Abstract
It is documented that chronic renal diseases are gender related. The protective role of angiotensin II receptor 1 (AT1) blocker losartan against cisplatin (CP)-induced nephrotoxicity was reported in males, but the role of gender is not well known. Six groups of Wistar rats were studied. Rats were divided into two groups of males and females to receive losartan for 9 days plus a single dose of CP (7 mg/kg) at day 3. Two positive control groups of males and females received the same regimen, except that they received saline instead of losartan. The negative control groups received saline instead of CP at day 3 and also saline instead of losartan. The blood samples were obtained, and the kidneys underwent histopathological investigations. All the CP-treated animals lost weight, but losartan promoted weight loss in females (p < 0.05). Coadministration of losartan and CP in females, but not in males, significantly increased the serum levels of blood urea nitrogen and creatinine when compared with the negative and positive control groups (p < 0.05). No significant differences were observed in serum levels of total proteins, magnesium, and nitrite between the groups. Administration of CP increased the kidney tissue damage score (KTDS) and normalized kidney weight (p < 0.05). However, in the presence of AT1 blockade, the KTDS (nonsignificantly) and normalized kidney weight (significantly, p < 0.05) increased in the CP-treated females. Such an observation was not seen in males. Losartan may prevent CP-induced nephrotoxicity in males, but it promotes the CP-induced damage in females, which may be related to the renin–angiotensin system receptors in the kidneys.
INTRODUCTION
Women have a lower risk of developing chronic renal diseases.Citation1 Cisplatin (CP), as an antitumor drug, is accompanied by nephrotoxicity in both genders.Citation2–5 To avoid the CP-induced nephrotoxicity, some antioxidants or nutrient agents were investigated in animal models.Citation6–10 Losartan, as an angiotensin II receptor 1 (AT1) blocker, has an antioxidant effect,Citation11,12 and its preventive role against CP-induced nephrotoxicity was reported in male animals.Citation13,14 The role of gender in CP-induced nephrotoxicity is not well documented yet, but it is reported that urinary sodium excretion after CP administration is different in males and females.Citation15
Renin–angiotensin system (RAS) includes different receptors with different vascular responses. In addition to AT1, there are two other receptors; angiotensin II receptor 2 (AT2) and angiotensin-(1-7) receptor (Mas) in RAS, which are gender dependent, and the role of these receptors in females is more important than in males, especially in the absence of AT1.Citation16 These gender differences may influence renal circulation.Citation17 Therefore, the inhibition of AT1 with losartan may cause different responses from AT2 and Mas receptors in females and males. Accordingly, we hypothesized that AT1 blockade with losartan may provide a gender-related nephroprotective role against CP-induced nephrotoxicity. To test this hypothesis, losartan accompanied by CP were administrated in two different gender groups of rats and were compared with the positive and negative control groups.
MATERIALS AND METHODS
Animals
Adult female (166.95 ± 4.07 g) and male (182.06 ± 5.05 g) Wistar rats (Animal Centre, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran) were individually housed at a temperature range of 23–25°C. Rats had free access to water and rat chow. The rats were acclimatized to the diet for at least 1 week prior to the experiment. The experimental procedures were approved by the Isfahan University of Medical Sciences Ethics Committee in advance.
Experimental Protocol
The animals were randomly divided into six groups as follows:
Group 1 (L + CPM group, n = 5). Male rats received losartan (10 mg/kg, i.p., per day) for 9 days plus a single dose of CP (7 mg/kg, IP) at day 3.
Group 2 (L + CPF group, n = 5). Female rats received the same regimen as group 1.
Group 3 (CPM group, n = 5). Male rats received the same regimen as group 1 except that they received saline instead of losartan and they were considered as male positive control group.
Group 4 (CPF group, n = 5). Female rats received the same regimen as group 3 and they were considered as female positive control group.
Groups 5 and 6. These groups (male, n = 5; female, n = 5) received saline instead of CP at day 3 and also saline instead of losartan during the study and they were considered as negative control groups.
cis-Diamminedichloroplatinum (II) (CP, code P4394) was purchased from Sigma (St. Louis, MO, USA). The body weights of animals were daily recorded. Blood samples were obtained from each animal at day 9 (or 6 days after CP administration), and the serum was collected and stored at −20°C until measurement. Then, all animals were killed, and the kidneys were removed, immediately weighed, and fixed in formalin for histopathological procedures.
Measurements
The levels of serum creatinine (Cr), blood urea nitrogen (BUN), total proteins (TP), and magnesium (Mg) were determined using quantitative diagnostic kits (Pars Azmoon Co, Tehran, Iran). The serum level of nitrite (NO stable metabolite) was measured using a colorimetric enzyme-linked immunosorbent assay (ELISA) kit (Promega Corporation, Madison, WI, USA) that involves the Griess reaction. Briefly, after adding sulfanilamide solution and incubation, N-1-naphthyl ethylenediamine dihydrochloride solution was added. Then, absorbance was measured with a microreader, and the nitrite concentration of samples was determined by comparison with the nitrite standard reference curve.
Histopathological Procedures
The removed left kidneys were fixed in 10% neutral formalin solution and embedded in paraffin for histopathological staining. The hematoxylin and eosin stains were applied to examine the tubular damage. The damage was evaluated and graded by two independent pathologists. The kidney tissue damage score (KTDS) was obtained based on the intensity of tubular lesions in each kidney. The presence of acute tubular injuries, such as tubular dilation and simplification, tubular cell swelling and necrosis, tubular casts, and intraluminal cell debris with infiltration of inflammatory cells, was considered. The KTDS was graded from 1 to 4, while the score of zero was assigned to normal tubules without damage.
Statistical Analysis
Data are expressed as mean ± SEM. To compare the percentage changes in body weight; kidney weight; and the serum levels of BUN, Cr, Mg, TP, and nitrite between the groups, one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) test (post hoc multiple comparison) was applied. To compare the KTDSs between the groups, Kruskal–Wallis test and Mann–Whitney U test were used. To determine the correlation between kidney weight and KTDS, the nonparametric Spearman correlation test was used. The values of p < 0.05 were considered as statistically significant.
RESULTS
Effect of CP on Serum BUN and Cr Levels
The serum levels of BUN and Cr significantly increased in CPM and L + CPF groups when compared with the negative control group (p < 0.05). In the presence of AT1 blockade, the serum levels of BUN and Cr in females (not in males) were significantly higher than those in the positive control group (p < 0.05). These data indicate that coadministration of CP and losartan promotes the serum levels of BUN and Cr in females but not in males ().
Figure 1. (A) and (B) The serum levels of BUN and Cr in male and female animals, respectively, treated with losartan + CP, CP (positive control group), and saline (negative control group). (C) The percentage weight changes in male and female animals, respectively. The percentage weight change was calculated as [(last day weight – weight on the CP injection day)/weight on the CP injection day] × 100. The asterisk (*) indicates significant difference (p < 0.05) when compared with the negative control group. The sign (#) indicates significant difference (p < 0.05) when compared with the positive control group.
![Figure 1. (A) and (B) The serum levels of BUN and Cr in male and female animals, respectively, treated with losartan + CP, CP (positive control group), and saline (negative control group). (C) The percentage weight changes in male and female animals, respectively. The percentage weight change was calculated as [(last day weight – weight on the CP injection day)/weight on the CP injection day] × 100. The asterisk (*) indicates significant difference (p < 0.05) when compared with the negative control group. The sign (#) indicates significant difference (p < 0.05) when compared with the positive control group.](/cms/asset/ef2b34cd-ca73-43bf-b281-3a5122f8f37f/irnf_a_700886_f0001_b.jpg)
Effect of CP on Body Weight
The percentage (%) body weight change (from day 3 to day 9) was obtained. All CP-treated animals lost weight during the experiment. Males treated with CP alone (CPM group) and females treated with losartan + CP (L + CPF group) had statistically significant weight changes compared with the negative control groups (p < 0.05). On the contrary, the percentage weight change in males treated with losartan + CP (L + CPM group) was significantly lower than that in the positive control group (p < 0.05, ). Such an observation was not seen in females, when AT1 was blocked. This result indicates that coadministration of CP and losartan promotes weight loss in females more than in males.
Effect of CP on Mg, TP, and Nitrite Levels
No significant changes were observed in serum levels of TP, Mg, and nitrite in all experiment groups ().
Table 1. The serum levels of Mg, TP, and nitrite in both genders.
Effect of CP on Kidney Damage
The CP-induced kidney damage was evaluated and scored by two independent pathologists. These data are presented in . Significantly higher kidney damage was detected in the CP-treated groups when compared with the negative control group in both genders (p < 0.05). However, in the presence of AT1 blockade, the KTDS in females (not in males) was higher than that in the positive control group, but the difference was not statistically significant. Normalized kidney weight (kidney weight/100 g of body weight in the last day) significantly increased in the L + CPF group in comparison with the negative control group (p < 0.05), but no significant difference in KTDSs was observed between the L + CPM and negative control groups. The results also indicate significant correlations between normalized kidney weight and KTDS in both genders (, p < 0.05). The sample images of the kidney tissue for each group are shown in .
Figure 2. (A) and (B) Total kidney weight/100 g of body weight values and KTDSs in male and female animals, respectively, treated with losartan + CP, CP (positive control group), and saline (negative control group). The asterisk (*) indicates significant difference (p < 0.05) in comparison with the negative control group. The sign (#) indicates significant difference (p < 0.05) in comparison with the positive control group. (C) Correlations between total kidney weight/100 g of body weight and KTDS in male and female animals, respectively. Significant correlations (p < 0.05) were observed between KTDS and kidney weight in both genders.
Figure 3. The images (magnification × 100) of the kidney tissue. (A) group 1: male, losartan + CP; (B) group 2: female, losartan + CP; (C) group 3: male, CP; (D) group 4: female, CP; (E) group 5: male, saline; and (F) group 6: female, saline. More tissue damage was observed in female losartan + CP and male CP-treated groups.
DISCUSSION
CP, as an antineoplastic drug, is mainly used in clinic for the treatment of solid tumors in ovary, testis, neck, and head. The most important limitation to the use of CP is its nephrotoxicity.Citation2–4 The main finding of this research was the negative role of losartan as a nephroprotectant in female (not in male) rats when it is accompanied by CP.
It seems that this difference is probably related to RAS, which acts differently in male and female kidneys. RAS plays an important role in renal circulation. Furthermore, renal circulation can potentially be involved in the development of nephrotoxicity induced by CP. The main product of RAS, angiotensin II, binds to main receptors AT1 and AT2.Citation18 The vast majority of angiotensin II actions are mediated via the AT1 receptor,Citation18,19 including antinatriuresis, vascular resistance, growth development, aldosterone secretion, inhibition of thirst, salt appetite, and renin biosynthesis and release.Citation19,20 The AT1 and AT2 receptors are differently distributed in tissues. The AT1 receptor is widely expressed in tissues, while the AT2 receptor is mostly expressed in fetal tissues, and AT1 receptor is expressed more than AT2 receptor after birth.Citation19,21,22 It is reported that the expression of AT2 receptors in females is more than that in males, and actually this receptor is gender relatedCitation16,23 because estrogen partially upregulates AT2 receptorsCitation24 and downregulates AT1 receptor gene expression.Citation25 The impact of Mas receptor on RAS, which has vasodilatation effects, is different in males and females,Citation26,27 and the differences may influence renal circulation. The preventive role of losartan in CP-induced nephrotoxicity could be gender related. The inhibition of AT1 with losartan may cause AT2 and Mas vasodilatation responses in females more than in males.Citation28,29 Therefore, a greater blood flow may transport more CP into the kidney, and causes more tissue damage.
As seen in , the presence of tubular dilation, tubular cell swelling and necrosis, tubular casts, and intraluminal cell debris in the female group treated with losartan and CP is higher than that in the similar male group. Other researches indicate that RAS has an effective role in the pathophysiology of renal diseases and suggest that the inhibition of the RAS through AT1 receptor blockade delays progressive renal failure in patients.Citation30–32 Other studies showed that in animals treated with losartan as prophylaxis, the expression of AT2 receptor significantly increases.Citation33 Likewise, AT2 receptors are upregulated in injured tissues such as in renal failure and vascular injury during inflammation.Citation19,34 Therefore, the intensity of CP-induced nephrotoxicity in females cannot be limited by losartan, due to different expression ratios of AT2 and Mas receptors in females compared with males.
Losartan also has an antioxidant effect,Citation11,12 and the effects of some antioxidants are gender related.Citation35 Accordingly, the antioxidant effect of losartan may also be gender related.
It is concluded that although losartan is a nephroprotectant in CP-induced nephrotoxicity in experimental male animals,Citation14 the coadministration of losartan and CP causes more kidney dysfunction in female rats. Therefore, it is suggested to avoid coadministration of AT1 antagonists with CP in humans until otherwise approved.
ACKNOWLEDGMENT
This research was supported by Isfahan University of Medical Sciences.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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