Objective: To investigate high-salt exposure-induced polarization of mononuclear macrophages and the changes in proliferation and phenotypic transformation of renal fibroblasts in a co-culture system.
Methods: Cultured mononuclear macrophages were exposed to high salt (161 mmol/L Na +) for 2 h and the surface markers of M0, M1 and M2-type macrophages were detected with RT-qPCR. The culture medium of the macrophages in normal and high-salt groups was collected for detection of the mRNA and protein levels of IL-6 and TGF-β1 using RT-qPCR and ELISA. A co-culture system of high salt-exposed macrophages and renal fibroblasts (NRK-49F) was established using a Transwell chamber, and the changes in proliferation and migration of NRK-49F cells were examined using EdU assay and Transwell assay, respectively. Western blotting was performed to detect the expressions of collagen I, collagen III and collagen α-SMA in NRK-49F cells.
Results: The high salt-exposed macrophages showed significantly increased mRNA levels of M2-type macrophage surface markers mannose receptor and arginase (P < 0.05). The results of EdU and Transwell assays showed that NRK-49F cells co-cultured with high salt-exposed macrophages exhibited significantly increased proliferation and migration ability (P < 0.05). Co-culture with high salt-exposed macrophages resulted in significantly enhanced protein expressions of collagen I, collagen III and α-SMA in NRK-49F cells (P < 0.05) and significantly increased levels of IL-6 and TGF-β1 in the culture medium (P < 0.05).
Conclusions: High-salt exposure induces polarization of mononuclear macrophages into M2-type macrophages and promotes secretion of IL-6 and TGF-β1 by the macrophages to induce the proliferation and phenotypic transformation of NRK-49F cells.
目的: 探讨高盐诱导单核巨噬细胞极化，以及极化后巨噬细胞对肾脏纤维细胞株(NRK-49F)增殖及表型转化的影响。
方法: 培养大鼠骨髓单核巨噬细胞及NRK-49F，随后予以高盐(Na+161 mmol/L)刺激单核巨噬细胞2 h。采用RT-qPCR检测M0、M1、M2各型巨噬细胞表面标记物，同时分别收集正常及高盐组巨噬细胞培养基，予以RT-qPCR及Elisa分别检测培养基中IL-6及TGF-β的mRNA表达与蛋白表达。随后建立巨噬细胞与NRK-49F的Transwell小室共培养体系。采用EdU及Transwell分别检测NRK-49F增殖及迁移能力。予以Western blot检测NRK-49F中collagen I、collagen III及α-平滑肌肌动蛋白(α-SMA)相对表达情况。
结果: RT-qPCR结果显示，与对照组相比，高盐组细胞中表达M2型巨噬细胞表面标记物甘露糖受体(MR)和精氨酸酶(Arg)基因的mRNA水平显著升高(P < 0.05)。EdU及Transwell结果显示，共培养体系中，高盐处理巨噬细胞组上层NRK-49F增殖、迁移能力增强(P < 0.05)。Western blot结果显示，高盐处理组细胞培养基可诱导NRK-49F中collagen I、collagen III及和α-SMA蛋白表达增强(P < 0.05)。此外，RT-qPCR及Elisa结果显示高盐处理组单核巨噬细胞培养基中IL-6与TGF-β1均显著高表达(P < 0.05)。
结论: 高盐处理可诱导单核巨噬细胞像M2型极化并分泌IL-6与TGF-β1，从而诱导NRK-49F增殖及表型转化。
Keywords: cell proliferation; high salt; macrophages; phenotypic transformation; renal fibroblasts.