Sirt1对高糖诱导的足细胞外泌体释放的影响
Effect of Sirt1 on high glucose-induced exosome release from podocytes
丁琳 1周燕 1刘珊珊 1刘南池 1马瑞霞1
作者信息
- 1. 青岛大学附属医院肾病科,山东 青岛 266003
- 折叠
摘要
目的 探讨烟酰胺腺嘌呤二核苷酸依赖的去乙酰化酶 1(Sirt1)对高糖诱导的足细胞外泌体释放的影响.方法 将永生化小鼠足细胞 MPC5 分为正常糖组(5.5 mmol/L葡萄糖,A 组)、高渗组(5.5 mmol/L 葡萄糖+ 24.5 mmol/L甘露醇,B组)、高糖组(30.0 mmol/L葡萄糖,C 组)、高糖+Sirt1 过表达慢病毒转染组(Sirt1 过表达慢病毒转染+30.0 mmol/L葡萄糖,D组)、高糖+阴性慢病毒转染组(阴性慢病毒转染+30.0 mmol/L 葡萄糖,E组)、高糖+外泌体分泌抑制剂组(GW4869+30.0 mmol/L 葡萄糖,F 组)6 组.采用免疫印迹法检测各组细胞Sirt1、足细胞裂孔膜蛋白(Nephrin、Podocin)及CD63、CD81、Alix的表达水平,采用实时荧光定量 PCR(RT-qPCR)检测D、E组细胞Sirt1 mRNA表达水平,使用透射电子显微镜观察足细胞外泌体形态,采用纳米粒子跟踪分析技术检测外泌体的粒径和浓度.结果 RT-qPCR结果显示,D组足细胞Sirt1 mRNA相对表达量显著高于E组(t= 14.580,P＜0.01).纳米粒子跟踪分析及免疫印迹结果显示,A～C组间足细胞 Sirt1、Nephrin和 Podocin蛋白相对表达量比较差异均具有显著性(F=49.84～106.40,P＜0.01);与 A 组相比,C 组足细胞外泌体分泌显著增加(t= 14.550,P＜0.01),Nephrin、Podocin、Sirt1 相对表达量显著减少(t=7.446～15.110,P＜0.01);与 E组相比,D组足细胞外泌体分泌显著减少(t=74.610,P＜0.01),Nephrin、Podocin、Sirt1 相对表达量显著增加(t=4.657～32.860,P＜0.05);与C组相比,F组足细胞外泌体分泌显著减少(t=16.300,P＜0.05),Nephrin、Podocin相对表达量显著增加(t=3.790、8.151,P＜0.01),Sirt1 表达水平无统计学差异(P＞0.05).结论 高糖诱导的足细胞 Sirt1 减少可促进外泌体分泌及足细胞损伤.
Abstract
Objective To investigate the effect of nicotinamide adenine dinucleotide-dependent deacetylase 1(Sirt1)on high glucose-induced exosome release from podocytes.Methods Immortalized mouse podocytes MPC5 were divided into six groups:normal glucose group(5.5 mmol/L glucose,group A),high mannitol group(5.5 mmol/L glucose+24.5 mmol/L manni-tol,group B),high glucose group(30.0 mmol/L glucose,group C),high glucose+Sirt1-overexpressed lentivirus transfection group(Sirt1-overexpressed lentivirus transfection+30.0 mmol/L glucose,group D),high glucose+negative lentivirus transfection group(negative lentivirus transfection+30.0 mmol/L glucose,group E),and high glucose+exosome secretion inhibitor group(GW4869+30.0 mmol/L glucose,group F).Western blot was used to analyze the expression levels of Nephrin,Podocin,Sirt1,CD63,CD81,and Alix in each group.Real-time quantitative polymerase chain reaction was used to analyze the expression level of Sirt1 mRNA in D and E group.The morphology of podocyte exosomes was observed by a transmission electron microscope.The particle size and concentration of exosomes were determined by nanoparticle tracking analysis.Results The results of RT-qPCR showed that the relative expression of Sirt1 mRNA was significantly increased in group D compared with that in group E(t= 14.580,P＜0.01).The results of nanoparticle tracking analysis and Western blot showed that the relative expression of Sirt1,Nephrin,and Podocin proteins in podocytes among groups A to C was significantly different(F=49.84-106.40,P＜0.01).Com-pared with group A,group C had significantly increased secretion of podocyte exosomes(t=14.550,P＜0.01)and significantly re-duced expression of Sirt1,Nephrin,and Podocin(t=7.446-15.110,P＜0.01).Compared with group E,group D had significantly reduced release of podocyte exosomes(t=74.610,P＜0.01)and significantly increased relative expression of Sirt1,Nephrin,and Podocin(t=4.657-32.860,P＜0.05).Compared with group C,group F had significantly reduced release of podocyte exosomes(t=16.300,P＜0.05)and significantly increased relative expression of Nephrin and Podocin(t=3.790,8.151,P＜0.01),but showed no significant change in the expression level of Sirt1(P＞0.05).Con-clusion Loss of Sirt1 in high glucose-treated podocytes promotes exosome secretion and podocyte injury.
关键词
糖尿病肾病/血糖/足细胞/外泌体/抗衰老酶1Key words
Diabetic nephropathies/Blood glucose/Podocytes/Exosomes/Sirtuin 1引用本文复制引用
基金项目
山东省自然科学基金(ZR2022MH161)
青岛市医药卫生科研计划项目(2021-WJZD189)
青岛市民生科技计划项目(16-6-2-20-snh)
青岛大学附属医院临床医学+X项目(3390)
青岛市医疗卫生重点学科建设项目()
出版年
2024
国家科技期刊平台
NSTL
维普
万方数据