Effects of secretome derived from macrophages exposed to calcium oxalate crystals

The association between kidney stone disease and renal fibrosis has been widely explored in recent years but its underlying mechanisms remain far from complete understanding. Using label-free quantitative proteomics (nanoLC-ESI-LTQ-Orbitrap MS/MS), this study identified 23 significantly altered secreted proteins from calcium oxalate monohydrate (COM)-exposed macrophages (COM-MP) compared with control macrophages (Ctrl-MP) secretome. Functional annotation and protein-protein interactions network analysis revealed that these altered secreted proteins were involved mainly in inflammatory response and fibroblast activation. BHK-21 renal fibroblasts treated with COM-MP secretome had more spindle-shaped morphology with greater spindle index.

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Fig: Cell death assay by flow cytometry with annexin V/propidium iodide co-staining: Macrophages were incubated in serum-free medium with or without 100 µg/ml COM crystals for 16, 24, and 48 h. The cells were then collected and co-stained with annexin V and propidium iodide followed by flow cytometric analysis. aScatter plots showing fluorescence signal of FITC-conjugated annexin V (X-axis) and propidium iodide (Y-axis). b Quantitative analysis of cell death calculated by using Formula. The data were obtained from three independent experiments using different biological replicates and the bars indicate mean ± SEM (all the source data are presented in Supplementary Data). Red-dashed rectangle indicates the optimal time-point that was chosen for all subsequent experiments.

Immunofluorescence study and gelatin zymography revealed increased levels of fibroblast activation markers (α-smooth muscle actin and F-actin) and fibrotic factors (fibronectin and matrix metalloproteinase-9 and -2) in the COM-MP secretome-treated fibroblasts. Our findings indicate that proteins secreted from macrophages exposed to COM crystals induce renal fibroblast activation and may play important roles in renal fibrogenesis in kidney stone disease.

Yoodee, S., Noonin, C., Sueksakit, K. et al. Effects of secretome derived from macrophages exposed to calcium oxalate crystals on renal fibroblast activation. Commun Biol 4, 959 (2021). https://doi.org/10.1038/s42003-021-02479-2

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