Figure 5

Burns septic acute renal failure (ARF) group plasma altered cytoskeleton, megalin and tight junction expression and polarity in tubular cells. (a, b) Representative micrographs showing normal distribution ((a) control healthy plasma) and marked re-arrangement of cytoskeleton actin on tubular cells exposed for 48 h to burns septic ARF group plasma (b) with formation of "heaps" (white arrows) visible via ultraviolet (UV) light microscopy after staining with fluorescein isothiocyanate (FITC)-conjugated phalloidin (magnification × 100). (c, d) Representative immunofluorescence of megalin expression on tubular cells incubated with control healthy plasma (c) and its down-regulation in presence of burns septic ARF group plasma (d) (magnification × 400, nuclei counterstained with 1 μg/ml propidium iodide). Similar results were observed with all tested plasma samples. (e) Representative Western blot analysis of megalin expression on tubular cells (vehicle alone in lane 1, control healthy plasma in lanes 2–5, burns septic ARF group plasma in lanes 6–9) and densitometric analysis. Beta-actin was used as reference for protein loading. (f) Significant loss of polarity of tubular cells evaluated by the decrease of trans-epithelial resistance (TER) normalized for the membrane area after incubation with burns septic ARF group plasma for 12 h (*p < 0.05 burns septic ARF group vs control healthy plasma). (g-i) Representative micrographs showing the expression of the tight junction protein ZO-1 on unstimulated tubular cells (g), in the presence of control healthy plasma (h) and its down-regulation after incubation with burns septic ARF group plasma (i) (magnification × 400, nuclei counterstained with 1 μg/ml propidium iodide). Similar results were obtained with all tested plasma samples. Values in (f) are expressed as averages ± standard error (SE). Each plasma sample was tested in triplicate. Analysis of variance (ANOVA) with Newman-Keuls multi-comparison test was performed.