INTIMP-03088; No of Pages 8 International Immunopharmacology xxx (2013) xxx–xxx
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Olmesartan protects against oxidative stress possibly through the Nrf2 signaling pathway and inhibits inflammation in daunorubicin-induced nephrotoxicity in rats Vengadeshprabhu Karuppa Gounder a, Somasundaram Arumugam a, Wawaimuli Arozal a, Rajarajan A. Thandavarayan a, Vigneshwaran Pitchaimani a, Meilei Harima a, Kenji Suzuki b, Mayumi Nomoto a, Kenichi Watanabe a,⁎ a b
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Department of Clinical Pharmacology, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata City 956-8603, Japan Department of Gastroenterology, Niigata University Graduate School of Medical and Dental Sciences, Niigata City 951-8510, Japan
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Article history: Received 6 June 2013 Received in revised form 14 November 2013 Accepted 18 November 2013 Available online xxxx
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Anthracycline anticancer drug daunorubicin (DNR) can induce chronic nephrotoxicity, which is believed to be based on oxidative injury. Olmesartan has significant blood pressure lowering effect via modulating renin-angiotensin system although its mechanism of action in DNR-induced renal injury is largely unknown. Transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) is an important regulator of cellular oxidative stress. This study examined the role of Nrf2 in olmesartan-mediated antioxidant effects in DNR induced kidney cells. In addition, key factors involved in promoting inflammation and oxidative stress were studied. Sprague-Dawley rats were treated with a cumulative dose of 9 mg/kg DNR (i.v.). Olmesartan was administered orally every day for 6 weeks. DNR treated rats showed nephrotoxicity as evidenced by worsening renal function, which was evaluated by measuring total cholesterol, triglyceride levels in kidney tissue and histopathological approaches; treatment with olmesartan reversed these changes. Furthermore, olmesartan treatment down-regulated phospho-MAPKAPK-2, caspase-12, p47phox, p67phox, upregulated renal expression of PPAR-γ, Bcl-xL, glutathione peroxidase and Nrf2. Furthermore, olmesartan down-regulated matrix metalloproteinase-2 and angiotensin II type I receptor expression in the kidney. In conclusion, the result demonstrated that angiotensin II and oxidative stress play a key role in DNR-induced nephrotoxicity. The present results indicated that olmesartan protects against oxidative stress, which may be possibly via the induction of Nrf2 signaling pathways. © 2013 Published by Elsevier B.V.
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Keywords: Daunorubicin Olmesartan Oxidative stress Chronic nephrotoxicity Nuclear factor-erythroid 2-related factor 2 Angiotensin II type I receptor
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1. Introduction
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Daunorubicin (DNR) is one of the anthracycline (ANT) anti-tumor agents widely used in the treatment of acute myeloid leukemia. However, the clinical use of DNR has been limited by its undesirable systemic toxicity, especially cardiotoxicity and nephrotoxicity, which eventually lead to congestive heart failure and renal failure, respectively [1–3]. ANT-induced changes in the renal tissue of rats include glomerular capillary permeability and glomerular atrophy [4]. It is believed that the biochemical mechanism for ANT-induced heart and kidney damage involves the generation of reactive oxygen species (ROS) as a consequence of redox cycling [5]. The excess ROS can damage cellular lipids, proteins, or DNA inhibiting their normal function [6]. Increased generation of ROS leads to tissue injury and dysfunction by attacking,
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⁎ Corresponding author at: Department of Clinical Pharmacology, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, 265-1 Higashijima, Akiha-ku, Niigata City 956-8603, Japan. Tel.: + 81 250 25 5267; fax: + 81 250 25 5021. E-mail address: [email protected] (K. Watanabe).
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denaturing, and modifying structural and functional molecules and by activating redox-sensitive transcription factors and signal transduction pathways. These events, in turn, promote necrosis, apoptosis, inflammation, fibrosis, and other disorders. Redox systems including antioxidant enzymes and phase II detoxifying and antioxidant agents provide protection against ROS-induced tissue injury [7]. Blockade of oxidative stress can ameliorate renal sclerosis through anti-inflammatory and antiapoptotic processes [8]. In the kidney, activation of the angiotensin II–angiotensin II type I receptor (AT1R) pathway results in hypertension, the production of proinflammatory mediators, cell proliferation, extracellular matrix synthesis, and release of ROS, which facilitate kidney damage and advance chronic kidney disease [9]. Blockade of renin angiotensin system (RAS) is highly effective in retarding progression of renal disease in humans and experimental animals [10]. The salutary effect of RAS blockade is, not only, due to its antihypertensive/hemodynamic actions, but also its ability to reduce oxidative stress, inflammation and fibrosis which are major mediators of progression of renal disease [11,12]. However, studies have suggested that angiotensin converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) exert a protective
1567-5769/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.intimp.2013.11.018
Please cite this article as: Gounder VK, et al, Olmesartan protects against oxidative stress possibly through the Nrf2 signaling pathway and inhibits inflammation in daunorubicin..., Int Immunopharmacol (2013), http://dx.doi.org/10.1016/j.intimp.2013.11.018
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DNR was kindly donated by Meiji Seika Kaisha Ltd. (Tokyo, Japan). Olmesartan was donated by Daichi-Sankyo Pharmaceutical (Tokyo, Japan). All antibodies were purchased from Santa Cruz Biotechnology Inc. (CA) or Cell signaling (USA). Unless otherwise stated all reagents were of analytical grade and were purchased from Sigma (Tokyo, Japan).
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2.2. Experimental animals
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Eight-weeks-old male Sprague-Dawley rats were obtained from Charles River Japan Inc. (Kanagawa, Japan). The animals were quarantined and acclimatized for additional 2 weeks prior to the initiation of the experiments. They were randomly divided into four groups, and the first two groups served as normal (N, n = 5) and normal treated with olmesartan (10 mg/kg/day, per oral) (N + Olm, n = 5). Other rats received a single intravenous injection of DNR at a dose of 3 mg/kg (i.v.) on day 0. The drug was administered in three equal injections at 48 h intervals for a period of one week to achieve an accumulative dose of 9 mg/kg, which is well documented to produce cardiotoxicity and nephrotoxicity [3,5]. DNR treated rats were divided into two groups and each received either vehicle (1% gum Arabic) alone (DNR, n = 12) or olmesartan (10 mg/kg/day, per oral) (DNR + Olm, n = 10). Administration of olmesartan was started on the same day as DNR administration and continued for 5 additional weeks after cessation of DNR administration (6 weeks total period). The dose of olmesartan and duration of study were chosen on the basis of previous reports [5,17,18]. On day 41, rats were placed individually in metabolic cages for 24-h urine collections for the measurement of protein concentrations. After the end of the study period (6 weeks), rats were sacrificed after measuring body weight (BW) and kidney tissue was harvested for semi-
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Blood samples collected by heart puncture during sacrifice were utilized for the subsequent determination of creatinine, total cholesterol (TC), triglyceride, and blood urea nitrogen (BUN) and were stored at −80 °C. TC and triglyceride levels were determined using standard enzymatic procedures. Serum creatinine level was determined by the Jaffe method [19]. BUN was determined by the diacetylmonoxime method [20].
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2.4. Histopathological studies
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2.3. Estimation of biochemical parameters
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The kidney tissue was decapsulated. Half of the kidney was immediately snapfrozen in liquid nitrogen for subsequent protein extraction and enzymatic assays. The remaining excised kidneys were cut into about 2-mm-thick transverse slices and fixed in 10% formalin. Sections of 3–5 μm thickness were stained with hematoxylin and eosin (HE) for histological examination. A histomorphological evaluation of all the kidney sections was carried out in a blinded fashion by a pathologist who was unaware of the treatment groups.
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2.5. Protein analysis by Western blotting
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quantitative immunoblotting and immunohistochemical studies. The 140 animal experiments were performed in accordance with national guide- 141 lines for the use and care of our institute. 142
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Protein lysate was prepared from renal tissue as described previously [21]. The total protein concentration in samples was measured by the bicinchoninic acid method [22]. For the determination of protein levels of Nrf2, Keap1, peroxisome proliferator-activated receptor gamma (PPAR-γ), B-cell lymphoma-extra-large (Bcl-xL), phospho-mitogen activated protein kinase-activated protein kinase (phospho-MAPKAPK)2, caspase-12, p47phox and p67phox, equal amounts of protein extracts (30 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad, CA) and transferred electrophoretically to nitrocellulose membranes. Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline Tween (20 mM Tris, pH 7.6, 137 mM NaCl and 0.1% Tween 20). The membrane was incubated overnight at 4 °C with the primary antibody and the bound antibody was visualized using the respective horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology Inc.) and chemiluminescence developing agents (Amersham Biosciences, Buckinghamshire, UK). The level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was estimated in every sample to check for equal loading of samples. Films were scanned and band densities were quantified with densitometric analysis using the Scion Image program (Epson GT-X700, Tokyo, Japan). All values were expressed as relative to their GAPDH content.
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2.6. Immunohistochemical assay
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Formalin-fixed, paraffin-embedded renal tissue sections were used for immunohistochemical staining. After deparaffinization and hydration, the slides were washed in Tris-buffered saline (TBS; 10 mM/l Tris HCl, 0.85% NaCl, pH 7.2). Endogenous peroxidase activity was quenched by incubating the slides in methanol and 0.3% H2O2 in methanol. Blocked in 10% normal serum with 1% BSA in TBS for 1 h at room temperature. After overnight incubation with the primary antibody, either rabbit polyclonal anti-AT1R (Peninsula Laboratories Inc., San Carlos, CA) or goat monoclonal anti-matrix metalloproteinase (MMP)-2 (diluted 1:100) (Santa Cruz Biotechnology, Inc.), at 4 °C, the slides were washed in TBS and then HRP conjugated secondary antibody was added and the slides were further incubated at room temperature for 45 min. The slides were washed in TBS and incubated with diaminobenzidine tetra hydrochloride as the substrate and counterstained with
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role toward doxorubicin (DOX) and DNR-induced cardiotoxicity and nephrotoxicity [1,12]. Nuclear factor-erythroid-2-related factor 2 (Nrf2) has also been found to be a critical transcription factor that binds to the antioxidant response element (ARE) in the promoter region of a number of genes encoding antioxidant and phase 2 enzymes, such as glutathione peroxidase (GPx), catalase, and superoxide dismutase, in several types of cells and tissues [13]. Nrf2 is held as an inactive complex bound to a repressor molecule known as Keap1 (Kelch-like ECH-associated protein1) which facilitates its ubiquitination. Keap1 contains several reactive cysteine residues that serve as sensors of the intra-cellular redox state. Oxidative or covalent modification of thiols in these cysteine residues or Nrf2 by protein kinases results in dissociation of Nrf2 from Keap1 and its migration and binding to the ARE in the promoter regions of genes encoding the antioxidant and phase 2 detoxifying enzymes [14]. The Nrf2mediated regulation of cellular antioxidant and anti-inflammatory machinery plays an important role in the defense against oxidative stress [15]. Within the ARB class, olmesartan medoxomil is a long acting AT1R antagonist approved for the treatment of mild to severe hypertension, alone or in combination with other agents. Moreover, olmesartan is therapeutically effective for the treatment of patients with heart failure by reducing cytokines and oxidative stress through its antiinflammatory effects [16]. Hence, the present study was aimed to investigate the ameliorative potential of olmesartan on proinflammatory cytokines, oxidative stress, renal dysfunction and the role of Nrf2 in olmesartan mediated antioxidant effects in chronic DNR-induced nephrotoxicity rats. To the best of our knowledge, we are the first to examine the effects of chronic administration of ARB olmesartan against oxidative stress possibly through the Nrf2 signaling pathway in DNR-induced nephrotoxicity.
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Please cite this article as: Gounder VK, et al, Olmesartan protects against oxidative stress possibly through the Nrf2 signaling pathway and inhibits inflammation in daunorubicin..., Int Immunopharmacol (2013), http://dx.doi.org/10.1016/j.intimp.2013.11.018
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N + Olm group revealed normal histological findings whereas DNR administration resulted in substantial damage to kidney as indicated by glomerular congestion, tubular necrosis, tubular dilatation, intertubular hemorrhage, and renal fibrosis. Renal changes in group DNR were improved in the rats treated with olmesartan (Fig. 1A and B).
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3.4. Renal expression of AT1R and MMP-2
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Kidney sections of group DNR showed stronger immune reactivity for AT1R and MMP-2 than those of group N and group N + Olm, whereas group DNR + Olm revealed a significant decrease in the renal level of AT1R (Fig. 1C and C1) and MMP-2 (Fig. 1D and D1).
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2.7. Measurement of glutathione peroxidase (GPx) activity
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Kidney samples were homogenized in six volumes (per wet weight of tissue) of cold GPx assay buffer, the mixture was centrifuged for 15 min at 4 °C, and 8000 rpm in accordance with the total GPx assay kit instructions (Oxitek, ZeptoMetrix Corporation, New York). GPx activity in kidney tissues was measured using a kinetic ultravioletvisible spectrophotometer (Ultraspec 3100, Amersham Biosciences). The oxidation of NADPH to NADP+ was measured by the decrease in absorbance at 340 nm.
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2.8. Statistical analysis
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Data are presented as mean ± SEM and were analyzed using oneway analysis of variance (ANOVA) followed by Tukey multiple comparison test or two-tailed “t”-test when appropriate. A value of p b 0.05 was considered statistically significant. For statistical analysis, GraphPad Prism 5 software (San Diego, CA) was used.
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3. Results
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3.1. Effect of olmesartan on BW and survival rate
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Throughout the study period, BW was significantly decreased in DNR rats with or without olmesartan treatment compared with that of group N rats. Although olmesartan treatment tended to prevent the reduction of BW compared with that in group DNR, the effect did not attain statistical significance and olmesartan treatment to N + Olm group did not show significant changes in body weight compared to normal group (Table 1). Four (33.3%) rats in group DNR died between days 16 and 42 (Table 1). None of the rats died in group N, N + Olm and group DNR + Olm.
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3.2. Effect of olmesartan on kidney function
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Serum creatinine levels were significantly increased in group DNR compared with those in group N and N + Olm group. In addition TC and triglyceride levels were also significantly increased in group DNR compared with those in group N and group N + Olm. Treatment of DNR-injected rats with olmesartan resulted in a significant decrease in serum creatinine, BUN, TC and triglyceride levels compared with those of DNR-treated rats (Table 1).
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3.3. Effect of olmesartan against renal histological damage
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The severity of kidney injury was investigated by examination of HE and Azan-Mallory stained kidney sections. Renal tissues of group N and
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Table 1 Changes in survival rate, body weight and biochemical parameters after 6 weeks of treatment with olmesartan in DNR treated rats.
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The protein levels of caspase-12, NADPH oxidase sub-units p47phox and p67phox were significantly increased in group DNR compared with those in group N, as well as N + Olm group and these changes were significantly attenuated in group DNR + Olm. Moreover, renal protein expression of Bcl-xL was significantly decreased in group DNR compared with that in group N rats. In contrast, treatment with olmesartan increased the protein level of Bcl-xL compared with that in group DNR rats, but no significant changes in normal and normal treated with olmesartan group (Fig. 2A, B, C, D and E).
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3.5. Effects of olmesartan on oxidative stress and other signaling proteins
3.6. Effects of olmesartan on Nrf2 and Keap1 expression
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hematoxylin. A negative control without primary antibody was included in the experiment to verify the antibody specificity. Brown colored immunopositive AT1R and MMP-2 spots were counted at 400-fold magnification.
Nrf2 protein expression was significantly decreased in group DNR compared with that in group N and normal treated with olmesartan group, but when DNR rats treated with olmesartan its expression was significantly increased on comparison with group DNR rats (Fig. 3A and B). Moreover, no change in the renal protein expression of Keap1 in group DNR was observed when compared with other groups. Further, to confirm the effect of olmesartan on Nrf2 levels in normal rats, we have analyzed the normal rats treated with olmesartan alone and observed no significant change in its protein level when compared with the normal rats (Fig. 3A and C).
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3.7. Effects of olmesartan on PPAR-γ and phospho-MAPKAPK-2 expression 270 Western blotting analysis showed that PPAR-γ protein expression was significantly increased in group DNR compared with that in group N, which exhibited a further increase with the administration of olmesartan (Fig. 3A and D). Moreover, renal protein expression of phospho-MAPKAPK-2 was significantly increased in group DNR compared with that in group N and N + Olm rats. In contrast, treatment with olmesartan decreased the protein level of phospho-MAPKAPK-2 compared with that in group DNR rats (Fig. 3A and E).
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0 100 533 ± 8.9 1.7 20 ± 7.5 57.6 ± 3.7 47.3 ± 6.3
4 66.7 362 ± 8.0⁎⁎⁎ 3.2 ± 2.0⁎ 54 ± 7.1⁎ 581.8 ± 36.5⁎⁎⁎ 2691 ± 316.1⁎⁎⁎
0 100 385 ± 9.0 2.1 ± 0.9# 35 ± 9.2# 436.5 ± 31.3# 631.7 ± 156.5###
Results are presented as the mean ± SEM. BUN, blood urea nitrogen; BW, body weight; TC, total cholesterol; group N, aged-matched normal rat; group N + Olm, normal rats administered with olmesartan (10 mg/kg/day for 5 weeks, per oral); group DNR, DNR-treated rats administered with vehicle; Group DNR + Olm, DNR-treated rats administered with olmesartan (10 mg/kg/day for 5 weeks, per oral). ⁎p b 0.05 and ⁎⁎⁎p b 0.001 vs group N or N + Olm; #p b 0.05 and ###p b 0.001 vs group DNR.
Please cite this article as: Gounder VK, et al, Olmesartan protects against oxidative stress possibly through the Nrf2 signaling pathway and inhibits inflammation in daunorubicin..., Int Immunopharmacol (2013), http://dx.doi.org/10.1016/j.intimp.2013.11.018
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Fig. 1. (A), Hematoxylin and eosin staining of the cross-sectional tissue slices of kidney depicting glomerular congestion, tubular casts, and interstitial hemorrhage (200×). (B), Azan-Mallory staining for fibrosis of the cross-sectional tissue slices of kidney. Fibrosis is indicated by the blue area as opposed to the red renal tissue (200×). (C&C1), Renal levels of AT1R by immunohistochemical staining and the positive immunostaining of AT1R exhibit brown (400×) and its quantification data. (D&D1), Photomicrograph of kidney tissue sections showing MMP-2 staining and its quantification data. MMP-2 staining is shown in brown. All nuclei stained by hematoxylin are shown in blue (400×). Each bar represents mean ± SEM. Group N, agematched normal rats; group N + Olm, normal rats treated with olmesartan (10 mg/kg/day); group DNR, DNR-treated rats administered with vehicle; group DNR + Olm, DNR treated rats administered with olmesartan (10 mg/kg/day). ***p b 0.001 vs group N or N + Olm; #p b 0.05 and ###p b 0.001 vs group DNR. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: Gounder VK, et al, Olmesartan protects against oxidative stress possibly through the Nrf2 signaling pathway and inhibits inflammation in daunorubicin..., Int Immunopharmacol (2013), http://dx.doi.org/10.1016/j.intimp.2013.11.018
V.K. Gounder et al. / International Immunopharmacology xxx (2013) xxx–xxx
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Fig. 2. Renal expressions of p67phox, p47phox, caspase-12 and Bcl-xL. (A), Representative Western blots showing specific bands for p67phox, p47phox, caspase-12, Bcl-xL and GAPDH as an internal control. Equal amounts of protein sample obtained from whole renal homogenate were applied in each lane. (B–E), Densitometric data of protein analysis. The mean density values of p67phox, p47phox, caspase-12 and Bcl-xL were expressed as ratios relative to that of GAPDH. Each bar represents mean ± SEM. Group N, age-matched normal rats; group N + Olm, normal rats treated with olmesartan (10 mg/kg/day); group DNR, DNR-treated rats administered with vehicle; group DNR + Olm, DNR-treated rats administered with olmesartan (10 mg/kg/day). *p b 0.05, **p b 0.01 and ***p b 0.001 vs group N or N + Olm; #p b 0.05 and ###p b 0.001 vs group DNR.
3.8. Effects of olmesartan on renal GPx activity
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Administration of DNR caused a significant reduction in GPx activity in renal tissue compared with that in the group N and group N + Olm. Treatment with olmesartan significantly increased GPx activity compared with those in group DNR (Fig. 4).
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ANT nephrotoxicity presents the most unfavorable side effect of these highly efficient anticancer drugs. The present study indicated that a cumulative dose of DNR exhibited nephrotoxicity as evidenced by worsening renal function, as well as elevated serum creatinine,
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Fig. 3. Renal expressions of Nrf2, Keap1, PPAR-γ and phospho-MAPKAPK-2. (A), Representative Western blots showing specific bands for Nrf2, Keap1, PPAR-γ, phospho-MAPKAPK-2 and GAPDH as an internal control. Equal amounts of protein sample obtained from whole kidney homogenate were applied in each lane. (B–E), Densitometric data of protein analysis. The mean density values of Nrf2, Keap1, PPAR-γ and phospho-MAPKAPK-2 were expressed as ratios relative to that of GAPDH. Each bar represents mean ± SEM. Group N, age-matched normal rats; group N + Olm, normal rats treated with olmesartan (10 mg/kg/day); group DNR, DNR-treated rats administered with vehicle; group DNR + Olm, DNR-treated rats administered with olmesartan (10 mg/kg/day). *p b 0.05 vs group N or N + Olm; #p b 0.05 and ##p b 0.01 vs group DNR.
Please cite this article as: Gounder VK, et al, Olmesartan protects against oxidative stress possibly through the Nrf2 signaling pathway and inhibits inflammation in daunorubicin..., Int Immunopharmacol (2013), http://dx.doi.org/10.1016/j.intimp.2013.11.018
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Fig. 4. Effect of olmesartan on GPx activities in kidney homogenates from DNR-treated rats. The values are mean ± SEM. Group N, age-matched normal rats; group N + Olm, normal rats treated with olmesartan (10 mg/kg/day); group normal + Olm, normal rat treated with olmesartan (10 mg/kg/day); group DNR, DNR-treated rats administered with vehicle; group DNR + Olm, DNR-treated rats administered with olmesartan (10 mg/kg/day). **p b 0.01 vs group N or N + Olm; #p b 0.05 vs group DNR.
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BUN, TC and triglyceride (Table 1). These changes were associated with oxidative stress and inflammation in renal tissues. Chronic treatment with olmesartan showed protective effects against DNR-induced nephrotoxicity by reducing oxidative stress and inflammation and as a consequence, the renal functions were improved and mortality rate was reduced. To the best of our knowledge, our study is the first that identifies the protective effect of 6 weeks of treatment with olmesartan against nephrotoxicity induced by a cumulative dose of DNR. Angiotensin II is the major effector hormone of the RAS and contributes to a variety of renal and cardiovascular physiologic and pathologic mechanisms through stimulation of AT1R and AT2R. Up-regulation of AT1R was coupled with a marked increase in the numbers of angiotensin II-positive cells. Angiotensin II is known to raise arterial pressure, induce renal cell hypertrophy, and matrix protein accumulation, events that are linked to progression of renal disease [23]. In this study, we confirmed that the renal expression of its receptor AT1R was increased in DNR treated rats, and treatment with olmesartan mediated the down-regulation of AT1R (Fig. 1C and C1). Moreover, previous clinical and experimental studies showed that AT1R-mediated NADPH oxidase activation leads to generation of ROS [24]. A recent study showed that NADPH oxidase makes an important contribution to renal oxidative stress and inflammation, as well as renal damage and dysfunction [25]. To address the role of oxidative stress, we next measured in the renal protein expression of p67phox and p47phox, which are cytosolic subunits of NADPH oxidase, by Western blotting. Our results showed that DNR treatment increased the expression levels of p67phox and p47phox significantly and are inhibited by treatment with olmesartan. In addition, previous studies have shown that ANT induces apoptosis, which is supposed to occur through the production of ROS [26]. Our results showed that olmesartan treatment significantly reduced the level of activated caspase-12 and increased the level of Bcl-xL (Fig. 2A, B, C, D and E). MMPs are a family of proteases best known for their capacity to proteolyze the extracellular matrix proteins. MMPs, such as MMP-2 degrade the type IV collagen, the major structural component of basement membrane. In a previous study, Chang et al. [27] suggested that increased MMP-2 levels contribute to the pathogenesis of chronic kidney diseases. More recent studies found that increased MMP-2 levels correlate with proteinuria, oxidative stress, cardiovascular disease prevalence and the atherosclerosis marker-intima media thickness in patients with chronic kidney disease [28]. Over the last decade, a large number of studies have shown an increased MMP-2 activity/level in response to oxidative stress in various tissues from different species [29]. In agreement with these results, we could observe an increase in renal levels of MMP-2 and which was normalized by olmesartan treatment. Moreover, ROS have been recognized as important mediators that regulate signal transduction pathways, including MAPK [30]. The
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chronic treatment with aldosterone/salt induces MAPK activation via a ROS dependent pathway. MAPKAPK-2, a member of the MAPK family is activated by physical and chemical stress factors resulting in growth promotion, apoptosis, oxidative stress, and vasoconstriction [31]. Previous studies in vitro have implicated that ROS also regulate other signaling molecules; it is possible that ROS-mediated MAPK activation was involved, at least in part, in the progression of renal injury [30]. In the present study, treatment with olmesartan significantly down regulated the renal protein expressions of phospho-MAPKAPK-2 (Fig. 3A and E). Activation of PPAR-γ exerts anti-inflammatory [32], antifibrotic [33], anti-oxidative and vasculo-protective effect [34] on different renal diseases, but little has been known about the effect of PPAR-γ agonist in ANT-induced nephrotoxicity. Furthermore, it has been demonstrated that telmisartan suppressed the AT1R expression in both mRNA and protein levels through PPAR-γ mediated pathway. Involvement of PPAR-γ in AT1R suppression has been further confirmed by using GW9662, a PPAR-γ antagonist [35]. Recent studies suggest that telmisartan, but not other ARBs, could act as a partial PPARγ agonist [36]. In the present study, we demonstrated, that treatment with olmesartan significantly upregulated the renal protein expressions of PPAR-γ. Since, we have only measured the expression of PPAR-γ at protein level and not its activity, the net effect of overall PPAR-γ activity in our model is unclear. In light of this study limitation, further experiments will be required to understand whether the effects of olmesartan in our model are mediated by alterations in PPAR-γ activity. Nrf2, a member of the cap-N-collar family, is the key transcription factor that regulates antioxidant response element-mediated expression of detoxifying antioxidant enzymes [37]. Under basal conditions, Nrf2 is sequestered in the cytoplasm by an actin binding repressor protein Keap1, which facilitates its ubiquitination. In fact, an experimental study of spontaneous focal glomerulosclerosis rats has indicated that ARB treatment prevented nephropathy, suppressed oxidative stress, inflammatory process, and endoplasmic reticulum stress-induced apoptosis as well as restored Nrf2 activation and expression of the antioxidant enzymes [38]. A very recent study has revealed that telmisartan, could suppress NADPH oxidase and subsequently upregulate Nrf2, leading to the amelioration of renal oxidative stress [39]. In addition, Nrf2 can be activated by phosphorylation of some of its threonine or serine residues by upstream kinases such as protein kinase C, MAPKs, phosphatidylinositol-3-kinase/Akt, and casein kinase-2 [40]. In the present study, we have identified no significant changes in the renal protein expression levels of Nrf2 and Keap1 in between normal and N + Olm group rats. But interestingly, Nrf2 protein expression was significantly decreased in group DNR compared with that in group N and N + Olm, but when DNR rats treated with olmesartan its expression was significantly increased on comparison with group DNR rats (Fig. 3A and B). Recently, the Nrf2 is found to be a critical transcription factor that binds to the ARE in the promoter region of a number of genes encoding for antioxidative and phase 2 enzymes in several types of cells and tissues [41,42]. In the present study, the DNR-induced increase in ROS generation was potentially exacerbated by a significant reduction in antioxidant reserves in renal tissues. This might have caused a state of redox imbalance that enhanced the susceptibility of biological membranes to reaction with ROS, as indicated by decrease in GPx in the tissues, while administration of olmesartan with DNR significantly restored GPx activity (Fig. 4). Whether this effect also attributes to the Nrf2-mediated antioxidative stress in the advanced phase of renal toxicity in this model is uncertain and requires further investigation. In conclusion, progressive nephrotoxicity in the DNR-treated rats is associated with upregulation of oxidative stress, inflammation and conspicuous impairment of Nrf2 activation. Long-term therapy with olmesartan prevents development of renal toxicity by reducing inflammation and oxidative stress, which may be possibly via the induction of Nrf2 signaling pathways in this model. The possible mechanism (Fig. 5) behind this renoprotective action of olmesartan is attenuation of
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Fig. 5. The possible mechanism by which olmesartan afforded protection against DNR-induced nephrotoxicity. The major adverse effect of DNR treatment is the activation of renin angiotensin system and via activation of AT1R, DNR induces the production of free radicals such as reactive oxygen species possibly via activated NADPH oxidase and thereby causing oxidative stress and inflammation. Further, possibly via inhibition of antioxidant defense system such as Nrf2 signaling and depleting glutathione system it aggravates oxidative stress and inflammation, which eventually lead to renal injury and dysfunction. Olmesartan treatment effectively prevented oxidative stress and inflammation induced by DNR that are identified by the changes in the protein levels of oxidative stress markers such NADPH oxidase subunits, Nrf2, glutathione peroxidase and other signaling proteins.
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This research was supported by a Yujin Memorial Grant, Ministry of Education, Culture, Sports and Technology of Japan and by a grant from the Promotion and Mutual Aid Corporation for Private Schools, Japan.
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Olmesartan protects against oxidative stress possibly through the Nrf2 signaling pathway and inhibits inflammation in daunorubicin-induced nephrotoxicity in rats Vengadeshprabhu Karuppa Gounder a, Somasundaram Arumugam a, Wawaimuli Arozal a, Rajarajan A. Thandavarayan a, Vigneshwaran Pitchaimani a, Meilei Harima a, Kenji Suzuki b, Mayumi Nomoto a, Kenichi Watanabe a,⁎ a b
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Department of Clinical Pharmacology, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata City 956-8603, Japan Department of Gastroenterology, Niigata University Graduate School of Medical and Dental Sciences, Niigata City 951-8510, Japan
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Article history: Received 6 June 2013 Received in revised form 14 November 2013 Accepted 18 November 2013 Available online xxxx
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Anthracycline anticancer drug daunorubicin (DNR) can induce chronic nephrotoxicity, which is believed to be based on oxidative injury. Olmesartan has significant blood pressure lowering effect via modulating renin-angiotensin system although its mechanism of action in DNR-induced renal injury is largely unknown. Transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) is an important regulator of cellular oxidative stress. This study examined the role of Nrf2 in olmesartan-mediated antioxidant effects in DNR induced kidney cells. In addition, key factors involved in promoting inflammation and oxidative stress were studied. Sprague-Dawley rats were treated with a cumulative dose of 9 mg/kg DNR (i.v.). Olmesartan was administered orally every day for 6 weeks. DNR treated rats showed nephrotoxicity as evidenced by worsening renal function, which was evaluated by measuring total cholesterol, triglyceride levels in kidney tissue and histopathological approaches; treatment with olmesartan reversed these changes. Furthermore, olmesartan treatment down-regulated phospho-MAPKAPK-2, caspase-12, p47phox, p67phox, upregulated renal expression of PPAR-γ, Bcl-xL, glutathione peroxidase and Nrf2. Furthermore, olmesartan down-regulated matrix metalloproteinase-2 and angiotensin II type I receptor expression in the kidney. In conclusion, the result demonstrated that angiotensin II and oxidative stress play a key role in DNR-induced nephrotoxicity. The present results indicated that olmesartan protects against oxidative stress, which may be possibly via the induction of Nrf2 signaling pathways. © 2013 Published by Elsevier B.V.
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Keywords: Daunorubicin Olmesartan Oxidative stress Chronic nephrotoxicity Nuclear factor-erythroid 2-related factor 2 Angiotensin II type I receptor
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1. Introduction
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Daunorubicin (DNR) is one of the anthracycline (ANT) anti-tumor agents widely used in the treatment of acute myeloid leukemia. However, the clinical use of DNR has been limited by its undesirable systemic toxicity, especially cardiotoxicity and nephrotoxicity, which eventually lead to congestive heart failure and renal failure, respectively [1–3]. ANT-induced changes in the renal tissue of rats include glomerular capillary permeability and glomerular atrophy [4]. It is believed that the biochemical mechanism for ANT-induced heart and kidney damage involves the generation of reactive oxygen species (ROS) as a consequence of redox cycling [5]. The excess ROS can damage cellular lipids, proteins, or DNA inhibiting their normal function [6]. Increased generation of ROS leads to tissue injury and dysfunction by attacking,
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⁎ Corresponding author at: Department of Clinical Pharmacology, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, 265-1 Higashijima, Akiha-ku, Niigata City 956-8603, Japan. Tel.: + 81 250 25 5267; fax: + 81 250 25 5021. E-mail address: [email protected] (K. Watanabe).
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denaturing, and modifying structural and functional molecules and by activating redox-sensitive transcription factors and signal transduction pathways. These events, in turn, promote necrosis, apoptosis, inflammation, fibrosis, and other disorders. Redox systems including antioxidant enzymes and phase II detoxifying and antioxidant agents provide protection against ROS-induced tissue injury [7]. Blockade of oxidative stress can ameliorate renal sclerosis through anti-inflammatory and antiapoptotic processes [8]. In the kidney, activation of the angiotensin II–angiotensin II type I receptor (AT1R) pathway results in hypertension, the production of proinflammatory mediators, cell proliferation, extracellular matrix synthesis, and release of ROS, which facilitate kidney damage and advance chronic kidney disease [9]. Blockade of renin angiotensin system (RAS) is highly effective in retarding progression of renal disease in humans and experimental animals [10]. The salutary effect of RAS blockade is, not only, due to its antihypertensive/hemodynamic actions, but also its ability to reduce oxidative stress, inflammation and fibrosis which are major mediators of progression of renal disease [11,12]. However, studies have suggested that angiotensin converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) exert a protective
1567-5769/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.intimp.2013.11.018
Please cite this article as: Gounder VK, et al, Olmesartan protects against oxidative stress possibly through the Nrf2 signaling pathway and inhibits inflammation in daunorubicin..., Int Immunopharmacol (2013), http://dx.doi.org/10.1016/j.intimp.2013.11.018
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DNR was kindly donated by Meiji Seika Kaisha Ltd. (Tokyo, Japan). Olmesartan was donated by Daichi-Sankyo Pharmaceutical (Tokyo, Japan). All antibodies were purchased from Santa Cruz Biotechnology Inc. (CA) or Cell signaling (USA). Unless otherwise stated all reagents were of analytical grade and were purchased from Sigma (Tokyo, Japan).
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Eight-weeks-old male Sprague-Dawley rats were obtained from Charles River Japan Inc. (Kanagawa, Japan). The animals were quarantined and acclimatized for additional 2 weeks prior to the initiation of the experiments. They were randomly divided into four groups, and the first two groups served as normal (N, n = 5) and normal treated with olmesartan (10 mg/kg/day, per oral) (N + Olm, n = 5). Other rats received a single intravenous injection of DNR at a dose of 3 mg/kg (i.v.) on day 0. The drug was administered in three equal injections at 48 h intervals for a period of one week to achieve an accumulative dose of 9 mg/kg, which is well documented to produce cardiotoxicity and nephrotoxicity [3,5]. DNR treated rats were divided into two groups and each received either vehicle (1% gum Arabic) alone (DNR, n = 12) or olmesartan (10 mg/kg/day, per oral) (DNR + Olm, n = 10). Administration of olmesartan was started on the same day as DNR administration and continued for 5 additional weeks after cessation of DNR administration (6 weeks total period). The dose of olmesartan and duration of study were chosen on the basis of previous reports [5,17,18]. On day 41, rats were placed individually in metabolic cages for 24-h urine collections for the measurement of protein concentrations. After the end of the study period (6 weeks), rats were sacrificed after measuring body weight (BW) and kidney tissue was harvested for semi-
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Blood samples collected by heart puncture during sacrifice were utilized for the subsequent determination of creatinine, total cholesterol (TC), triglyceride, and blood urea nitrogen (BUN) and were stored at −80 °C. TC and triglyceride levels were determined using standard enzymatic procedures. Serum creatinine level was determined by the Jaffe method [19]. BUN was determined by the diacetylmonoxime method [20].
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The kidney tissue was decapsulated. Half of the kidney was immediately snapfrozen in liquid nitrogen for subsequent protein extraction and enzymatic assays. The remaining excised kidneys were cut into about 2-mm-thick transverse slices and fixed in 10% formalin. Sections of 3–5 μm thickness were stained with hematoxylin and eosin (HE) for histological examination. A histomorphological evaluation of all the kidney sections was carried out in a blinded fashion by a pathologist who was unaware of the treatment groups.
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quantitative immunoblotting and immunohistochemical studies. The 140 animal experiments were performed in accordance with national guide- 141 lines for the use and care of our institute. 142
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Protein lysate was prepared from renal tissue as described previously [21]. The total protein concentration in samples was measured by the bicinchoninic acid method [22]. For the determination of protein levels of Nrf2, Keap1, peroxisome proliferator-activated receptor gamma (PPAR-γ), B-cell lymphoma-extra-large (Bcl-xL), phospho-mitogen activated protein kinase-activated protein kinase (phospho-MAPKAPK)2, caspase-12, p47phox and p67phox, equal amounts of protein extracts (30 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad, CA) and transferred electrophoretically to nitrocellulose membranes. Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline Tween (20 mM Tris, pH 7.6, 137 mM NaCl and 0.1% Tween 20). The membrane was incubated overnight at 4 °C with the primary antibody and the bound antibody was visualized using the respective horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology Inc.) and chemiluminescence developing agents (Amersham Biosciences, Buckinghamshire, UK). The level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was estimated in every sample to check for equal loading of samples. Films were scanned and band densities were quantified with densitometric analysis using the Scion Image program (Epson GT-X700, Tokyo, Japan). All values were expressed as relative to their GAPDH content.
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Formalin-fixed, paraffin-embedded renal tissue sections were used for immunohistochemical staining. After deparaffinization and hydration, the slides were washed in Tris-buffered saline (TBS; 10 mM/l Tris HCl, 0.85% NaCl, pH 7.2). Endogenous peroxidase activity was quenched by incubating the slides in methanol and 0.3% H2O2 in methanol. Blocked in 10% normal serum with 1% BSA in TBS for 1 h at room temperature. After overnight incubation with the primary antibody, either rabbit polyclonal anti-AT1R (Peninsula Laboratories Inc., San Carlos, CA) or goat monoclonal anti-matrix metalloproteinase (MMP)-2 (diluted 1:100) (Santa Cruz Biotechnology, Inc.), at 4 °C, the slides were washed in TBS and then HRP conjugated secondary antibody was added and the slides were further incubated at room temperature for 45 min. The slides were washed in TBS and incubated with diaminobenzidine tetra hydrochloride as the substrate and counterstained with
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role toward doxorubicin (DOX) and DNR-induced cardiotoxicity and nephrotoxicity [1,12]. Nuclear factor-erythroid-2-related factor 2 (Nrf2) has also been found to be a critical transcription factor that binds to the antioxidant response element (ARE) in the promoter region of a number of genes encoding antioxidant and phase 2 enzymes, such as glutathione peroxidase (GPx), catalase, and superoxide dismutase, in several types of cells and tissues [13]. Nrf2 is held as an inactive complex bound to a repressor molecule known as Keap1 (Kelch-like ECH-associated protein1) which facilitates its ubiquitination. Keap1 contains several reactive cysteine residues that serve as sensors of the intra-cellular redox state. Oxidative or covalent modification of thiols in these cysteine residues or Nrf2 by protein kinases results in dissociation of Nrf2 from Keap1 and its migration and binding to the ARE in the promoter regions of genes encoding the antioxidant and phase 2 detoxifying enzymes [14]. The Nrf2mediated regulation of cellular antioxidant and anti-inflammatory machinery plays an important role in the defense against oxidative stress [15]. Within the ARB class, olmesartan medoxomil is a long acting AT1R antagonist approved for the treatment of mild to severe hypertension, alone or in combination with other agents. Moreover, olmesartan is therapeutically effective for the treatment of patients with heart failure by reducing cytokines and oxidative stress through its antiinflammatory effects [16]. Hence, the present study was aimed to investigate the ameliorative potential of olmesartan on proinflammatory cytokines, oxidative stress, renal dysfunction and the role of Nrf2 in olmesartan mediated antioxidant effects in chronic DNR-induced nephrotoxicity rats. To the best of our knowledge, we are the first to examine the effects of chronic administration of ARB olmesartan against oxidative stress possibly through the Nrf2 signaling pathway in DNR-induced nephrotoxicity.
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Please cite this article as: Gounder VK, et al, Olmesartan protects against oxidative stress possibly through the Nrf2 signaling pathway and inhibits inflammation in daunorubicin..., Int Immunopharmacol (2013), http://dx.doi.org/10.1016/j.intimp.2013.11.018
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N + Olm group revealed normal histological findings whereas DNR administration resulted in substantial damage to kidney as indicated by glomerular congestion, tubular necrosis, tubular dilatation, intertubular hemorrhage, and renal fibrosis. Renal changes in group DNR were improved in the rats treated with olmesartan (Fig. 1A and B).
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3.4. Renal expression of AT1R and MMP-2
244
Kidney sections of group DNR showed stronger immune reactivity for AT1R and MMP-2 than those of group N and group N + Olm, whereas group DNR + Olm revealed a significant decrease in the renal level of AT1R (Fig. 1C and C1) and MMP-2 (Fig. 1D and D1).
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2.7. Measurement of glutathione peroxidase (GPx) activity
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Kidney samples were homogenized in six volumes (per wet weight of tissue) of cold GPx assay buffer, the mixture was centrifuged for 15 min at 4 °C, and 8000 rpm in accordance with the total GPx assay kit instructions (Oxitek, ZeptoMetrix Corporation, New York). GPx activity in kidney tissues was measured using a kinetic ultravioletvisible spectrophotometer (Ultraspec 3100, Amersham Biosciences). The oxidation of NADPH to NADP+ was measured by the decrease in absorbance at 340 nm.
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2.8. Statistical analysis
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Data are presented as mean ± SEM and were analyzed using oneway analysis of variance (ANOVA) followed by Tukey multiple comparison test or two-tailed “t”-test when appropriate. A value of p b 0.05 was considered statistically significant. For statistical analysis, GraphPad Prism 5 software (San Diego, CA) was used.
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3. Results
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3.1. Effect of olmesartan on BW and survival rate
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227
Throughout the study period, BW was significantly decreased in DNR rats with or without olmesartan treatment compared with that of group N rats. Although olmesartan treatment tended to prevent the reduction of BW compared with that in group DNR, the effect did not attain statistical significance and olmesartan treatment to N + Olm group did not show significant changes in body weight compared to normal group (Table 1). Four (33.3%) rats in group DNR died between days 16 and 42 (Table 1). None of the rats died in group N, N + Olm and group DNR + Olm.
228
3.2. Effect of olmesartan on kidney function
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234 235
Serum creatinine levels were significantly increased in group DNR compared with those in group N and N + Olm group. In addition TC and triglyceride levels were also significantly increased in group DNR compared with those in group N and group N + Olm. Treatment of DNR-injected rats with olmesartan resulted in a significant decrease in serum creatinine, BUN, TC and triglyceride levels compared with those of DNR-treated rats (Table 1).
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3.3. Effect of olmesartan against renal histological damage
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The severity of kidney injury was investigated by examination of HE and Azan-Mallory stained kidney sections. Renal tissues of group N and
t1:1 t1:2
Table 1 Changes in survival rate, body weight and biochemical parameters after 6 weeks of treatment with olmesartan in DNR treated rats.
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t1:11 t1:12 t1:13
Number of death Survival rate (%) BW (g) Serum creatinine (mg/dl) BUN (mg/dl) TC (mg/dl) Triglyceride (mg/dl)
247 248
250 251
R O
O
The protein levels of caspase-12, NADPH oxidase sub-units p47phox and p67phox were significantly increased in group DNR compared with those in group N, as well as N + Olm group and these changes were significantly attenuated in group DNR + Olm. Moreover, renal protein expression of Bcl-xL was significantly decreased in group DNR compared with that in group N rats. In contrast, treatment with olmesartan increased the protein level of Bcl-xL compared with that in group DNR rats, but no significant changes in normal and normal treated with olmesartan group (Fig. 2A, B, C, D and E).
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P
t1:3 t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10
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E
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C
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E
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R
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3.5. Effects of olmesartan on oxidative stress and other signaling proteins
3.6. Effects of olmesartan on Nrf2 and Keap1 expression
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N C O
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U
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F
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hematoxylin. A negative control without primary antibody was included in the experiment to verify the antibody specificity. Brown colored immunopositive AT1R and MMP-2 spots were counted at 400-fold magnification.
Nrf2 protein expression was significantly decreased in group DNR compared with that in group N and normal treated with olmesartan group, but when DNR rats treated with olmesartan its expression was significantly increased on comparison with group DNR rats (Fig. 3A and B). Moreover, no change in the renal protein expression of Keap1 in group DNR was observed when compared with other groups. Further, to confirm the effect of olmesartan on Nrf2 levels in normal rats, we have analyzed the normal rats treated with olmesartan alone and observed no significant change in its protein level when compared with the normal rats (Fig. 3A and C).
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3.7. Effects of olmesartan on PPAR-γ and phospho-MAPKAPK-2 expression 270 Western blotting analysis showed that PPAR-γ protein expression was significantly increased in group DNR compared with that in group N, which exhibited a further increase with the administration of olmesartan (Fig. 3A and D). Moreover, renal protein expression of phospho-MAPKAPK-2 was significantly increased in group DNR compared with that in group N and N + Olm rats. In contrast, treatment with olmesartan decreased the protein level of phospho-MAPKAPK-2 compared with that in group DNR rats (Fig. 3A and E).
N (n = 5)
N + Olm (n = 5)
DNR (n = 12)
DNR + Olm (n = 10)
0 00 542 ± 9.1 1.9 ± 0.5 21 ± 2.4 53.3 ± 6.1 46 ± 19.3
0 100 533 ± 8.9 1.7 20 ± 7.5 57.6 ± 3.7 47.3 ± 6.3
4 66.7 362 ± 8.0⁎⁎⁎ 3.2 ± 2.0⁎ 54 ± 7.1⁎ 581.8 ± 36.5⁎⁎⁎ 2691 ± 316.1⁎⁎⁎
0 100 385 ± 9.0 2.1 ± 0.9# 35 ± 9.2# 436.5 ± 31.3# 631.7 ± 156.5###
Results are presented as the mean ± SEM. BUN, blood urea nitrogen; BW, body weight; TC, total cholesterol; group N, aged-matched normal rat; group N + Olm, normal rats administered with olmesartan (10 mg/kg/day for 5 weeks, per oral); group DNR, DNR-treated rats administered with vehicle; Group DNR + Olm, DNR-treated rats administered with olmesartan (10 mg/kg/day for 5 weeks, per oral). ⁎p b 0.05 and ⁎⁎⁎p b 0.001 vs group N or N + Olm; #p b 0.05 and ###p b 0.001 vs group DNR.
Please cite this article as: Gounder VK, et al, Olmesartan protects against oxidative stress possibly through the Nrf2 signaling pathway and inhibits inflammation in daunorubicin..., Int Immunopharmacol (2013), http://dx.doi.org/10.1016/j.intimp.2013.11.018
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N
N+Olm
DNR
DNR+Olm
F
A
D
P
R O
O
B
E
C
T
E
C
6
*** ###
N
8
C
10
D1
U
Average number of AT1R positive cells / field
C1
4 2 0
N
N+Olm
DNR
DNR+Olm
Average number of MMP2 positive cells / field
O
R
R
D
20
*** #
15
10
5
0
N
N+Olm
DNR
DNR+Olm
Fig. 1. (A), Hematoxylin and eosin staining of the cross-sectional tissue slices of kidney depicting glomerular congestion, tubular casts, and interstitial hemorrhage (200×). (B), Azan-Mallory staining for fibrosis of the cross-sectional tissue slices of kidney. Fibrosis is indicated by the blue area as opposed to the red renal tissue (200×). (C&C1), Renal levels of AT1R by immunohistochemical staining and the positive immunostaining of AT1R exhibit brown (400×) and its quantification data. (D&D1), Photomicrograph of kidney tissue sections showing MMP-2 staining and its quantification data. MMP-2 staining is shown in brown. All nuclei stained by hematoxylin are shown in blue (400×). Each bar represents mean ± SEM. Group N, agematched normal rats; group N + Olm, normal rats treated with olmesartan (10 mg/kg/day); group DNR, DNR-treated rats administered with vehicle; group DNR + Olm, DNR treated rats administered with olmesartan (10 mg/kg/day). ***p b 0.001 vs group N or N + Olm; #p b 0.05 and ###p b 0.001 vs group DNR. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: Gounder VK, et al, Olmesartan protects against oxidative stress possibly through the Nrf2 signaling pathway and inhibits inflammation in daunorubicin..., Int Immunopharmacol (2013), http://dx.doi.org/10.1016/j.intimp.2013.11.018
V.K. Gounder et al. / International Immunopharmacology xxx (2013) xxx–xxx
B N+Olm
N
Relative optical density (p67phox/GAPDH)
A
5
DNR+Olm
DNR
p67phox p47phox Caspase-12 Bcl-xL
2.0
**
1.5 1.0
###
0.5 0.0 N
GAPDH
0.5
0.0 N
N+Olm
DNR
Relative optical density (Bcl-xL/GAPDH)
** 3 #
2 1 0 N
DNR+Olm
N+Olm
DNR
1.5
#
O
#
DNR+Olm
E 4
1.0
R O
***
1.0
DNR
F
D 1.5
Relative optical density (caspase-12/GAPDH)
Relative optical density (p47phox/GAPDH)
C
N+Olm
*
0.5
0.0
DNR+Olm
N
N+Olm
DNR
DNR+Olm
E
D
P
Fig. 2. Renal expressions of p67phox, p47phox, caspase-12 and Bcl-xL. (A), Representative Western blots showing specific bands for p67phox, p47phox, caspase-12, Bcl-xL and GAPDH as an internal control. Equal amounts of protein sample obtained from whole renal homogenate were applied in each lane. (B–E), Densitometric data of protein analysis. The mean density values of p67phox, p47phox, caspase-12 and Bcl-xL were expressed as ratios relative to that of GAPDH. Each bar represents mean ± SEM. Group N, age-matched normal rats; group N + Olm, normal rats treated with olmesartan (10 mg/kg/day); group DNR, DNR-treated rats administered with vehicle; group DNR + Olm, DNR-treated rats administered with olmesartan (10 mg/kg/day). *p b 0.05, **p b 0.01 and ***p b 0.001 vs group N or N + Olm; #p b 0.05 and ###p b 0.001 vs group DNR.
3.8. Effects of olmesartan on renal GPx activity
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Administration of DNR caused a significant reduction in GPx activity in renal tissue compared with that in the group N and group N + Olm. Treatment with olmesartan significantly increased GPx activity compared with those in group DNR (Fig. 4).
C
4. Discussion
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ANT nephrotoxicity presents the most unfavorable side effect of these highly efficient anticancer drugs. The present study indicated that a cumulative dose of DNR exhibited nephrotoxicity as evidenced by worsening renal function, as well as elevated serum creatinine,
285 286
E
N+Olm
N
B
DNR+Olm
R
Nrf2
DNR
Relative optical density (Nrf2/GAPDH)
R
A
N C O
Keap1 PPAR-γ
phospho-MAPKAPK-2
2.5 2.0 ##
1.5 1.0
0.0 N
2.0
Relative optical density (PPAR-γ /GAPDH)
2.5
1.5 1.0 0.5 0.0 N
N+Olm
DNR
DNR+Olm
N+Olm
DNR
DNR+Olm
E
D 2.5
##
2.0
*
1.5 1.0 0.5 0.0 N
N+Olm
DNR
DNR+Olm
Relative optical density (phospho-MAPKAPK-2/GAPDH)
C
*
0.5
GAPDH
U
283
Relative optical density (Keap1/GAPDH)
281 282
T
279
1.5
* 1.0 #
0.5
0.0 N
N+Olm
DNR
DNR+Olm
Fig. 3. Renal expressions of Nrf2, Keap1, PPAR-γ and phospho-MAPKAPK-2. (A), Representative Western blots showing specific bands for Nrf2, Keap1, PPAR-γ, phospho-MAPKAPK-2 and GAPDH as an internal control. Equal amounts of protein sample obtained from whole kidney homogenate were applied in each lane. (B–E), Densitometric data of protein analysis. The mean density values of Nrf2, Keap1, PPAR-γ and phospho-MAPKAPK-2 were expressed as ratios relative to that of GAPDH. Each bar represents mean ± SEM. Group N, age-matched normal rats; group N + Olm, normal rats treated with olmesartan (10 mg/kg/day); group DNR, DNR-treated rats administered with vehicle; group DNR + Olm, DNR-treated rats administered with olmesartan (10 mg/kg/day). *p b 0.05 vs group N or N + Olm; #p b 0.05 and ##p b 0.01 vs group DNR.
Please cite this article as: Gounder VK, et al, Olmesartan protects against oxidative stress possibly through the Nrf2 signaling pathway and inhibits inflammation in daunorubicin..., Int Immunopharmacol (2013), http://dx.doi.org/10.1016/j.intimp.2013.11.018
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0.0 N
N+Olm
DNR
DNR+Olm
Fig. 4. Effect of olmesartan on GPx activities in kidney homogenates from DNR-treated rats. The values are mean ± SEM. Group N, age-matched normal rats; group N + Olm, normal rats treated with olmesartan (10 mg/kg/day); group normal + Olm, normal rat treated with olmesartan (10 mg/kg/day); group DNR, DNR-treated rats administered with vehicle; group DNR + Olm, DNR-treated rats administered with olmesartan (10 mg/kg/day). **p b 0.01 vs group N or N + Olm; #p b 0.05 vs group DNR.
305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335
T
C
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E
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R
299 300
R
297 298
O
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C
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N
291 292
BUN, TC and triglyceride (Table 1). These changes were associated with oxidative stress and inflammation in renal tissues. Chronic treatment with olmesartan showed protective effects against DNR-induced nephrotoxicity by reducing oxidative stress and inflammation and as a consequence, the renal functions were improved and mortality rate was reduced. To the best of our knowledge, our study is the first that identifies the protective effect of 6 weeks of treatment with olmesartan against nephrotoxicity induced by a cumulative dose of DNR. Angiotensin II is the major effector hormone of the RAS and contributes to a variety of renal and cardiovascular physiologic and pathologic mechanisms through stimulation of AT1R and AT2R. Up-regulation of AT1R was coupled with a marked increase in the numbers of angiotensin II-positive cells. Angiotensin II is known to raise arterial pressure, induce renal cell hypertrophy, and matrix protein accumulation, events that are linked to progression of renal disease [23]. In this study, we confirmed that the renal expression of its receptor AT1R was increased in DNR treated rats, and treatment with olmesartan mediated the down-regulation of AT1R (Fig. 1C and C1). Moreover, previous clinical and experimental studies showed that AT1R-mediated NADPH oxidase activation leads to generation of ROS [24]. A recent study showed that NADPH oxidase makes an important contribution to renal oxidative stress and inflammation, as well as renal damage and dysfunction [25]. To address the role of oxidative stress, we next measured in the renal protein expression of p67phox and p47phox, which are cytosolic subunits of NADPH oxidase, by Western blotting. Our results showed that DNR treatment increased the expression levels of p67phox and p47phox significantly and are inhibited by treatment with olmesartan. In addition, previous studies have shown that ANT induces apoptosis, which is supposed to occur through the production of ROS [26]. Our results showed that olmesartan treatment significantly reduced the level of activated caspase-12 and increased the level of Bcl-xL (Fig. 2A, B, C, D and E). MMPs are a family of proteases best known for their capacity to proteolyze the extracellular matrix proteins. MMPs, such as MMP-2 degrade the type IV collagen, the major structural component of basement membrane. In a previous study, Chang et al. [27] suggested that increased MMP-2 levels contribute to the pathogenesis of chronic kidney diseases. More recent studies found that increased MMP-2 levels correlate with proteinuria, oxidative stress, cardiovascular disease prevalence and the atherosclerosis marker-intima media thickness in patients with chronic kidney disease [28]. Over the last decade, a large number of studies have shown an increased MMP-2 activity/level in response to oxidative stress in various tissues from different species [29]. In agreement with these results, we could observe an increase in renal levels of MMP-2 and which was normalized by olmesartan treatment. Moreover, ROS have been recognized as important mediators that regulate signal transduction pathways, including MAPK [30]. The
U
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F
** 0.1
O
0.2
R O
#
P
0.3
chronic treatment with aldosterone/salt induces MAPK activation via a ROS dependent pathway. MAPKAPK-2, a member of the MAPK family is activated by physical and chemical stress factors resulting in growth promotion, apoptosis, oxidative stress, and vasoconstriction [31]. Previous studies in vitro have implicated that ROS also regulate other signaling molecules; it is possible that ROS-mediated MAPK activation was involved, at least in part, in the progression of renal injury [30]. In the present study, treatment with olmesartan significantly down regulated the renal protein expressions of phospho-MAPKAPK-2 (Fig. 3A and E). Activation of PPAR-γ exerts anti-inflammatory [32], antifibrotic [33], anti-oxidative and vasculo-protective effect [34] on different renal diseases, but little has been known about the effect of PPAR-γ agonist in ANT-induced nephrotoxicity. Furthermore, it has been demonstrated that telmisartan suppressed the AT1R expression in both mRNA and protein levels through PPAR-γ mediated pathway. Involvement of PPAR-γ in AT1R suppression has been further confirmed by using GW9662, a PPAR-γ antagonist [35]. Recent studies suggest that telmisartan, but not other ARBs, could act as a partial PPARγ agonist [36]. In the present study, we demonstrated, that treatment with olmesartan significantly upregulated the renal protein expressions of PPAR-γ. Since, we have only measured the expression of PPAR-γ at protein level and not its activity, the net effect of overall PPAR-γ activity in our model is unclear. In light of this study limitation, further experiments will be required to understand whether the effects of olmesartan in our model are mediated by alterations in PPAR-γ activity. Nrf2, a member of the cap-N-collar family, is the key transcription factor that regulates antioxidant response element-mediated expression of detoxifying antioxidant enzymes [37]. Under basal conditions, Nrf2 is sequestered in the cytoplasm by an actin binding repressor protein Keap1, which facilitates its ubiquitination. In fact, an experimental study of spontaneous focal glomerulosclerosis rats has indicated that ARB treatment prevented nephropathy, suppressed oxidative stress, inflammatory process, and endoplasmic reticulum stress-induced apoptosis as well as restored Nrf2 activation and expression of the antioxidant enzymes [38]. A very recent study has revealed that telmisartan, could suppress NADPH oxidase and subsequently upregulate Nrf2, leading to the amelioration of renal oxidative stress [39]. In addition, Nrf2 can be activated by phosphorylation of some of its threonine or serine residues by upstream kinases such as protein kinase C, MAPKs, phosphatidylinositol-3-kinase/Akt, and casein kinase-2 [40]. In the present study, we have identified no significant changes in the renal protein expression levels of Nrf2 and Keap1 in between normal and N + Olm group rats. But interestingly, Nrf2 protein expression was significantly decreased in group DNR compared with that in group N and N + Olm, but when DNR rats treated with olmesartan its expression was significantly increased on comparison with group DNR rats (Fig. 3A and B). Recently, the Nrf2 is found to be a critical transcription factor that binds to the ARE in the promoter region of a number of genes encoding for antioxidative and phase 2 enzymes in several types of cells and tissues [41,42]. In the present study, the DNR-induced increase in ROS generation was potentially exacerbated by a significant reduction in antioxidant reserves in renal tissues. This might have caused a state of redox imbalance that enhanced the susceptibility of biological membranes to reaction with ROS, as indicated by decrease in GPx in the tissues, while administration of olmesartan with DNR significantly restored GPx activity (Fig. 4). Whether this effect also attributes to the Nrf2-mediated antioxidative stress in the advanced phase of renal toxicity in this model is uncertain and requires further investigation. In conclusion, progressive nephrotoxicity in the DNR-treated rats is associated with upregulation of oxidative stress, inflammation and conspicuous impairment of Nrf2 activation. Long-term therapy with olmesartan prevents development of renal toxicity by reducing inflammation and oxidative stress, which may be possibly via the induction of Nrf2 signaling pathways in this model. The possible mechanism (Fig. 5) behind this renoprotective action of olmesartan is attenuation of
D
Renal Gpx activity (U/mg)
0.4
E
6
Please cite this article as: Gounder VK, et al, Olmesartan protects against oxidative stress possibly through the Nrf2 signaling pathway and inhibits inflammation in daunorubicin..., Int Immunopharmacol (2013), http://dx.doi.org/10.1016/j.intimp.2013.11.018
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Olmesartan Daunorubicin PPAR-γ AT1R Nrf2 NADPH oxidase subunits
F
Antioxidant defenses
Inflammatory stress
P
Oxidative stress
R O
ROS
O
Gpx
Nephrotoxicity
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E
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Fig. 5. The possible mechanism by which olmesartan afforded protection against DNR-induced nephrotoxicity. The major adverse effect of DNR treatment is the activation of renin angiotensin system and via activation of AT1R, DNR induces the production of free radicals such as reactive oxygen species possibly via activated NADPH oxidase and thereby causing oxidative stress and inflammation. Further, possibly via inhibition of antioxidant defense system such as Nrf2 signaling and depleting glutathione system it aggravates oxidative stress and inflammation, which eventually lead to renal injury and dysfunction. Olmesartan treatment effectively prevented oxidative stress and inflammation induced by DNR that are identified by the changes in the protein levels of oxidative stress markers such NADPH oxidase subunits, Nrf2, glutathione peroxidase and other signaling proteins.
407 Q2
Acknowledgment
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This research was supported by a Yujin Memorial Grant, Ministry of Education, Culture, Sports and Technology of Japan and by a grant from the Promotion and Mutual Aid Corporation for Private Schools, Japan.
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oxidative stress by simultaneously lowering production of ROS and raising production of endogenous antioxidants. In spite of these positive results, we could not reveal the precise mechanism of olmesartan action in this model and thus further studies are warranted to determine the effect of pharmacological activation of Nrf2 in this model.
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