）。结论脓毒症急性肾损伤是Klotho减少，自噬增强的一个过程，Klotho和自噬的表达具有一定的时间依赖性。

Objective To investigate the relationship between Klotho and autophagy in sepsis- induced acute kidney injury mice model. Methods The male healthy Balb/c mice were used to establish the model of sepsis-induced acute kidney injury by using cecal ligation and puncture （CLP）. Mice were sacrificed at3 h, 6 h, 12 h, 1 d, 2 d, 3 d, and5 d after CLP （n =12 for each interval） and on 1 d6 mice in sham group as well as 6 mice in normal group were sacrificed at the same time. Scr and BUN in the blood serum were detected. The HE and PAS staining were employed for observation on the histopathotogical changes in kidney tissues under light microscope. The autophagosomes were observed under transmission electron microscope （TEM）. The renal protein of Klotho, LC3 and P62 were detected by using Western blot and Immunohistochemistry. Statistical analyses were performed using Student＇s t-test by SPSS 23. 0. software. Results Scr and BUN increased significantly after CLP, especially on 1 d, respectively （165.64 ±20. 56） μmol/L and （45.51±4. 05） mmol/L. HE and PAS staining showed renal tissue was damaged obviously 1 d after CLP, as indicated by desquamation of the brush border of proximal tubular epithelial cells, appearance of bare basement membrane, and interstitial inflammatory cell infiltration. Under TEM, autophagosomes and phagocytosis were observed. Compared with sham group, the expression of Klotho protein decreased gradually from 3 h to 1 d and dropped to the trough at 1 d （t =51. 851, P 〈0. 01 ）, then resumed gradually from 2 d to 5 d. On the contrary, the activation of autophagy increased as indicated by the expression of LC3-Ⅱ/L3-I and p62. Autophagy was induced gradually from 3 h to 1 d and reached peak at 1 d, then declined gradually from 2 d to 5 d （ P 〈 0. 01 ）. The protein of Klotho and LC3- I1 mainly distributed in renal tubular cytoplasm, and Klotho was reduced significantly （t = -8. 371, P 〈 0. O1 ） and LC3-Ⅱ appeared in high density remarkably （ t = 4. 995, P = 0. 001 ） on 1 d after CLP. Conehlsions Klotho protein reduction and autophagy protein increase were observed in sepsis-induced acute kidney injury, and the expressions of Klotho and autophagy acted out in certain extent of time dependence.