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reperfusion injury in rats
Regulatory Peptides 140 (2007) 101 – 108 www.elsevier.com/locate/regpep
The protective effect of oxytocin on renal ischemia/reperfusion injury in rats Halil Tuğtepe a,⁎, Göksel Şener b , Neşe Karaaslan Bıyıklı c , Meral Yüksel d , Şule Çetinel e , Nursal Gedik f , Berrak Ç. Yeğen g a
Marmara University, School of Medicine, Department of Pediatric Surgery, Istanbul, Turkey Marmara University, School of Pharmacy Department of Pharmacology, Istanbul, Turkey c Marmara University, School of Medicine, Department of Pediatric Nephrology, Istanbul, Turkey d Marmara University, Vocational School of Health Related Professions, Istanbul, Turkey Marmara University, School of Medicine, Department of Histology and Embryology, Istanbul, Turkey f Kasimpasa Military Hospital, Division of Biochemistry, Istanbul, Turkey g Marmara University, School of Medicine, Department of Physiology, Istanbul, Turkey b
e
Received 7 September 2006; received in revised form 9 November 2006; accepted 11 November 2006 Available online 29 January 2007
Abstract Aim: Oxytocin was previously shown to have anti-inflammatory effects in different inflammation models. The major objective of the present study was to evaluate the protective role of oxytocin (OT) in protecting the kidney against ischemia/reperfusion (I/R) injury. Materials and methods: Male Wistar albino rats (250–300 g) were unilaterally nephrectomized, and subjected to 45 min of renal pedicle occlusion followed by 6 h of reperfusion. OT (1 mg/kg, ip) or vehicle was administered 15 min prior to ischemia and was repeated immediately before the reperfusion period. At the end of the reperfusion period, rats were decapitated and kidney samples were taken for histological examination or determination of malondialdehyde (MDA), an end product of lipid peroxidation; glutathione (GSH), a key antioxidant; and myeloperoxidase (MPO) activity, an index of tissue neutrophil infiltration. Creatinine and urea concentrations in blood were measured for the evaluation of renal function, while TNF-α and lactate dehydrogenase (LDH) levels were determined to evaluate generalized tissue damage. Formation of reactive oxygen species in renal tissue samples was monitored by chemiluminescence technique using luminol and lucigenin probes. Results: The results revealed that I/R injury increased (p b 0.01–0.001) serum urea, creatinine, TNF-α and LDH levels, as well as MDA, MPO and reactive oxygen radical levels in the renal tissue, while decreasing renal GSH content. However, alterations in these biochemical and histopathological indices due to I/R injury were attenuated by OT treatment (p b 0.05–0.001). Conclusions: Since OT administration improved renal function and microscopic damage, along with the alleviation of oxidant tissue responses, it appears that oxytocin protects renal tissue against I/R-induced oxidative damage. © 2006 Elsevier B.V. All rights reserved. Keywords: Oxytocin; Ischemia/reperfusion; Kidney; Lipid peroxidation; Myeloperoxidase; Glutathione
1. Introduction Ischemia/reperfusion injury (I/R) of the kidney, which occurs during renal transplantation, surgical revascularization of the renal artery, treatment of suprarenal aortic aneurysms [1,2], is a complex pathophysiological process and a major cause of acute renal failure, which initiates a complex sequence of events [3]. Although reperfusion is essential for the survival of ischemic ⁎ Corresponding author. Mazharbey Evsan Sok, Aytac Ap. No: 20/6, Göztepe, İstanbul, Turkey. Tel.: +90 216 428 02 50; fax: +90 216 325 72 17. E-mail address: [email protected] (H. Tuğtepe). 0167-0115/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2006.11.026
renal tissue, the prognosis of the organ dysfunction is further complicated by the additional damage due to the formation of oxygen-derived free radicals [4]. The mechanisms underlying I/R damage to kidneys are most likely multifactorial and interdependent, involving hypoxia, vascular endothelial injury, inflammatory responses, radical-induced damage, tubular obstruction, apoptosis and endothelial–epithelial cell dysfunction [5]. A better understanding of the cellular and molecular mechanisms of the injury is required for the improvement of current therapeutical approaches. Ischemic acute renal failure, primarily due to the breakdown of the cellular junctional complexes, leads to detachment of tubular cells from the extracellular matrix [6]. In cultured
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kidney cells, ischemia was shown to result in disruption and dysregulation of the actin cytoskeleton, and loss of intercellular and cell-to-extracellular matrix interactions and depletion of adenosine triphosphate [7]. The interactions between leukocytes and resident tissue cells in host defense and inflammation highlight many of the complexities of cell–cell and cell–matrix interactions. Leukocyte adhesion is an important event in leukocyte recruitment as it causes dynamic interactions among cells that promote antigen presentation, leukocyte activation, transcellular generation of inflammatory mediators and maintenance phases of inflammation [8–11]. Indeed, leukocyte adhesion has emerged as an attractive target for specific pharmacologic interventions in renal inflammatory diseases including ischemic acute renal failure. Recent studies of I/R injury have focused on neutrophil function, the actions of inflammatory cytokines, tissue factor and oxygen free radicals [12]. The activated neutrophils release reactive oxygen metabolites and cytotoxic proteins (e.g. proteases, myeloperoxidase, lactoferrin) into the extracellular fluid [13]. Studies concerning the preventive and inflammationdiminishing effects of antioxidants and anti-inflammatory peptides are gaining attention in I/R injury of the kidney. Most studies have shown the protective effects of antioxidant in I/R injury in the kidney [14–19]. Oxytocin (OT), a nonapeptide produced in the paraventricular and supraoptical nuclei in the hypothalamus, exerts a wide spectrum of central and peripheral effects. OT is mainly associated with uterine contraction during parturition and milk ejection reflex during lactation. It has also effects on cardiovascular and hydroelectrolitic regulation, various behaviors such as maternal, social and sexual behavior, as well as modulation of release of adenohypophyseal hormones [14,20– 23]. OT receptors have also been identified in other tissues, including the kidney, heart, thymus, pancreas and adipocytes [24]. OT was recently shown to facilitate wound healing and modulate the immune and inflammatory processes, since OT and OT receptors are found in the thymus and OT receptors contain response elements for acute phase reactants and interleukins [25]. It was found that OT may decrease the release of some interleukins and increase the survival of ischemic skin flaps in rats via the activation of the growth factors or antiinflammatory mechanisms [26]. In relation to this background, the present study was designed to investigate the possible protective effect of OT against I/R injury in the kidney, using functional, biochemical and histological parameters for the evaluation of the extent of oxidative damage. 2. Materials and methods 2.1. Animals Male Wistar rats (250–300 g) were housed in an air-conditioned room with 12-hour light and dark cycles, where the temperature (22 ± 2 °C) and relative humidity (65–70%) were kept constant. Rats were fed with standard laboratory chow with free access to water. All experimental protocols were approved by the Marmara University School of Medicine Animal Care and Use Committee.
2.2. Surgery and experimental design Under anesthesia (100 mg/kg ketamine and 0.75 mg/kg chlorpromazine; intraperitoneally; ip), an upper abdominal midline incision was made and right nephrectomy was performed. The left renal pedicle was occluded for 45 min to induce ischemia and then subjected to reperfusion for 6 h (I/R groups). The rats were treated with either oxytocin (OT; 1 mg/kg; ip; Ibrahim Ethem Ulagay Ilaç AŞ, Istanbul) or saline at 30 min prior to ischemia, and the dose was repeated immediately before the reperfusion period (OT-treated I/R or I/R groups, respectively). None of the animals died during the I/R period. Another group of rats underwent only laparotomy, where the kidneys were manipulated without nephrectomy or occlusion, and they were treated with either saline (control group) or OT (OT group) at similar time points. 2.3. Biochemical analysis The animals were decapitated at 6 h of reperfusion, and trunk blood samples were collected for the analysis of serum urea, creatinine, TNF-α and LDH levels. Renal tissue samples obtained from each animal were stored at − 70 °C for the measurement of malondialdehyde, glutathione and myeloperoxidase activity, and luminol and lucigenin chemiluminescences. Additional kidney samples were placed in 10% formaldehyde for histopathological evaluation of renal injury and for the determination of tissue collagen content. 2.4. Measurement of serum LDH, urea, creatinine and TNF-α levels Serum LDH [27], urea [28] and creatinine [29] levels were determined spectrophotometrically using an automated analyzer. Serum levels of tumor necrosis factor alpha (TNF-α) were quantified according to the manufacturer's instructions and guidelines using enzyme-linked immunosorbent assay (ELISA) kit specific for rats (Biosource International, Nivelles, Belgium). This particular assay kit was selected because of its high degree of sensitivity, specificity, inter- and intraassay precision, and small amount of sample required to conduct the assay. TNF-α in the serum samples was expressed as pg/ml. 2.5. Renal malondialdehyde (MDA) and glutathione (GSH) assays Tissue samples were homogenized with ice-cold trichloroacetic acid (1 g tissue plus 10 ml 10% TCA) in an Ultra Turrax tissue homogenizer. The MDA levels were assayed for products of lipid peroxidation by monitoring thiobarbituric acid reactive substance formation as described previously [30]. Lipid peroxidation was expressed in terms of MDA equivalents using an extinction coefficient of 1.56 × 105 M− 1 cm− 1 and the results are expressed as nmol MDA/g tissue. Glutathione measurements were performed using a modification of the Ellman procedure [31]. Briefly, after centrifugation at 2000 g for 10 min, 0.5 ml of supernatant was added to 2 ml of 0.3 mol/l Na2HPO4·2H2O solution. A 0.2 ml solution of dithiobisnitrobenzoate (0.4 mg/ml
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1% sodium citrate) was added and the absorbance at 412 nm was measured immediately after mixing. Glutathione levels were calculated using an extinction coefficient of 1.36 × 105 M− 1 cm− 1. The results are expressed in μmol GSH/g tissue.
Table 2 Luminol and lucigenin chemiluminescence levels in control and I/R groups treated with either saline or oxytocin Chemiluminescence (rlu/mg)
Control
OT
I/R + saline
I/R + OT
2.6. Measurement of myeloperoxidase activity
Luminol Lucigenin
10.3 ± 0.6 15.1 ± 1.1
11.8 ± 0.7 15.4 ± 1.4
18.5 ± 2.4⁎⁎ 26.3 ± 1.5⁎⁎⁎
14.10 ± 0.8+ 18.3 ± 1.6++
Activity of tissue MPO, an enzyme that is found predominantly in the azurophilic granules of polymorphonuclear leukocytes (PMN), correlates with the number of PMN determined histochemically in inflamed tissues and, thus, it is used as an indication of accumulation of neutrophils [32]. Tissue MPO activity was assessed by measuring the H2O2-dependent oxidation of o-dianizidine 2HCl. Briefly, tissue samples (0.2–0.3 g) were homogenized in 10 vol of ice-cold potassium phosphate buffer (50 mmol/l K2HPO4, pH 6.0) containing hexadecyltrimethylammonium bromide (HETAB; 0.5%, w/v). The homogenate was centrifuged at 41,400 g for 10 min at 4 °C, and the supernatant was discarded. The pellet was then rehomogenized with an equivalent volume of 50 mmol/l K2HPO4 containing 0.5% (w/v) hexadecyltrimethylammonium bromide and 10 mmol/l ethylenediaminetetraacetic acid (EDTA, Sigma). MPO activity was assessed by measuring the H2O2-dependent oxidation of o-dianizidine 2HCl. One unit of enzyme activity was defined as the amount of the MPO present per gram of tissue weight that caused a change in absorbance of 1.0/min at 460 nm and 37 °C [33].
Each group consists of 8 rats. ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001; compared with control group. + p b 0.05, ++p b 0.01; compared with saline-treated I/R group.
1,4-phthalazinedione) were obtained from Sigma (St. Louis, MO). Measurements were made at room temperature using Junior LB 9509 luminometer (EG&G Berthold, Germany). Specimens were put into vials containing PBS-HEPES buffer (0.5 M PBS containing 20 mM HEPES, pH 7.2). ROS were quantitated after the addition of enhancers such as lucigenin or luminol for a final concentration of 0.2 mM. Luminol detects a group of reactive species, i.e. UOH, H2O2, HOCl radicals and lucigenin is selective for O2−. Counts were obtained at 1 min intervals and the results were given as the area under curve for a counting period of 5 min. Counts were corrected for wet tissue weight (rlu/mg tissue) [35]. 2.9. Histopathological examination For light microscopic investigations, renal tissue specimens were fixed in 10% formaldehyde, dehydrated in alcohol series,
2.7. Tissue collagen measurement Tissue collagen was measured as a free radical-induced fibrosis marker. Tissue samples were cut with a razor blade, immediately fixed in 10% formalin then samples were embedded in paraffin, and sections, approximately 15 μm thick were obtained. The evaluation of collagen content was based on the method published by Lopez De Leon and Rojkind [34], which is based on selective binding of the dyes Sirius Red and Fast Green FCF to collagen and noncollagenous components, respectively. Both dyes were eluted readily and simultaneously by using 0.1 N NaOH–methanol (1:1, v/v). Finally, the absorbances at 540 and 605 nm were used to determine the amount of collagen and protein, respectively. 2.8. Measurement of luminol and lucigenin chemiluminescences To assess the role of reactive oxygen species in I/R-induced tissue damage, luminol and lucigenin chemiluminescences were measured as indicators of radical formation. Lucigenin (bis-Nmethylacridiniumnitrate) and luminol (5-amino-2,3-dihydroTable 1 Blood urea nitrogen (BUN) and serum creatinine levels of experimental groups
Creatinine BUN
Control
OT
I/R + saline
I/R + OT
0.4 ± 0.06 21.6 ± 5.2
0.52 ± 0.22 29.0 ± 9.0
8.61 ± 1.79⁎⁎⁎ 123.3 ± 5.8⁎⁎⁎
1.73 ± 0.13++ 84.2 ± 7.0⁎⁎⁎,++
OT: Oxytocin; I/R: Ischemia/reperfusion. ⁎⁎⁎p b 0.001, compared to sham-operated control group. ++ p b 0.01, compared to saline-treated I/R group.
Fig. 1. a) TNF-α and b) lactate dehyrogenase (LDH) levels in serum samples of control (C), oxytocin-treated control (OT), saline-treated ischemia/reperfusion (I/R) and oxytocin-treated I/R (I/R + OT) groups. Each group consists of 8 animals. ⁎⁎⁎: p b 0.001 versus control group; +++: p b 0.001 versus I/R group.
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Fig. 2. a) Malondialdehyde (MDA) and b) glutathione (GSH) levels in the kidney tissue of control (C), oxytocin-treated control (OT), saline-treated ischemia/ reperfusion (I/R) and oxytocin-treated I/R (I/R+ OT) groups. Each group consists of 8 animals. ⁎⁎⁎p b 0.001; compared to control group. ++p b 0.01, +++p b 0.001; compared to saline-treated I/R group.
( p b 0.01 and 0.001). Chemiluminescence levels in the kidney samples detected by both luminol and lucigenin probes showed significant increases in the saline-treated I/R group as compared to the chemiluminescence levels of the control group (p b 0.001; Table 2). On the other hand, OT treatment in the I/R group decreased the elevations in chemiluminescence values of luminol and lucigenin (p b 0.05–0.01). In the I/R group treated with saline, serum TNF-α level showed a dramatic increase (53.6 ± 5.2 pg/ml) as compared to control group (8.8 ± 1.2 pg/ml, p b 0.001) and OT administration reduced the concentration of this inflammatory cytokine significantly (31.7 ± 2.9 pg/ml, p b 0.001; Fig. 1A). Similarly, serum LDH activity was found to be higher in the I/R group (4833 ± 230 U/l) compared to control (1888 ± 212 U/l, p b 0.001), indicating generalized tissue damage, while OT treatment attenuated the I/R-induced LDH response (1697 ± 64 U/l, p b 0.001; Fig. 1B). The renal tissue MDA content in the control group (19.8 ± 2.4 nmol/g) was elevated by I/R injury (49.1 ± 5.4 nmol/g, p b 0.001); however, OT treatment significantly decreased the I/ R-induced elevation in renal MDA level (26.5 ± 2.6 nmol/g, p b 0.001; Fig. 2A). In accordance with that, ischemia and reperfusion caused a significant decrease in renal GSH level (0.4 ± 0.06 μmol/g; p b 0.001) when compared to control group (1.5 ± 0.11 μmol/g), while in the OT-treated I/R group, renal GSH content was found to be preserved (1.2 ± 0.15 μmol/g;
cleared in toluene and embedded in paraffin. Paraffin sections (5 μm) were stained with hematoxylin and eosin (H&E) and examined under a photomicroscope (Olympus BH 2, Tokyo, Japan). All tissue sections were examined microscopically for the characterization of histopathological changes by an experienced histologist who was unaware of the treatment conditions. 2.10. Statistical analysis Statistical analysis was carried out using GraphPad Prism 3.0 (GraphPad Software, San Diego, CA, USA). Each group consisted of 8 animals. All data were expressed as means± S.E.M. Groups of data were compared with an analysis of variance (ANOVA) followed by Tukey's multiple comparison tests. Values of p b 0.05 were regarded as significant. 3. Results Regarding all the studied parameters, oxytocin treatment in the sham-operated control group was found to be not different than the saline-treated control group. The blood urea nitrogen (BUN) and creatinine levels in the saline-treated I/R group were found to be significantly higher than those in the control rats ( p b 0.001; Table 1). When OT was administered before ischemia and the subsequent reperfusion period, the elevations in BUN and serum creatinine levels were significantly depressed
Fig. 3. a) Myeloperoxidase (MPO) activity and b) collagen content of control (C), oxytocin-treated control (OT), saline-treated ischemia/reperfusion (I/R) and oxytocin-treated I/R (I/R + OT) groups. Each group consists of 8 animals. ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001; compared to control group. +p b 0.05 compared to saline-treated I/R group.
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level (9 ± 1.1 U/g, p b 0.01), which was found to be not different than that of the control group. Collagen content of renal tissue was found to be significantly higher in the I/R group (24.2 ± 1.1 μg/mg protein) with respect to the control group (18.1 ± 0.8 μg/mg; Fig. 3B). OT treatment, however, significantly abolished the increase in the renal tissue collagen content (19.2 ± 1.3 μg/mg), which was found to be close to control values. Light microscopic evaluation of the control groups, treated with either OT or saline, revealed a regular morphology of renal parenchyma with well-designated glomeruli and tubuli (Fig. 4A). In the saline-treated I/R group, the interstitial hemorrhage, dilated tubuli and prominent glomerular degeneration followed by atrophy revealed that I/R caused a severe glomerular, tubular and interstitial damage. The tubular dilation was present throughout the tissue (Fig. 4B). In the OT-treated I/R group, there was a significant regeneration in all the features of the injury. The reduced tubular dilation, loss of the interstitial hemorrhage and glomerular atrophy were the features indicating regeneration (Fig. 4C). 4. Discussion
Fig. 4. a) Control kidney, regular morphology with glomeruli (arrows) and tubuli (⁎), b) saline-treated I/R group, prominent interstitial hemorrhage (arrows) and tubular degeneration (⁎), c) OT-treated I/R group, with reduction in the interstitial hemorrhage (arrows) and tubular degeneration (⁎), H&E × 200.
p b 0.01), being not significantly different from that of the control group (Fig. 2B). Myeloperoxidase activity, which is accepted as an indicator of neutrophil infiltration, was significantly higher in the kidney tissue of the saline-treated I/R group (15.5 ± 2.1 U/g, p b 0.001) than that of the control group (6.6 ± 0.6 U/g; Fig. 3A). On the other hand, OT treatment in the I/R group significantly decreased renal tissue MPO
The results of the present study reveal that OT reduces I/R injury in rat kidney, with improved renal function, as evidenced by decreased serum creatinine and BUN levels, normal renal histopathology, improved antioxidant status (increased levels of GSH) along with decreases in lipid peroxidation (reduced MDA), PMN infiltration (reduced MPO activity) and reactive oxygen species (reduced lucigenin). Moreover, increases in serum TNF-α and LDH levels, which play important roles in systemic inflammatory processes, were also attenuated by oxytocin treatment. Renal I/R injury is the major cause of acute renal failure in both native and transplanted organs. The severity of the injury depends on the duration of ischemia and subsequent reperfusion periods. Reperfusion, although essential for the survival of ischemic renal tissue, causes additional damage. Acute ischemia leads to the activation of the endothelium with an increase in permeability and expression of different adhesion molecules [36] and elicits an acute inflammatory response characterized by activation of neutrophils [37]. Activated neutrophils induce tissue injury through the production and release of reactive oxygen metabolites and cytotoxic proteins (e.g. proteases, myeloperoxidase, lactoferrin) into the extracellular fluid [38], which constitute the inflammatory cascades that trigger the radical-induced I/R injury. In the present study, renal I/R caused an elevation in tissue MPO activity indicating the presence of enhanced PMN recruitment in the inflamed tissue, while the increased renal malondialdehyde level, an indicator of lipid peroxidation, verified the oxidative damage in the renal tissue. In our study, oxytocin administration before I/R prevented both oxidative renal injury and tissue neutrophil accumulation. A possible mediator behind the decrease in MPO activity in response to oxytocin could be nitric oxide [39]. Nitric oxide, released by oxytocin, may inhibit the adhesion and accumulation of neutrophils [40–42]. On the other hand, GSH, which provides a cellular defense against oxidative injury, is frequently used as a measure of tissue antioxidant status. Therefore, reduced GSH
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levels shown in the present study may be considered as a sign of diminished antioxidant pool in the renal tissue with I/R injury. The replenishment of renal GSH content by OT therapy may be considered as the antioxidant function of OT. Our recent studies confirmed the antioxidant property of OT in colitis-, pyelonephritis- and sepsis-induced inflammatory models [43–45]. It was also shown that in brain membranes OT displayed antioxidant properties in aqueous medium, scavenging free peroxyl radicals, preventing LDL oxidation and inhibiting lipid peroxidation [46]. In accordance with these, the present study supports the antioxidant effect of OT on the renal tissue through the inhibition of tissue neutrophil accumulation and associated production of reactive oxygen species. Much of the tubular and glomerular dysfunction in acute renal failure is due to I/R and is postulated to occur during the reperfusion period following anoxia. Generation of oxygenderived radicals has been considered as one of the major factors contributing to this reperfusion injury [47]. Reactive oxygen species can generate hypochlorous acid in the presence of neutrophil-derived MPO and initiate the deactivation of antiproteases and activation of latent proteases, which lead to tissue damage [38]. In addition, oxidative stress stimulates the accumulation of macrophages and monocytes in I/R kidney. Peripheral monocytes infiltrating the kidney have traditionally been considered the primary source of renal TNF. It has been shown that exogenous TNF induces renal cell apoptosis, glomerular endothelial damage, fibrin deposition, cellular infiltration and renal failure [48,49]. In this study, the tissue associated MPO activity and serum TNF-α levels were increased in I/R injury. The reversal of these parameters by oxytocin treatment suggests that the mechanism of the protective effect of oxytocin involves the inhibition of inflammatory cell infiltration and the release of TNF-α. In the present study, acute renal failure that occurred following I/R injury was characterized by a rise in serum urea and creatinine levels, tubular injury, invasion of leukocytes and upregulation of pro-inflammatory mediators. Impairment in renal function was shown by increased serum creatinine and BUN levels and elevation of fibrotic activity was assessed by high renal collagen content in the injured tissue. Treatment with oxytocin significantly prevented the development of acute renal failure and resulted in normal renal function and normal renal morphology. These findings suggest that OT may have an additional protective effect on oxidative injury-induced production and deposition of extracellular matrix components, which may then result in tissue fibrosis. According to our results, it is clear that oxytocin treatment reduced inflammatory response to renal I/R injury and suppressed the generation of reactive oxygen species. On the other hand, it is known that OT administration causes hemodynamic alterations and reduces cardiac output [21,23]. Since a gradual increase in blood flow upon reperfusion is expected to reduce the reperfusion-induced renal injury [50], it is possible that cardiovascular action of oxytocin may contribute to its protective effect in I/R injury through its inhibitory effect on renal blood flow. Additionally, it is also possible that OT, at high concentrations, may interact with
vasopressin receptors [21,23,51], which also contribute to the anti-inflammatory effects of OT. Another possibility to consider is that OT may release ANP [52], and thereby increase the glomerular filtration rate. Thus, it seems likely that reduced plasma creatinine levels by OT treatment in the present study may be attributed to the actions of ANP. Recent reports suggest that cardiovascular and renal effects of OT are related, at least in part, to nitric oxide (NO) synthesis [23,40,41,53]. There are many articles demonstrating a relationship between NO and I/R injury. Although Kurata et al. have demonstrated the protective action of NO and have shown that the pre-treatment with NO donors improves renal function after I/R [54], others have shown that NO treatment after I/R worsens it [55,56]. Accordingly, inducible nitric oxide synthase inhibition reduces renal I/R injury [57–60]. Acute renal failure, a complex syndrome involving renal vasoconstriction, extensive tubular damage, tubular cell necrosis, glomerular filtration failure and glomerular injury [39], does not have a specific therapy regimen except for supportive care. Different experimental models have shown that antioxidant therapy can protect against I/R-induced oxidative damage [61]. With the data in hand, we can conclude that oxytocin, as an antioxidant, protects the kidney from I/R-induced oxidative injury. However, further experimental and clinical studies are required to consider antioxidants, including oxytocin, in the treatment protocols of I/R injury. The protective effect of oxytocin can be attributed, at least in part, to its availability to balance oxidant status, to inhibit neutrophil infiltration and to regulate the activation of pro-inflammatory mediators. References [1] Bird JE, Milhoan K, Wilson CB, Young SG, Mundy CA, Parthasarathy S, Blantz RC. Ischemic acute renal failure and antioxidant therapy in the rat. The relation between glomerular and tubular dysfunction. J Clin Invest 1988;81:1630–8. [2] Joosten SA, Sijpkens YW, van Kooten C, Paul LC. Chronic renal allograft rejection: pathophysiologic considerations. Kidney Int 2005;68:1–13. [3] Thadhani R, Pascual M, Bonventre JV. Acute renal failure. N Engl J Med 1996;360:1448–60. [4] Weight SC, Bell PR, Nicholson ML. Renal ischemia–reperfusion injury. Br J Surg 1996;83:162–70. [5] Willams P, Lopez H, Britt D, Chan C, Ezrin A, Hottendorf R. Characterization of renal ischemia–reperfusion injury in rats. J Pharmacol Toxicol Methods 1997;37:1–7. [6] Molitoris BA, Mars J. The role of cell adhesion molecules in ischemic acute renal failure. Am J Med 1999;106:583–92. [7] Molitoris BA, Falk SA, Dahl RH. Ischemia induced loss of epithelial polarity. Role of tight junction. J Clin Invest 1989;84:1334–9. [8] Brady HR. Leucocyte adhesion molecules: potential targets for therapeutic intervention in kidney diseases. Curr Opin Nephrol Hypertens 1993;2:171–82. [9] Brady HR. Leucocyte adhesion molecules and kidney diseases. Kidney Int 1994;45:1285–300. [10] Springer TA. Adhesion receptors of the immune system. Nature 1990;346:425–34. [11] Arnaout MA. Leucocyte adhesion molecules deficiency: its structural basis, pathophysiology and implications for modulating the inflammatory response. Immunol Rev 1990;114:145–80. [12] Matsuyama M, Yoshimura R, Akioka K, Okamoto M, Ushigome H, Kadotani Y, Nakatani T, Yoshimura N. Tissue factor antisense oligonucleotides prevent renal ischemia–reperfusion injury. Transplantation 2003;76:786–91.
H. Tuğtepe et al. / Regulatory Peptides 140 (2007) 101–108 [13] Kettle AJ, Winterbourn CC. Myeloperoxidase: a key regulator of neutrophil oxidant production. Redox Rep 1997;3:3–15. [14] Huang W, Lee S-L, Sjöquist M. Natriuretic role of endogenous oxytocin in male rats infused with hypertonic NaCl. Am J Physiol 1995;268:R634–40. [15] Sehirli AO, Sener G, Satiroglu H, Ayanoglu-Dulger G. Protective effect of N-acetylcysteine on the renal ischemia/reperfusion injury in the rat. J Nephrol 2003;16:75–80. [16] Sener G, Sehirli AO, Keyer-Uysal M, Arbak S, Ersoy Y, Yegen BC. The protective of melatonin on renal ischemia–reperfusion in rat. J Pineal Res 2002;32:120–6. [17] Sener G, Sehirli O, Erkanlı G, Cetinel S, Gedik N, Yegen BC. 2-Mercaptoethane sulfonate (MESNA) protects against burn-induced renal injury in rats. Burns 2004;30:557–64. [18] Sener G, Sener E, Sehirli AÖ, Velioglu A, Cetinel S, Gedik N, Sakarcan A. Ginkgo biloba extract ameliorates ischemia reperfusion-induced renal injury in rats. Pharmacol Res 2005;52:216–22. [19] Sener G, Tugtepe H, Yuksel M, Cetinel S, Gedik N, Yegen BC. Resveratrol ameliorates ischemia/reperfusion induced renal injury in rats. Arch Med Res 2006;37:822–9. [20] Peterssson M, Lundeberg T, Uvnas-Moberg K. Short term increase and long term decrease of blood pressure in response to oxytocin potentiating effect of female steroid hormones. J Cardiovasc Pharmacol 1999;33:102–8. [21] Petty MA, Lang RE, Unger T, Ganten D. The cardiovascular effects of oxytocin in conscious male rats. Eur J Pharmacol 1985;112:203–10. [22] Antunes-Rodrigues J, de Castro M, Elias LL, Valenca MM, McCann SM. Neuroendocrine control of body fluid metabolism. Physiol Rev 2004;84:169–208. [23] Costa-E-Sousa RH, Pereira-Junior PP, Oliveira PF, Olivares EL, Werneckde-Castro JP, Mello DB, Nascimento JH, Campos-de-Carvalho AC. Cardiac effects of oxytocin: is there a role for this peptide in cardiovascular homeostasis? Regul Pept 2005;132:107–12. [24] Gimpl G, Fahrenholz F. The oxytocin receptor system: structure, function and regulation. Physiol Rev 2001;81:629–83. [25] Kimura T. Investigation of the oxytocin receptor at the molecular level. In: Ivell R, Russell JA, editors. Oxytocin: cellular and molecular approaches in medicine and research. New York: Plenum Press; 1995. p. 259–68. [26] Peterssson M, Lundeberg T, Sholström A, Wiberg U, Uvnas-Moberg K. Oxytocin increases the survival of musculocutaneous flaps. NaunynSchmiedeberg's Arch Pharmacol 1998;357:701–4. [27] Martinek RG. A rapid ultraviolet spectrophotometric lactic dehydrogenase assay. Clin Chim Acta 1972;40:91–9. [28] Talke H, Schubert GE. Enzymatic urea determination in the blood and serum in the Warburg optical test. Klin Wochenschr 1965;43:174–5. [29] Slot C. Plasma creatinine determination. A new and specific Jaffe reaction method. Scand J Clin Lab Invest 1965;17:381–7. [30] Beuge JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymol 1978;52:302–11. [31] Beutler E. Glutathione in red blood cell metabolism. A manual of biochemical methods. New York: Grune & Stratton; 1975. p. 112–4. [32] Bradley PP, Priebat DA, Christersen RD, Rothstein G. Measurement of cutaneous inflammation. Estimation of neutrophil content with an enzyme marker. J Invest Dermatol 1982;78:206–9. [33] Hillegas LM, Griswold DE, Brickson B, Albrightson-Winslow C. Assessment of myeloperoxidase activity in whole rat kidney. J Pharmacol Methods 1990;24:285–95. [34] Lopez De Leon A, Rojkind M. A simple micromethod for collagen and total protein determination in formalin-fixed paraffin-embedded sections. J Histochem Cytochem 1985;33:737–43. [35] Haklar U, Yuksel M, Velioglu A, Turkmen M, Haklar G, Yalcın AS. Oxygen radicals and nitric oxide levels in chondral or meniscal lesions or both. Clin Orthop Relat Res 2002;403:135–42. [36] Halloran PF, Homik J, Goes N, Lui SL, Urmson J, Ramassar V, Cockfield SM. The “injury response”: a concept linking nonspecific injury, acute rejection and long-term transplant outcomes. Transplant Proc 1997;29:79–81. [37] Heinzelmann M, Mercer-Jones MA, Passmore JC. Neutrophils and renal failure. Am J Kidney Dis 1999;34:384–99.
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[38] Kettle AJ, Winterbourn CC. Myeloperoxidase: a key regulator of neutrophil oxidant production. Redox Rep 1997;3:3–15. [39] Rhoden EL, Pereira-Lima L, Rhoden CR, Lucas ML, Teloken C, BellKlein A. Role of L-arginine/nitric oxide pathway in renal ischemia– reperfusion in rats. Eur J Surg 2001;167:224–8. [40] Thibonnier M, Conarty DM, Preston JA, Plesnicher CL, Dweik RA, Erzurum SC. Human vascular endothelial cells express oxytocin receptors. Endocrinology 1999;140:1301–9. [41] Soares TJ, Coimbra TM, Martins AR, Pereira AG, Carnio EC, Branco LG, Albuquerque-Araujo WI, deNucci G, Favaretto AL, Gutkowska J, McCann SM, Antunes-Rodriguez J. Atrial natriuretic peptide and oxytocin induce natriuresis by release of cGMP. Proc Natl Acad Sci U S A 1999;96:278–83. [42] Dal Secco D, Moreira AP, Freitas A, Silva JS, Rossi MA, Ferreira SH, Cunha FQ. Nitric oxide inhibits neutrophil migration by a mechanism dependent on ICAM-1: role of soluble guanylate cyclase. Nitric Oxide 2006;15:77–86. [43] Iseri SO, Sener G, Saglam B, Gedik N, Ercan F, Yegen BC. Oxytocin ameliorates oxidative colonic inflammation by a neutrophil-dependent mechanism. Peptides 2005;26:483–91. [44] Biyikli NK, Tugtepe H, Sener G, Velioglu-Ogunc A, Cetinel S, Midillioglu S, Gedik N, Yegen BC. Oxytocin alleviates oxidative renal injury in pyelonephritic rats via a neutrophil-dependent mechanism. Peptides 2006;27:2249–57. [45] Iseri SÖ, Sener G, Saglam B, Gedik N, Ercan F, Yegen BC. Oxytocin protects against sepsis-induced multiple organ damage: role of neutrophils. J Surg Res 2005;126:73–81. [46] Moosman B, Behl C. Secretory peptide hormones are biochemical antioxidants; structure–activity relationship. Mol Pharmacol 2002;61:260–8. [47] Paller MS, Hoidal JR, Ferritis TF. Oxygen free radicals in ischemic acute renal failure in the rat. J Clin Invest 1984;74:1156–64. [48] Beratni T, Zoja C, Corna D, Perico N, Ghezzi P, Remuzzi G. Tumor necrosis factor induces glomerular damage in the rabbit. Am J Pathol 1989;134:419–30. [49] Liebertal W, Koh JS, Levine JS. Necrosis and apoptosis in acute renal failure. Semin Nephrol 1998;18:505–18. [50] Durani NK, Yavuzer R, Mittal V, Bradford MM, Labocki C, Siberberg B. The effect of gradually increased blood flow on ischemia–reperfusion injury in rat kidney. Am J Surg 2006;191:334–7. [51] Miller ME, Davidge ST, Mitchell BF. Oxytocin does not directly affect vascular tone in vessels from nonpregnant and pregnant rats. Am J Physiol 2002;282:H1223–8. [52] Gutkowska J, Jankowski M, Lambert C, Mukaddam-Daher S, Zingg HH, McCann SH. Oxytocin releases atrial natriuretic peptide by combining with oxytocin receptors in the heart. Proc Natl Acad Sci U S A 1997;94:11704–9. [53] Jankowski M, Wang D, Mukaddam-Daher S, Gutkowska J. Pregnancy alters nitric oxide synthase and natriuretic peptide systems in the rat left ventricle. J Endocrinol 2005;184:209–17. [54] Kurata H, Takaoka M, Kubo Y, Katayama T, Tsutsui H, Takayama J, Ohkita M, Matsumura Y. Protective effect of nitric oxide on ischemia/ reperfusion-induced renal injury and endothelin-1 overproduction. Eur J Pharmacol 2005;517:232–9. [55] Kucuk HF, Kaptanoglu L, Ozalp F, Kurt N, Bingul S, Torlak OA, Colak E, Akyol H, Gul AE. Role of glyceryl trinitrate, a nitric oxide donor, in the renal ischemia–reperfusion injury of rats. Eur Surg Res 2006;38:431–7. [56] Nakajima A, Ueda K, Takaoka M, Yoshima Y, Matsumura Y. Opposite effects of pre and postischemic treatments with nitric oxide donor on ischemia/reperfusion-induced renal injury. J Pharmacol Exp Ther 2006;316:1038–46. [57] Walker LM, Walker PD, Imam SZ, Ali SF, Mayeux PR. Evidence for peroxynitrite formation in renal ischemia–reperfusion injury: studies with the inducible nitric oxide synthase inhibitor L-N(6)-(1-iminoethyl)lysine. J Pharmacol Exp Ther 2000;295:417–22. [58] Chatterjee PK, Patel NS, Kvale EO, Cuzzocrea S, Brown PA, Stewart KN, Mota-Filipe H, Thiemermann C. Inhibition of inducible nitric oxide synthase reduces renal ischemia/reperfusion injury. Kidney Int 2002;61:862–71.
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The protective effect of oxytocin on renal ischemia/reperfusion injury in rats Halil Tuğtepe a,⁎, Göksel Şener b , Neşe Karaaslan Bıyıklı c , Meral Yüksel d , Şule Çetinel e , Nursal Gedik f , Berrak Ç. Yeğen g a
Marmara University, School of Medicine, Department of Pediatric Surgery, Istanbul, Turkey Marmara University, School of Pharmacy Department of Pharmacology, Istanbul, Turkey c Marmara University, School of Medicine, Department of Pediatric Nephrology, Istanbul, Turkey d Marmara University, Vocational School of Health Related Professions, Istanbul, Turkey Marmara University, School of Medicine, Department of Histology and Embryology, Istanbul, Turkey f Kasimpasa Military Hospital, Division of Biochemistry, Istanbul, Turkey g Marmara University, School of Medicine, Department of Physiology, Istanbul, Turkey b
e
Received 7 September 2006; received in revised form 9 November 2006; accepted 11 November 2006 Available online 29 January 2007
Abstract Aim: Oxytocin was previously shown to have anti-inflammatory effects in different inflammation models. The major objective of the present study was to evaluate the protective role of oxytocin (OT) in protecting the kidney against ischemia/reperfusion (I/R) injury. Materials and methods: Male Wistar albino rats (250–300 g) were unilaterally nephrectomized, and subjected to 45 min of renal pedicle occlusion followed by 6 h of reperfusion. OT (1 mg/kg, ip) or vehicle was administered 15 min prior to ischemia and was repeated immediately before the reperfusion period. At the end of the reperfusion period, rats were decapitated and kidney samples were taken for histological examination or determination of malondialdehyde (MDA), an end product of lipid peroxidation; glutathione (GSH), a key antioxidant; and myeloperoxidase (MPO) activity, an index of tissue neutrophil infiltration. Creatinine and urea concentrations in blood were measured for the evaluation of renal function, while TNF-α and lactate dehydrogenase (LDH) levels were determined to evaluate generalized tissue damage. Formation of reactive oxygen species in renal tissue samples was monitored by chemiluminescence technique using luminol and lucigenin probes. Results: The results revealed that I/R injury increased (p b 0.01–0.001) serum urea, creatinine, TNF-α and LDH levels, as well as MDA, MPO and reactive oxygen radical levels in the renal tissue, while decreasing renal GSH content. However, alterations in these biochemical and histopathological indices due to I/R injury were attenuated by OT treatment (p b 0.05–0.001). Conclusions: Since OT administration improved renal function and microscopic damage, along with the alleviation of oxidant tissue responses, it appears that oxytocin protects renal tissue against I/R-induced oxidative damage. © 2006 Elsevier B.V. All rights reserved. Keywords: Oxytocin; Ischemia/reperfusion; Kidney; Lipid peroxidation; Myeloperoxidase; Glutathione
1. Introduction Ischemia/reperfusion injury (I/R) of the kidney, which occurs during renal transplantation, surgical revascularization of the renal artery, treatment of suprarenal aortic aneurysms [1,2], is a complex pathophysiological process and a major cause of acute renal failure, which initiates a complex sequence of events [3]. Although reperfusion is essential for the survival of ischemic ⁎ Corresponding author. Mazharbey Evsan Sok, Aytac Ap. No: 20/6, Göztepe, İstanbul, Turkey. Tel.: +90 216 428 02 50; fax: +90 216 325 72 17. E-mail address: [email protected] (H. Tuğtepe). 0167-0115/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2006.11.026
renal tissue, the prognosis of the organ dysfunction is further complicated by the additional damage due to the formation of oxygen-derived free radicals [4]. The mechanisms underlying I/R damage to kidneys are most likely multifactorial and interdependent, involving hypoxia, vascular endothelial injury, inflammatory responses, radical-induced damage, tubular obstruction, apoptosis and endothelial–epithelial cell dysfunction [5]. A better understanding of the cellular and molecular mechanisms of the injury is required for the improvement of current therapeutical approaches. Ischemic acute renal failure, primarily due to the breakdown of the cellular junctional complexes, leads to detachment of tubular cells from the extracellular matrix [6]. In cultured
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kidney cells, ischemia was shown to result in disruption and dysregulation of the actin cytoskeleton, and loss of intercellular and cell-to-extracellular matrix interactions and depletion of adenosine triphosphate [7]. The interactions between leukocytes and resident tissue cells in host defense and inflammation highlight many of the complexities of cell–cell and cell–matrix interactions. Leukocyte adhesion is an important event in leukocyte recruitment as it causes dynamic interactions among cells that promote antigen presentation, leukocyte activation, transcellular generation of inflammatory mediators and maintenance phases of inflammation [8–11]. Indeed, leukocyte adhesion has emerged as an attractive target for specific pharmacologic interventions in renal inflammatory diseases including ischemic acute renal failure. Recent studies of I/R injury have focused on neutrophil function, the actions of inflammatory cytokines, tissue factor and oxygen free radicals [12]. The activated neutrophils release reactive oxygen metabolites and cytotoxic proteins (e.g. proteases, myeloperoxidase, lactoferrin) into the extracellular fluid [13]. Studies concerning the preventive and inflammationdiminishing effects of antioxidants and anti-inflammatory peptides are gaining attention in I/R injury of the kidney. Most studies have shown the protective effects of antioxidant in I/R injury in the kidney [14–19]. Oxytocin (OT), a nonapeptide produced in the paraventricular and supraoptical nuclei in the hypothalamus, exerts a wide spectrum of central and peripheral effects. OT is mainly associated with uterine contraction during parturition and milk ejection reflex during lactation. It has also effects on cardiovascular and hydroelectrolitic regulation, various behaviors such as maternal, social and sexual behavior, as well as modulation of release of adenohypophyseal hormones [14,20– 23]. OT receptors have also been identified in other tissues, including the kidney, heart, thymus, pancreas and adipocytes [24]. OT was recently shown to facilitate wound healing and modulate the immune and inflammatory processes, since OT and OT receptors are found in the thymus and OT receptors contain response elements for acute phase reactants and interleukins [25]. It was found that OT may decrease the release of some interleukins and increase the survival of ischemic skin flaps in rats via the activation of the growth factors or antiinflammatory mechanisms [26]. In relation to this background, the present study was designed to investigate the possible protective effect of OT against I/R injury in the kidney, using functional, biochemical and histological parameters for the evaluation of the extent of oxidative damage. 2. Materials and methods 2.1. Animals Male Wistar rats (250–300 g) were housed in an air-conditioned room with 12-hour light and dark cycles, where the temperature (22 ± 2 °C) and relative humidity (65–70%) were kept constant. Rats were fed with standard laboratory chow with free access to water. All experimental protocols were approved by the Marmara University School of Medicine Animal Care and Use Committee.
2.2. Surgery and experimental design Under anesthesia (100 mg/kg ketamine and 0.75 mg/kg chlorpromazine; intraperitoneally; ip), an upper abdominal midline incision was made and right nephrectomy was performed. The left renal pedicle was occluded for 45 min to induce ischemia and then subjected to reperfusion for 6 h (I/R groups). The rats were treated with either oxytocin (OT; 1 mg/kg; ip; Ibrahim Ethem Ulagay Ilaç AŞ, Istanbul) or saline at 30 min prior to ischemia, and the dose was repeated immediately before the reperfusion period (OT-treated I/R or I/R groups, respectively). None of the animals died during the I/R period. Another group of rats underwent only laparotomy, where the kidneys were manipulated without nephrectomy or occlusion, and they were treated with either saline (control group) or OT (OT group) at similar time points. 2.3. Biochemical analysis The animals were decapitated at 6 h of reperfusion, and trunk blood samples were collected for the analysis of serum urea, creatinine, TNF-α and LDH levels. Renal tissue samples obtained from each animal were stored at − 70 °C for the measurement of malondialdehyde, glutathione and myeloperoxidase activity, and luminol and lucigenin chemiluminescences. Additional kidney samples were placed in 10% formaldehyde for histopathological evaluation of renal injury and for the determination of tissue collagen content. 2.4. Measurement of serum LDH, urea, creatinine and TNF-α levels Serum LDH [27], urea [28] and creatinine [29] levels were determined spectrophotometrically using an automated analyzer. Serum levels of tumor necrosis factor alpha (TNF-α) were quantified according to the manufacturer's instructions and guidelines using enzyme-linked immunosorbent assay (ELISA) kit specific for rats (Biosource International, Nivelles, Belgium). This particular assay kit was selected because of its high degree of sensitivity, specificity, inter- and intraassay precision, and small amount of sample required to conduct the assay. TNF-α in the serum samples was expressed as pg/ml. 2.5. Renal malondialdehyde (MDA) and glutathione (GSH) assays Tissue samples were homogenized with ice-cold trichloroacetic acid (1 g tissue plus 10 ml 10% TCA) in an Ultra Turrax tissue homogenizer. The MDA levels were assayed for products of lipid peroxidation by monitoring thiobarbituric acid reactive substance formation as described previously [30]. Lipid peroxidation was expressed in terms of MDA equivalents using an extinction coefficient of 1.56 × 105 M− 1 cm− 1 and the results are expressed as nmol MDA/g tissue. Glutathione measurements were performed using a modification of the Ellman procedure [31]. Briefly, after centrifugation at 2000 g for 10 min, 0.5 ml of supernatant was added to 2 ml of 0.3 mol/l Na2HPO4·2H2O solution. A 0.2 ml solution of dithiobisnitrobenzoate (0.4 mg/ml
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1% sodium citrate) was added and the absorbance at 412 nm was measured immediately after mixing. Glutathione levels were calculated using an extinction coefficient of 1.36 × 105 M− 1 cm− 1. The results are expressed in μmol GSH/g tissue.
Table 2 Luminol and lucigenin chemiluminescence levels in control and I/R groups treated with either saline or oxytocin Chemiluminescence (rlu/mg)
Control
OT
I/R + saline
I/R + OT
2.6. Measurement of myeloperoxidase activity
Luminol Lucigenin
10.3 ± 0.6 15.1 ± 1.1
11.8 ± 0.7 15.4 ± 1.4
18.5 ± 2.4⁎⁎ 26.3 ± 1.5⁎⁎⁎
14.10 ± 0.8+ 18.3 ± 1.6++
Activity of tissue MPO, an enzyme that is found predominantly in the azurophilic granules of polymorphonuclear leukocytes (PMN), correlates with the number of PMN determined histochemically in inflamed tissues and, thus, it is used as an indication of accumulation of neutrophils [32]. Tissue MPO activity was assessed by measuring the H2O2-dependent oxidation of o-dianizidine 2HCl. Briefly, tissue samples (0.2–0.3 g) were homogenized in 10 vol of ice-cold potassium phosphate buffer (50 mmol/l K2HPO4, pH 6.0) containing hexadecyltrimethylammonium bromide (HETAB; 0.5%, w/v). The homogenate was centrifuged at 41,400 g for 10 min at 4 °C, and the supernatant was discarded. The pellet was then rehomogenized with an equivalent volume of 50 mmol/l K2HPO4 containing 0.5% (w/v) hexadecyltrimethylammonium bromide and 10 mmol/l ethylenediaminetetraacetic acid (EDTA, Sigma). MPO activity was assessed by measuring the H2O2-dependent oxidation of o-dianizidine 2HCl. One unit of enzyme activity was defined as the amount of the MPO present per gram of tissue weight that caused a change in absorbance of 1.0/min at 460 nm and 37 °C [33].
Each group consists of 8 rats. ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001; compared with control group. + p b 0.05, ++p b 0.01; compared with saline-treated I/R group.
1,4-phthalazinedione) were obtained from Sigma (St. Louis, MO). Measurements were made at room temperature using Junior LB 9509 luminometer (EG&G Berthold, Germany). Specimens were put into vials containing PBS-HEPES buffer (0.5 M PBS containing 20 mM HEPES, pH 7.2). ROS were quantitated after the addition of enhancers such as lucigenin or luminol for a final concentration of 0.2 mM. Luminol detects a group of reactive species, i.e. UOH, H2O2, HOCl radicals and lucigenin is selective for O2−. Counts were obtained at 1 min intervals and the results were given as the area under curve for a counting period of 5 min. Counts were corrected for wet tissue weight (rlu/mg tissue) [35]. 2.9. Histopathological examination For light microscopic investigations, renal tissue specimens were fixed in 10% formaldehyde, dehydrated in alcohol series,
2.7. Tissue collagen measurement Tissue collagen was measured as a free radical-induced fibrosis marker. Tissue samples were cut with a razor blade, immediately fixed in 10% formalin then samples were embedded in paraffin, and sections, approximately 15 μm thick were obtained. The evaluation of collagen content was based on the method published by Lopez De Leon and Rojkind [34], which is based on selective binding of the dyes Sirius Red and Fast Green FCF to collagen and noncollagenous components, respectively. Both dyes were eluted readily and simultaneously by using 0.1 N NaOH–methanol (1:1, v/v). Finally, the absorbances at 540 and 605 nm were used to determine the amount of collagen and protein, respectively. 2.8. Measurement of luminol and lucigenin chemiluminescences To assess the role of reactive oxygen species in I/R-induced tissue damage, luminol and lucigenin chemiluminescences were measured as indicators of radical formation. Lucigenin (bis-Nmethylacridiniumnitrate) and luminol (5-amino-2,3-dihydroTable 1 Blood urea nitrogen (BUN) and serum creatinine levels of experimental groups
Creatinine BUN
Control
OT
I/R + saline
I/R + OT
0.4 ± 0.06 21.6 ± 5.2
0.52 ± 0.22 29.0 ± 9.0
8.61 ± 1.79⁎⁎⁎ 123.3 ± 5.8⁎⁎⁎
1.73 ± 0.13++ 84.2 ± 7.0⁎⁎⁎,++
OT: Oxytocin; I/R: Ischemia/reperfusion. ⁎⁎⁎p b 0.001, compared to sham-operated control group. ++ p b 0.01, compared to saline-treated I/R group.
Fig. 1. a) TNF-α and b) lactate dehyrogenase (LDH) levels in serum samples of control (C), oxytocin-treated control (OT), saline-treated ischemia/reperfusion (I/R) and oxytocin-treated I/R (I/R + OT) groups. Each group consists of 8 animals. ⁎⁎⁎: p b 0.001 versus control group; +++: p b 0.001 versus I/R group.
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Fig. 2. a) Malondialdehyde (MDA) and b) glutathione (GSH) levels in the kidney tissue of control (C), oxytocin-treated control (OT), saline-treated ischemia/ reperfusion (I/R) and oxytocin-treated I/R (I/R+ OT) groups. Each group consists of 8 animals. ⁎⁎⁎p b 0.001; compared to control group. ++p b 0.01, +++p b 0.001; compared to saline-treated I/R group.
( p b 0.01 and 0.001). Chemiluminescence levels in the kidney samples detected by both luminol and lucigenin probes showed significant increases in the saline-treated I/R group as compared to the chemiluminescence levels of the control group (p b 0.001; Table 2). On the other hand, OT treatment in the I/R group decreased the elevations in chemiluminescence values of luminol and lucigenin (p b 0.05–0.01). In the I/R group treated with saline, serum TNF-α level showed a dramatic increase (53.6 ± 5.2 pg/ml) as compared to control group (8.8 ± 1.2 pg/ml, p b 0.001) and OT administration reduced the concentration of this inflammatory cytokine significantly (31.7 ± 2.9 pg/ml, p b 0.001; Fig. 1A). Similarly, serum LDH activity was found to be higher in the I/R group (4833 ± 230 U/l) compared to control (1888 ± 212 U/l, p b 0.001), indicating generalized tissue damage, while OT treatment attenuated the I/R-induced LDH response (1697 ± 64 U/l, p b 0.001; Fig. 1B). The renal tissue MDA content in the control group (19.8 ± 2.4 nmol/g) was elevated by I/R injury (49.1 ± 5.4 nmol/g, p b 0.001); however, OT treatment significantly decreased the I/ R-induced elevation in renal MDA level (26.5 ± 2.6 nmol/g, p b 0.001; Fig. 2A). In accordance with that, ischemia and reperfusion caused a significant decrease in renal GSH level (0.4 ± 0.06 μmol/g; p b 0.001) when compared to control group (1.5 ± 0.11 μmol/g), while in the OT-treated I/R group, renal GSH content was found to be preserved (1.2 ± 0.15 μmol/g;
cleared in toluene and embedded in paraffin. Paraffin sections (5 μm) were stained with hematoxylin and eosin (H&E) and examined under a photomicroscope (Olympus BH 2, Tokyo, Japan). All tissue sections were examined microscopically for the characterization of histopathological changes by an experienced histologist who was unaware of the treatment conditions. 2.10. Statistical analysis Statistical analysis was carried out using GraphPad Prism 3.0 (GraphPad Software, San Diego, CA, USA). Each group consisted of 8 animals. All data were expressed as means± S.E.M. Groups of data were compared with an analysis of variance (ANOVA) followed by Tukey's multiple comparison tests. Values of p b 0.05 were regarded as significant. 3. Results Regarding all the studied parameters, oxytocin treatment in the sham-operated control group was found to be not different than the saline-treated control group. The blood urea nitrogen (BUN) and creatinine levels in the saline-treated I/R group were found to be significantly higher than those in the control rats ( p b 0.001; Table 1). When OT was administered before ischemia and the subsequent reperfusion period, the elevations in BUN and serum creatinine levels were significantly depressed
Fig. 3. a) Myeloperoxidase (MPO) activity and b) collagen content of control (C), oxytocin-treated control (OT), saline-treated ischemia/reperfusion (I/R) and oxytocin-treated I/R (I/R + OT) groups. Each group consists of 8 animals. ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001; compared to control group. +p b 0.05 compared to saline-treated I/R group.
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level (9 ± 1.1 U/g, p b 0.01), which was found to be not different than that of the control group. Collagen content of renal tissue was found to be significantly higher in the I/R group (24.2 ± 1.1 μg/mg protein) with respect to the control group (18.1 ± 0.8 μg/mg; Fig. 3B). OT treatment, however, significantly abolished the increase in the renal tissue collagen content (19.2 ± 1.3 μg/mg), which was found to be close to control values. Light microscopic evaluation of the control groups, treated with either OT or saline, revealed a regular morphology of renal parenchyma with well-designated glomeruli and tubuli (Fig. 4A). In the saline-treated I/R group, the interstitial hemorrhage, dilated tubuli and prominent glomerular degeneration followed by atrophy revealed that I/R caused a severe glomerular, tubular and interstitial damage. The tubular dilation was present throughout the tissue (Fig. 4B). In the OT-treated I/R group, there was a significant regeneration in all the features of the injury. The reduced tubular dilation, loss of the interstitial hemorrhage and glomerular atrophy were the features indicating regeneration (Fig. 4C). 4. Discussion
Fig. 4. a) Control kidney, regular morphology with glomeruli (arrows) and tubuli (⁎), b) saline-treated I/R group, prominent interstitial hemorrhage (arrows) and tubular degeneration (⁎), c) OT-treated I/R group, with reduction in the interstitial hemorrhage (arrows) and tubular degeneration (⁎), H&E × 200.
p b 0.01), being not significantly different from that of the control group (Fig. 2B). Myeloperoxidase activity, which is accepted as an indicator of neutrophil infiltration, was significantly higher in the kidney tissue of the saline-treated I/R group (15.5 ± 2.1 U/g, p b 0.001) than that of the control group (6.6 ± 0.6 U/g; Fig. 3A). On the other hand, OT treatment in the I/R group significantly decreased renal tissue MPO
The results of the present study reveal that OT reduces I/R injury in rat kidney, with improved renal function, as evidenced by decreased serum creatinine and BUN levels, normal renal histopathology, improved antioxidant status (increased levels of GSH) along with decreases in lipid peroxidation (reduced MDA), PMN infiltration (reduced MPO activity) and reactive oxygen species (reduced lucigenin). Moreover, increases in serum TNF-α and LDH levels, which play important roles in systemic inflammatory processes, were also attenuated by oxytocin treatment. Renal I/R injury is the major cause of acute renal failure in both native and transplanted organs. The severity of the injury depends on the duration of ischemia and subsequent reperfusion periods. Reperfusion, although essential for the survival of ischemic renal tissue, causes additional damage. Acute ischemia leads to the activation of the endothelium with an increase in permeability and expression of different adhesion molecules [36] and elicits an acute inflammatory response characterized by activation of neutrophils [37]. Activated neutrophils induce tissue injury through the production and release of reactive oxygen metabolites and cytotoxic proteins (e.g. proteases, myeloperoxidase, lactoferrin) into the extracellular fluid [38], which constitute the inflammatory cascades that trigger the radical-induced I/R injury. In the present study, renal I/R caused an elevation in tissue MPO activity indicating the presence of enhanced PMN recruitment in the inflamed tissue, while the increased renal malondialdehyde level, an indicator of lipid peroxidation, verified the oxidative damage in the renal tissue. In our study, oxytocin administration before I/R prevented both oxidative renal injury and tissue neutrophil accumulation. A possible mediator behind the decrease in MPO activity in response to oxytocin could be nitric oxide [39]. Nitric oxide, released by oxytocin, may inhibit the adhesion and accumulation of neutrophils [40–42]. On the other hand, GSH, which provides a cellular defense against oxidative injury, is frequently used as a measure of tissue antioxidant status. Therefore, reduced GSH
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levels shown in the present study may be considered as a sign of diminished antioxidant pool in the renal tissue with I/R injury. The replenishment of renal GSH content by OT therapy may be considered as the antioxidant function of OT. Our recent studies confirmed the antioxidant property of OT in colitis-, pyelonephritis- and sepsis-induced inflammatory models [43–45]. It was also shown that in brain membranes OT displayed antioxidant properties in aqueous medium, scavenging free peroxyl radicals, preventing LDL oxidation and inhibiting lipid peroxidation [46]. In accordance with these, the present study supports the antioxidant effect of OT on the renal tissue through the inhibition of tissue neutrophil accumulation and associated production of reactive oxygen species. Much of the tubular and glomerular dysfunction in acute renal failure is due to I/R and is postulated to occur during the reperfusion period following anoxia. Generation of oxygenderived radicals has been considered as one of the major factors contributing to this reperfusion injury [47]. Reactive oxygen species can generate hypochlorous acid in the presence of neutrophil-derived MPO and initiate the deactivation of antiproteases and activation of latent proteases, which lead to tissue damage [38]. In addition, oxidative stress stimulates the accumulation of macrophages and monocytes in I/R kidney. Peripheral monocytes infiltrating the kidney have traditionally been considered the primary source of renal TNF. It has been shown that exogenous TNF induces renal cell apoptosis, glomerular endothelial damage, fibrin deposition, cellular infiltration and renal failure [48,49]. In this study, the tissue associated MPO activity and serum TNF-α levels were increased in I/R injury. The reversal of these parameters by oxytocin treatment suggests that the mechanism of the protective effect of oxytocin involves the inhibition of inflammatory cell infiltration and the release of TNF-α. In the present study, acute renal failure that occurred following I/R injury was characterized by a rise in serum urea and creatinine levels, tubular injury, invasion of leukocytes and upregulation of pro-inflammatory mediators. Impairment in renal function was shown by increased serum creatinine and BUN levels and elevation of fibrotic activity was assessed by high renal collagen content in the injured tissue. Treatment with oxytocin significantly prevented the development of acute renal failure and resulted in normal renal function and normal renal morphology. These findings suggest that OT may have an additional protective effect on oxidative injury-induced production and deposition of extracellular matrix components, which may then result in tissue fibrosis. According to our results, it is clear that oxytocin treatment reduced inflammatory response to renal I/R injury and suppressed the generation of reactive oxygen species. On the other hand, it is known that OT administration causes hemodynamic alterations and reduces cardiac output [21,23]. Since a gradual increase in blood flow upon reperfusion is expected to reduce the reperfusion-induced renal injury [50], it is possible that cardiovascular action of oxytocin may contribute to its protective effect in I/R injury through its inhibitory effect on renal blood flow. Additionally, it is also possible that OT, at high concentrations, may interact with
vasopressin receptors [21,23,51], which also contribute to the anti-inflammatory effects of OT. Another possibility to consider is that OT may release ANP [52], and thereby increase the glomerular filtration rate. Thus, it seems likely that reduced plasma creatinine levels by OT treatment in the present study may be attributed to the actions of ANP. Recent reports suggest that cardiovascular and renal effects of OT are related, at least in part, to nitric oxide (NO) synthesis [23,40,41,53]. There are many articles demonstrating a relationship between NO and I/R injury. Although Kurata et al. have demonstrated the protective action of NO and have shown that the pre-treatment with NO donors improves renal function after I/R [54], others have shown that NO treatment after I/R worsens it [55,56]. Accordingly, inducible nitric oxide synthase inhibition reduces renal I/R injury [57–60]. Acute renal failure, a complex syndrome involving renal vasoconstriction, extensive tubular damage, tubular cell necrosis, glomerular filtration failure and glomerular injury [39], does not have a specific therapy regimen except for supportive care. Different experimental models have shown that antioxidant therapy can protect against I/R-induced oxidative damage [61]. With the data in hand, we can conclude that oxytocin, as an antioxidant, protects the kidney from I/R-induced oxidative injury. However, further experimental and clinical studies are required to consider antioxidants, including oxytocin, in the treatment protocols of I/R injury. The protective effect of oxytocin can be attributed, at least in part, to its availability to balance oxidant status, to inhibit neutrophil infiltration and to regulate the activation of pro-inflammatory mediators. References [1] Bird JE, Milhoan K, Wilson CB, Young SG, Mundy CA, Parthasarathy S, Blantz RC. Ischemic acute renal failure and antioxidant therapy in the rat. The relation between glomerular and tubular dysfunction. J Clin Invest 1988;81:1630–8. [2] Joosten SA, Sijpkens YW, van Kooten C, Paul LC. Chronic renal allograft rejection: pathophysiologic considerations. Kidney Int 2005;68:1–13. [3] Thadhani R, Pascual M, Bonventre JV. Acute renal failure. N Engl J Med 1996;360:1448–60. [4] Weight SC, Bell PR, Nicholson ML. Renal ischemia–reperfusion injury. Br J Surg 1996;83:162–70. [5] Willams P, Lopez H, Britt D, Chan C, Ezrin A, Hottendorf R. Characterization of renal ischemia–reperfusion injury in rats. J Pharmacol Toxicol Methods 1997;37:1–7. [6] Molitoris BA, Mars J. The role of cell adhesion molecules in ischemic acute renal failure. Am J Med 1999;106:583–92. [7] Molitoris BA, Falk SA, Dahl RH. Ischemia induced loss of epithelial polarity. Role of tight junction. J Clin Invest 1989;84:1334–9. [8] Brady HR. Leucocyte adhesion molecules: potential targets for therapeutic intervention in kidney diseases. Curr Opin Nephrol Hypertens 1993;2:171–82. [9] Brady HR. Leucocyte adhesion molecules and kidney diseases. Kidney Int 1994;45:1285–300. [10] Springer TA. Adhesion receptors of the immune system. Nature 1990;346:425–34. [11] Arnaout MA. Leucocyte adhesion molecules deficiency: its structural basis, pathophysiology and implications for modulating the inflammatory response. Immunol Rev 1990;114:145–80. [12] Matsuyama M, Yoshimura R, Akioka K, Okamoto M, Ushigome H, Kadotani Y, Nakatani T, Yoshimura N. Tissue factor antisense oligonucleotides prevent renal ischemia–reperfusion injury. Transplantation 2003;76:786–91.
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