Active Phagocytosis and Diachronic Processing of Calcium Oxalate Monohydrate Crystals in an in vitro Macrophage Model
Active Phagocytosis and Diachronic Processing of Calcium Oxalate Monohydrate Crystals in an in vitro Macrophage Model
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DOI：
10.1159/000501965
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发表时间：
2019-10-01
影响因子：
2.8
通讯作者：
Yasui, Takahiro
中科院分区：
文献类型：
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作者：
Okada, Atsushi;Aoki, Hiromasa;Yasui, Takahiro
背景：我们之前发现肾巨噬细胞（M phi s）吞噬肾草酸钙一水合物（COM）晶体。这项研究使用体外模型研究了吞噬晶体的处理。方法：将 J774.1 小鼠 M phi 暴露于 COM 晶体，并使用偏光显微镜在有/无细胞松弛素 B (CB)（一种吞噬作用抑制剂）的情况下观察 24 小时，以确认活跃的晶体吞噬作用。使用 LysoTracker 和使用透射电子显微镜对溶酶体相关膜蛋白 1 进行免疫组织化学染色来确认被吞噬的 COM 晶体被溶酶体摄取。使用偏光显微镜对特定 M phi 进行历时跟踪，以捕获吞噬 COM 晶体处理的整个过程。在存在和不存在尼日利亚霉素的情况下，使用成像细胞术对荧光 COM (f-COM) 晶体进行了后续研究，以消除酸性细胞器中的 pH 梯度。结果：吞噬率随着 COM 密度的增加而增加，并且在用 CB 处理的细胞中显着降低 (p < 0.01)。我们观察到，被吞噬的晶体共定位在 M phi 的溶酶体内；此外，历时观察表明，被吞噬的COM晶体在M phi分裂过程中被细分，并在培养第7天被消除。此外，成像流式细胞仪显示，48 h后尼日利亚素（-）组f-COM晶体的荧光水平明显低于尼日利亚素（+）组。结论：本研究证实了 M phi s 对吞噬 COM 晶体的主动吞噬作用和溶酶体加工作用。这一发现有望有助于未来增强M phi s COM晶体吞噬能力的药物的开发。
Background: We previously discovered that renal macrophages (M phi s) phagocytose renal calcium oxalate monohydrate (COM) crystals. This study investigated the processing of engulfed crystals using in vitro models. Methods: J774.1 mouse M phi s were exposed to COM crystals and observed for 24 h using polarized light microscopy with/without cytochalasin B (CB), an inhibitor of phagocytosis, to confirm active crystal phagocytosis. LysoTracker and immunohistochemical staining using transmission electron microscopy for lysosomal-associated membrane protein 1 were used to confirm engulfed COM crystal uptake into lysosomes. Diachronic tracking of specific M phi s was performed to capture the entire course of engulfed COM crystal processing using polarized light microscopy. Follow-up studies of fluorescent COM (f-COM) crystals using imaging cytometry were performed in the presence and absence of nigericin to dissipate the pH gradient in acidic organelles. Results: Phagocytosis rates increased with COM density and were significantly lower in cells treated with CB (p < 0.01). We observed that engulfed crystals colocalized within lysosomes of the M phi s; moreover, diachronic observation indicated that the engulfed COM crystals were subdivided during M phi division and eliminated by the 7th day of culture. Additionally, imaging cytometry showed that the fluorescence level of f-COM crystals in the nigericin (-) group after 48 h was significantly lower than that in the nigericin (+) group. Conclusions: This study confirmed active phagocytosis and lysosomal processing of engulfed COM crystals by M phi s. This discovery is expected to contribute to the development of future drugs that enhance the COM crystal phagocytic ability of M phi s.