Fig 2: Vtn activates integrin αvβ5/Src signaling in vitro. (A) The protein-protein interaction (PPI) network of Vtn revealed by STRING analysis. (B) Representative Western blots show integrin αv, β1, β3 and β5 expression in human Vtn (50 ng/ml)-stimulated NRK-49F cells at different time points. (C) Quantitative data of protein bands show integrin β5 (Itg β5) expression in Vtn-stimulated NRK-49F cells. *P < 0.05 vs control cells (n = 3). (D) The PPI networks of integrin β5 revealed by STRING analysis. (E, F) Representative images of protein bands (E) and densitometric quantification (F) show p-Src (Tyr419) and total Src expression. *P < 0.05 vs. control cells (n = 3). (G-I) Co-immunoprecipitation demonstrates that Vtn formed a complex with integrin β5 and p-Src (Tyr419) in NRK-49F cells. (J, K) Representative protein expression bands (J) and densitometric quantification (K) show p-Src (Tyr419) and total Src expression in Vtn-stimulated NRK-49F cells in the absence or presence of integrin αvβ5 blocking antibody for 1 h. *P < 0.05 vs normal NRK-49F cells, †P < 0.05 vs Vtn alone (n = 3). (L, M) Representative protein expression bands (L) and densitometric quantification (M) show an increased expression of proliferation-related proteins. *P < 0.05 vs normal NRK-49F cells, †P < 0.05 vs Vtn alone (n = 3). (N, O) Western blots (N) and quantification (O) show p-Src (Tyr419) and total Src expression in Vtn-stimulated NRK-49F cells with or without of Src inhibitor Saracatinib for 1 h. *P < 0.05 vs normal NRK-49F cells, †P < 0.05 vs Vtn alone (n = 3). (P, Q) Western blots (P) and densitometric quantification (Q) show the proliferation-related proteins. *P < 0.05 vs normal NRK-49F cells, †P < 0.05 vs Vtn alone (n = 3). (R) Representative images of EdU-positive NRK-49F cells. (S) Diagram shows the Raw264.7/NRK49F cells co-culture experiment. Raw264.7 cells were induced to secrete Vtn by priming with TGF-β1. After 6 h of incubation, TGF-β1 was removed by changing to fresh media. NRK-49F cells were co-cultured with Raw264.7 cells in different chambers in the absence or presence of Saracatinib for 48 h. (T, U) Western blots (T) and densitometric quantification (U) show fibrosis-related and cell proliferation-related proteins expression in NRK-49F cells after co-culture with Raw264.7 cells. *P < 0.05 vs normal NRK-49F cells, †P < 0.05 vs NRK-49F cells co-culture with TGF-β1-treated Raw264.7 cells (n = 3). Scale bar, 100 µm.

Fig 3: Vitronectin is induced in chronic kidney disease in mice and human. (A) The schematic presentation shows the discovery of vitronectin (Vtn) as one of the most upregulated proteins in the KTS of mice with unilateral ischemia-reperfusion injury (UIRI). Identification of protein expression in KTS samples was analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). (B) Relative expression level of Vtn in the KTS of Sham and UIRI groups. *P < 0.05 vs. sham group (n = 3). (C, D) Vtn expression in UIRI kidneys as detected by Western blot (C). Densitometric quantification (D) of renal Vtn expression in UIRI mice (n = 6). (E) Representative Vtn immunohistochemical staining in the kidney section from Sham and UIRI group. Arrow indicates positive Vtn expression. (F, G) Vtn expression in UUO kidneys as detected by Western blot (F). Densitometric quantification (G) of Vtn protein in the obstructed kidneys. (H) Immunohistochemical staining shows the expression of Vtn in Sham and UUO mice. The black arrow indicates Vtn-positive area. (I) Representative Vtn immunohistochemical staining in normal kidney and renal biopsies of various human CKD. Ctrl, the paraffin-tissues were derived from the non-tumorous kidney tissues of patients with renal cell carcinoma. Specimens from CKD patients with various etiologies, such as membranous nephritis (MN), diabetic nephropathy (DN), lupus nephritis (LN), and IgA nephropathy (IgAN). The black arrowheads indicate Vtn-positive staining. (J) Quantification of kidney Vtn expression in human biopsy tissues from healthy subjects and CKD patients. (K) Vtn was detected in urine in healthy subjects (n = 13) and CKD patients with various etiologies (n = 116). Urinary Vtn protein concentrations were corrected by urinary creatinine (µg/mg creatinine). (L-O The) Linear regression revealed that urinary Vtn were correlated with kidney dysfunction (estimated glomerular filtration rate [eGFR]) (L), serum creatinine (Scr) (M), urinary albumin creatinine ratio (UACR) (N) and serum cystatin C (O). *P < 0.05 vs. sham controls or healthy subjects (n = 6-12). Scale bar, 50 µm.

Fig 4: Pharmacologic blockade of Src signaling inhibits fibroblast proliferation and attenuates renal fibrosis in vivo. (A) A schematic diagram illustrating experimental protocol. Red, green, and black arrowheads show the points of time for UIRI, UNx and sacrifice, respectively. The yellow arrowhead denotes the mouse tail-vein injection of empty vector (pcDNA3) or Flag-tagged Vtn expression plasmid (pFlag-Vtn). Repeated black arrowheads indicate the points of time when mice were treated with different doses of Saracatinib by intraperitoneal injection. (B) Histogram displays Scr levels in five groups as indicated. (C, D) Western blots and densitometric quantification demonstrate Fn, a-SMA and PCNA expression. (E, F) Western blots (E) and densitometric analysis (F) show p-Src (Tyr419) and total Src expression in different groups. (G) Immunohistochemical staining for Fn, a-SMA, Ki-67, p-Src (Tyr419) and MTS for collagen deposition. Arrowheads represent positive area. (H-K) Graphs depict the percentage of Fn, a-SMA (H) and p-Src (Tyr419) (J) positive area and the total number of Ki-67+ (I) cells. Quantitative data of kidney fibrosis area (K) in each group. At least 10 fields of kidney sections were randomly selected, and the average of positive area per mouse was calculated. *P < 0.05 vs sham group, #P < 0.05 vs UIRI plus pcDNA3 group, †P < 0.05 vs UIRI plus pFlag-Vtn group (n = 6). Scale bar, 50 µm.

Fig 5: Knockout of Vtn inhibits integrin avß5/Src signaling in UIRI model. (A) Western blots show integrin av, integrin ß5, integrin ß1, p-Src (Tyr419) and total Src expression in WT and Vtn KO mice. (B) Immunohistochemical staining on adjacent serial sections showed co-localization of integrin ß5 and p-Src (Tyr419). Arrowheads point to positive staining. Scale bar, 50 µm. (C, D) Histograms show integrin av, integrin ß5, p-Src (Tyr419) (C) and integrin ß1 (D) expression in WT and Vtn KO mice. (E) Immunohistochemical staining for integrin ß5 and vimentin on adjacent serial sections. Arrowheads point to co-localization. (F) Immunohistochemical staining for PCNA and vimentin on adjacent serial sections. (G, H) Protein expression bands (G) and densitometric quantification (H) show proliferation-related proteins expression. (I, J) Immunohistochemical staining (I) and the positive cell numbers (J) of PCNA and Ki-67 in each group. *P < 0.05 vs WT Sham group, †P < 0.05 vs WT UIRI group, n.s, not significant (n = 6). Scale bar, 50 µm.