The mechanism underlying calcium oxalate (CaOx) stone formation remains unclear, although recurrent kidney stone formation has been linked to injury of kidney tubular cells in response to their exposure to higher oxalate and CaOx crystals. We hypothesized that CaOx induces the externalization and membrane enrichment of the negatively charged phospholipid phosphatidylserine (PS), while simultaneously enhancing the expression of calcium-binding proteins as a protective mechanism against cell death in inner medullary collecting duct (IMCD) cells. Annexin A2, a calcium-dependent phospholipid-binding protein, may play a protective role in CaOx stone formation. Mouse IMCD (mIMCD3) cells were treated with CaOx before performing a targeted lipidomic analysis of membrane fractions. Additionally, mIMCD3 cells were challenged with methyl-beta-cyclodextrin (MBCD) PS liposomes, Annexin A2 was evaluated by Western blotting and densitometric analysis. Lipidomic analysis revealed an enrichment of multiple PS lipid species in membrane fractions of mIMCD3 cells treated with CaOx. Western blotting and densitometric analysis further demonstrated a significant increase in Annexin A2 protein expression in the membrane fractions of mIMCD3 cells treated with MBCD PS liposomes. CaOx and MBCD PS liposomes were found to differentially affect membrane fluidity in mIMCD3 cells. Importantly, CaOx treatment caused an increase in ACSL4 and caspase 9 protein expression, indicating initiation of ferroptosis and apoptosis, respectively. The siRNA mediated knockdown of annexin A2 further augmented the upregulation of caspase 9 after CaOx treatment. Collectively, these findings suggest that Annexin A2, a calcium-binding and plasma membrane repair protein, is upregulated in response to CaOx-induced PS enrichment in mIMCD3 cells.
Keywords: Annexin A2; Calcium oxalate; Inner medullary collecting duct cells; Kidney stones; Phosphatidylserine.
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