1. Inhibition of cytochrome P450 2E1 and activation of transcription factor Nrf2 are renoprotective in myoglobinuric acute kidney injury 
Zhe Wang, Sudhir V. Shah, Hua Liu, Radhakrishna Baliga 
Kidney International 
Volume 86, Issue 2, Pages (August 2014)
DOI: /ki
Copyright © 2014 International Society of Nephrology Terms and Conditions

2. Figure 1
Glycerol-induced nuclear factor erythroid 2–related factor 2 (Nrf2) nuclear translocation and activation of its targets in the kidney. Western blotting analysis to detect (a) nuclear, (b) cytoplasmic Nrf2 level, and (c) Heme oxygenase-1 (HO-1) level in the kidney cortex of rats following treatment with glycerol at different time points. The blots are representative of four independent experiments. The summary bar graph represents mean±s.e. pixel densitometry average of four experiments. (d) Heme oxygenase-1 (HO-1) mRNA levels were determined by real-time PCR analysis. Values are mean±s.e., n=4, *P<0.05 compared with control (0h). Assessment of Nrf2-dependent and -independent gene expressions in the kidneys of rats treated with glycerol made by real-time PCR analysis. (e) Superoxide dismutase-1 (SOD-1), (f) Glutamate-cysteine ligase catalytic subunit (GCLC), (g) Glutathione peroxidase-1 (Gpx-1), (h) catalase. The data are mean±s.e., n=4, *P<0.05 compared with control (0h).
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

3. Figure 2
Myoglobin-induced nuclear factor erythroid 2–related factor 2 (Nrf2) nuclear translocation and oxidative stress in vitro. LLC-PK1 cells were treated with myoglobin. (a) Nuclear fraction and (b) cytoplasmic fraction of the cells were collected and subjected to western blotting analysis. (c) Immunofluorescence localization of Nrf2 in LLC-PK1 cells treated for 24h with vehicle (dimethylsuphoxide (DMSO)) or myoglobin (10mg/ml) or 1h with H2O2 (2.5mM). Cells were fixed with methanol before incubation with an Nrf2 primary antibody followed by incubation with a fluorescein isothiocyanate-conjugated secondary antibody. For each panel, an image of the cell nuclei stained with DNA-specific dye 4’,6-diamidino-2-phenylindole (DAPI) is also presented. Bar=20μM. (d) The effects of sulforaphane (SFN) or retinoic acid (RA) on myoglobin-induced H2O2 generation in LLC-PK1 cells exposed to myoglobin. The difference in the mean fluorescence between treated and untreated (control) cells was calculated as fluorescence decrease due to H2O2 generation. Each value represents the mean±s.e. of five determinations. *P<0.05 compared with LLC-PK1 cells treated with myoglobin alone. (e) The effects of SFN, RA, or Nrf2 siRNA knockdown on myoglobin-induced cytotoxicity in LLC-PK1 cells. Cells were exposed to myoglobin for 24h in the absence or presence of SFN or RA. Each value represents the mean±s.e. of five independent experiments. *P<0.05. (f) Western blot of proteins extracted from LLC-PK1 cells untransfected or transfected with either Nrf2-specific short interfering RNA (siRNA) or scramble siRNA. (g) The effects of Nrf2 siRNA knockdown on myoglobin induced on H2O2 generation in LLC-PK1 cells exposed to myoglobin. The difference in the mean fluorescence between scramble siRNA and Nrf2 siRNA cells was calculated as fluorescence decrease due to H2O2 generation. Each value represents the mean±s.e. of five determinations. *P<0.05 compared with LLC-PK1 cells treated with myoglobin alone. LDH, lactate dehydrogenase.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

4. Figure 3
Effect of glycerol on renal CYP2E1 and AKI in rats. (a) Myoglobin levels in the kidney or liver of glycerol-treated rats at different time points. Values are mean±s.e., n=4, *P<0.05 compared with untreated controls, +P<0.05 compared with liver. (b) Kidney injury molecule (Kim)-1 mRNA levels were determined by real-time PCR analysis. Values are mean±s.e., n=4, *P<0.05 compared with control (0h). (c) Protein levels of Kim-1 were determined by western blotting. The blots are representative of four independent experiments. The summary bar graph represents the mean (±s.e.) pixel densitometry average of four experiments. (d) Effect of glycerol on lipid peroxidation in rat kidney. Immunostaining of malondialdehyde demonstrated a gradual increase in positive staining in the tubules throughout the time period of the study. Bar=50μM. (e) Renal function as measured by plasma creatinine. Values are mean±s.e., n=4, *P<0.05 comparing glycerol-treated rats with control animals. (f) Protein levels of CYP2E1 in the kidney cortex were determined by western blotting. The blots are representative of four independent experiments. The summary bar graph represents the mean (±s.e.) pixel densitometry average of four experiments. *P<0.05 compared with control (0h). (g) Western blotting analysis to detect the CYP2E1 level in the liver at 24h following glycerol treatment. The values are mean±s.e., n=4. (h) CYP2E1 mRNA levels in the kidney cortex were determined by real-time PCR analysis. Values are mean±s.e., n=4, *P< 0.05 compared with control (0h).
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

5. Figure 4
Effect of chlormethiazole (CMZ) on glycerol-induced acute kidney injury (AKI). (a) Real-time PCR analysis to detect CYP2E1 mRNA level in the kidney cortex at 3, 6, 12, and 24h following a single injection of 75mg/kg of CYP2E1 inhibitor CMZ. Values are mean±s.e., n=4, *P<0.05 compared with the control (0h). (b) Western blotting analysis to detect the CYP2E1 level in the kidney following CMZ treatment. The blots are representative of four independent experiments. The summary bar graph represents the mean (±s.e.) pixel densitometry average of four experiments. *P<0.05 compared with control (0h). (c and d) Effect of CMZ on glycerol-induced AKI as measured by plasma urea nitrogen and creatinine. The values are mean±s.e., n=4, *P<0.05 compared with control, +P<0.05 compared with glycerol treatment. (e) Effect of CMZ on myoglobin levels in the kidney in glycerol-treated rats. There was no significant statistical difference between glycerol-treated and glycerol+CMZ–treated animals. *P<0.05 compared with control. (f) Effect of CMZ on glycerol-induced lipid peroxidation. Immunostaining of tetraethoxypropane (MDA) demonstrated that CMZ was able to ameliorate glycerol-induced lipid peroxidation. Bar=50μM. (g) Effect of CMZ on thiobarbituric acid-reactive species (TBARS) concentration in the kidney of glycerol-treated rats. The values are mean±s.e., n=4, *P<0.05 compared with control, +P<0.05 compared with glycerol treatment. (h) Effect of CMZ on bleomycin-detectable iron content in the kidney of glycerol-treated rats. The values are mean±s.e., n=4, *P<0.05 compared with control, +P<0.05 compared with glycerol treatment.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

6. Figure 5
Effect of chlormethiazole (CMZ) on nuclear translocation of nuclear factor erythroid 2–related factor 2 (Nrf2) and induction of heme oxygenase-1 (HO-1). (a) Western blot analysis to detect the effect of CMZ on nuclear Nrf2 levels in the kidney cortex of glycerol-treated rats. (b) Western blot analysis to detect the effect of CMZ on HO-1 protein level in the kidney cortex of glycerol-treated rats. The blots are representative of four independent experiments. The summary bar graph represents the mean (±s.e.) pixel densitometry average of four experiments. (c) Real-time PCR analysis was performed to detect the effect of CMZ on HO-1 mRNA level in the kidneys of glycerol-treated rats. The values are mean±s.e., n=4, *P<0.05 compared with control, +P<0.05 compared with glycerol treatment.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

7. Figure 6
Effect of CYP2E1 inhibition/knockdown on myoglobin-induced oxidative damage in vitro. (a) Effect of myoglobin on CYP2E1 protein level in LLC-PK1 cells. Cells were treated with 10mg/ml of myoglobin for 0, 0.5, 1, and 2h before being harvested and subjected to western blot analysis. (b) Time- and concentration-dependent H2O2 generation in LLC-PK1 cells exposed to myoglobin. The difference in the mean DCF fluorescence between treated and untreated (control) cells was calculated as fluorescence increase due to H2O2 generation (expressed as % of control). The values are mean±s.e., n=5. (c) Time- and concentration-dependent cytotoxicity as measured by lactate dehydrogenase (LDH) release in LLC-PK1 cells exposed to myoglobin. LDH release was measured in cells treated with myoglobin at 0, 6, 12, and 24h. The values are mean±s.e., n=5. (d) Effect of CYP2E1 inhibitors chlormethiazole (CMZ) or diethyldithiocarbamate (DEDC) on H2O2 generation in LLC-PK1 cells exposed to myoglobin (10mg/ml). The difference in the mean fluorescence between treated and untreated (control) cells was calculated as fluorescence decrease due to H2O2 generation. The values are mean±s.e., n=5, *P<0.05 compared with LLC-PK1 cells treated with myoglobin alone. (e) Effect of CYP2E1 inhibitors or CYP2E1 siRNA knockdown on myoglobin-induced cytotoxicity as measured by LDH release in LLC-PK1 cells. LLC-PK1 cells were treated with the CYP2E1 inhibitors diethyldithiocarbamate (DEDC) or CMZ for 2h followed by the addition of 10mg/ml of myoglobin for 24h. The values are mean±s.e., n=5, *P<0.05 compared with control, +P<0.05 compared with myoglobin treatment. (f) Western blot of proteins extracted from LLC-PK1 cells either untransfected or transfected with CYP2E1-specific siRNA or scramble siRNA. (g) The effects of CYP2E1 siRNA knockdown on myoglobin-induced H2O2 generation in LLC-PK1 cells. The difference in the mean fluorescence between scramble siRNA and CYP2E1 siRNA cells was calculated as fluorescence decrease due to H2O2 generation. Each value represents the mean±s.e. of five determinations. *P<0.05 compared with LLC-PK1 cells treated with myoglobin.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

8. Figure 7
Role of CYP2E1 and its interrelationship with Nrf2 in rhabdomyolysis-induced acute kidney injury (AKI).
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions