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Abstract：
OBJECTIVE: To explore the activation of tumor necrosis factor-α (TNF-α) signaling pathway and the expressions of the associated inflammatory factors in NPHP1-defective renal tubular epithelial cells.
METHODS: A human proximal renal tubular cell (HK2) model of lentivirus-mediated NPHP1 knockdown (NPHP1KD) was constructed, and the expressions of TNF-α, p38, and C/EBPβ and the inflammatory factors CXCL5, CCL20, IL-1β, IL-6 and MCP-1 were detected using RT-qPCR, Western blotting or enzyme-linked immunosorbent assay. A small interfering RNA (siRNA) was transfected in wild-type and NPHP1KDHK2 cells, and the changes in the expressions of TNF-α, p38, and C/EBPβ and the inflammatory factors were examined.
RESULTS: NPHP1KDHK2 cells showed significantly increased mRNA expressions of TNF-α, C/EBPβ, CXCL5, IL-1β, and IL-6 (P < 0.05), protein expressions of phospho-p38 and C/EBPβ (P < 0.05), and IL-6 level in the culture supernatant (P < 0.05), and these changes were significantly blocked by transfection of cells with siRNA-C/EBPβ (P < 0.05).
CONCLUSIONS: TNF-α signaling pathway is activated and its associated inflammatory factors are upregulated in NPHP1KDHK2 cells, and C/EBPβ may serve as a key transcription factor to mediate these changes.
METHODS: A human proximal renal tubular cell (HK2) model of lentivirus-mediated NPHP1 knockdown (NPHP1KD) was constructed, and the expressions of TNF-α, p38, and C/EBPβ and the inflammatory factors CXCL5, CCL20, IL-1β, IL-6 and MCP-1 were detected using RT-qPCR, Western blotting or enzyme-linked immunosorbent assay. A small interfering RNA (siRNA) was transfected in wild-type and NPHP1KDHK2 cells, and the changes in the expressions of TNF-α, p38, and C/EBPβ and the inflammatory factors were examined.
RESULTS: NPHP1KDHK2 cells showed significantly increased mRNA expressions of TNF-α, C/EBPβ, CXCL5, IL-1β, and IL-6 (P < 0.05), protein expressions of phospho-p38 and C/EBPβ (P < 0.05), and IL-6 level in the culture supernatant (P < 0.05), and these changes were significantly blocked by transfection of cells with siRNA-C/EBPβ (P < 0.05).
CONCLUSIONS: TNF-α signaling pathway is activated and its associated inflammatory factors are upregulated in NPHP1KDHK2 cells, and C/EBPβ may serve as a key transcription factor to mediate these changes.
摘要：
目的：探讨NPHP1缺陷型肾小管上皮细胞肿瘤坏死因子-α(TNF-α)信号通路的激活及相关炎症因子的表达。
方法：构建慢病毒介导的NPHP1敲低（NPHP1KD）的人近端肾小管细胞（HK2）模型，和TNF-α的表达，p38，C/EBPβ和炎症因子CXCL5，CCL20，IL-1β，使用RT-qPCR检测IL-6和MCP-1，蛋白质印迹或酶联免疫吸附测定。在野生型和NPHP1KDHK2细胞中转染小干扰RNA(siRNA),TNF-α表达的变化,检测p38、C/EBPβ和炎症因子。
结果：NPHP1KDHK2细胞显示TNF-α的mRNA表达明显增加，C/EBPβ,CXCL5，IL-1β，IL-6（P<0.05），磷酸化p38和C/EBPβ的蛋白表达（P<0.05），培养上清液中IL-6水平(P<0.05),这些变化被siRNA-C/EBPβ转染细胞显著阻断(P<0.05)。
结论：NPHP1KDHK2细胞中TNF-α信号通路被激活，其相关炎症因子被上调，C/EBPβ可能是介导这些变化的关键转录因子。
方法：构建慢病毒介导的NPHP1敲低（NPHP1KD）的人近端肾小管细胞（HK2）模型，和TNF-α的表达，p38，C/EBPβ和炎症因子CXCL5，CCL20，IL-1β，使用RT-qPCR检测IL-6和MCP-1，蛋白质印迹或酶联免疫吸附测定。在野生型和NPHP1KDHK2细胞中转染小干扰RNA(siRNA),TNF-α表达的变化,检测p38、C/EBPβ和炎症因子。
结果：NPHP1KDHK2细胞显示TNF-α的mRNA表达明显增加，C/EBPβ,CXCL5，IL-1β，IL-6（P<0.05），磷酸化p38和C/EBPβ的蛋白表达（P<0.05），培养上清液中IL-6水平(P<0.05),这些变化被siRNA-C/EBPβ转染细胞显著阻断(P<0.05)。
结论：NPHP1KDHK2细胞中TNF-α信号通路被激活，其相关炎症因子被上调，C/EBPβ可能是介导这些变化的关键转录因子。
