Abstract
Objective To analyze and identify the differentially expressed proteins in human renal tubular epithelial cells (HK-2) after injury caused by oxalic acid and calcium oxalate monohydrate (COM) crystal, and to explore the potential role of renal tubular cell injury in kidney stone formation.Methods Normal HK-2 cells were cultured in vitro and the culture medium was changed with serum-free medium after cell growth to confluence. Oxalic acid and COM crystals (final concentration at 2 mmol/L and 200 mg/L, respectively) were added in the experimental group. Cells in both groups were then incubated at 37 ℃ for 12 h. The extracted proteins from both groups were separated by two dimensional electrophoresis followed by analysis, and the differentially expressed proteins were identified by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Two identified proteins were then verified by western blot. Results Reproducible two dimensional gel images of the proteins from both groups were successfully obtained. By using LC-ESI-MS/MS, 12 proteins: FK506-binding protein 4, isoform alpha-enolase of alpha-enolase, isoform M1 of pyruvate kinase isozymes M1/M2, ATP synthase subunit alpha, isoform 1 of 3′(2′), 5′-bisphosphate nucleotidase 1, isoform 2 of nucleophosmin, L-lactate dehydrogenase B chain, Budding uninhibited by benzimidazoles 3, Cofilin-1, Fascin, pyIsoform 1 of cytosol aminopeptidase, were identified. The deferentially expressed proteins were related to cellular processes including energy metabolism, cell multiplication, apoptosis, Ca2+ channel activity regulation, cell movement and signal transduction. Western blot verified that higher ENO1 but lower Cofilin-1 expressed in HK-2 cells after the injury. Conclusions High level oxalic acid and COM crystals can cause protein expression profile changes in normal human HK-2 cells. The changes of protein expression may not only protect HK-2 cells from being injured, but also be related to kidney stone formation. Key words: Renal tubular epithelial cell; Oxalate; Calcium oxalate monohydrate; Renal calculus; Proteomics
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