FIG. 3.

MSC-EVs promoted protection but not proliferation in PTECs after injury. After ATP depletion injury, MSC-EVs were incubated with PTECs for 24 h. (A) Number of viable cells by counting with Trypan blue staining. (B) Proliferation was performed by an ELISA for Brdu incorporation. (C) Cell death analysis by Muse Annexin V & Dead Cell Assay. Black bars indicate cell death rate by early apoptosis and white bars represent late apoptosis. (D) Apoptosis was also evaluated by TUNEL and expressed as percentage of positive cells (500 cells were counted in random fields using a fluorescent microscopy at a magnification of ×200). (E) Effect of MSC-EVs on TER of PTECs. TER was measured in all groups before submitted to the different conditions and no significant difference was observed (not shown). Final measures were performed 24 h after the cell incubation with antimycin A. Each group is indicated in the abscissa; in the control group (CTR) the cells were not submitted to injury. CTR/EV represents PTECs that were incubated with MSC-EVs; INJ indicates the PTECs submitted to injury, while INJ/EV is the group submitted to injury and then incubated with MSC-EVs. Statistical analysis was performed by ANOVA with Newman-Keuls multicomparison test: *statistical difference related to the control group; **statistical difference between injured group and injured group treated with MSC-EVs (P<0.05; n=5). ANOVA, analysis of variance; TER, transespithelial resistance.