Effects of calcium oxalate crystals on neutrophil cellular proteome and functions: implications for nephrolithiasis

Abstract

Background: The majority of stone formers (87.5–95.9%) exhibit mild to moderate interstitial inflammation surrounding the stone. Neutrophils and neutrophil-derived genes/proteins have been found in renal papillae, stone matrix and urine of calcium oxalate monohydrate (COM) stone formers. However, neutrophil-crystal interactions, especially responses of neutrophils to COM crystals, remained unknown. Methods: This study addressed the effects of COM crystals on neutrophil cellular proteome and functions, including phagocytosis, activation/degranulation, neutrophil extracellular traps (NETs) formation and reactive oxygen species (ROS) production. Results: Label-free quantitative (LFQ) proteomics using nanoLC-ESI-Qq-TOF MS/MS with highly stringent criteria revealed that COM caused altered levels of 22 neutrophil proteins involved mainly in immune responses. Investigating neutrophil innate immune functions using flow cytometry, immunofluorescence/fluorescence imaging, ELISA and dichlorodihydrofluorescein diacetate (DCFH-DA) assay revealed that COM enhanced neutrophil phagocytic activity, NETs formation, activation/degranulation and ROS production. Moreover, secretome (a set of secretory products) from COM-treated neutrophils induced the recruitment of macrophages to phagocytose the COM-treated neutrophils. Conclusions: These findings illustrate the expression and functional responses of neutrophils to COM crystals and implicate the important roles that neutrophils play in nephrolithiasis.