Radical scavenger edaravone developed for clinical use ameliorates ischemia/reperfusion injury in rat kidney  Kent Doi, Yoshifumi Suzuki, Akihide Nakao, Toshiro Fujita, Eisei Noiri  Kidney International  Volume 65, Issue 5, Pages 1714-1723 (May 2004) DOI: 10.1111/j.1523-1755.2004.00567.x Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 1 Blood urea nitrogen (BUN) (A), serum creatinine (Cr) (B) in rats subjected to 45-minute ischemia 24hours after surgery. Ischemia, control ischemia group; Edaravone, edaravone-treated groups; DMTU, dimethylthiourea-treated group; Sham, sham-operated group. *P < 0.05 versus control ischemia group; **P < 0.01 versus control ischemia group. Kidney International 2004 65, 1714-1723DOI: (10.1111/j.1523-1755.2004.00567.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 2 Time-course of serum creatinine (Cr) showing the continuous protective effect of edaravone. All the animals treated by edaravone at the doses of 5mg/kg and 10mg/kg and by dimethylthiourea (DMTU) survived for 72hours after surgery. However, only three (33.3%) of nine rats in the control ischemia group and three (50%) of six rats treated by edaravone at the dose of 1mg/kg survived for 72hours after surgery. The analysis was performed only on animals that survived at the examination of renal function. Symbols are: (•) control ischemia; (▪) edaravone 1mg/kg; (○) edaravone 5mg/kg; (□) edaravone 10mg/kg; and (▴) DMTU. Kidney International 2004 65, 1714-1723DOI: (10.1111/j.1523-1755.2004.00567.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 3 Histologic findings. Findings stained with hematoxylin-eosin (A), fluorescence microscopy detecting reactive oxygen species (ROS) by dichlorodihydrofluorescein (CM-H2DCFDA) (B), immunohistochemistry using antibody to 4-hydroxy-2-hexenal (HHE)-modified proteins (C), and histologic scoring of the intensity of ischemic acute renal failure (D). ATN, acute tubular necrosis; sham, sham-operated group; ischemia, control ischemia group; edaravone, edaravone-treated group. *P < 0.05 versus control ischemia; **P < 0.01 versus control ischemia. Kidney International 2004 65, 1714-1723DOI: (10.1111/j.1523-1755.2004.00567.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 3 Histologic findings. Findings stained with hematoxylin-eosin (A), fluorescence microscopy detecting reactive oxygen species (ROS) by dichlorodihydrofluorescein (CM-H2DCFDA) (B), immunohistochemistry using antibody to 4-hydroxy-2-hexenal (HHE)-modified proteins (C), and histologic scoring of the intensity of ischemic acute renal failure (D). ATN, acute tubular necrosis; sham, sham-operated group; ischemia, control ischemia group; edaravone, edaravone-treated group. *P < 0.05 versus control ischemia; **P < 0.01 versus control ischemia. Kidney International 2004 65, 1714-1723DOI: (10.1111/j.1523-1755.2004.00567.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 4 Western analysis of 4-hydroxy-2-hexenal (HHE)-modified protein expression. Sham, sham-operated group; ischemia, control ischemia group; Edaravone, edaravone-treated group. Histogram demonstrates the relative density of bands compared with alpha-tubulin (N = 3). Representative image is demonstrated in upper panel. Kidney International 2004 65, 1714-1723DOI: (10.1111/j.1523-1755.2004.00567.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 5 Measurement of intracellular reactive oxygen species (ROS) production in human renal proximal tubular cells (HKC-8) using the fluorogenic probe of intracellular ROS production, dichlorodihydrofluorescein (CM-H2DCFDA), in vitro. Fluorescence intensity (relative unit) was measured 60 minutes after exposure to H2O2 at the concentration of 0.5mmol/L. HKC-8 cells were incubated with edaravone at the concentration of 10−5 mol/L and 10−4 mol/L. *P < 0.05 versus control. Kidney International 2004 65, 1714-1723DOI: (10.1111/j.1523-1755.2004.00567.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 6 Chronological analysis of intracellular reactive oxygen species (ROS) production in human renal proximal tubular cells (HKC-8) by dichlorodihydrofluorescein (CM-H2DCFDA) in vitro. Fluorescence intensity (relative unit) was measured in six independent wells in a 96-well microplate. *P < 0.005 versus control. Symbols are: (○) H2O2 0.5mmol/L; (•) edaravone 10−4 mol/L + H2O2 0.5mmol/L. Kidney International 2004 65, 1714-1723DOI: (10.1111/j.1523-1755.2004.00567.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 7 Cell viability assay with Alamar blue. Human renal proximal tubular cells (HKC-8) loaded with hydrogen peroxide at the concentration of 0.5mmol/L were examined. *P < 0.05 versus control. Symbols are: (○) H2O2 0.5mmol/L; (•) edaravone 10−4 mol/L + H2O2 0.5mmol/L; (▪) edaravone 10−5 mol/L + H2O2 0.5mmol/L. Kidney International 2004 65, 1714-1723DOI: (10.1111/j.1523-1755.2004.00567.x) Copyright © 2004 International Society of Nephrology Terms and Conditions