Figure 4

Characterization of dysfunctional ECs in endotoxemia. Immunohistochemical analysis for caspase-3 showed rare apoptotic EC in endotoxemic pigs within glomeruli (D), at vascular (E) and peritubular levels (F, zoomed image, thin black arrow). Moreover, an increased number of tubular cells underwent apoptosis (F, zoomed image, thick black arrow). Caspase-3 nuclear expression in tubular cells (G) was analyzed as reported in the Methods section and expressed as median ± IQR of at least five independent pigs for each group. Magnification 10× (A, B, D, E) and 20× (C, F). Double immunofluorescence analysis (CD31 red; Ki-67 green) revealed Ki-67⁺/CD31⁺ proliferating ECs in endotoxemic pigs (I) compared with the T9 control group (H). Single-color images (J, K) underlined the co-localization of C31 (red J) and Ki-67 (green K) markers on ECs. Results are expressed as median ± IQR of the numbers of Ki-67⁺/CD31⁺ cells/HPF of at least five independent pigs for each group (L). Magnification, 630×. To-pro 3 was used to counterstain nuclei (blue).