Fig. 1: Injured tubular cells-derived exosomes protect fibroblasts from apoptosis induced by cisplatin in vitro.

a Diagram shows the experimental design. Human kidney proximal tubular epithelial cells (HK-2) were treated with TGF-β1 (2 ng/ml) for 6 h, and then removed TGF-β1 and continued to incubate for additional 48 h in serum-free medium. Exosomes were isolated from conditioned media by ultracentrifugation and used to stimulate normal rat kidney interstitial fibroblasts (NRK-49F), followed by incubating with cisplatin (25 μg/ml) for 24 h. b Western blotting analyses demonstrate protein expression of CD63, TSG101 and Calnexin in TGF-β1-treated HK-2 cells. Numbers (1–3) indicate each individual culture plate in a given group. c Representative transmission electron microscopy (TEM) shows the exosomes isolated from conditioned media of HK-2 cells. Scale bar, 200 nm. d Western blotting analyses show induction of CD63 and TSG101 expression in EVs isolated from TGF-β1-treated HK-2 cells. Numbers (1–3) indicate EVs isolated from each individual treatment of HK-2 cells in a given group. e Average size distribution curve of exosomes released from HK-2 cells with or without TGF-β1 treatment. Exosomes size was determined by Nanoparticle Tracking Assay (NTA). Three 60 s videos were recorded for each sample, and NTA analysis settings kept constant between samples. The average concentration of vesicles was plotted against their size, with the black lines and the color areas representing the fitting curve and the error bar, respectively. f Fluorescent staining confirms the intracellular uptake of HK-2 cell-derived exosomes by NRK-49F cells. HK-2 cells were incubated and labeled with Dil-C18 (red), then exosomes were isolated. HK-2 cell-derived exosomes (30 μg/ml) were incubated with NRK-49F cells for 24 h, followed by immunofluorescence staining for α-tubulin (green). Arrows indicate HK-2 cell-derived exosomes. Scale bar, 25 µm. g Quantitative data of Dil⁺ fluorescence intensity in NRK-49F cells. (n = 3). h, i Representative FACS analyses (h) and quantitative data (i) show that exosomes derived from TGF-β1-treated HK-2 cells (CTL-Exo or TGFβ-Exo, 30 μg/ml) reduced cisplatin-caused fibroblast apoptosis. The PE-labeled Annexin V-positive cells were counted by flow cytometry. ^**P < 0.01, ^††P < 0.01 (n = 3). j Representative micrographs show TUNEL-positive cells in different groups as indicated. Scale bar, 50 µm. k 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). l Western blotting analyses demonstrate protein expression of p53, cleaved caspase-3, FasL, FADD, PARP-1, fibronectin and α-SMA after various treatments in NRK-49F cells. Numbers (1–3) indicate each individual treatment in a given group. m Representative micrographs show immunofluorescence staining of fibronectin in different groups as indicated. Arrows indicate positive staining. Data presented as mean ± S.E.M. of three independent experiments. p values were calculated using unpaired Student’s t test for two groups comparison or Student-Newman-Kuels test for mutiple groups comparison.