Fig. 3: I/R injury shifts tubular VEGF-A secretion from free form to sEV. | npj Regenerative Medicine

Fig. 3: I/R injury shifts tubular VEGF-A secretion from free form to sEV.

From: Tubular epithelial cells-derived small extracellular vesicle-VEGF-A promotes peritubular capillary repair in ischemic kidney injury

Fig. 3

a Schematic illustration of the experimental design. In brief, renal sEV were purified using differential centrifugation from the same weight of digested renal cortex tissues. To quantify the VEGF-A levels in renal sEV, concentrations of total VEGF-A and free VEGF-A were first detected in the supernatants as indicated in the illustration, and then the content of VEGF-A in sEV was calculated by total VEGF-A subtracted from free VEGF-A. Schematic created with BioRender.com. b Representative TEM image of renal sEV purified from Sham mice. c Size distribution of sEV isolated from Sham (Sham-sEV) and IRI (IRI-sEV) mice. d Quantification of the particle concentrations of Sham-sEV and IRI-sEV (n = 3). e Western blotting analysis of sEV markers (CD9, Alix, and CD63) and VEGF-A in Sham-sEV and IRI-sEV (n = 6). f NanoFCM analysis of the VEGF-A + IRI-sEV. g ELISA analysis of the secreted VEGF-A in total, free, and sEV forms of the kidneys (n = 3). h Proportion of free VEGF-A and VEGF-A + sEV. i A transwell culture system was used to mimic the milieu of polarized TECs. mTECs were cultured in the upper chamber with or without hypoxia. The contents of VEGF-A in the upper (apical) and lower (basolateral) chambers were detected by ELISA (n = 5). j ELISA analysis of the VEGF-A showing hypoxia increased the secretion of VEGF-A in mTECs, which was inhibited by GW4869 administration (n = 3). mTECs were co-transfected with VEGF-A-GFP (green) and CD63-mCherry (red) to visualize the trafficking of VEGF-A + sEV under a confocal microscope. k Live-cell imaging showing the co-localization of VEGF-A and CD63. Scale bar, 20 μm. Related to Supplementary Movie 1. l Representative ultra-sensitive SIM image of the purified sEV from the transfected mTECs. Scale bar, 200 nm. Data are presented as means ± SD. **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. Sham or Sham-sEV group (a–h), Ctrl (i), or DMSO+GW4869-Hypo- group (j); ####p < 0.001 vs. DMSO+GW4869-Hypo+ group (j). Two-tailed Student’s t test (d–i), One-way ANOVA (j). U.D., undetected.

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