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Abstract：
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 diet-induced 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 150 μmol/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 150 μmol/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-1β in 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 150 μmol/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-1β in 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-1β secretion after EGR3 silencing in human podocytes (both P<0.05). Overexpression or silencing of EGR3 negatively regulated the expression of PRMT1 and p-STAT3.
CONCLUSIONS: EGR3 may reduce ORG podocyte inflammatory damage 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150 μmol/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(nephrosis 1)、NPHS2(nephrosis 2)、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150 μmol/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)，150 μmol/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。.
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 150 μmol/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 150 μmol/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-1β in 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 150 μmol/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-1β in 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-1β secretion after EGR3 silencing in human podocytes (both P<0.05). Overexpression or silencing of EGR3 negatively regulated the expression of PRMT1 and p-STAT3.
CONCLUSIONS: EGR3 may reduce ORG podocyte inflammatory damage 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150 μmol/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(nephrosis 1)、NPHS2(nephrosis 2)、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150 μmol/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)，150 μmol/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。.
摘要：
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