Calcium Oxalate Augments Phosphatidylserine Concentration in Murine Inner Medullary Collecting Duct Cell Membranes, Leading to Increased Annexin A2 Protein Expression

Background: Although the exact mechanism of calcium oxalate (CaOx) stone formation remains unclear, it is generally accepted that recurrent kidney stone formation is linked to injury to renal tubular epithelial cells. It is postulated that exposure of renal epithelial cells to high oxalate and CaOx crystals causes enrichment of phosphatidylserine (PS) at the surface promoting crystal attachment. We hypothesized that CaOx exposure to inner medullary collecting duct (IMCD) cells increases membrane concentration of the negatively charged PS and augments expression of the calcium-dependent phospholipid-binding protein annexin A2 to protect against cell death and allow for membrane repair. Methods: Monolayers of mouse IMCD (mIMCD3) cells were treated with CaOx or vehicle for 8 hours and for 24 hours, and then targeted mass spectrometry-based lipidomics was performed using the membrane fractions from each group. In other experiments, monolayers of mIMCD3 cells were treated with methyl-beta-cyclodextrin (MBCD) PS liposomes or vehicle for 24 hours, and changes in annexin A2 protein expression were assessed by Western blotting and densitometric analysis. Next, siRNA mediated knockdown studies were performed to corroborate a role for annexin A2 at the plasma membrane. Results: Lipidomic analysis revealed an enrichment of multiple PS lipid species, including PS(20:3_20:3), PS(20:3_20:4), PS(20:3_22:5), PS(20:4_20:4), PS(20:4_22:5), and PS(20:5_20:5) in membrane fractions of mIMCD3 cells treated with CaOx compared to cells treated with vehicle alone for 24 hours. Western blotting and densitometric analysis showed a significant increase in Annexin A2 protein expression in the membrane fractions of mIMCD3 cells treated with either CaOx or MBCD PS liposomes compared to vehicle treatment for 24 hours. Changes in transepithelial resistance were observed in mIMCD3 cells treated with CaOx compared to cells transiently transfected with annexin A2 siRNA and non-targeting siRNA. Conclusion: Taken together, these data suggest that the upregulation of annexin A2 protein in response to an increase in the concentration of PS in IMCD membranes may serve to protect against cell death and/or aid in calcium-dependent plasma membrane repair. Funding: NIDDK Support