Experimental Nephrology Laboratory, Basic Pathology Department, Universidade Federal do Paraná, 81.531-980, Curitiba, PR, Brazil.
Experimental Nephrology Laboratory, Clinical Analysis Department, Universidade Federal do Paraná, Curitiba, PR, Brazil.
Toxicol Lett. 2021 Sep 1;347:12-22. doi: 10.1016/j.toxlet.2021.04.019. Epub 2021 May 1.
p-Cresyl sulfate (PCS), indoxyl sulfate (IS), and inorganic phosphate (Pi) are uremic toxins found in chronic kidney disease (CKD) that are closely related to endothelial extracellular vesicles (EVs) formation. The present study aimed to understand the role of EVs and their role in cell adhesion and migration, inflammation, and oxidative stress. Human endothelial cells were treated with PCS, IS, and Pi in pre-established uremic and kinetic recommendations. EVs were characterized using scanning electron microscopy, flow cytometry, and NanoSight assays. The concentrations of EVs were established using Alamar Blue and MTT assays. Cell adhesion to extracellular matrix proteins was analyzed using an adhesion assay. Inflammation and oxidative stress were assessed by vascular cell adhesion molecule-1 expression/monocyte migration and reactive oxygen species production, respectively. The capacity of EVs to stimulate endothelial cell migration was evaluated using a wound-healing assay. Our data showed that endothelial cells stimulated with uremic toxins can induce the formation of EVs of different sizes, quantities, and concentrations, depending on the uremic toxin used. Cell adhesion was significantly (P < 0.01) stimulated in cells exposed to PCS-induced extracellular vesicles (PCSEVs) and inorganic phosphate-induced extracellular vesicles (PiEVs). Cell migration was significantly (P < 0.05) stimulated by PCSEVs. VCAM-1 expression was evident in cells treated with PCSEVs and IS-induced extracellular vesicles (ISEVs). EVs are not able to stimulate monocyte migration or oxidative stress. In conclusion, EVs may be a biomarker of endothelial injury and the inflammatory process, playing an important role in cell-to-cell communication and pathophysiological processes, although more studies are needed to better understand the mechanisms of EVs in uremia.
对甲酚硫酸盐（PCS）、吲哚硫酸酯（IS）和无机磷酸盐（Pi）是慢性肾脏病（CKD）中发现的尿毒症毒素，它们与内皮细胞外囊泡（EVs）的形成密切相关。本研究旨在了解 EVs 的作用及其在细胞黏附与迁移、炎症和氧化应激中的作用。用预先建立的尿毒症和动力学推荐的 PCS、IS 和 Pi 处理人内皮细胞。使用扫描电子显微镜、流式细胞术和 NanoSight 分析对 EVs 进行了表征。使用 Alamar Blue 和 MTT 分析测定 EVs 的浓度。使用黏附测定分析细胞对细胞外基质蛋白的黏附。通过血管细胞黏附分子-1 表达/单核细胞迁移和活性氧物质产生分别评估炎症和氧化应激。使用划痕愈合测定评估 EVs 刺激内皮细胞迁移的能力。我们的数据表明，用尿毒症毒素刺激的内皮细胞可以根据所用的尿毒症毒素诱导形成不同大小、数量和浓度的 EVs。在暴露于 PCS 诱导的细胞外囊泡（PCSEVs）和无机磷酸盐诱导的细胞外囊泡（PiEVs）的细胞中，细胞黏附明显受到刺激（P<0.01）。PCSEVs 显著刺激细胞迁移（P<0.05）。在用 PCSEVs 和 IS 诱导的细胞外囊泡（ISEVs）处理的细胞中，VCAM-1 表达明显。EVs 不能刺激单核细胞迁移或氧化应激。总之，EVs 可能是内皮损伤和炎症过程的生物标志物，在细胞间通讯和病理生理过程中发挥重要作用，尽管需要进一步研究以更好地了解尿毒症中 EVs 的机制。
