Department of Urology, The First Affiliated Hospital of Guangxi Medical University, Nanning, China.
Department of Urology, The First Affiliated Hospital of Guangxi Medical University, Nanning, China,
Kidney Blood Press Res. 2019;44(4):777-791. doi: 10.1159/000501558. Epub 2019 Aug 13.
M2 macrophages have important roles in diseases such as tumours, cardiovascular diseases and renal diseases. This study aimed to determine the effects and protective mechanism of M2 macrophages against oxidative stress injury and apoptosis induced by calcium oxalate crystals (CaOx) in renal tubular epithelial cells (HK-2) under coculture conditions.
THP-1 cells were induced to differentiate into M2 macrophages by using phorbol-12-myristate-13-acetate, IL-4 and IL-13. Morphological features were observed by microscopy. Phenotypic markers were identified by reverse transcription-polymerase chain reaction, Western blot and enzyme-linked immunosorbent assay (ELISA). HK-2 cells were treated with 0.5 mg/mL CaOx crystals and co-cultured with M2 macrophages or apocynin. The viability of HK-2 cells was detected by CCK-8 assay. The lactate dehydrogenase (LDH) activity of HK-2 cells was analysed using a microplate reader. The apoptosis of HK-2 cells was examined by flow cytometry and Hoechst 33258 staining. Reactive oxygen species (ROS) expression and mitochondrial membrane potential in HK-2 cells were detected by a fluorescence microplate reader. Western blot analysis was conducted to detect the expression of p47phox, Bcl-2, cleaved caspase-3, cytochrome c, p38 MAPK, phospho-p38 MAPK, Akt and phospho-Akt.
The results of morphology, reverse transcription-polymerase chain reaction, Western blot and ELISA showed that THP-1 cells were successfully polarised to M2 macrophages. The results of co-culture suggested that M2 macrophages or apocynin significantly increased the cell viability and decreased the LDH activity and apoptosis rate after HK-2 cells were challenged with CaOx crystals. The expression of the p47phox protein and the concentration of ROS were reduced, the release of mitochondrial membrane potential and the expression of the Bcl-2 protein were upregulated and the protein expression of cleaved caspase-3 and cytochrome c was downregulated. The expression of the phosphorylated form of p38 MAPK increased. Under coculture conditions with M2 macrophages, the Akt protein of HK-2 cells treated with CaOx crystals was dephosphorylated, but the phosphorylated form of Akt was not reduced by apocynin.
M2 macrophages reduced the oxidative stress injury and apoptosis of HK-2 cells by downregulating the activation of NADPH oxidase, reducing the production of ROS, inhibiting the phosphorylation of p38 MAPK and enhancing the phosphorylation of Akt. We have revealed one of the possible mechanisms by which M2 macrophages reduce the formation of kidney stones.
M2 巨噬细胞在肿瘤、心血管疾病和肾脏疾病等疾病中具有重要作用。本研究旨在探讨在共培养条件下，M2 巨噬细胞对草酸钙晶体（CaOx）诱导的肾小管上皮细胞（HK-2）氧化应激损伤和凋亡的作用及保护机制。
用佛波醇 12-肉豆蔻酸 13-乙酸酯、白细胞介素-4 和白细胞介素-13 诱导 THP-1 细胞分化为 M2 巨噬细胞。通过显微镜观察细胞形态。采用逆转录-聚合酶链反应、Western blot 和酶联免疫吸附试验（ELISA）鉴定表型标志物。用 0.5mg/ml CaOx 晶体处理 HK-2 细胞，并与 M2 巨噬细胞或 apocynin 共培养。用 CCK-8 法检测 HK-2 细胞的活力。用微孔板读数仪分析 HK-2 细胞的乳酸脱氢酶（LDH）活性。用流式细胞术和 Hoechst 33258 染色检测 HK-2 细胞的凋亡。用荧光微孔板读数仪检测 HK-2 细胞中活性氧（ROS）的表达和线粒体膜电位。采用 Western blot 分析检测 p47phox、Bcl-2、cleaved caspase-3、细胞色素 c、p38 MAPK、磷酸化 p38 MAPK、Akt 和磷酸化 Akt 的表达。
形态学、逆转录-聚合酶链反应、Western blot 和 ELISA 结果表明，THP-1 细胞成功极化成为 M2 巨噬细胞。共培养结果表明，M2 巨噬细胞或 apocynin 可显著提高 HK-2 细胞在草酸钙晶体刺激后的细胞活力，降低 LDH 活性和凋亡率。p47phox 蛋白的表达和 ROS 的浓度降低，线粒体膜电位的释放和 Bcl-2 蛋白的表达上调，cleaved caspase-3 和细胞色素 c 的蛋白表达下调。磷酸化 p38 MAPK 的表达增加。在与 M2 巨噬细胞共培养的条件下，用 CaOx 晶体处理的 HK-2 细胞中的 Akt 蛋白去磷酸化，但 apocynin 并未降低磷酸化 Akt 的表达。
M2 巨噬细胞通过下调 NADPH 氧化酶的激活、减少 ROS 的产生、抑制 p38 MAPK 的磷酸化和增强 Akt 的磷酸化，减少 HK-2 细胞的氧化应激损伤和凋亡。我们揭示了 M2 巨噬细胞减少肾结石形成的可能机制之一。
