Adaptive Response of HK-2 Cells to High Levels of COM Crystals Highlights CaSR as an Enhancer of Crystal Adhesion

Background: Calcium oxalate monohydrate (COM) is an aetiologic factor for urolithiasis. However, how Human Kidney-2 (HK-2) cells respond to a high COM has not yet been completely elucidated. Materials: and methods: A gel-based proteomics approach was applied to investigate COM-induced cellular proteomic changes. The COM-induced upregulation of calcium-sensing receptor (CaSR) in HK-2 cells was studied. Surface phospholipids (PS), which play a role in urolithiasis formation by mediating adhesion of HK-2 cells, were labelled in the inner or outer leaflet of the plasma membrane of HK-2 cells with fluorescent nitrobenzoxadiazole (NBD) to form NBD-PS to detect transmembrane movements of PS. After labelling, HK-2 cells were exposed to COM in the presence of the CaSR-specific agonist gadolinium chloride (GdCl 3 ) or the CaSR-specific antagonist NPS2390. Inward and outward transmembrane movements of PS were tracked with a fluorescence quenching assay. Surface-expressed PS was detected by an annexin V binding assay. Changes in aminophospholipid translocase (APLT), oxidative stress (OS), levels of apoptosis-related proteins in HK-2 cells and crystal adhesion were also assessed. Results: : COM increased CaSR and surface-expressed PS levels, decreased APLT activity, impaired inward transport of PS, and enhanced outward transport of PS. However, pretreatment with GdCl 3 further effectively inhibited the inward movement of PS and APLT activity and increased surface-expressed PS levels compared with COM treatment alone. In contrast, NPS2390 promoted the inward movement of PS and APLT activity and decreased surface-expressed PS levels compared with COM treatment alone. COM increased OS, apoptosis of HK-2 cells and crystal adhesion onto cells, and this increase was further enhanced by GdCl 3 pretreatment but attenuated by NPS2390 treatment. Conclusions: : These results strongly suggest that COM-induced CaSR generation may affect crystal adhesion by regulating PS externalization, apoptosis and OS in HK-2 cells.