Abstract
In kidney stone disease, macrophages secrete various mediators via classical secretory pathway and cause renal interstitial inflammation. However, whether their extracellular vesicles, particularly exosomes, are involved in kidney stone pathogenesis remained unknown. This study investigated alterations in exosomal proteome of U937-derived macrophages (by phorbol-12-myristate-13-acetate activation) after exposure to calcium oxalate monohydrate (COM) crystals for 16-h using 2-DE-based proteomics approach. Six significantly altered proteins in COM-treated exosomes were successfully identified by nanoscale liquid chromatography–electrospray ionization–electron transfer dissociation tandem mass spectrometry as proteins involved mainly in immune processes, including T-cell activation and homeostasis, Fcγ receptor-mediated phagocytosis, interferon-γ (IFN-γ) regulation, and cell migration/movement. The decreased heat shock protein 90-beta (HSP90β) and increased vimentin were confirmed by Western blotting. ELISA showed that the COM-treated macrophages produced greater level of interleukin-1β (IL-1β), one of the markers for inflammasome activation. Functional studies demonstrated that COM-treated exosomes enhanced monocyte and T-cell migration, monocyte activation and macrophage phagocytic activity, but on the other hand, reduced T-cell activation. In addition, COM-treated exosomes enhanced production of proinflammatory cytokine IL-8 by monocytes that could be restored to its basal level by small-interfering RNA targeting on vimentin (si-Vimentin). Moreover, si-Vimentin could also abolish effects of COM-treated exosomes on monocyte and T-cell migration as well as macrophage phagocytic activity. These findings provided some implications to the immune response during kidney stone pathogenesis via exosomal pathway of macrophages after exposure to COM crystals.
Highlights
During an initial phase of kidney stone formation, the causative chemical crystals, such as calcium oxalate (CaOx), can be deposited in the renal interstitium, where macrophages are recruited to eliminate these crystals via phagocytosis [1,2,3]
Due to differences in adhesive capability, binding kinetics, atomic lattice, and surface ionic pattern, calcium oxalate monohydrate (COM) crystals are more pathogenic during the kidney stone pathogenesis than CaOx dihydrate (COD) crystals, which can be found in the normal urine of healthy individuals [5,6,7,8,9]
Exosomes are originated from the internalized vesicles via endocytosis that subsequently form multivesicular bodies (MVB) [29]
Summary
During an initial phase of kidney stone formation, the causative chemical crystals, such as calcium oxalate (CaOx), can be deposited in the renal interstitium, where macrophages are recruited to eliminate these crystals via phagocytosis [1,2,3]. Several lines of evidence have shown that macrophages exposed to COM crystals increase secretion of reactive oxygen species (ROS), chemokines, proinflammatory cytokines, and several fibrotic factors to promote renal interstitial inflammation in kidney stone disease [10,11,12]. Macrophages exposed to naturally occurred kidney stone fragments secrete greater levels of several chemokines, macrophage inhibitory protein-1, monocyte chemoattractant protein-1, and interleukin-8 (IL-8) [17]. These chemokines enhance recruitment of various immune cells, i.e., monocytes, macrophages, neutrophils, dendritic cells, and T-cells into the inflammatory locale [18]
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