Calcium oxalate monohydrate crystals increase protein cargo loading in small extracellular vesicles secreted from renal proximal tubular epithelial cells that further induce renal fibroblast activation via endocytosis

Calcium oxalate monohydrate (COM), the main crystalline type in kidney stones, can induce a wide spectrum of renal deteriorations, from tubular cell injury to renal fibrosis. Kidney stone disease (KSD) is thus considered one of the risks for chronic kidney disease (CKD), but with unclear mechanisms. Herein, we addressed the effects of COM crystals on the secretion of small extracellular vesicles (EVs) from renal proximal tubular epithelial cells (PTECs) that might further affect renal fibroblasts, as the final effectors related to renal fibrogenesis. After 24-h exposure, the amount of total proteins of small EVs secreted from the COM-treated PTECs (HK-2 cells) (termed “COM-treated small EVs”) was significantly greater than that of small EVs secreted from control (untreated) HK-2 cells (termed “control small EVs”). However, the total number of small EVs was comparable between groups, indicating the increased protein cargo loading into COM-treated small EVs. Incubation of renal fibroblasts (BHK-21 cells) with COM-treated small EVs caused an increase in cell spindle index compared with incubation with control small EVs and untreated cells. COM-treated small EVs also increased levels of markers for activated fibroblasts (i.e. F-actin, α-SMA, fibronectin and collagen I). Additionally, BHK-21 cells exposed to COM-treated small EVs secreted greater levels of MCP-1 (proinflammatory) and TGF-β (profibrotic) cytokines. However, all of the stimulatory effects of COM-treated small EVs on BHK-21 cells were abolished by co-incubation with heparin or chlorpromazine (CPZ), both of which are endocytosis (small EVs uptake) inhibitors. These results indicate that COM crystals elevate protein cargo loading into small EVs secreted from renal PTECs that further induce renal fibroblast activation via endocytosis. Not applicable (This is not a clinical trial).