Figure Legends
Figure 1 Progressive renal dysfunction and kidney fibrosis in hyperuricemia-related CKD mice. (A) Brief instruction on model establishment and sample collection; (B) Biochemical measurements from day 0 to day 21, including serum uric acid, creatinine, urea, and urine albumin-to-creatinine ratio; (C) Periodic acid-Schiff (PAS) and Masson trichrome staining of renal tissue. PAS stain indicates progressively tubular atrophy and glomerulosclerosis. Trichrome stain indicates progressively interstitial collagen deposition. Tubular injuries and interstitial fibrosis were quantified and presented; (D-E) From day 0 to day 21, renal tissue is characterized with progressively exacerbated inflammation and fibrosis. n=6; ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.
Figure 2 Bioinformatic analyses revealed ITGAM as the hub gene associated with downstream FAK pathway. (A) Sketch map of how we incorporated mRNA and protein sequencing data to select core genes and pathways related. In brief, we found out 1,337 DEGs both at mRNA and protein levels and further adopted hub gene analyses (MCODE followed by CentiScape in Cytoscape) to acquire simplified 49 genes. Finally, we ranked the top genes by degrees of enrichment to pathways. (B) There are 6 of 49 hub genes corresponding to top canonical pathways conducted by IPA. Itgam and Itgb2, as two heterodimers of Mac-1, ranked the 1st. Itgam on day 21, at mRNA and protein level, was upregulated to 19 and 3.3 times of day 0. (D) KEGG results based on genes significantly positively correlated with Itgam at both mRNA and protein levels; (E) Schematic diagram of ITGAM and related pathway in macrophage after exposure to uric acid.
Figure 3 ITGAM expression and macrophage M2 polarization in vivo. (A) ITGAM was increasingly upregulated at mRNA and protein levels; (B) ITGAM co-locates with macrophage marker F4/80; (C) ITGAM/FAK/Akt1/β-catenin pathway was activated in vivo (day 21 vs day 0); (D) Location of two core molecules in pathway, including p-FAK and β-catenin; (E-F) M2 polarization was activated in hyperuricemia-related CKD as revealed by omics data and PCR quantification in vivo. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.
Figure 4 Two experimental models in vitro validated overexpression of ITGAM, pathway activation, and M2 polarization of macrophages. (A) study design of uric acid at 800 μM stimulating Raw 264.7 vs. Raw 264.7 +TCMK1; (B) Raw 264.7 alone, under stimulation of uric acid, greatly overexpresses ITGAM and the expression levels in two models at 48 hours are not significantly different. (C) Either Raw 264.7 or co-culture system presented obvious activation of macrophage M2 activation after uric acid exposure. (D) Two models at different time points shows activation of ITGAM/FAK/Akt/β-catenin pathway. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.
Figure 5 Interventions of ITGAM, FAK, and Akt verified the participation of pathway ITGAM/FAK/Akt/β-catenin in macrophage M2 polarization in hyperuricemia-related CKD. (A) Design of ITGAM and pathway intervention; (B-C) Itgam silencing significantly inhibited pathway and attenuated M2 polarization; (D-E) Inhibition of FAK greatly inhibited downstream pathway and macrophage M2 polarization; (F-G) Inhibition of Akt downregulated β-catenin and macrophage M2 polarization. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.