Active Phagocytosis and Diachronic Processing of Calcium Oxalate Monohydrate Crystals in an in vitro Macrophage Model

Abstract

Background: We previously discovered that renal macrophages (Mφs) phagocytose renal calcium oxalate monohydrate (COM) crystals. This study investigated the processing of engulfed crystals using in vitro models. Methods: J774.1 mouse Mφs 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 Mφs 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 Mφs; moreover, diachronic observation indicated that the engulfed COM crystals were subdivided during Mφ 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 Mφs. This discovery is expected to contribute to the development of future drugs that enhance the COM crystal phagocytic ability of Mφs.

Macrophage Function in Calcium Oxalate Kidney Stone Formation: A Systematic Review of Literature

“…Williams et al further investigated monocyte mitochondrial function in human serum specimens and reported significantly reduced monocyte mitochondrial maximal respiration, reserve 2). Internalized COM crystals become surrounded by phagosomes that fuse with lysosomes to promote dissolution and spontaneous elimination (29). Mj functions have also been investigated in RTEC.…”

“…The authors confirmed the diachronic elimination of engulfed COM crystals in Mφs ( Figure 2 ). Internalized COM crystals become surrounded by phagosomes that fuse with lysosomes to promote dissolution and spontaneous elimination ( 29 ). Mφ functions have also been investigated in RTEC.…”

Identification of the pivotal role of SPP1 in kidney stone disease based on multiple bioinformatics analysis

“…Non-polarized macrophages were designated as M0 phenotype. In vitro studies have demonstrated that M0 macrophages were able to internalize and eliminate COM crystals via phagocytosis [ 35 , 36 ]. M0 macrophages could also affect the function of other cells.…”

Bisphosphonate Use May Reduce the Risk of Urolithiasis in Astronauts on Long‐Term Spaceflights

“…Finally, we did not assess bone resorptive inflammatory cytokines, such as IL-1β, IL-6, and TNF-α. We have reported that these cytokines can trigger inflammatory macrophage activation and induce renal stone formation in mice and humans (24)(25)(26)(27)(28)(29) . In addition, IL-1β and TNF-α are increased during spaceflight, with a trend for increased IL-6 (30).…”

“…We have reported that these cytokines can trigger inflammatory macrophage activation and induce renal stone formation in mice and humans. ( 24 , 25 , 26 , 27 , 28 , 29 ) In addition, IL‐1β and TNF‐α are increased during spaceflight, with a trend for increased IL‐6. ( 30 ) Further studies are needed to assess changes in these blood and urinary cytokines as risk factors for renal stone formation during spaceflight.…”