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The protective activity of noscapine on renal ischemia-reperfusion injury in male Wistar rat.

Objective(s): Bradykinin is a part of the kinin-kallikrein system which is involved in ischemia-reperfusion injury via B1 and B2 receptors. Noscapine is a non-competitive antagonist of bradykinin receptors. Noscapine has been reported to to be able to protect some organs against ischemia-reperfusion injury but its effect on renal ischemia-reperfusion injury (RIR) in rats is unknown. Therefore, the present study was designed to evaluate the effect of noscapine on renal ischemia-reperfusion injury in rats. Materials and Methods: Twenty four rats were randomly assigned to four groups; sham, RIR control, pre-and post-treatment with noscapine. To induce RIR injury, 20 days after right nephrectomy, animals underwent a midline laparotomy and the renal artery was clamped for 40 min to induce ischemia, and the clamp was then removed to allow reperfusion for 48 hr. Animals received noscapine or vehicle 1 hr before RIR or just prior to reperfusion. At the end of the experiment, animals were killed by cardiac exsanguination. Blood samples were collected to assess blood urea nitrogen (BUN) and creatinine. The kidneys were also removed for histopathlogical and western-blot analysis. Results: Noscapine treatment 1 hr before RIR or just prior to reperfusion protects the renal tissue structure as compared with the control. The expression levels of the studied inflammatory mediators, TNF-α and MCP-1in pretreated-, and treated-noscapine groups decreased as compared with the control group. The levels of BUN and creatinine in pre-, and post-treated noscapine groups were significantly lower than in control animals. Conclusion: Noscapine protects renal tissue structure and function against RIR through down-regulation of the inflammatory mediators.


Introduction
One of the most common reasons for renal ischemia-reperfusion (RIR) is renal transplantation (1). It has been shown that ischemia-reperfusion (I/R) injury affects a variety of renal cells such as tubular epithelial cells and endothelial cells. Following the activation of the inflammatory reactions during I/R injury, tumor necrosis factor-α (TNF-α) and interleukin 1β (IL-1β) are released (2). There are many reports about the effect of the activation of bradykinin receptor (B1 and B2) on I/R injuries in different organs. Many reports have shown that bradykinin plays a deleterious effect in the lung (3), liver (4) and intestine (5,6) after I/R injury. Additionally, early activation of B2 receptor has been shown to increase the production of reactive oxygen species and disturb renal function following RIR in rats (7).
Noscapine is an iso-qinulinin alkaloid and a noncompetitive bradykinin receptors antagonist which has been shown to reduce cerebral edema after brain hypoxic ischemia (8).The effect of noscapine on RIR is unknown. Therefore, the present study was designed to evaluate the effect of noscapine on RIR injury in male Wistar rats.

Animals
Male Wistar rats (body weight: 220-280 g) were purchased from the animal house of Institute Pasteur, Tehran, Iran. The animals were fed on  (1-4) ×400 magnifcation. Asterisk indicates a significant decrease (*P<0.01) as compared to the control group. β indicates a significant increase ( β P<0.001) as compared to sham-operated group. Data are expressed as mean±SEM conventional diets and had free access to water. They were maintained under standard conditions of humidity, temperature (22±2°C) and light/dark cycle (12 hr: 12 hr). All experiments were carried out in accordance with the ethics committee of Iran University of Medical Sciences, Tehran, Iran.

Induction of renal ischemia-reperfusion injury
Animal in all experimental groups were anesthetized using intraperitoneally administered pentobarbital (50 µg/kg) and then underwent right nephrectomy. To induce RIR injury, 20 days after right nephrectomy, animals underwent a midline laparotomy, and the renal pedicle was carefully isolated. Then, the renal artery was clamped for 40 min using an arterial clamp to induce ischemia, and the clamp was then removed to allow reperfusion for 48 hr. Sham-operated rats underwent laparotomy without inducing RIR injury. In all groups, the abdominal incision was double-sutured immediately after beginning reperfusion (7).

Animal grouping and treatment
Twenty four male Wistar rats were randomly assigned to 4 groups; sham, RIR control, pre-and post-treatment with noscapine. Sham-operated and RIR control rats received saline (0.5 ml, IP); pretreated noscapine animals received noscapine 2 mg/kg (IP) 1 hr before RIR and post-treated animals received noscapine 2 mg/kg (IP) just prior to reperfusion. Forty hours after RIR, animals were killed by cardiac exsanguination. To assess the renal function, blood samples were collected in chilled tubes and were centrifuged at 3500 rpm for 10 min. Serum samples were kept at -20°C until the measurement of creatinine. Pars Azmoon (Iran) kits were used to measure the serum level of creatinine and blood urea nitrogen (BUN). One part of the explanted kidney was immediately snap frozen in liquid nitrogen and stored at -80°C until protein analysis and the other part was fixed in 10% formalin for histologic evaluation.

Histological evaluation
For histological evaluation, as mentioned earlier, the explanted kidneys from all studied groups were fixed in 10% formalin, kept for 3 weeks, dehydrated in graded ethanol, and embedded in paraffin. Thereafter, sections of tissue were cut at 4 µm using a microtome, stained with hematoxylin and eosin. Tubular damage was scored using a scoring system ranging 0-3 by an observer who was unaware of the study. The score was as follows: normal=0; mild=1; moderate=2 and severe=3. The following variables were assessed: casts formation, tubular dilation swelling, diffuse intertitial edema, epithelial detachment, expansion of Bowman's capsule, loss of nuclei and loss of brush border (9).

Protein extraction and western blot analysis
The frozen kidney tissue was homogenized in homogenization buffer containing protease inhibitors and protein extracted using TriPure isolation reagent (Roche Diagnostics, Germany). The concentration of the extracted proteins was determined using Bradford method and the protein expression was evaluated by western blotting as previously described (10). The protein pellets were mixed with SDS-buffer, boiled for 5 min and then centrifuged at 15000 rpm for 5 min at 4°C. The extracted proteins were separated using SDS-PAGE on 12% acrylamide gels and transferred to a PVDF membrane. The membranes were blocked with 5% non-fat dry milk dissolved in tris-buffered saline with 0.1% tween 20 (TBST, pH 7.6) for 3 hr and then incubated overnight at 4°C with anti-MCP-1; monocyte chemoattractant protein-1[ab7202] (dilution 1:1000; Abcam, USA), anti-TNF-α antibody (ab66579) (dilution 1:1000; Abcam, USA), or anti-β-actin antibody [ab20272] (dilution 1:5000; Abcam, USA). After three times washing with TBST, the secondary antibody was applied for 1 hr at room temperature. Goat Anti-Rabbit IgG H&L (HRP) (ab6721, USA) was used as the secondary antibody for TNF-α, MCP-1and β-actin. Band intensity was measured densitometrically using UV.doc software (Bio-Rad, USA) and the values were normalized to ß-actin.

Statistical analysis
Data are shown as mean ± SEM Statistical analysis was performed by one-way ANOVA and followed by post hoc Tukey's test. Significance was set at a P<0.05 level.

Noscapine protects the renal tissue after RIR
Forty min renal ischemia followed by 48 hr reperfusion significantly increased cast formation, tubular dilation swelling, diffuse intertitial edema, epithelial detachment and expansion of Bowman's capsule as compared with the sham-operated rats (Figure1 A-B; P<0.001). As shown in Figures 1C and D, administration of noscapine just prior to reperfusion and also one hour before RIR protects the renal tissue structure. Cast formation, tubular dilation swelling, diffuse intertitial edema, epithelial detachment and expansion of Bowman's capsule in noscapine-treated and pretreated animals were significantly lower than those of control group (P<0.01).

Administration of noscapine decreased the level of inflammatory mediators on RIR in rats
According to Figure 2 A and B, the results of western blotting analysis showed that administration of noscapine just prior to reperfusion and also 1 hr before RIR, decreased the expression of MCP-1 and TNF-α (P<0.01).

Administration of noscapine improves the renal function after RIR in rats
The reults showed that 48 hr after RIR, serum creatinine and BUN were significantly increased in control rats as compared with the sham-operated animals (P<0.001, Figures 3A and B). Noscapine treatment improves the renal function. As shown in Figures 3A and B, serum creatinine and BUN levels were significantly decreased in noscapine-treated and pretreated rats as compared with the control rats.

Discussion
The protective effects of bradykinin receptor antagonists [FR173657 and Hoe 140] have been shown in different models of tissue injuries such as the intestine and renal ischemia/reperfusion injuries (6,7). Noscapine, a non-competitive bradykinin receptors antagonist, has been shown to decrease brain edema and damage against hypoxic/ischemic insult in neonatal rats (8). Moreover, it has been reported that oral administration of noscapine to acute ischemic stroke patients improves clinical prognosis and decreases the mortality rates (12). Other well-known beneficial effects of noscapine are antitussive (15) and anti-tumor activity (13,14,16,17). Consistent with these reports, the present study showed the protective role of noscapine against RIR injury in male Wistar rats, for the first time. On the basis of the histological analysis, inhibition of bradikinin receptors by an intraperitoneal injection of noscapine 1 hr before RIR as pretreatment and also just prior to reperfusion as treatment, protects the renal tissue structure against RIR injury in rats ( Figures 1A and B).
Tumor necrosis factor-α has been shown to be secreted at early stages of ischemia-reperfusion injuries and mediate the induction of other chemokines such as monocyte chemoattractant protein1 (MCP-1) to attract leukocytes migrating to the inflammatory site which finally results in the inflammation (18). Additionally, it has been reported that over expression of TNF-α stimulates the expression of chemokines like MCP-1 (19). Moreover, it has been reported that MCP-1 contributes to the recruitment of macrophages which is associated with glomerulosclerosis and fibrosis (20). Jiang et al reported that a single administration of mycophenolate mofetil, a relatively novel immunosuppressant, improved the renal function and inhibited the formation of fibrosis in I/R injury in Sprague-Dawley rats which may be due to a decrease in MCP-1 and TGF-β1 expression as well as macrophage infiltration (21). Also, Jia et al showed that short hairpin RNA of endothelin A receptor, ETaR shRNA, preserve the renal function in RIR in rats. In their model, shRNA down-regulated ETaR which in turn reduces the mRNA expression of ET-1 as well as other cytokines and chemokines including MCP-1 and TNF-α subsequently improved renal function and structure (22). Moreover, Chiang and his co-workers also showed that Hoe 140, an antagonist of bradykinin receptors, protects the renal function against RIR by down-regulating the gene expression of MCP-1 and TNF-α, parallel with reducing their plasma levels (7). Consistent with these results, our findings showed that a single injection of noscapine decreased the expression of TNF-α and MCP-1 which proposes the reduction in expression of inflammatory cytokine and chemokine as a possible mechanism for the renoprotective activity of noscapine. Therefore, it can be concluded that the renoprotective effect of noscapine on I/R injury is partly mediated by down-regulation of the inflammatory protein expression.
In the present study, in order to assess the protective effect of noscapine on renal function, we measured the levels of BUN and creatinine. Forty eight hours after RIR, the levels of BUN and creatinine in control animals were significantly higher than those in sham-operated rats which shows that the renal function was impaired by I/R injury ( Figures 3A and B). According to Figures 3 A  and B, the administration of noscapine just prior to reperfusion and 60 min before the onset of ischemia significantly reversed these levels. These findings suggest that noscapine effectively downregulates the expression of inflammatory mediators and subsequently improves the renal tissue structure and renal function. The findings also showed that the levels of BUN and creatinine in noscapine-pretreated rats that received it 60 min before the onset of I/R were significantly lower as compared with noscapine-treated animals. Therefore, to achieve better protective results, the time of noscapine administration is an important factor. We suggest that earlier inhibition of bradykinin receptors can be more effective. Consistent with these results, Chiang and his co-workers showed that early activation of bradykinin receptors by kallikrein had more deleterious effects in the corresponding animals on renal tissue function and structure as compared with the kallikrein-treated rats with RIR (7).

Conclusion
The present study, showed the renoprotective effect of noscapine on I/R injury, for the first time. The findings of this study demonstrated that: Pretreatment with noscapine improved the levels of BUN and creatinine and the renal function following RIR.
The protein expression of TNF-α and MCP-1 in noscapine-pretreated rats were lower as compared with control rats.