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.