Comparison of the Efficacy of Melatonin and 1400W on Renal Ischemia/Reperfusion Injury: A Role for Inhibiting iNOS

Abstract

Introduction. We investigated the roles of melatonin (a powerful antioxidant, iNOS inhibitor, and a scavenger of peroxynitrite) and 1400W (a strong and selective inhibitor of inducible nitric oxide) on renal dysfunction and injury induced by ischemia/reperfusion (I/R) of rat kidney, since oxidative and nitrosative injury are believed to be the major causes. Materials and methods. Thirty-two male Sprague-Dawley rats were divided into four groups of sham-operated, I/R, I/R + Melatonin and I/R + 1400W. Rats were given either melatonin (10 mg/kg) or 1400W (10 mg/kg) in the I/R + Melatonin and I/R + 1400W groups respectively at 6 h prior to ischemia and at the beginning of reperfusion via intraperitoneal route. I/R injury was induced by 60 min of bilateral renal ischemia followed by 6 h of reperfusion. After reperfusion, kidneys and blood were obtained for histopathologic and biochemical evaluation. Results. Melatonin and 1400W had an ameliorative effect on both oxidative and nitrosative stress in the kidneys against renal I/R injury in rats. In addition, melatonin significantly reduced elevated nitro-oxidative stress product, restored decreased antioxidant enzymes and attenuated histological alterations when compared with 1400W. Conclusions. Both Melatonin and 1400W were efficient in ameliorating experimental I/R injury of the kidneys. Moreover, melatonin was more effective than 1400W possibly through inhibiting iNOS as well as scavenging free oxygen radicals and peroxynitrite.

Keywords:

• melatonin
• 1400W
• renal ischemia/reperfusion
• oxidative stress
• peroxynitrite

INTRODUCTION

Cessation of kidney blood supply leads to acute renal failure (ARF), causing failure of the kidneys over a period of hours or days.^[1–3] The causes of ARF are multifactorial, but ischemic ARF caused by hypotension followed by resuscitation is frequent, the etiology of which is reflected in animal models of renal ischemia/reperfusion (I/R).^[1,4]

Reperfusion of the kidneys causes the activation and adhesion of polymorphonuclear neutrophils (PMNL), with the release of proinflammatory substances and the formation of free radicals, which are nitrogen-derived reactive nitrogen species (RNS) or oxygen-derived reactive oxygen species (ROS), such as superoxide (O^•₂⁻), hydrogen peroxide (H₂O₂), and hydroxyl radicals (^•OH).^[1,4–8]

It is presumed that ROS production reduces the transcription of endothelial nitric oxide synthase (eNOS), which provides nitric oxide (NO) under physiologic circumstances. On the other hand, ROS/RNS activates inducible nitric oxide synthase (iNOS), which causes an almost 1,000-fold higher NO production than eNOS. Intensified expression of iNOS has been detected in virtually all cell types tested, including macrophages, fibroblasts, chondrocytes, osteoclasts, and epithelial cells, and results in the production of large amounts of NO in animals and patients with inflammatory diseases.^[9–12] In addition, the level of iNOS expression is well correlated with the degree of inflammation. Once iNOS is activated, because of NO's affinity for O•₂⁻, neither enzymatic nor pharmacologic levels of conventional antioxidants are able to compete with NO for O•₂⁻; as a result, high peroxynitrite (ONOO⁻) levels are elevated.^[5,13,14] Because NO derived from iNOS also has an important role in I/R process, it is crucial to determine whether NO or ONOO⁻ exerted these effects.^[7,15] Various in vivo studies have shown that NO biosynthesis and its action are closely related to the pathogenesis of renal I/R injury. In addition, it has been shown that selective or non-selective iNOS inhibitors prevent I/R injury in skeletal muscle, myocardial infarction, brain, liver, and kidney tissues,^[13,16–21] and that iNOS–knockout mice show reduced I/R injury in the kidney.^[20]

Melatonin is mainly secreted from pineal gland and a variety of non-pineal tissues and organs, including the kidney.^[22] Non-pineal melatonin is believed to function as an antioxidant and free radical scavenger within the cells and has been shown to have protective effects against I/R injury in kidney, heart, and intestine, among others.^[22–24] Additionally, several of melatonin's metabolites are highly effective radical scavengers.^[25,26] N-[3(aminomethyl) benzyl) acetamidine] (1400W) is a highly selective iNOS inhibitor that decreases I/R injury in both kidney and heart in experimental studies.^[27,28]

We hypothesized that the melatonin as a strong antioxidant and iNOS inhibitor agent as well as a scavenger of peroxynitrite may counteract more efficiently than 1400W, which is an iNOS inhibitor only. Based on this hypothesis, this study was designed to investigate whether melatonin or 1400W has greater protective effects on kidney damage induced by renal I/R.

MATERIALS AND METHODS

Animals and Surgery

The project was approved by the Experimental Ethics Committee of Gulhane Military Medical Academy, Ankara, Turkey, and the National Institute of Health's Guide for the Care and Use of Laboratory Animals was followed.

Thirty-two male Sprague-Dawley rats, weighing 250–300 g, were provided by the Gulhane Military Medical Academy, Experimental Research Council, and housed in standard cages at a constant temperature (24°C) and light-dark cycle in a controlled environment. Rats were fed with standard rat chow and water ad libitum.

Rats were randomly divided into four groups: sham-operated (n = 8), renal I/R (n = 8), renal I/R + melatonin (n = 8), and renal I/R + 1400W (n = 8).

Both chemicals—namely, melatonin (M5250, Sigma Chemical, St Louis, Missouri, USA) and 1400W (W4262, Sigma Chemical)—were administered at a dose of 10 mg/kg at 60 min prior to ischemia and at the beginning of reperfusion via intraperitoneal route.

Following a 12 h fasting period, animals were anesthetized with an intraperitoneal injection of ketamine hydrochloride (50 mg/kg) and xylazine (10 mg/kg). The rats were placed on a heating pad kept at 39°C to maintain constant body temperature. A midline incision was made, the renal pedicle observed and arteries bilaterally occluded with an atraumatic microvascular clamp (Bulldog Artery Clamp, Harvard Apparatus, Massachusetts, USA) for 60 min. The time of ischemia was chosen to maximize reproducibility of renal functional impairment while minimizing mortality in these animals. After 60 min of renal ischemia, clamps were removed and the kidneys were inspected for restoration of blood flow. The abdomen was closed in two layers. Sham-operated animals underwent the same surgical procedure without clamp application. Following 6 h of reperfusion period, animals were killed by cervical dislocation. At the time of death, blood was collected by heart puncture for measurement of biochemical analysis. Both kidneys were harvested for histopathological evaluation and biochemical examination.

Biochemical Analysis

Serum samples were used for the measurement of blood urea nitrogen (BUN) and serum creatinine (S_Cr) levels, which were used as indicators of impaired glomerular function, and aspartate aminotransferase (AST), which was used as an indicator of renal I/R injury.^[29] BUN, S_Cr, and AST were determined with an Abbott-Aeroset autoanalyzer (Chicago, Illinois, USA) using original kits.

The frozen tissues were homogenized in phosphate buffer (pH 7.4) by means of homogenization (Heidolph Diax 900; Heidolph Elektro GmbH, Kelhaim, Germany) in an ice cube. The supernatant was allocated into 2–3 separate tubes and stored at −70°C again. First, the protein content of tissue homogenates was measured by the method of Lowry et al. with bovine serum albumin as the standard. Efficacy of treatment was assessed by tissue level of malondialdehyde (MDA) using the method of Ohkawa et al., protein carbonyl content (PCC) using the method of Levine et al., superoxide dismutase (SOD) using the method of Sun et al., and glutathione peroxidase (GPx) using the method of Paglia and Valentine.^[13,30,40] Nitrate and nitrite (NO_x) levels, end products of nitric oxide degradation, were measured using the method described by Miranda et al., as we described in our previous works.^[13,30]

Histopathologic Evaluation

Both kidneys of each animal were taken for histopathologic evaluation. In all groups, samples of kidney were placed in formalin and processed through to paraffin. They were subsequently sectioned at 5 μm and stained with Hematoxylin-Eosin (H&E). The sections were scored with a previously described semiquantitative scale designed to evaluate the degree of renal damage (tubular cell necrosis, cytoplasmic vacuole formation, hemorrhage, and tubular dilatation).^[31] A minimum of 10 fields for each kidney slide were examined and assigned for severity of changes. The scoring system used was 0 = absent, 1 = present, and 2 = marked. Total histopathologic injury score per kidney was calculated by addition of all scores. Blind analysis of the histological samples was performed by two independent experts.

Statistical Analysis

All biochemical data are expressed as mean ± standard error of the mean (SEM). All statistical analyses were carried out using SPSS statistical software (SPSS for Windows, Version 15.0, Chicago, Illinois, USA). Differences in measured parameters among the three groups were analyzed by Kruskal-Wallis test. Dual comparisons between groups that present significant values were evaluated with Mann-Whitney U test. Statistical significance was accepted at a value of p < 0.05.

RESULTS

All animals survived throughout the experimental period.

Renal Function Markers

There was a significant increase in the S_Cr and BUN levels in the I/R group compared to sham-operated groups, suggesting a significant degree of glomerular dysfunction (see Table 1). Administration of melatonin or 1400W significantly decreased the S_Cr and BUN levels. Renal I/R produced a significant increase in the serum AST level, used as a marker of renal injury. On the other hand, serum concentration of AST was significantly decreased in the rats administered either melatonin or 1400W. It was seen that melatonin is more efficient with renal function than 1400W based on serum biochemical markers (see Table 1).

Table 1  Biochemical evaluation of serum for each group

	Creatinine (mg/dL)	BUN (mg/dL)	AST (IU/L)
Control (n = 8)	0.8 ± 0.3	32 ± 5	145 ± 36
I/R (n = 8)	1.7 ± 0.5^‡^§	65 ± 6^‡^§	214 ± 53^‡^§
I/R + melatonin (n = 8)	1.1 ± 0.4^†^§	47 ± 8^†^§	186 ± 76^†
I/R + 1400W (n = 8)	1.3 ± 0.6^†^‡	56 ± 7^†^‡	180 ± 84^†

*Significantly different from control.

^†Significantly different from I/R group.

^‡Significantly different from I/R + M group.

^§Significantly different from I/R + 1400W group.

Antioxidant Enzyme Activities

The renal I/R injury significantly reduced antioxidant enzyme activities (SOD and GSH-Px). Both melatonin and 1400W significantly increased antioxidant enzyme activities, but melatonin did more than 1400W (see Table 2).

Table 2  Biochemichal evaluation of kidney for each groups

	MDA (nmol/g-protein)	PCC (nmol/g-protein)	NOx (μmol/g-tissue)	SOD (U/g-protein)	GSH-Px (U/g-protein)
Control (n = 8)	0.61 ± 0.10	46.3 ± 4.1	52.2 ± 4.5	1432 ± 215	5.27 ± 1.8
I/R (n = 8)	1.35 ± 0.32^‡^§	94.8 ± 6.4^‡^§	82.2 ± 7.6^‡^§	2352 ± 285^‡^§	9.51 ± 2.4^‡^§
I/R+melatonin (n = 8)	0.94 ± 0.42^†^§	67.5 ± 4.9^§	65.2 ± 6.3	1745 ± 282^†^§	7.35 ± 2.3^†
I/R+1400W (n = 8)	1.12 ± 0.52*^†‡	84.3 ± 3.8^†^‡	68.4 ± 5.7^†	1934 ± 317^†^‡	7.44 ± 2.8^†

*Significantly different from control.

†Significantly different from I/R group.

^‡Significantly different from I/R + M group.

^§Significantly different from I/R + 1400W group.

Oxidative and Nitrosative Stress Markers

The rats subjected to renal I/R revealed a strike increase in the tissue MDA and PCC levels, suggesting increased lipid peroxidation and protein oxidation. The administration of melatonin or 1400W revealed a significant decrease in the levels of MDA and PCC in the rats subjected to renal I/R (see Table 2).

NO_x level (nitrite/nitrite concentration) in the renal tissue, as as indicator of NO and peroxynitrite production, was significantly increased in the rats subjected to renal I/R. Increased tissue NOx levels were significantly decreased in the I/R + melatonin and I/R + 1400W (see Table 2).

Histopathologic Evaluation

Histopathologic grading of renal injury is displayed as median (min-max) (see Table 3). The total injury score was significantly increased in the I/R group, indicating significant renal injury. Conversely, total histological injury score was significantly decreased in the treatment groups. Although significant glomerular and tubular injury with hemorrhage and accumulation of PMNL was seen in the rats subjected to I/R, a lower degree of injury was seen in the groups treated with either melatonin and 1400W. Representative histological samples from all groups are displayed in Figure 1.

Table 3  Histopathologic evaluation of kidney sections for each group

	Histopathologic scores of renal injury
Control (n = 8)	0 (0–0)
I/R (n = 8)	3 (2–4)^‡^§
I/R + melatonin (n = 8)	1 (1–2)^†
I/R + 1400W (n = 8)	1 (1–3)^†

*Significantly different from control.

^†Significantly different from I/R group.

^‡Significantly different from I/R + M group.

^§Significantly different from I/R + 1400W group.

Figure 1.  Representative histological photographs of kidney tissues from sham operated (control), renal ischemia reperfusion (IR) injury, renal ischemia reperfusion + melatonin (IR + Mel), and renal ischemia reperfusion + 1400W (IR + 1400W) groups. Kidney tissues were taken from cortex (right column) and medulla (left column). Upper row: Sham-operated animals show normal histological characteristic of glomeruli and tubules. Second row: Rats subjected to renal IR injury show marked necrosis with tubular dilation (D), swelling, luminal congestion, and medullar hemorrhage (H). Third row: Rats subjected to renal IR injury plus melatonin show moderate kidney damage and moderate dilatation (D) of the tubular structure. In comparison with the IR + Mel group, IR + 1400W group shows preservation of tissue histology of the kidney. (H&E, Scale bars: 50 micron).

DISCUSSION

In this study, we used melatonin as a strong antioxidant, selective iNOS inhibitor, and scavenger of ONOO⁻, and 1400W as a selective iNOS inhibitor, to protect kidneys against renal I/R injury in an experimental rat model. Our results clearly show that melatonin and 1400W ameliorate both oxidative and nitrosative stress in the kidneys against renal I/R injury in the rats. Interestingly, treatment with melatonin was much more effective than treatment with 1400W. The reason for this might be that melatonin is not only a strong anti-oxidants, but also an iNOS inhibitor and ONOO⁻ scavenger.^[25] Apart from this, several intracellular metabolites of melatonin are well-known free radical scavengers.^[25]

Administration of melatonin or 1400W improved renal function, as evidenced by reduction in the levels of S_Cr, BUN, and AST, as compared to sham-operated animals. These findings were also confirmed with histological analyses.

In this study, we demonstrate that melatonin attenuated the degree of lipid peroxidation and protein oxidation in the kidneys of rats subjected to renal I/R injury. One of the possible explanation for this finding is that melatonin and its metabolites have the ability to scavenge many of the oxygen-based and nitrogen-based radicals, including ONOO⁻.^[25,32] In addition, when melatonin enters cellular membranes, it mainly localizes in a superficial position in lipid bilayers near the polar heads of membrane phospholipids.^[22,33,34] While in this position, it is obviously capable of functioning as a free radical scavenger, and it may also provide an indirect means by which the membranes resist oxidative damage. Another possible explanation is that melatonin supports several intracellular antioxidant enzymes, including SOD and GSH-Px,^[35,36] and induces the activity of glutamylcysteine ligase (GCL, formerly referred to as gamma-glutamylcysteine synthetase), thereby stimulating the production of another crucial intracellular antioxidant, glutathione.^[37] In addition, melatonin has been shown to stimulate gene expression of SOD and GSH-Px.^[22,33]

Regarding the ameliorative effects of 1400W, there are conflicting data for both a beneficial and a harmful effect of NO on I/R injury.^[38] NO is one of the most important mediators in pathophysiological changes of tissues. Under physiologic conditions, NO maintains vascular tone and inhibits aggregation and adhesion of neutrophils and platelets to vascular endothelium; these are beneficial aspects of NO function.^[25] Low levels of NO production protect an organ in the early stages of injury, whereas elevated and prolonged NO production by iNOS during the later stages of the insult result in or potentiate organ injury. Increased expression of iNOS was shown to contribute to I/R injury in the kidney.^[4,31] On the other hand, our previous work showed that inhibition of iNOS improves functional recovery in reperfused kidneys, in agreement with reports on other tissues. Taken together, the data in this study support the contention that NO produced from iNOS plays a deleterious role in kidneys subjected to renal I/R injury. The mechanism of NO-induced I/R injury may also be a result of the development of ONOO⁻, as NO couples with O•₂⁻, which is also increased in response to ischemia. The production of ONOO⁻ (nitrosative stress) occurs almost instantaneously and causes the nitration of cellular proteins with subsequent loss of protein structure and function.^[15,39,40] Therefore, we conclude that ONOO⁻ is critical in renal I/R injury, and that the reduction of ONOO⁻ may be a potential mechanism to prevent kidneys.

NO and ONOO⁻ are eventually converted to nitrite (NO₂) and/or nitrate (NO₃) (i.e., NO_x).^[21,39] Therefore, NO_x levels are used as an indirect but reliable indicator for NO and ONOO⁻ formation in vivo.^[39,41] In this study, we found that renal I/R caused a significant increase in tissue NO_x and melatonin significantly inhibited I/R induced increases in tissue NO_x. Because melatonin neutralizes ONOO⁻, we speculate that melatonin's protective effects were a consequence of its versatile ability to scavenge ROS and ONOO⁻.^[22,32] In addition, we believe that free oxygen radicals, NO and ONOO⁻, have a crucial role in injury during inflammation-induced renal I/R, and this should be kept in mind when managing or planning new studies.

In conclusion, the findings of this study demonstrated clearly that melatonin or 1400W prevents I/R injury in the kidney. NO and/or ONOO⁻ and free oxygen radicals are involved in renal I/R injury, and the scavenging of both free oxygen radicals and peroxynitrite are crucial in reducing renal damage in ARF. Future studies may consider experimental and clinical application of melatonin^[42] in protecting the kidney against I/R injury.