Department of Environmental Toxicology, The Institute of Environmental and Human Health (TIEHH), Texas Tech University, Lubbock, Texas 79409, United States.
Chem Res Toxicol. 2022 Nov 21;35(11):2168-2179. doi: 10.1021/acs.chemrestox.2c00258. Epub 2022 Nov 10.
Kidney fibrosis is a common step during chronic kidney disease (CKD), and its incidence has been increasing worldwide. Aberrant recovery after repeated acute kidney injury leads to fibrosis. The mechanism of fibrogenic changes in the kidney is not fully understood. Folic acid-induced kidney fibrosis in mice is an established in vivo model to study kidney fibrosis, but the mechanism is poorly understood. Moreover, the effect of higher concentrations of folic acid on kidney epithelial cells in vitro has not yet been studied. Oxidative stress is a common property of nephrotoxicants. Therefore, this study evaluated the role of folic acid-induced oxidative stress in fibrogenic changes by using the in vitro renal proximal tubular epithelial cell culture model. To obtain comprehensive and robust data, three different cell lines derived from human and mouse kidney epithelium were treated with higher concentrations of folic acid for both acute and long-term durations, and the effects were determined at the cellular and molecular levels. The result of cell viability by the MTT assay and the measurement of reactive oxygen species (ROS) levels by the DCF assay revealed that folic acid caused cytotoxicity and increased levels of ROS in acute exposure. The cotreatment with antioxidant -acetyl cysteine (NAC) protected the cytotoxic effect, suggesting the role of folic acid-induced oxidative stress in cytotoxicity. In contrast, the long-term exposure to folic acid caused increased growth, DNA damage, and changes in the expression of marker genes for EMT, fibrosis, oxidative stress, and oxidative DNA damage. Some of these changes, particularly the acute effects, were abrogated by cotreatment with antioxidant NAC. In summary, the novel findings of this study suggest that higher concentrations of folic acid-induced oxidative stress act as the driver of cytotoxicity as an acute effect and of fibrotic changes as a long-term effect in kidney epithelial cells.
肾纤维化是慢性肾脏病（CKD）的常见步骤，其发病率在全球范围内呈上升趋势。反复急性肾损伤后的异常恢复导致纤维化。肾脏纤维化发生的机制尚未完全阐明。叶酸诱导的小鼠肾纤维化是研究肾纤维化的一种成熟的体内模型，但机制尚不清楚。此外，尚未研究体外更高浓度叶酸对肾上皮细胞的影响。氧化应激是肾毒性物质的共同特性。因此，本研究通过体外肾近端肾小管上皮细胞培养模型，评估叶酸诱导的氧化应激在纤维化变化中的作用。为了获得全面而稳健的数据，用三种不同的人源和鼠源肾上皮细胞系，用更高浓度的叶酸进行急性和长期处理，并在细胞和分子水平上测定其作用。MTT 法测定细胞活力和 DCF 法测定活性氧（ROS）水平的结果表明，叶酸在急性暴露时引起细胞毒性并增加 ROS 水平。抗氧化剂 -乙酰半胱氨酸（NAC）的共处理保护了细胞毒性作用，表明叶酸诱导的氧化应激在细胞毒性中的作用。相比之下，长期暴露于叶酸导致细胞生长增加、DNA 损伤以及 EMT、纤维化、氧化应激和氧化 DNA 损伤相关标志物基因表达的改变。这些变化中的一些，特别是急性效应，可被共处理抗氧化剂 NAC 所消除。总之，本研究的新发现表明，较高浓度的叶酸诱导的氧化应激作为急性效应的细胞毒性和长期效应的纤维化变化的驱动因素，在肾上皮细胞中起作用。
