Analysis of HK-2 cells exposed to oxalate and calcium oxalate crystals: proteomic insights into the molecular mechanisms of renal injury and stone formation

Abstract

Exposure to high levels of oxalate and calcium oxalate monohydrate (COM) crystals is injurious to renal epithelial cells and triggers serial responses related to stone formation. Multiple molecules and proteins are involved in this process, but previous studies have generally been limited, without an overall understanding of protein expression alteration after oxalate and/or crystal exposure as well as its role in stone formation. We used proteomic analysis to reveal the changes in the proteome of HK-2 cells induced by oxalate and COM crystals, so as to provide candidate proteins involved in the molecular mechanisms concerning HK-2 cell injury and kidney stone formation. HK-2 cells were exposed to oxalate plus COM crystals at different concentrations in various samples. Cell viability was determined using a Cell Counting Kit-8 assay kit. For proteomic analysis, cells were exposed to oxalate (2 mM) and COM crystals (200 ug/ml) for 12 h. The proteins were separated by two-dimensional electrophoresis and the differentially expressed proteins were identified by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Validation of protein expression was further performed by Western blot analysis. Oxalate and COM crystals showed concentration-dependent toxicity on HK-2 cells. A total of 12 differentially expressed proteins in HK-2 cells induced by oxalate and COM crystals were identified, which were involved in various aspects of cellular processes. Our study provides a platform for further studying the molecular mechanism of renal epithelial cell injury and kidney stone formation.