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Published on: August 22, 2025
Active Phagocytosis and Diachronic Processing of Calcium Oxalate Monohydrate Crystals in an in vitro Macrophage Model
Atsushi Okada1, Hiromasa Aoki2, Daichi Onozato2
1Department of Nephro-Urology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan, a-okada@med.nagoya-cu.ac.jp.
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.
Insights
Renal macrophages actively phagocytose and process calcium oxalate monohydrate (COM) crystals within lysosomes. These crystals are eliminated by the 7th day, suggesting potential therapeutic targets for enhancing COM crystal clearance.
Area of Science:
- Nephrology
- Immunology
- Cell Biology
Background:
- Renal macrophages (Mφs) are known to phagocytose calcium oxalate monohydrate (COM) crystals.
- This study investigates the in vitro processing mechanisms of engulfed COM crystals by Mφs.
Purpose of the Study:
- To confirm active phagocytosis of COM crystals by Mφs.
- To elucidate the intracellular processing and degradation pathways of COM crystals within Mφs.
- To explore the potential for therapeutic interventions targeting Mφs for COM crystal clearance.
Main Methods:
- J774.1 mouse Mφs were exposed to COM crystals and observed using polarized light microscopy.
- Cytochalasin B (CB) was used to inhibit phagocytosis and confirm active uptake.
- LysoTracker and transmission electron microscopy (TEM) were employed to identify lysosomal localization of crystals.
- Diachronic tracking and imaging cytometry with fluorescent COM (f-COM) crystals were utilized to monitor crystal processing over time.
Main Results:
- Phagocytosis rates of COM crystals by Mφs increased with crystal density.
- Active phagocytosis was confirmed by reduced uptake in CB-treated cells.
- Engulfed COM crystals were localized within macrophage lysosomes.
- Diachronic observation revealed crystal subdivision during Mφ division and elimination by day 7.
- Imaging cytometry indicated lysosomal processing of f-COM crystals, with lower fluorescence in acidic conditions.
Conclusions:
- This study confirms that renal macrophages actively phagocytose and process COM crystals via lysosomal pathways.
- Engulfed COM crystals are effectively degraded and eliminated by Mφs within a week.
- These findings suggest that enhancing Mφ phagocytic capacity could be a therapeutic strategy for kidney stone diseases.
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