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Interact CardioVasc Thorac Surg 2008;7:235-239. doi:10.1510/icvts.2007.161356 © 2008 European Association of Cardio-Thoracic Surgery
The influence of levosimendan and iloprost on renal ischemia–reperfusion: an experimental study ye Lafcp OrtacDepartment of Cardiovascular Surgery, Atatürk Education and Research Hospital, Izmir, Turkey Received 2 July 2007; received in revised form 3 October 2007; accepted 4 October 2007
*Corresponding author. 2/11 sok. No: 7 D: 18 Oyak Sitesi, Izmir, Turkey. Fax: +90 232 2434848.
The effects of iloprost on ischemia–reperfusion injury have been studied on the skeletal, muscle, liver, myocardium, kidney, and spinal cord. However, no sufficient data exist about effects of levosimendan on renal ischemia–reperfusion injury. The purpose of this experimental study was to investigate and compare effectiveness of levosimendan and iloprost on renal injury induced by ischemia and reperfusion. Fifty rabbits were divided into five groups. Levosimendan was continuously infused starting half an hour before the cross-clamp. Cross-clamp time was one hour. After one hour ischemia, levosimendan was continued for 4 h in Group A whereas Group B took iloprost in the same protocol. Group C was the control group which did not receive any medication. Group D was sham group and Group E was medicated both iloprost and levosimendan. Renal tissues were histologically and biochemically evaluated. The histological scores were obtained according to presence of tubuler necrosis and atrophy, regenerative atypia, hydropic degeneration (Group A vs. Group C<0.001, Group B vs. Group C<0.001, Group D vs. Group C<0.01, Group E vs. Group C<0.001). Mean malondialdehyde levels were 114±12 nmol/g tissue; in Group A 121±13 nmol/g tissue, in Group B 134±13 nmol/g tissue, in Group E 130±11 nmol/g tissue, in Group D 134±11 nmol/g tissue (Group A vs. Group B; P=0.003, Group B vs. Group D; P=0.132, Group A vs. Group E; P=0.132). Malondialdehyde levels and histologic scores of all of the groups were significantly different from the control group. Iloprost and pentoxyfillin reduced renal ischemia–reperfusion injury in rabbit model. There was no significant difference between these two medications.
Key Words: Iloprost; Levosimendan; Ischemia/reperfusion injury; Rabbit kidney
Reperfusion of a previously ischemic organ triggers a cascade, collectively termed ischemia–reperfusion injury that leads to production of reactive oxygen species and causes to massive secretion of systemic inflammatory mediators [1, 2]. The effects of ischemia–reperfusion can result in development of the systemic inflammatory response syndromes and multiple organ dysfunctions, both of which account for 30–40% of the mortalities in the intensive care units [3, 4]. Iloprost, a stable prostacyclin analog was shown to decrease neutrophil activation and aggregation beside inhibition of oxygen-free radical production and release of lysosomal enzymes [5, 6]. Levosimendan is a recently synthesized positive inotropic drug which improves myocardial contraction without increasing calcium concentration in myocardial cells and has also a vasodilating effect by opening adenosine triphosphate-sensitive potassium channels [7]. It was reported that a single dose levosimendan administration seems to have anti-inflammatory and anti-apoptotic properties, reducing circulating proinflammatory cytokines and soluble apoptosis mediators [8]. The effects of iloprost on ischemia–reperfusion injury have been studied on the skeletal, muscle, liver, myocardium, kidney, and spinal cord [5, 6, 9]. However, no sufficient data exist about effects of levosimendan on renal ischemia–reperfusion injury. The purpose of this experimental study was to investigate and compare effectiveness of levosimendan and iloprost on renal injury induced by ischemia and reperfusion.
2.1. Animal preparation After approval of the study by the local animal care committee, experiments were performed on 50 New Zealand white rabbits of both sexes, each weighing approximately 3 kg. Anesthesia was induced by intramuscular ketamine (50 mg/kg) and xylasine; then followed by 25 mg/kg fractionally as needed during the intervention to allow for spontaneous respiration, and without endotracheal intubation. A catheter (24 gauge) was placed in an ear vein to give maintenance fluid of 0.9% NaCl (20 ml/h). An arterial catheter (20 gauge) was placed in an ear artery to monitor blood pressure (Peta  Kma 375). The room temperature was kept between 28–30 °C. The experiment was done according to the ethics of Pamukkale University Medical School, Denizli, Turkey. The study was funded by the authors. 2.2. Study groups and operative technique Animals were randomly allocated into five groups each consisting of ten rabbits. The experiment was continued until ten animals in each group survived during the entire procedure and postoperative 4 h. In Group A, rabbits received a loading dose of 25 µg/kg levosimendan over 15 min, followed by a continuous infusion of 0.1–0.2 mg/kg/min ceased after renal artery declamping. In Group B, iloprost was infused at a rate of 25 ng/kg/min at the same period. A similar value of saline solution was given in the control group, Group C correspondingly. In sham Group D, the operation was performed in the same fashion, but without artery occlusion, in Group E, both iloprost and levosimendan were infused simultaneously.After sterile surgical preparation, a midline laparotomy was performed, and the abdominal aorta was reached through a transperitoneal approach. The left renal artery was mobilized from the aorta. Each animal was anticoagulated with 150 U/kg heparin intravenously. Renal ischemia was induced by clamping the artery with an arterial bulldog clamp. The clamp was removed after 60 min and restoration of blood flow was verified visually. Abdominal layers were closed and catheters were removed. When the animals awakened from anesthesia, they were taken to their cages. A total of four animals died during the procedure, one at the anesthesia induction, and three in surgical intervention. They were excluded from the study. After 4 h reperfusion, left nephrectomy was done and renal biopsies were taken from the cortex of the kidneys for microscopic, histological and biochemical evaluation. 2.3. Histopathologic evaluation After the last 4th hour reperfusion, animals were anesthetized with intramuscular ketamine, 50 mg/kg in dose, and euthanized by intravenous administration of a high concentration of potassium (K).Portions of the fixed renal biopsy materials were examined microscopically (Nikon model Eclipse E600W). The specimens were fixed in 10% formalin. Paraffin blocks were cut at 5 µm and stained with hematoxylin-eosin. Microscopic renal injury was considered in the presence of tubuler necrosis and atrophy, regenerative atypia, hydropic degeneration, interstitial fibrosis, loss of supranuclear cytoplasms and brush border disappearance. Renal injury was scored semiquantatively according to these characteristics as; grade 0 as normal, grade 1 as mild (focal), grade 2 as moderate (multifocal) and grade 3 as severe (diffuse) pathologic chances (Tables 3, 4) by the same pathologist, who was blinded to the study [23].
2.4. Tissue MDA analysis Lipid peroxidation measurement was measured from the renal cortical malondialdehyde (MDA) content as Uchiama and Mihara described [24]. Renal cortical tissues were homogenized by using Ultra-Turrax T25 homogenisator in 1.15% ice-cold potassium chloride solution containing 50 ml/l of Triton X-100. A 0.5 ml homogenate was mixed with 3 ml of 1% phosphoric acid and 1 ml of 0.6% thiobarbituric acid. The mixture was heated on boiling water for 45 min. After addition of 4 ml of n-butanol, the contents were centrifuged at 4000 gyration per minute for 10 min. The upper organic layer absorbance was measured at 532 nm spectrophotometrically. The tissue MDA levels were expressed as nmoles per gram renal tissue. Statistical analysis was done with SPSS 10.0 statistical software program (SPSS Inc, Chicago, IL). Continuous variables were expressed as the mean±1 S.D. Differences between the experimental and control groups were analyzed for their statistical significance by use of the two-tailed Student t-test. The P-values <0.05 were considered to be statistically significant.
There were no statistically significant differences in the physiological parameters between five groups in preischemic and postischemic periods. Hemodynamic variables of all five groups are shown in Table 1. Preischemic and postischemic mean arterial pressure was minimally lower in iloprost and levosimendan group when compared with control group. Levosimendan was well tolerated in all animals, and mean arterial pressure decreased by 8% from baseline. There was no significant alteration in the heart rate recordings. Table 2 shows mean values and S.D. of pH, pO2, pCO2, HCO3. Blood pH, pO2, and HCO3 levels of medicated groups were found significantly higher than the levels of control group.
Histological evidence of reperfusion injury was the presence of tubuler necrosis and atrophy, regenerative atypia, hydropic degeneration, interstitial fibrosis, loss of supranuclear cytoplasms and brush border disappearance. The mean histopathologic scores of Group A, B, and D, E were significiantly lower than the control group (Group A vs. Group C; P<0.001, Group B vs. Group C; P<0.001, Group D vs. Group C; P<0.001, Group E vs. Group C; P<0.001, Group B vs. Group E; P=0.311, Group A vs. Group E; P=0.312). There was no significant differences between levosimendan and iloprost groups (P=0.315) (Table 4). The MDA levels of the medicated groups were significantly lower than the levels of the control group. Mean malondialdehyde levels were 114±12 nmol/g tissue; in Group A 121±13 nmol/g tissue, in Group B 134±13 nmol/g tissue, in Group E 130±11 nmol/g tissue, in Group D 134±11 nmol/g tissue (Group A vs. Group B; P=0.003, Group B vs. Group D; P=0.132, Group A vs. Group E; P=0.132). Fig. 1 demonstrates a normal renal histology whereas Figs. 2–4 demonstrate abnormal renal morphologies.
Mean MDA level was 35±6 nmol/g tissue in Group C (Group A vs. Group C; P<0.001, Group B vs. Group C; P<0.01, Group E vs. Group C; P<0.001, Group D vs. Group C P<0.001). This showed that the ischemia reperfusion model was well formed in the rabbits.
The results did not confirm the primary hypothesis that levosimendan showed a better protection than iloprost. However, it was proven that levosimendan and iloprost significantly reduced the ischemia/reperfusion injury on the light microscopic basis. It can be said that levosimendan and iloprost significantly improved the ischemia/reperfusion injury in the renal tubules. Reperfusion of a previously ischemic organ triggers a cascade, termed ischemia–reperfusion injury [10]. The reperfusion of endothelial cells results in production of reactive oxygen species, such as hydroxyl radicals, hydrogen peroxide, and superoxide anions, and causes an imbalance which in turn leads to secretion of a systemic inflammatory radical, such as interleukin-1, -8, platelet-activating factor and tumor-necrosis factor alpha [11]. The ischemia–reperfusion process results in leukocyte dependent micro-vascular dysfunction, leukocyte plugging, impaired endothelium-dependent dilatation, enhanced capillary fluid filtration, and plasma protein extravasations all of which affect distal organs [12]. Influence of this process on kidneys is an increase in vascular permeability, leading to the reduction of renal cortical blood flow [13]. Iloprost, a stable prostacyclin analog acts as membrane stabilization and inhibits neutrophil functions which are potential mediators of ischemia–reperfusion injury [9]. Iloprost also decreases white blood cell aggregation and adhesion to vascular endothelium superoxide radical production from stimulated canine and human neutrophils, and free radical formation in myocardium subject to ischemia–reperfusion injury [14]. Emrecan and co-workers reported that iloprost protected the kidneys against ischemia–reperfusion [15]. Our study also reveals that iloprost has a protective effect on kidneys after transient ischemia. Levosimendan is a recently synthesized positive inotropic drug that improves myocardial contraction without increasing calcium concentration in myocardial cells and has also a vasodilatation effect by opening adenosine triphosphate-sensitive potassium channels [7]. It was reported that a single dose of levosimendan administration seems to have anti-inflammatory and anti-apoptotic properties, reducing circulating proinflammatory cytokines and soluble apoptosis mediators [8]. Animal studies have demonstrated that the vasodilatation by levosimendan may be partially related to lowering of intracellular free calcium through potential inhibition of phosphodiesterase III, calcium desensitization, or opening of adenosine triphosphate-sensitive potassium channels [15–18]. However, no sufficient data exist about the role of levosimendan on kidneys after ischemia–reperfusion. Ischemia–reperfusion injury attributed to the development of reactive oxygen species mediated lipid peroxidation, and this process can be measured through its products like malondialdehyde (MDA) [19–22]. In this study, a cortical MDA level was significantly lower in iloprost and levosimendan groups than in control group. These low MDA levels in this recent study demonstrated that lipid peroxidation was avoided in the iloprost and levosimendan groups. There were no significant differences between iloprost and levosimendan treated groups.
According to our results, iloprost and levosimendan are useful therapeutic agents for the treatment of acute renal ischemic conditions.
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Abstract
1. Introduction
Mβ2 integrin. Eur J Clin Invest 1996;26:860–866.
lu H, Ormen M, Atila K, Sario