FIGURE 5.

Complement activation in tubular and endothelial cells culture after AMR‐derived EV exposition. (A, B) C3 and CFH gene transcript level in RPTEC after AMR‐derived EV stimulation (5e⁺⁴ EV/cells target for 24 h). Gene expression was assessed by qPCR and compared with normal untreated RPTEC cultured for 24 h. LPS, IFNγ and H₂O₂ exposed cells were used as positive control of C3 complement increase (not showed). Gene expression levels were normalized to the housekeeping gene β‐actin. Data are displayed as means ± SD, n = 5, one‐way ANOVA, *p < .05. (C) Primary human endothelial cells were cultured in serum‐free media, then exposed to EV 5e⁺⁴ EV/cells target for 24 h. (C) Complement activation in cells culture, supernatants were assessed by complement functional assay with a protocol adapted for cell culture, data are displayed as mean ± SD of percentage of complement activation compared with a positive control, medians were compared with a Mann–Whitney U test **p < .01, n = 5 per group. (D, E) Immunofluorescence analysis for C4d complement fragment. Endothelial cell grown in serum‐free conditions were exposed to AMR‐derived EV for 24 h (5e⁺⁴ EV/cells target) then labeled by immunofluorescence for C4d. Scale bar: 50 μm. Magnification, 630×. (D) Data are shown as mean ± SD and were analyzed by one‐way ANOVA test (n = 3 per group), *p < .01 versus TX Ctrl group, §p < .001 versus untreated cells.