Introduction

We previously reported that tubular cell damage facilitates kidney crystal formation. Although recent evidence shows that damaged cells could induce autophagosomes and autolysosomes to perform autophagy, engulfing and removing damaged organelles, the association between autophagy and kidney crystal formation remains unclear. Hence, we analyzed the role of autophagy in renal crystal formation.

Methods

In vitro study We exposed M1 cells derived from the cortical collecting duct of the mouse to calcium oxalate monohydrate (COM) crystals at a concentration of 20 μg/cm² and the levels of autophagy-related proteins (LC3-B, Beclin-1, p62) were assessed using fluorescent immunostaining and western blotting. Additionally, immunostaining of the organelles was carried out to determine mitochondrial and lysosomal damage, and the COM crystal adhesion ratio to cells was measured. Furthermore, using tandem fluorescent-tagged LC3 (tfLC3) assay, we examined autophagy behavior._x000D_ In vivo study Kidney crystal formation in C57BL/6J mice was induced by daily intra-abdominal injection of 80 mg/kg^-1 glyoxylic acid, and the relationship between crystal formation and the ultrastructure of autophagosomes and autolysosomes was observed using polarized light microscopy and transmission electron microscopy (TEM). Western blotting and immunostaining of autophagy-related proteins were performed, and GFP-LC3 transgenic mice were created to check autophagy in kidneys._x000D_

Results

In vitro study COM exposure damaged many organelles, in response of which autophagy increased. After 8 hours, the COM crystal adhesion ratio to M1 cells had increased and tfLC3 assay showed a slight increase in autophagy._x000D_ In vivo study Remarkable accumulation of autophagosomes and autolysosomes in proximal renal tubular cells of mice without renal crystal deposits was observed. As crystal deposits increased, autophagy expression decreased (Figure 1). Similarly, western blot analysis results from GFP-LC3 mice showed crystal deposits increase as autophagy decreases. Furthermore, crystals deposits tended to decrease to promote autophagy. _x000D_

Conclusions

Results indicate that autophagy removes damaged organelles and maintains cellular homeostasis in renal tubular cells. Consequently, autophagy prevents renal crystal formation.

Funding

none