Fig. 2: Inhibition of the exosome release from tubular cells promotes fibroblast apoptosis in vitro.

a Experimental design. HK-2 cells were treated with TGF-β1 (2 ng/ml) for 6 h in the absence or presence of dimethyl amiloride (DMA) or shRab27a transfection, and then washed to remove all the treatments and continued to incubate for additional 48 h in serum-free medium. Exosomes were isolated from conditioned media by ultracentrifugation. b Representative Western blotting show CD63, TSG101 and Calnexin expression in HK-2 cells after DMA treatment. Numbers (1–3) indicate each individual treatment in a given group. c Representative Western blotting indicates that DMA attenuated the induction of CD63 and TSG101 expression in exosomes isolated from TGF-β1-treated HK-2 cells. Numbers (1–3) indicate exosomes isolated from each individual treatment in a given group. d, e Blockade of exosome generation by DMA abolished the cytoprotection elicited by exosomes derived from TGF-β1-treated HK-2 cells. Representative FACS analyses (d) and quantitative data (e) show the abundance of apoptotic cells in different groups. ^**P < 0.01, ^††P < 0.01 (n = 3). f Western blotting analyses demonstrate protein expression of p53, cleaved caspase-3, FasL, PARP-1, fibronectin and α-SMA after various treatments in NRK-49F cells. Numbers (1–3) indicate each individual treatment in a given group. g Representative micrographs show immunofluorescence staining of fibronectin in different groups as indicated. Arrows indicate positive staining. Scale bar, 50 µm. h Graphic presentation shows the quantitative determination of fibronectin-positive staining. Each point indicates one of three different random fields of view in one micrograph. ^**P < 0.01, ^††P < 0.01 (n = 3). i Western blotting indicates that knockdown of Rab27a reduced CD63 and Calnexin induction in TGF-β1-treated HK-2 cells. Numbers (1–3) indicate each individual treatment in a given group. j Western blotting shows the reduction of CD63 and TSG101 expression in exosomes isolated from TGF-β1-treated HK-2 cells after Rab27a knockdown. Numbers (1–3) indicate exosomes isolated from each individual treatment in a given group. k Representative micrographs show TUNEL-positive cells in different groups as indicated. Scale bar, 50 µm. l Graphic presentation shows the quantitative determination of TUNEL-positive fibroblast cells in different groups. Each point indicates one of three different random fields of view in one micrograph. ^**P < 0.01, ^††P < 0.01 (n = 3). m Western blotting analyses demonstrate protein expression of p53, cleaved caspase-3, FasL, PARP-1, fibronectin and α-SMA after various treatments in NRK-49F cells. Numbers (1–3) indicate each individual treatment in a given group. Data presented as mean ± S.E.M. of three independent experiments. p values were calculated using Student-Newman-Kuels test for multiple groups comparison.