|Year : 2015 | Volume
| Issue : 1 | Page : 2-6
Nonprotective role of losartan in cisplatin-induced nephrotoxicity in ovariectomized rat model treated with estradiol
Fereshteh Ghadirian1, Mehdi Nematbakhsh2, Shahla Roozbehani3, Safoora Mazaheri4, Ardeshir Talebi5, Zahra Pezeshki4, Farzaneh Ashrafi6
1 Water and Electrolytes Research Center; Department of Biology, Falavarjan Branch, Islamic Azad University, Iran
2 Water and Electrolytes Research Center; Department of Physiology, University of Medical Sciences; Isfahan MN Institute of Basic and Applied Sciences Research, Isfahan, Iran
3 Department of Biology, Falavarjan Branch, Islamic Azad University, Isfahan, Iran
4 Water and Electrolytes Research Center, University of Medical Sciences, Isfahan, Iran
5 Department of Clinical Pathology, University of Medical Sciences, Isfahan, Iran
6 Department of Internal Medicine, University of Medical Sciences, Isfahan, Iran
|Date of Web Publication||20-May-2015|
Department of Physiology, Water and Electrolytes Research Center, Kidney Diseases Research Center, Isfahan University of Medical Sciences, Isfahan
Source of Support: Isfahan University of Medical Sciences, Conflict of Interest: None
| Abstract|| |
Objective: The aim was to study the role of losartan on cisplatin (CP)-induced nephrotoxicity in an estradiol replacement rat model. Materials and Methods: Forty-seven female Wistar rats were ovariectomized and divided into seven groups. Groups 1 and 2 were treated with placebo (sesame oil) on a weekly basis for 4 weeks, and then they received saline or CP (2.5 mg/kg/day) for the next 7 days, respectively. Group 3 received estradiol (250 μg/kg/week) for 4 weeks, and then treated with losartan (10 mg/kg/day) for the next 10 days while from day 3; they also received CP for 7 days. Group 4 had treatment regimen the same as Group 3, except saline instead of losartan. Groups 5 and 6 were treated similar to Groups 3 and 4, respectively, but the dose of estradiol was doubled. Finally, Group 7 was treated as Group 3 except vehicle instead of estradiol. At the end of the experiment, blood samples were obtained to measure blood urea nitrogen (BUN), creatinine (Cr), nitric oxide metabolite (nitrite) and malondialdehyde (MDA). The kidney tissues were also subjected to pathology investigations. Results: Cisplatin significantly increased the serum level of BUN, Cr, and MDA (P < 0.05). CP also increased kidney weight (KW) and kidney tissue damage score (KTDS), and decreased bodyweight significantly (P < 0.05). There is a significant difference in KTDS, weight changes and serum level of MDA between positive control groups compared with the groups received combination of losartan and CP (P < 0.05). Conclusions: It seems that the estrogen could promote CP-induced nephrotoxicity. Losartan also did not intensify CP-induced nephrotoxicity. Accordingly, supplementation of losartan to attenuate CP-induced nephrotoxicity in ovariectomized model treated with estradiol is not suggested.
Keywords: Cisplatin, estrogen, losartan, nephrotoxicity, ovariectomized rats
|How to cite this article:|
Ghadirian F, Nematbakhsh M, Roozbehani S, Mazaheri S, Talebi A, Pezeshki Z, Ashrafi F. Nonprotective role of losartan in cisplatin-induced nephrotoxicity in ovariectomized rat model treated with estradiol. J Pharm Negative Results 2015;6:2-6
|How to cite this URL:|
Ghadirian F, Nematbakhsh M, Roozbehani S, Mazaheri S, Talebi A, Pezeshki Z, Ashrafi F. Nonprotective role of losartan in cisplatin-induced nephrotoxicity in ovariectomized rat model treated with estradiol. J Pharm Negative Results [serial online] 2015 [cited 2020 Jan 21];6:2-6. Available from: http://www.pnrjournal.com/text.asp?2015/6/1/2/157375
| Introduction|| |
Cisplatin (Cis-Diamminedichloroplatinum, CP) is an antineoplastic drug used for treatment of solid tumors.  Its chief dose-limiting side-effect is nephrotoxicity.  CP may change renal circulation,  and it binds to DNA and causes cytotoxic lesions in tumors and other dividing cells.  The quiescent proximal tubule cells are selectively damaged by CP. The mechanism underlying this renal cell injury has been the focus of severe investigations for many years, and it indicated that inflammation, oxidative stress injury, and apoptosis probably describe part of this injury.  Conversion of CP to nephrotoxic molecules in the proximal tubule cells is required for cell injury whereas the highest concentration of CP was found in mitochondria, microsome, cytosol, and nuclei. 
Recent studies have provided evidences for sex-based differences in CP-induced nephrotoxicity.  The sex-based difference in nephroprotective effect of losartan against CP-induced nephrotoxicity was also reported.  Losartan acts as a blocker for angiotensin II receptor type 1 (ATR1) and facilitates renal circulation.  The protective role of losartan against CP-induced nephrotoxicity was reported before. , On the other hand, some studies indicated that estrogen enhances oxidative stress in kidney,  and CP-induced nephrotoxicity is intensified in ovariectomized rats treated with estrogen.  Therefore, in this study, the effect of losartan on CP-induced nephrotoxicity in ovariectomized rat model treated with estradiol was investigated. In order to achieve this objective, the ovariectomized rats were treated with two different doses of estradiol valerate, and then losartan was administrated accompanied with CP.
| Materials and methods|| |
The experiment was performed on 47 adult female Wistar rats (150 ± 15 g, Animal Centre, Isfahan University of Medical Sciences, Isfahan, Iran). The rats were housed at a temperature of 23-25°C. This research was considered and approved by official Ethics Committee of the Isfahan University of Medical Sciences.
Cisplatin, and estradiol valerate were purchased from EBEWE Pharma Ges.m.b.H (Unterach, Austria) and Aburaihan (Tehran, Iran) respectively.
The animals were anesthetized with ketamine and xylazine (75 and 20 mg/kg, i.p). An incision was made in the abdominal middle line to expose and remove the ovaries from the retroperitoneal space. One week later the animals were assigned to 7 groups.
Group 1 (n = 5) received sesame oil (placebo) intramuscularly as placebo once a week for 4 weeks. At the end of the 4 th week, they received 0.5 ml saline daily for 7 days. This group was considered as a negative control group (Ovariectomized (OV) + saline) which OV stand for ovariectomized rats.
Group 2 (n = 7) received regimen the same as Group 1 except CP (2.5 mg/kg/day, i.p) instead of saline. This group was named positive control group (OV + CP).
Group 3 (n = 7) received estradiol valerate (250 μg/kg/week) solved in sesame oil intramuscularly for 4 weeks. After last enjection of estradiol valerate, the rats received losartan (10 mg/kg/day, i.p) for 10 days and at 3 th day, CP (2.5 mg/kg/day, i.p) was accompanied with losartan. This group was named OVE250 + L + CP which OVE stand for ovariectomized rats received estradiol (250 mg/kg).
Group 4 (n = 7) was treated similar to Group 3 except saline instead of losartan, and was named OVE250 + CP.
Group 5 (n = 7, OVE500 + L + CP) and Group 6 (n = 7, OVE500 + CP) were treated as Groups 4 and 5, respectively, but the dose of estradiol was doubled.
Group 7 (n = 7, OV + L + CP) had treatment regimen the same as Group 3 except placebo instead of estradiol valerate.
At the end of the experiment, all animals were sacrificed, and blood samples were obtained by heart puncture.  After centrifuging serum samples were removed and kept in − 20 until measurement. Finally, the animals were killed; kidneys were removed and weighed immediately. For pathological investigation left kidney was fixed in formalin. Also, right kidney was homogenized and centrifuged at 15000 g. Removed supernatant was kept in − 20 until measurement.
The serum levels of creatinine (Cr) and blood urea nitrogen (BUN) were determined using quantitative diagnostic kits (Pars Azmoon, Iran). The serum and kidney levels of nitrite (stable nitric oxide metabolite) were measured using a colorimetric assay that involves the Griess reaction. The serum and kidney levels of malondialdehyde (MDA) were determined by thiobarbituric acid (TBA) 0.67% and trichloroacetic acid (TCA) 10%.
Hematoxylin and eosin staining was performed to determine the kidney tissue damages. The intensity of damage was scored from 1 (small damage) to 4 (severe damage). The score zero was assigned for normal tissue.
Data are expressed as mean ± standard error of the mean. The data in positive and negative control groups were compared by Student's t-test (for quantitative data) and Mann-Whitney (for kidney tissue damage score, [KTDS]). To compare the data between positive control group and other treated groups, one way ANOVA and Dunnett's test as posthoc (for quantitative data) and Kruskal-Wallis test (for KTDS data). P <0.05 were considered as statistically significant.
| Results|| |
Cisplatin significantly increased the serum levels of BUN, Cr, and MDA; kidney weight (KW) and KTDS (P < 0.05) in ovariectomized rats. These changes indicated the toxicity effect of CP on kidney. CP also significantly decreased bodyweight (P < 0.05) while it did not alter the level of nitrite [Table 1].
|Table 1: ΔBW, KW, KTDS, Cr, BUN, SMDA, SN and KMDA and KN and ΔUter in negative (group 1) and positive (group 2) control groups |
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On the other hand, Groups 3-7 were compared with the positive control group (Group 2). The bodyweight significantly decreased in Groups 3 and 7 when compared with the positive control group (P < 0.05) [Figure 1]. The KTDS in Groups 3 and 6 also decreased significantly (P < 0.05) [Figure 2]. The level of kidney nitrite and serum MDA in the groups treated with estradiol valerate (Groups 3-6) decreased significantly when compared with the positive control group (P < 0.05), and as expected the uterus weight in these groups increased significantly (P < 0.05). There were no significant changes in serum levels of BUN, Cr, and nitrite, and kidney MDA in the treated groups when compared with the positive control group [Figure 1]. In general, these data indicate that co-administration of losartan and estradiol did not protect the kidney against CP-induced nephrotoxicity.
|Figure 1: The data for bodyweight change (ΔWeight), total kidney weight/100 (gr) bodyweight (KW), kidney tissue damage score (KTDS), serum level of creatinine (Cr), serum level of BUN, serum and kidney levels of MDA and nitrite, and uterus weight change (UT) in five experiment groups (3-7). One-way ANOVA was applied for analyses. The (*) indicates significant difference (P < 0.05) when compared with the positive control group (Group 2). Group 2: OV+CP, Group 3: OVE250+L+CP, Group 4: OVE250+CP, Group 5: OVE500+L+CP, Group 6: OVE500 +CP, and Group 7: OV+L+CP|
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|Figure 2: The pathology images (magnification X100) of kidney tissue in seven experimental groups (see text for group detail). No damage was seen in negative control group (group 1). The tissue damage in Groups 3 and 6 is less than that in the positive control group (P< 0.05). Group 2: OV+CP, Group 3: OVE250+L+CP, Group 4: OVE250+CP, Group 5: OVE500+L+CP, Group 6: OVE500 +CP, and Group 7: OV+L+CP|
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| Discussion|| |
The main purpose of this study was to investigate the role of losartan against CP-induced nephrotoxicity in ovariectomized rats model treated with estradiol. We found that administration of losartan alone and estradiol alone, or their combination has no protective role against CP-induced nephrotoxicity in ovariectomized rats model treated with estradiol.
Other studies explained that CP increased serum levels of BUN and Cr. , Our findings are consistent with these results. CP induces weight loss  due to gastrointestinal disturbances. Reduction of bodyweight is not related to estradiol administration because bodyweight did not decrease in the negative control group. CP increased the KW due to retention of water and minerals, renal circulation changes, or apoptosis.  Our previous study indicated that KW have a significant correlation with kidney injuries; such that the heavier kidney, the more injury developed. 
Recently, it was observed that response to CP-induced nephrotoxicity is sex-dependent,  and this gender difference was not related to estrogen, because estrogen itself promotes CP-induced nephrotoxicity.  Furthermore, previous reports indicated that the losartan is a nephroprotectant agent against CP-induced nephrotoxicity.  In a study carried out by Saleh et al., CP was administrated as a single dose and single dose administration of losartan reduced the kidney toxicity.  In our previous study, we also administrated CP as a single dose, and we found that the losartan is nephroprotective in male but not in female. , It should be mentioned, here again, that CP was administrated daily not as a single dose, and this treatment may alter the expected result. So, according to these findings, it seems that administration of losartan alone and estradiol alone, or their combination has no protective role against CP-induced nephrotoxicity.
Results of uterus weight and nitrite level indicate that the estradiol was effective in estradiol-treated groups. Uterus weight has a significant increase resulting from the proliferation of uterus cells.  Estrogen causes vessels dilation, and nitric oxide formation, which is also a vasodilating agent. Losartan blocks ATR1, which leads to an increase in renal blood flow. Therefore, all these parameters may promote CP transport to the kidneys and develop the kidney tissue damage. No protective role of losartan was seen in CP-induced nephrotoxicity model.  Moreover, reduction of serum MDA level in the experiment groups that received estrogen is related to antioxidant property of estrogen and its effect on CP in kidney tissue.  Estradiol as an antioxidant could reduce the MDA level and oxidative stress, but as a hormone could not reduce CP-induced nephrotoxicity. It should be mentioned some antioxidant may not alter the oxidative stress.  The KTDS in Groups 3 and 6 was less than that in the positive control group while the serum levels of BUN and Cr, KW, and weight changes were not in agreement with such findings. It seems this discrepancy does not affect the conclusion because the given mean KTDS in Groups 3 and 6 is greater than 2 out of 4, which indicates about 50% of the tubules were damaged.
| Conclusion|| |
It seems that the estrogen could promote CP-induced nephrotoxicity, and losartan could not intensify the induced nephrotoxicity in ovariectomized rats. In addition, combination of losartan and estrogen could not improve CP-induced nephrotoxicity.
| Acknowledgments|| |
This research was supported by Isfahan University of Medical Sciences.
| References|| |
Yao X, Panichpisal K, Kurtzman N, Nugent K. Cisplatin nephrotoxicity: A review. Am J Med Sci 2007;334:115-24.
Miller RP, Tadagavadi RK, Ramesh G, Reeves WB. Mechanisms of cisplatin nephrotoxicity. Toxins (Basel) 2010;2:2490-518.
Haghighi M, Nematbakhsh M, Talebi A, Nasri H, Ashrafi F, Roshanaei K, et al.
The role of angiotensin II receptor 1 (AT1) blockade in cisplatin-induced nephrotoxicity in rats: Gender-related differences. Ren Fail 2012;34:1046-51.
Barabas K, Milner R, Lurie D, Adin C. Cisplatin: A review of toxicities and therapeutic applications. Vet Comp Oncol 2008;6:1-18.
Merouani A, Shpall EJ, Jones RB, Archer PG, Schrier RW. Renal function in high dose chemotherapy and autologous hematopoietic cell support treatment for breast cancer. Kidney Int 1996;50:1026-31.
Kuhlmann MK, Burkhardt G, Köhler H. Insights into potential cellular mechanisms of cisplatin nephrotoxicity and their clinical application. Nephrol Dial Transplant 1997;12:2478-80.
Eshraghi-Jazi F, Nematbakhsh M, Pezeshki Z, Nasri H, Talebi A, Safari T, et al.
Sex differences in protective effect of recombinant human erythropoietin against cisplatin-induced nephrotoxicity in rats. Iran J Kidney Dis 2013;7:383-9.
Nematbakhsh M, Pezeshki Z, Eshraghi-Jazi F, Ashrafi F, Nasri H, Talebi A, et al
. Vitamin E, vitamin C, or losartan is not nephroprotectant against cisplatin-induced nephrotoxicity in presence of estrogen in ovariectomized rat model. Int J Nephrol 2012;2012:284896.
Deegan PM, Nolan C, Ryan MP, Basinger MA, Jones MM, Hande KR. The role of the renin-angiotensin system in cisplatin nephrotoxicity. Ren Fail 1995;17:665-74.
Beleh MA, Lin YC, Brueggemeier RW. Estrogen metabolism in microsomal, cell, and tissue preparations of kidney and liver from Syrian hamsters. J Steroid Biochem Mol Biol 1995;52:479-89.
Pezeshki Z, Nematbakhsh M, Nasri H, Talebi A, Pilehvarian AA, Safari T, et al.
Evidence against protective role of sex hormone estrogen in cisplatin-induced nephrotoxicity in ovarectomized rat model. Toxicol Int 2013;20:43-7.
Parasuraman S, Raveendran R, Kesavan R. Blood sample collection in small laboratory animals. J Pharmacol Pharmacother 2010;1:87-93.
Mazaheri S, Nematbakhsh M, Bahadorani M, Pezeshki Z, Talebi A, Ghannadi AR, et al.
Effects of fennel essential oil on cisplatin-induced nephrotoxicity in ovariectomized rats. Toxicol Int 2013;20:138-45.
Jilanchi S, Nematbakhsh M, Bahadorani M, Talebi A, Eshraghi-Jazi F, Mansouri A, et al.
Vitamin e is a nephroprotectant agent in male but not in female in a model of cisplatin-induced nephrotoxicity. ISRN Nephrol 2013;2013:280395.
Endo Y, Kanbayashi H. Modified rice bran beneficial for weight loss of mice as a major and acute adverse effect of cisplatin. Pharmacol Toxicol 2003;92:300-3.
Nematbakhsh M, Ashrafi F, Nasri H, Talebi A, Pezeshki Z, Eshraghi F, et al.
A model for prediction of cisplatin induced nephrotoxicity by kidney weight in experimental rats. J Res Med Sci 2013;18:370-3.
Nematbakhsh M, Ebrahimian S, Tooyserkani M, Eshraghi-Jazi F, Talebi A, Ashrafi F. Gender difference in cisplatin-induced nephrotoxicity in a rat model: Greater intensity of damage in male than female. Nephrourol Mon 2013;5:818-21.
Saleh S, Ain-Shoka AA, El-Demerdash E, Khalef MM. Protective effects of the angiotensin II receptor blocker losartan on cisplatin-induced kidney injury. Chemotherapy 2009;55:399-406.
Ashrafi F, Nematbakhsh M, Safari T, Talebi A, Nasri H, Khazaei M, et al.
A combination of vitamin C and losartan for cisplatin-induced nephrotoxicity in rats. Iran J Kidney Dis 2012;6:361-5.
Branham WS, Zehr DR, Sheehan DM. Differential sensitivity of rat uterine growth and epithelium hypertrophy to estrogens and antiestrogens. Proc Soc Exp Biol Med 1993;203:297-303.
Rastghalam R, Nematbakhsh M, Bahadorani M, Eshraghi-Jazi F, Talebi A, Moeini M, et al.
Angiotensin type-1 receptor blockade may not protect kidney against cisplatin-induced nephrotoxicity in rats. ISRN Nephrol 2014;2014:479645.
Miura T, Muraoka S, Ogiso T. Inhibition of lipid peroxidation by estradiol and 2-hydroxyestradiol. Steroids 1996;61:379-83.
de Bairros AV, Roehrs M, Ribeiro G, de Freitas F, Moreira AP, Tonello R, et al.
Treatment with N-acetylcysteine does not alter blood glucose levels and the oxidative stress status in diabetic rats. J Pharm Negat Results 2013;4:5-12.
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