LSD1在急性肾损伤肾纤维化中的保护作用及机制

Effects of LSD1 on renal fibrosis after acute kidney injury

目的探讨组蛋白赖氨酸特异性去甲基化酶1(LSD1)在急性肾损伤(AKI)大鼠肾纤维化中的保护作用,并探讨机制。方法取30只SD大鼠,随机分为对照组、AKI 1d组、AKI 1周组、AKI 2周组、AKI 4周组、AKI 8周组;对照组切除左侧肾脏,右肾不做处理;AKI 1 d组、AKI 1周组、AKI 2周组、AKI 4周组、AKI 8周组切除左侧肾脏后,夹闭右侧肾蒂40 min,分别于1 d、1周、2周、4周、8周后心脏采血并处死大鼠留取肾组织。将人肾小管上皮细胞(HK-2)分为对照组、AKI组、AKI+空质粒组、AKI+LSD1过表达组,对照组正常培养HK-2细胞;AKI组HK-2细胞缺氧培养12 h后正常培养1 h;AKI+空质粒组HK-2细胞转染空白质粒后缺氧培养12 h后正常培养1 h;AKI+LSD1过表达组HK-2细胞转染LSD1质粒后缺氧培养12 h正常培养1 h。应用免疫印迹技术检测肾组织及HK-2细胞中LSD1、H3K4me1、H3K4me2、TGF-β1及纤维化指标的表达,观察AKI后肾纤维化发生发展过程中LSD1表达、组蛋白甲基化情况及其变化对纤维化的影响;应用染色体免疫共沉淀(Ch IP)技术探索LSD1对肾脏纤维化影响的机制。结果动物实验中,与对照组相比,AKI 1、2、4、8周组纤维化指标均升高(P均<0.05),TGF-β1、LSD1及H3K4甲基化标志物H3K4me1、H3K4me2表达均增多(P均<0.05),而AKI 1 d组较对照组差异无统计学意义(P>0.05);细胞实验中,与AKI组、AKI+空质粒组相比,AKI+LSD1过表达组纤维化指标水平下降,TGF-β1、H3K4me1及H3K4me2表达均下降(P均<0.05);细胞实验中,与对照组相比,AKI组及AKI+空质粒组TGF-β1启动子上H3K4me1及H3K4me2表达增加(P均<0.05);与AKI组及AKI+空质粒组相比,AKI+LSD1过表达组TGF-β1启动子上H3K4me1及H3K4me2表达下降(P<0.05)。结论 LSD1通过引起H3K4me1及H3K4me2去甲基化使TGF-β1表达下降,从而在AKI后肾纤维化中起保护作用。

bjective To investigate the effects of histone lysine-specific demethylase1(LSD1)on the development and progression of renal fibrosis after acute kidney injury(AKI).Methods Thirty SD rats were randomly divided into the control group,AKI 1-day group,AKI 1-week(AKI 1W)group,AKI 2-week(AKI 2W)group,AKI 4-week(AKI 4W)group,and AKI 8-week(AKI 8W)group;in the control group,the left kidney was resected and the right kidney was not treated;in the AKI groups,the left kidney was resected and we clamped the right renal pedicle for 40 min,then collected the heart blood and kidney tissues of rats at 1 d,1 w,2 w,4 w and 8 w,respectively.The human renal tubular epithelial cells(HK-2)were divided into the control group,AKI group,AKI+empty plasmid group,and AKI+LSD1 over-expression group.In the control group,we normally cultured the HK-2 cells;in the AKI group,HK-2 cells were cultured for 12 h under hypoxia and then normally cultured for 1 h;in the AKI+empty plasmid group,after the HK-2 cells were transfected with blank plasmid,they were first cultured for 12 h under hypoxia and then normally cultured for 1 h;in the AKI+LSD1 over-expression group,after the HK-2 cells were transfected with LSD1 plasmid,they were first cultured for 12 h under hypoxia and then normally cultured for 1 h.The expression levels of LSD1,H3K4me1,H3K4me2,TGF-β1,and fibrosis markers were detected by Western blotting in the renal tissues and HK-2 cells.We observed the LSD1 expression changes in the occurrence and development of renal fibrosis after AKI,as well as histone methylation and its impact on fibrosis.The specific mechanism of LSD1 on renal fibrosis was explored by chromosome immunoprecipitation(ChIP)technique.Results In the animal experiments,compared with the control group,the fibrosis indexes of AKI 1W,2W,4W and 8W groups were significantly higher,and the expression of TGF-β1,LSD1,and H3K4 methylation markers H3K4me1 and H3K4me2 increased(all P<0.05);there was no significant differences between AKI 1d group and control group(P>0.05).In the cell experiments,compared with the AKI group and AKI+empty plasmid group,the fibrosis indexes decreased significantly,and the expression of TGF-β1,H3K4 methylation markers H3K4me1 and H3K4me2 decreased in the AKI+LSD1 over-expression group(all P<0.05).In the cell experiment,compared with the control group,the expression of H3K4me1 and H3K4me2 increased on TGF-β1 promoters in the AKI group and AKI+plasmid group(all P<0.05);compared with the AKI group and AKI+plasmid group,the expression of H3K4me1 and H3K4me2 on TGF-β1 promoters in the AKI+LSD1 over-expression group significantly decreased(P<0.05).Conclusion LSD1 plays a protective role in renal fibrosis after AKI by inducing demethylation of H3K4me1 and H3K4me2 to decrease the expression of TGF-β1.