FIGURE 3.

Overexpression of OPN promotes renal fibrosis but blocking with exosome inhibitor dimethyl amiloride (DMA) inhibits kidney fibrosis in vivo. (a) Diagram showing the experimental design. Black arrows indicate the injection of pcDNA3 or OPN overexpression plasmid (pCMV‐OPN). Red arrows indicate the time points undergoing UIRI, UNx and sacrifice. Green arrows indicate dimethyl amiloride (DMA) treatment (10 mg/kg body weight). (b and c) Graphic presentation showing the serum creatinine levels (b) and BUN levels (c) in different groups after UIRI. ***p < 0.001 versus sham controls; ^† p < 0.05, ^†† p < 0.01 versus UIRI (n = 5); ^# p < 0.05, ^## p < 0.01 versus UIRI + pCMV‐OPN (n = 5). Western blot (d) and quantitative data (e–j) showing the upregulation of renal Flag, CD63, TSG101, OPN, N‐OPN and CD44 expression after overexpression OPN, but these effects were abolished after DMA treatment. *p < 0.05, ***p < 0.001 versus sham controls; ^† p < 0.05, ^†† p < 0.01, ^††† p < 0.001 versus UIRI; ^# p < 0.05, ^### p < 0.001 versus UIRI + pCMV‐OPN (n = 5). (k) Kidney tissues from different groups were subjected to Masson's trichrome staining, immunofluorescence staining of OPN, collagen I, vimentin and fibronectin, and immunostaining staining for CD44, as indicated respectively. Arrows indicate positive staining; scale bar: 50 μm. (l–r) Western blots analyses show renal expression of fibrosis‐related proteins. Representative western blot (l) and quantitative data for fibronectin (m), α‐SMA (n), PDGFR‐β (o), collagen I (p), vimentin (q) and active β‐catenin (r) proteins are shown. Numbers (1 to 2) indicate individual animal in a given group. *p < 0.05, **p < 0.01, ***p < 0.001 versus sham controls; ^† p < 0.05, ^†† p < 0.01, ^††† p < 0.001 versus UIRI; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 versus UIRI + pCMV‐OPN (n = 5)