Fig 2: Depletion of endogenous MMP-10 reduces kidney injury and β-catenin activation after ischemia-reperfusion injury.A Experimental design. The timing of UIRI surgery, plasmid injection, unilateral nephrectomy, and sacrifice were indicated by red and black arrow or arrowhead as indicated, respectively. B, C Western blotting showed the protein level of MMP-10 in different groups as indicated. Representative Western blotting (B) and quantitative data (C) were shown. Data are presented as the mean ± SEM. **P < 0.01 versus sham controls (n = 5); ††P < 0.01 versus UIRI injected with Ctrl-shRNA (n = 5). D Representative images showed the expression and localization of MMP-10 in the kidney after different treatments. Scale bar, 50 µm. E, F Graphic presentation showed serum creatinine (SCr) and blood urea nitrogen (BUN) levels in three groups as indicated. Data are presented as the mean ± SEM. ***P < 0.001 versus sham controls (n = 5). †††P < 0.001 versus UIRI injected with Ctrl-shRNA (n = 5). G, H Western blotting showed the protein levels of fibronectin, collagen I, α-SMA, and KIM-1 in different groups. Representative Western blotting (G) and quantitative data (H) were shown. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus sham controls (n = 5); †P < 0.05, ††P < 0.01, †††P < 0.001 versus UIRI injected with Ctrl-shRNA (n = 5). I, J Knockdown of MMP-10 ameliorated kidney injury and fibrosis. Renal fibrotic lesions were evaluated using Sirius red staining and immunohistochemical staining for α-SMA. The tubular injury was assessed through immunohistochemical staining for KIM-1. Scale bar, 50 µm. Data are presented as the mean ± SEM. ***P < 0.001 versus sham controls (n = 5), †††P < 0.001 versus UIRI injected with Ctrl-shRNA (n = 5).

Fig 3: Identification of β-catenin as the major effector mediating MMP-10 action.A–C Western blotting showed that rhMMP-10 (100 ng/ml) induced fibronectin and α-smooth muscle actin (α-SMA) and repressed E-cadherin in HK-2 cells. Representative Western blotting (A) and quantitative data (B, C) are reported. Data are presented as the mean ± SEM. ***P < 0.001 versus the controls (n = 6). D STRING analysis revealed the protein-protein interaction (PPI) network involving MMP-10 (https://cn.string-db.org/, accessed on 8 August 2023). The interaction between MMP-10 and β-catenin was noticed. E Analysis of the Cancer Dependency Map (https://depmap.org, accessed on 10 August 2023) revealed the correlation between MMP-10 expression and alteration in β-catenin. Linear regression (left) and volcano plot (right) demonstrated a positive correlation between MMP-10 and β-catenin. F, H RhMMP-10 activated β-catenin signaling in vitro, leading to the induction of active β-catenin, PAI-1, and MMP-7 proteins. Data are presented as the mean ± SEM. **P < 0.01, ***P < 0.001 versus the controls (n = 6). I Representative micrographs showed the nuclear translocation of β-catenin after rhMMP-10 treatment for 12 h. White arrows indicate nuclear staining of β-catenin. Scale bar, 50 µm. J, K ICG-001 (10 μM) abolished MMP-10-mediated fibronectin and α-SMA expression. Data are presented as the mean ± SEM. ***P < 0.001 versus the controls (n = 6); †††P < 0.001 versus rhMMP-10 alone (n = 6). L Representative immunofluorescence staining showed that ICG-001 inhibited fibronectin expression and deposition induced by MMP-10. Scale bar, 50 µm. M, N ICG-001 inhibited MMP-10-mediated β-catenin activation and its downstream MMP-7 and PAI-1 induction. Representative Western blotting (M) and quantitative data (N) were presented. Data are presented as the mean ± SEM. **P < 0.01, ***P < 0.001 versus the controls (n = 6); ††P < 0.01, †††P < 0.001 versus rhMMP-10 alone (n = 6). O qRT-PCR analysis showed that rhMMP-10 did not significantly affect the expression of Wnt ligands except Wnt10a. Data are presented as the mean ± SEM. *P < 0.05 versus controls (n = 6).

Fig 4: MMP-10 transactivates β-catenin via HB-EGF/EGFR/ERK1/2/GSK-3β cascade.A Representative micrographs showed the colocalization of MMP-10 (Red) and β-catenin (Green) in the obstructed kidney after UUO. Arrows indicate positive staining. Scale bar, 50 µm. B–D Knockdown of endogenous MMP-10 abolished the expression of β-catenin and its downstream genes in UUO model. Representative Western blotting results (B) and corresponding quantitative data (C, D) were shown. Data are presented as the mean ± SEM. ***P < 0.001 versus sham controls (n = 5); †P < 0.05, ††P < 0.01, †††P < 0.001 versus UUO injected with Ctrl-shRNA (n = 5). E, F Representative immunohistochemical staining for β-catenin further demonstrated that knockdown of MMP-10 inhibited β-catenin expression in renal tubules. Scale bar, 50 µm. Data are presented as the mean ± SEM. ***P < 0.001 versus sham controls (n = 5). †††P < 0.001 versus UUO injected with Ctrl-shRNA (n = 5). G Double immunofluorescence staining showed the colocalization between MMP-10 (Red) and β-catenin (Green) in patients with various CKD as indicated. Scale bar, 25 µm. H–L Western blotting showed that knockdown of MMP-10 inhibited the expression of cleaved HB-EGF, p-EGFR (Tyr845), p-ERK (Thr202/Tyr204), and p-GSK-3β (Ser9) in the obstructed kidney after UUO. Representative Western blotting (H) and quantitative data (I–L) were shown. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus sham controls (n = 5); †P < 0.05, ††P < 0.01, †††P < 0.001 versus UUO injected with Ctrl-shRNA (n = 5). M, N Immunohistochemical staining for p-EGFR (Tyr845) demonstrated a decreased EGFR phosphorylation in renal tubules after MMP-10 knockdown. Scale bar, 50 µm. Data are presented as the mean ± SEM. ***P < 0.001 versus sham controls (n = 5), ††P < 0.01 versus UUO injected with Ctrl-shRNA (n = 5). O Representative micrographs showed the colocalization of p-EGFR (Red) and β-catenin (Green) in UUO. Scale bar, 50 µm.

Fig 5: Knockdown of endogenous MMP-10 ameliorates kidney injury and fibrosis in obstructive nephropathy.A Experimental design. The red and green arrows show the timing of UUO surgery and plasmid injection, respectively. B, C Representative micrographs (B) and quantitative data (C) demonstrated MMP-10 protein expression in different groups as indicated. Scale bar, 50 µm. Data are presented as the mean ± SEM. **P < 0.01 versus sham controls (n = 5); †P < 0.05 versus UUO (n = 5). D, E Western blot analysis showed MMP-10 protein levels in various groups as indicated. Data are presented as the mean ± SEM. **P < 0.01 versus sham controls (n = 5), ††P < 0.01 versus UUO (n = 5). F, G Western blot analysis showed the expression of several fibrosis-related proteins such as fibronectin, α-SMA, and vimentin. Representative Western blotting (F) and quantitative data (G) are presented. Data are presented as the mean ± SEM. **P < 0.01, ***P < 0.001 versus sham controls (n = 5); ††P < 0.01, †††P < 0.001 versus UUO (n = 5). H, I Representative Western blotting (H) and quantitative data (I) showed KIM-1 and E-cadherin protein in various groups as indicated. Data are presented as the mean ± SEM. **P < 0.01 versus sham controls (n = 5), ††P < 0.01 versus UUO (n = 5). J, K Representative micrographs (J) and quantitative data (K) showed immunohistochemical staining for α-SMA and KIM-1 and Sirius red staining for collagen deposition. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01 versus sham controls (n = 5); †P < 0.05, ††P < 0.01 versus UUO (n = 5). Scale bar, 50 µm.