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Figure 4 | Critical Care

Figure 4

From: Circulating plasma factors induce tubular and glomerular alterations in septic burns patients

Figure 4

Burns septic ARF group plasma activated caspases, up-regulated Fas/CD40 and modulated Bax/Bcl-2 in tubular cells. (a) Significant increased activities of caspases 3, 8 and 9 on tubular cells incubated for 48 h with burns septic acute renal failure (ARF) group plasma (n = 19) in comparison to control healthy plasma (n = 10). All plasma samples were tested in triplicate. Student t test was performed: (p < 0.05 *caspase-3, §caspase-8 and #caspase-9 activities of burns septic ARF group vs control healthy plasma). (b-d) Representative images of fluorescence-activated cell sorting (FACS) and immunofluorescence (insets) analysis of Fas (CD95) expression on tubular cell surface after exposure to different stimuli. With respect to vehicle alone (b) or control healthy plasma (c), burns septic ARF group plasma induced a marked up-regulation of Fas (d) (magnification × 400, nuclei counterstained with 1 μg/ml propidium iodide). Similar results were obtained with all tested plasma. (e) Up-regulation of the pro-apoptotic protein Bax and down-regulation of the antiapoptotic protein Bcl-2 in representative Western blot analysis on lysates of tubular cells (vehicle alone in lane 1, control healthy plasma in lanes 2–5, burns septic ARF group plasma in lanes 6–9) and related densitometric analysis. Beta-actin was used as reference for protein loading. (f) Percentage variation of expression of genes involved in apoptosis of tubular cells exposed to burns septic ARF group plasma. Tubular cells showed an increased expression of genes related to receptor-mediated (Fas, Fas-Ligand) and mitochondrial apoptotic pathways (Bax, Bak), of the co-stimulatory molecule CD40, of the CD40-transducer protein TRAF-3 and of the positive regulator of nuclear factor (NF)-κB activator RIPK2. Results are given as ratio between densitometric analyses of gene expression in tubular cells exposed to burns septic ARF group plasma with respect to control healthy plasma. House-keeping genes (beta-actin, GAPDH) were used as reference for densitometric analysis. Three experiments were performed with similar results. These data are feely available from the ArrayExpress databank of the European Bioinformatics Institute (see Materials and methods). Representative FACS analysis of CD40 expression was performed on unstimulated tubular cells (Vehicle), or in the presence of control healthy plasma, or burns septic ARF group plasma. Similar results were obtained with all tested burns septic ARF group plasma. The grey areas show the isotypic control.

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