EGR3通过抑制PRMT1/p-STAT3通路减轻肥胖相关性肾病足细胞炎症损伤

EGR3 reduces podocyte inflammatory damage in obesity related glomerulopathy by inhibiting the PRMT1/p-STAT3 pathway

目的:肥胖会导致肥胖相关性肾病(obesity related glomerulopathy,ORG),但其发病机制并不明确。本研究拟检测早期生长反应蛋白3(early growth response protein 3,EGR3)在ORG患者和高脂饮食诱导的肥胖小鼠肾皮质组织中的表达,并探讨EGR3抑制棕榈酸(palmitic acid,PA)诱导的人足细胞炎症损伤的分子机制。方法:收集排除其他疾病导致的肾损害并经组织病理学证实的ORG患者(n=6)和高脂饮食诱导的肥胖小鼠的肾皮质组织(n=10)。使用150μmol/L PA干预人和小鼠足细胞48 h;人足细胞中分别过表达或沉默EGR3。采用酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测白细胞介素(interleukin,IL)-6和IL-1β的含量;real-time RT-PCR检测EGR3、足细胞分子标志NPHS1(nephrosis1)、NPHS2(nephrosis2)、足糖萼蛋白(podocalyxin,PODXL)、平足蛋白(podoplanin,PDPN)mRNA的表达;RNA-seq检测人足细胞过表达EGR3并150μmol/L PA干预后与对照组的差异表达基因(differentially expressed genes,DEGs);免疫共沉淀(co-immunoprecipitation,Co-IP)+液相色谱串联质谱(liquid chromatography tandem mass spectrometry,LC-MS)检测EGR3可能的相互作用蛋白质,并与RNA-seq的结果取交集;Co-IP验证EGR3与蛋白精氨酸甲基转移酶1(protein arginine methyltransferases 1,PRMT1)的相互作用;沉默EGR3和PRMT1抑制剂干预后检测PA诱导的足细胞培养液中IL-6和IL-1β的含量;蛋白质印迹法检测分别过表达或沉默EGR3后磷酸化信号转导及转录激活蛋白3(phosphorylated signal transducer and activator of transcription 3,p-STAT3)的蛋白质表达。结果:EGR3在ORG患者和高脂饮食诱导的肥胖小鼠肾皮质组织中的表达均显著上调(均P<0.01),150μmol/L PA干预人和小鼠足细胞48 h后显著上调2种细胞EGR3的表达(均P<0.05)。人足细胞过表达或沉默EGR3分别抑制或促进PA干预后细胞培养液中IL-6和IL-1β的分泌,并分别上调或下调NPHS1、PODXL、NPHS2及PDPN的表达(均P<0.05)。RNA-seq结果显示共有988个DEGs,Co-IP+LC-MS共发现238个可能与EGR3相互作用的蛋白质,且Co-IP证实PRMT1为EGR3的相互作用蛋白质。PRMT1抑制剂能部分减少人足细胞沉默EGR3后PA诱导的IL-6及IL-1β的分泌(均P<0.05);此外,过表达或沉默EGR3负调控PRMT1及p-STAT3的表达。结论:EGR3可能通过抑制PRMT1/p-STAT3通路减轻ORG足细胞炎症损伤。

Objective:Obesity related glomerulopathy(ORG)is induced by obesity,but the pathogenesis remains unclear.This study aims to investigate the expression of early growth response protein 3(EGR3)in the renal cortex tissues of ORG patients and high-fat dietinduced obese mice,and to further explore the molecular mechanism of EGR3 in inhibiting palmitic acid(PA)induced human podocyte inflammatory damage.Methods:Renal cortex tissues were collected from ORG patients(n=6)who have been excluded from kidney damage caused by other diseases and confirmed by histopathology,and from obese mice induced by high-fat diet(n=10).Human and mouse podocytes were intervened with 150μmol/L PA for 48 hours.EGR3 was overexpressed or silenced in human podocytes.Enzyme linked immunosorbent assay(ELISA)was used to detcet the levels of interleukin-6(IL-6)and interleukin-1β(IL-1β).Real-time RT-PCR was used to detect the mRNA expressions of EGR3,podocytes molecular markers nephrosis 1(NPHS1),nephrosis 2(NPHS2),podocalyxin(PODXL),and podoplanin(PDPN).RNA-seq was performed to detect differentially expressed genes(DEGs)after human podocytes overexpressing EGR3 and treated with 150μmol/L PA compared with the control group.Co-immunoprecipitation(Co-IP)combined with liquid chromatography tandem mass spectrometry(LC-MS)was used to detect potential interacting proteins of EGR3 and the intersected with the RNA-seq results.Co-IP confirmed the interaction between EGR3 and protein arginine methyltransferases 1(PRMT1),after silencing EGR3 and PRMT1 inhibitor intervention,the secretion of IL-6 and IL-1βin PA-induced podocytes was detected.Western blotting was used to detect the expression of phosphorylated signal transducer and activator of transcription 3(p-STAT3)after overexpression or silencing of EGR3.Results:EGR3 was significantly upregulated in renal cortex tissues of ORG patients and high-fat diet-induced obese mice(both P<0.01).In addition,after treating with 150μmol/L PA for 48 hours,the expression of EGR3 in human and mouse podocytes was significantly upregulated(both P<0.05).Overexpression or silencing of EGR3 in human podocytes inhibited or promoted the secretion of IL-6 and IL-1βin the cell culture supernatant after PA intervention,respectively,and upregulated or downregulated the expression of NPHS1,PODXL,NPHS2,and PDPN(all P<0.05).RNA-seq showed a total of 988 DEGs,and Co-IP+LC-MS identified a total of 238 proteins that may interact with EGR3.Co-IP confirmed that PRMT1 was an interacting protein with EGR3.Furthermore,PRMT1 inhibitors could partially reduce PA-induced IL-6 and IL-1βsecretion after EGR3 silencing in human podocytes(both P<0.05).Overexpression or silencing of EGR3 negatively regulated the expression of PRMT1 and p-STAT3.Conclusion:EGR3 may reduce ORG podocyte inflammatory damage by inhibiting the PRMT1/p-STAT3 pathway.