足细胞自噬在膜性肾病经典动物模型中的作用及其激活可能的细胞内机制研究
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摘要
目的探讨足细胞自噬在膜性肾病经典动物模型中的作用及其激活可能的细胞内机制。方法将50只SPF级健康雄性SD大鼠随机平均分为被动肾炎(PHN)2d组、PHN4d组、PHN7d组、PHN21d组以及对照组,建立大鼠PHN模型。观察足细胞形态的变化和自噬泡,并计算单个肾小球足细胞的数量。采用免疫组织化学染色法对肾小球内膜攻击复合物C5b-9的沉淀和半胱氨酸天冬氨酸蛋白酶(caspase)-3的表达进行检测,用TUNEL法对肾小球内细胞凋亡进行检测,Western印迹法对肾小球自噬相关蛋白LC3、GRP78、p-PERK、p-JNK、ATF6α的表达进行检测。结果至注射抗体后第21天可见典型模性肾病改变,而其尿蛋白量自建模3 d起有显著上升,至21 d可达到(51.4±5.9)mg/24 h。P62蛋白并无在PHN模型大鼠肾小球内发生堆积,故可确定LC3Ⅱ表达的提高非自噬流受阻导致。可观察到造模早期ATF6α、p-PERK、p-JNK有逐步上升的趋势,但21 d(即后期)又开始回落。结论膜性肾病免疫介导的损伤可通过激活内质网应激从而增强足细胞自噬作用以保持其内环境的稳定及生存。
Objective To explore the role of autophagy in the classic animal model of membranous nephropathy and its possible mechanism of cell activation.Methods Fifty healthy maleSD rats of SPF grade were randomly and averagely divided into PHN 2 d group,PHN4 d group,PHN 7 d group,PHN21 d group and control group,and the PHN model was established.The morphological changes of podocytes and autophagic vesicles were observed,and the number of podocytes in a single glomerulus was calculated.The expression of C5 b-9 in the renal tissue and the expression of caspase-3 was detected by immunohistochemistry staining.Apoptosis of glomerular cells was detected by TUNEL method,and expression of GRP78,p-PERK,p-JNK,p-JNK,ATF6 α,and LC3 were detected by Western blot.Results Typical model nephropathy was observed on the 21 st day after injection of antibody,and the urinary protein level increased significantly from the 3 d after injection,reaching(51.4 +5.9) mg/24 h on the 21 st day.P62 protein did not accumulate in the glomeruli of PHN model rats,so it can be determined that the increase of LC3 II expression is caused by non-autophagic flow blockage.It can be observed that ATF6 a,p-PERK and p-JNK increased gradually in the early stage of modeling,but began to decline again in 21 days(i.e.later stage).Conclusion Immune-mediated injury in membranous nephropathy can enhance podocyte autophagy by activating endoplasmic reticulum stress to maintain the stability of its internal environment and survival.
Objective To explore the role of autophagy in the classic animal model of membranous nephropathy and its possible mechanism of cell activation.Methods Fifty healthy maleSD rats of SPF grade were randomly and averagely divided into PHN 2 d group,PHN4 d group,PHN 7 d group,PHN21 d group and control group,and the PHN model was established.The morphological changes of podocytes and autophagic vesicles were observed,and the number of podocytes in a single glomerulus was calculated.The expression of C5 b-9 in the renal tissue and the expression of caspase-3 was detected by immunohistochemistry staining.Apoptosis of glomerular cells was detected by TUNEL method,and expression of GRP78,p-PERK,p-JNK,p-JNK,ATF6 α,and LC3 were detected by Western blot.Results Typical model nephropathy was observed on the 21 st day after injection of antibody,and the urinary protein level increased significantly from the 3 d after injection,reaching(51.4 +5.9) mg/24 h on the 21 st day.P62 protein did not accumulate in the glomeruli of PHN model rats,so it can be determined that the increase of LC3 II expression is caused by non-autophagic flow blockage.It can be observed that ATF6 a,p-PERK and p-JNK increased gradually in the early stage of modeling,but began to decline again in 21 days(i.e.later stage).Conclusion Immune-mediated injury in membranous nephropathy can enhance podocyte autophagy by activating endoplasmic reticulum stress to maintain the stability of its internal environment and survival.
引文
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[11] 张利,刘建璟,孙林春.晚期糖基化终末产物通过Wnt/β-catenin信号通路调控足细胞自噬和迁移[J].临床肾脏病杂志,2018,18(12):778-783
[12] Hu M,Crawford SA,Henstridge DC,et al.p32 protein levels are integral to mitochondrial and endoplasmic reticulum morphology,cell metabolism and survival[J].Biochem J,2013,453(3):381-391
[13] 宋诩,卢宏柱.自噬与足细胞病[J].广东医学,2018,39(23):3578-3581
[14] 凌春燕.足细胞自噬在肾小球疾病中的研究进展[J].重庆医学,2018,47(31):4053-4058
[15] 师朗,杨克举,刘阳,等.自噬活性在糖尿病肾病患者肾组织足细胞损伤中的作用研究[J].疑难病杂志,2018,17(12):1324-1327
[2] 陈生晓,林晓明,甘艳,等.膜性肾病组织中足细胞自噬体数量、自噬相关蛋白表达变化及其意义[J].山东医药,2019,59(8):73-75
[3] Colobran R,Pujol-Borrell R,Armengol MP,et al.The chemokine network.II on how polymorphisms and alternative splicing increase the number of molecular species and configure intricate patterns of disease susceptibility[J].Clin Exp Immunol,2007,150(1):1-12
[4] Hu Z,Hanley JR,Zhang R,et al.A conflict-based model of color categorical perception:evidence from a priming study[J].Psychon Bull Rev,2014,21(5):1214-1223
[5] 杨凤杰,周建华,吕倩影,等.足细胞自噬在被动Heymann肾炎发病中的作用[J].中华肾脏病杂志,2014,30(1):41-47
[6] 任晶晶,张伟,何强.自噬与特发性膜性肾病研究进展[J].浙江临床医学,2018,20(9):1604-1605
[7] 高尚.补体与特发性膜性肾病研究进展[J].医学综述,2016,22(9):1676-1679
[8] Phillips RS,Poteh P,Miller KA,et al.STM2360 encodes a d-ornithine/d-lysine decarboxylase in Salmonella enterica serovar typhimurium[J].Arch Biochem Biophys,2017,634(2):83-87
[9] 周翠,杨倩,杨英杰,等.自噬在高糖诱导的足细胞脂质蓄积中的作用[J].中华肾脏病杂志,2018,34(10):765-770
[10] Butler MR,Ma H,Yang F,et al.Endoplasmic reticulum (ER) Ca2+-channel activity contributes to ER stress and cone death in cyclic nucleotide-gated channel deficiency[J].J Biol Chem,2017,292(27):11189-11205
[11] 张利,刘建璟,孙林春.晚期糖基化终末产物通过Wnt/β-catenin信号通路调控足细胞自噬和迁移[J].临床肾脏病杂志,2018,18(12):778-783
[12] Hu M,Crawford SA,Henstridge DC,et al.p32 protein levels are integral to mitochondrial and endoplasmic reticulum morphology,cell metabolism and survival[J].Biochem J,2013,453(3):381-391
[13] 宋诩,卢宏柱.自噬与足细胞病[J].广东医学,2018,39(23):3578-3581
[14] 凌春燕.足细胞自噬在肾小球疾病中的研究进展[J].重庆医学,2018,47(31):4053-4058
[15] 师朗,杨克举,刘阳,等.自噬活性在糖尿病肾病患者肾组织足细胞损伤中的作用研究[J].疑难病杂志,2018,17(12):1324-1327