Figure 5
Free heme toxicity in renal epithelial cells. (a) Electrical monolayer resistance of confluent HK-2 renal epithelial cells was analyzed with an electric cell–substrate impedance sensing (ECIS) instrument before and after treatment with hemin (0 μM, 10 μM, 20 μM, 40 μM) in serum-free medium (n=4 biologic replicates per treatment). The arrowhead indicates the time point of hemin treatment. (b) Relative cellular ATP concentrations of HK-2 cells after treatment with heme (0–40 μM) for 8 h (n=8 biologic replicates per treatment). Experiments were performed in serum-free medium in the absence or presence of hemopexin. (c) Relative reduced glutathione (GSH) concentrations in control and heme-treated (4 h) HK-2 cells (n=8 replicates per treatment). (d) Stable isotope labeling by amino acids in cell culture (SILAC)-based mass spectrometry analysis of the heme-induced proteome. The two volcano plots illustrate differential protein expression of HK-2 cells that were treated with 10 μM heme (left plot) or 40 μM heme (right plot) for 12 h. The x axis indicates relative log2 protein expression ratios of treated versus control cells. The data are representative of six biologic samples per condition. Significantly regulated proteins (in any condition) are highlighted in blue (FDR 5%). Only proteins that were detected in at least three of six samples per condition were included in this analysis. (e) Box plot representation of selected signature protein regulation (n=6 biologic replicas per condition)
