Uric acid directly induced macrophage M2 polarization and strongly activated ITGAM and FAK signaling
We performed in vitro experiments to explore how uric acid functions on macrophage. Since uric acid at high concentration causes tubular damages(Johnson et al., 2018; Milanesi et al., 2019), we designed two in vitro models: model1, using uric acid to stimulate Raw 264.7 macrophage; and model 2, using uric acid to stimulate the co-culture environment of Raw 264.7+proximal tubular cell TCMK1. Macrophages were collected after 24 hours and 48 hours of stimulation (Figure 4A ). The ITGAM mRNA expression in model 1 quickly increased to peak after 24 hours, and ITGAM in model 1 was higher or equal to that in model 2 at time points of 24 and 48 hours, respectively (Figure 4B ). Although not highly consistence in M2 polarization markers, two models both presented obvious M2 polarization especially after 48 hours (Figure 4C ). ITGAM and downstream FAK signaling were activated after 24 hours of uric acid stimulation. As indicated by KEGG enrichment, we confirmed the higher phosphorylation of FAK and Akt1, lower phosphorylation of GSK-3β, and less degraded β-catenin (Figure 4D ). Evidence above indicated that uric acid strongly activated ITGAM/FAK signaling in macrophage and M2 polarization, regardless of existence of tubular epithelial cells.