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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 28, 2003 as doi:10.1096/fj.02-0504fje. |
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INSERM U388, IFR31, Institut Louis Bugnard, CHU Rangueil, Toulouse 31403 Cedex 4, France;
* Department of Pathology, CHU Rangueil, Toulouse 31403 Cedex 4, France;
Institut de Recherches Internationales Servier, 6, 92415 Courbevoie, France; and
Laboratory of Biochemistry, CHU Rangueil, Toulouse 31403 Cedex 4, France
2Correspondence: INSERM U388, Institut Louis Bugnard, CHU Rangueil, Bat. L3, 31403 Toulouse Cedex 4, France. E-mail: parini{at}toulouse.inserm.fr
SPECIFIC AIM
The pineal hormone melatonin has been reported to protect the tissue from oxidative damage. This study was designed to determine whether melatonin could prevent cell events leading to tissue injury and renal dysfunction after ischemia/reperfusion (I/R).
PRINCIPAL FINDING
To examine the effects of melatonin on oxidative stress and caspase-3-dependent cell apoptosis, rats were subjected to unilateral renal ischemia for 45 min. Sham-operated and ischemic groups of animals were treated with vehicle or melatonin (5 mg/kg, i.v.) 15 min before ischemia. The kidneys were removed after 5 min and 6 h of reperfusion. To examine whether melatonin would improve the renal function after I/R, rats were subjected to bilateral ischemia for 45 min, followed by 24 and 48 h of reperfusion.
1. Effect of melatonin on lipid peroxidation induced by I/R
To determine whether melatonin attenuates oxidative stress induced by I/R, we measured the kidney levels of a marker of lipid peroxidation, malondialdehyde (MDA), in rats subjected to unilateral ischemia or sham surgery. After 5 min of reperfusion, renal content of MDA was increased in vehicle-treated ischemic rats compared with sham-operated animals. Melatonin treatment prevented postischemic MDA increase by 74% (P<0.01).
2. Effect of melatonin on caspase-3 activation and cell apoptosis
Apoptosis was evaluated by detection of fragmented chromosomal DNA by TUNEL assay shown in Fig. 1
. The kidneys from rat in the vehicle-treated ischemic group showed extensive TUNEL-positive staining at 6 h of reperfusion, predominantly in the outer medulla (Fig. 1A, B
). In contrast, kidneys from melatonin-treated rats were not different from that observed in sham-operated rats (Fig. 1A, B
).
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Melatonin also prevented activation of caspase-3, a key mediator of apoptotic death. As shown in Fig. 2
, caspase-3 activity was significantly increased in vehicle-treated ischemic group at 6 h of reperfusion compared with sham-operated groups. Pretreatment with melatonin fully inhibited the caspase-3 activation induced by I/R (Fig. 2)
.
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3. Effects of melatonin on renal function
To evaluate the effect of melatonin on recovery of renal function, experiments were performed in the bilateral model of renal ischemia. Kidney function was evaluated in animals subjected to ischemia or sham surgery by determining creatinine and blood urea nitrogen (BUN) levels 24 and 48 h after reperfusion. Ischemia, followed by reperfusion, produced significant increases in plasma concentrations of creatinine and BUN. However, rat treatment with melatonin 15 min before ischemia significantly decreased creatinine and BUN concentrations by 54% and 61%, respectively, at 24 h of reperfusion.
4. Effects of melatonin on tubular necrosis after I/R
Necrotic injury induced by bilateral renal ischemia was evaluated 24 and 48 h of reperfusion. Histological sections of kidneys from vehicle-treated ischemic rats showed extensive tubular necrosis mainly located at the proximal tubules after 24 and 48 h of reperfusion. The histology of ischemic kidneys from rats pretreated with melatonin 15 min before ischemia was normal and indistinguishable from sham-operated animals in 8 of 10 rats.
Treatment with melatonin in sham animals did not produce any detectable histological abnormalities.
CONCLUSIONS AND SIGNIFICANCE
In the present study, we demonstrate that melatonin inhibits caspase-3 activation and prevents postreperfusion apoptotic and necrotic cell death in the kidney. Furthermore, we have shown that melatonin reduces postreperfusion oxidative stress and improves the recovery of renal function. These results suggest that melatonin may represent a novel therapeutic approach for prevention of I/R injury.
Renal tubule cell apoptosis has been consistently observed after I/R in vivo and in vitro models of renal injury and may represent a direct mechanism by which tubule cells are damaged. Prevention of cell apoptosis has been demonstrated to reduce ischemic damage in many organ systems. An essential component of the apoptotic transduction pathway triggered by I/R is the intracellular generation of reactive oxygen species. Melatonin has been found to protect cells from oxidative stress induced by a variety of free radical-generating agents and processes. In fact, melatonin displays antioxidant properties and plays a critical role in preserving the functional integrity of membrane lipids. We have demonstrated that postreperfusion lipid peroxidation is prevented by melatonin treatment showing that the melatonin reduces the postreperfusion oxidative stress. We have also shown that melatonin reduces ischemia-induced cell apoptosis, suggesting that melatonin may act through apoptotic transduction pathways.
Some independent pathways have been implicated in the control of apoptotic death. Recent reports have indicated the importance of caspase families of proteins for apoptotic death. A member of this family, caspase-3, has been shown to be a major execution caspase that acts downstream in the apoptosis pathway and is involved in cleaving important substrates such as inhibitor of caspase-activated DNAse. The investigation of caspase-3-deficient mice suggested that this enzyme in particular might be an appropriate target for therapeutic intervention in diseases that result from apoptosis. The present study provides the first in vivo evidence that melatonin prevents activation of the proapoptotic factor caspase-3 after postischemic injury. The fact that rat pretreatment with melatonin prevents caspase-3-dependent cell apoptosis suggests that the mechanisms of melatonin’s action may involve caspase signaling cascade. There is some evidence suggesting that melatonin may act through signal transduction pathways to influence cellular activity. Melatonin receptors have been shown to inhibit signal transduction cascades including formation of cAMP, mobilization of calcium, release of arachidonic acid, and the synthesis of diacylglycerol. It has also been shown that melatonin receptors may activate mitogen-activated protein kinase cascade. Thus, the effects of melatonin are clearly not limited to its hypothetical direct free radical-scavenging ability and suggest that melatonin may signal through a variety of regulated pathways. However, it is unclear whether activation of melatonin receptors may participate in the prevention of postreperfusion renal injury by melatonin.
Although melatonin has been widely used in humans to treat jet lag and sleep disorder syndromes, we demonstrated that pretreatment with melatonin protects against the development of acute tubular necrosis and improves renal function after I/R. Our findings imply that melatonin may protect against oxidative stress-induced cell death. It is possible that prevention of necrotic cell death by melatonin confers a protective effect on overall tubular integrity and nephron function.
In conclusion, we report a novel finding regarding the protective effect of melatonin against renal ischemic injury in vivo. These results emphasize the importance of caspase signaling pathways to the protective effects of melatonin and represent a first step toward characterization of the cellular and molecular mechanisms by which melatonin prevents I/R injury. The demonstration that melatonin pretreatment prevents caspase-3-dependent cell apoptosis and renal dysfunction after I/R may provide new therapeutic implications in the treatment of kidney diseases characterized by apoptotic and necrotic cell death.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0504fje; to cite this article, use FASEB J. (March 5, 2003) 10.1096/fj.02-0504fje 
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