Diphenyleneiodium (DPI) reduces oxalate ion- and calcium oxalate monohydrate and brushite crystal-induced upregulation of MCP-1 in NRK 52E cells
Diphenyleneiodium (DPI) reduces oxalate ion- and calcium oxalate monohydrate and brushite crystal-induced upregulation of MCP-1 in NRK 52E cells
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DOI：
10.1093/ndt/gfh750
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发表时间：
2005-05-01
影响因子：
6.1
通讯作者：
Khan, SR
中科院分区：
文献类型：
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作者：
Umekawa, T;Byer, K;Khan, SR
背景我们早期的研究表明，暴露于草酸盐（Ox）离子和草酸钙一水合物（COM）晶体或透钙磷石（Br）形式的磷酸钙，在NRK 52 E大鼠肾上皮细胞中的单核细胞趋化蛋白-1（MCP-1）的上调。上调是由活性氧（ROS）介导。本研究旨在探讨NADPH氧化酶是否参与ROS的产生。将NRK 52 E细胞的汇合培养物暴露于Ox离子或COM和Br晶体。分别暴露1、3、6、12、24和48 h，分离MCP-1 mRNA，24 h进行酶联免疫吸附试验（ELISA），以确定蛋白质分泌到培养基中。我们还研究了自由基清除剂，过氧化氢酶，和NADPH氧化酶抑制剂氯化二苯乙炔（DPI），对Ox和晶体诱导的MCP-1 mRNA和蛋白表达的影响。采用实时荧光定量PCR法检测细胞内MCP-1 mRNA的转录水平。检测过氧化氢和8-异前列腺素的含量，以探讨ROS的参与。NRK 52 E细胞暴露于Ox离子以及晶体导致MCP-1 mRNA的表达增加和化学引诱物的产生。用过氧化氢酶处理降低了Ox和晶体诱导的MCP-1 mRNA和蛋白的表达。DPI可降低晶体诱导的基因表达和蛋白质生成，但对OX诱导的基因表达和蛋白质生成无影响。暴露于Ox离子、COM和Br晶体刺激ROS介导的MCP-1 mRNA表达和蛋白质产生的增加。在DPI存在下，ROS产生、脂质过氧化、低密度脂蛋白释放和诱导型MCP-1基因和蛋白的减少表明NADPH氧化酶参与ROS的产生。
Background. Our earlier studies have demonstrated upregulation of monocyte chemoattractant protein-1 (MCP-1) in NRK52E rat renal epithelial cells by exposure to oxalate (Ox) ions and crystals of calcium oxalate monohydrate (COM) or the brushite (Br) form of calcium phosphate. The upregulation was mediated by reactive oxygen species (ROS). This study was performed to investigate whether NADPH oxidase is involved in ROS production.Methods. Confluent cultures of NRK52E cells were exposed to Ox ions or COM and Br crystals. They were exposed for 1, 3, 6, 12, 24 and 48 h for isolation of MCP-1 mRNA and 24 h for enzyme-linked immunosorbent assay (ELISA) to determine the secretion of protein into the culture medium. We also investigated the effect of free radical scavenger, catalase, and the NADPH oxidase inhibitor diphenyleneiodium (DPI) chloride, on the Ox- and crystal-induced expression of MCP-1 mRNA and protein. The transcription of MCP-1 mRNA in the cells was determined using real-time polymerase chain reaction. Hydrogen peroxide and 8-isoprostane were measured to investigate the involvement of ROS.Results. Exposure of NRK52E cells to Ox ions as well as the crystals resulted in increased expression of MCP-1 mRNA and production of the chemoattractant. Treatment with catalase reduced the Ox- and crystal-induced expression of both MCP-1 mRNA and protein. DPI reduced the crystal-induced gene expression and protein production but not Ox-induced gene expression and protein production.Conclusions. Exposure to Ox ions, and COM and Br crystals stimulates a ROS-mediated increase in MCP-1 mRNA expression and protein production. Reduction in ROS production, lipid peroxidation, low-density lipoprotein release, and inducible MCP-1 gene and protein in the presence of DPI indicates an involvement of NADPH oxidase in the production of ROS.