NDT Advance Access published online on February 19, 2008
Nephrology Dialysis Transplantation, doi:10.1093/ndt/gfm779
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Postconditioning is an effective strategy to reduce renal ischaemia/reperfusion injury
1 Institute of Internal Medicine, Department of Medical and Occupational Sciences, University of Foggia, Foggia, Italy 2 Division of Nephrology, Department of Biomedical Sciences, University of Foggia, Foggia, Italy 3 IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, University of Foggia, Foggia, Italy
Correspondence and offprint requests to: Loreto Gesualdo, Division of Nephrology, Department of Biomedical Sciences, University of Foggia Viale Pinto, 71100 Foggia, Italy. Tel: +39-881-732054; Fax: +39-881-732054; E-mail: l.gesualdo{at}unifg.it
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Abstract |
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Background. Several recent studies have shown that a brief ischaemia applied during the onset of reperfusion (postconditioning) is cardioprotective in different animal models. The potential application of postconditioning to organs different from the heart, i.e. kidney, is not available and is investigated in the present study. We also tested the hypothesis that mitochondria play a central role in renal protection during reperfusion.
Methods. Wistar rats were subjected to left nephrectomy and 90-min right kidney occlusion. In controls, the blood flow was restored without intervention. In postconditioned rats, complete reperfusion was preceded by 3 min, 6 min and 12 min of reperfusion in a consecutive sequence, each separated by 5 min of reocclusion. Animals were studied for 48 h. Mitochondrial respiratory chain function, rate of hydroperoxide production and carbonyl proteins were measured at the end of postconditioning and 24 h and 48 h after reperfusion.
Results. BUN and creatinine significantly decreased in the postconditioning group as compared to control rats. Mitochondrial respiratory function was significantly impaired in control rats, mainly at the level of Complex II. Postconditioning significantly reduced this mitochondria impairment. The rate of mitochondrial peroxide production was higher in the control group than in the protected group at the end of postconditioning reperfusion. Moreover, mitochondrial protein oxidation was significantly higher in control rats than in the postconditioning group at the end of reperfusion.
Conclusions. In the present study, postconditioning reduced renal functional injury and reduces mitochondria respiratory chain impairment, mitochondria peroxide production and protein damage.
Keywords: ischaemia–reperfusion; mitochondria; oxidative stress; postconditioning
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Ischaemic injury to vital organs such as the heart, brain and kidneys contributes significantly to morbidity and mortality throughout the world. Renal ischaemia as a consequence of arterial occlusion, shock and organ transplantation is a common cause of renal cell death, renal failure, delayed graft function and renal graft rejection. Deprived of oxygen-carrying blood, cellular respiration is impaired with irreversible damage occurring virtually in every organelle and subcellular system of the affected cells. After an acute renal ischaemic event, early reperfusion remains the most effective strategy to limit organ damage. However, reperfusion of the kidney has the potential to cause lethal cell death similar to that observed in the heart. Novel protective strategies applied at the time of reperfusion are required to target this injury.
An area of increasing interest is the possibility of rendering an organ resistant to subsequent injury by a prior ischaemic insult, i.e. a preconditioning maneuver. Ischaemic preconditioning of the kidney confers protection against a subsequent ischaemic attack [1]. Nevertheless, the protective potential of ischaemic preconditioning has not been realized in clinical practice because it necessitates an intervention applied before the onset of ischaemia, which is often difficult to predict in non-surgical situation. A more amenable approach is to intervene at the onset of reperfusion, the timing of which is under the control of the operator. Recently, Zhao et al. showed that repetitive cycles of short ischaemia during reperfusion significantly reduce heart infarct size in a canine model [2]. Postconditioning is a mechanical maneuver imposed during the early moments of reperfusion that attenuates endothelial activation and dysfunction and apoptosis. From the first report of Zhao et al., several studies have clearly demonstrated that postconditioning marshals a variety of endogenous mechanisms that operate at numerous levels and target a broad range of cardioprotective mechanisms. To date, several tissue protective pathways have been identified that functionally relate to reduced cell death. Our group has recently reported that postconditioning limits mitochondria failure and prevents the production of reactive oxygen species (ROS) in isolated perfused hearts [3].
The potential application of postconditioning to organs other than the heart, i.e. kidney, has not been investigated in detail. The most technical problem is to establish the correct algorithm of postconditioning. From preconditioning studies it is well known that a critical interval of ischaemia and reperfusion exists and the ‘right window’ is sometimes difficult to define. Chien et al. have recently reported that the amount of ROS product correlates with the duration of ischaemia. More relevantly, they defined a minimal 5-min reperfusion required for detection of ROS, whereas no ROS was detected 10 and 24 h after reperfusion [4]. These data suggest that the first minutes of reperfusion are crucial for defining the final fate of the kidney after ischaemia, exactly as occurred in the heart model. In addition, it has been recently reported that the gradually increased blood flow on to the ischaemic kidney decreases ischaemic changes [5]. The present work shows, for the first time, that the application of a specific postconditioning algorithm protects the kidney from ischaemia/reperfusion injury through a mechanism that involves protection of mitochondria respiratory chain function, similar to the heart model. Our data suggest that postconditioning may be an effective strategy for various organs subjected to ischaemia/reperfusion.
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Experimental protocol
All animals received care in compliance with the Principles of Laboratory Animal Care formulated by the National Society of the Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86-23, revised 1985), as well as with Italian law on animal experimentation. Male Wistar rats weighing 200–250 g (Harlan, Italy) were anaesthetized by i.p. 100 mg/kg ketamine and 2.5 mg/kg acepromazine. The animals were placed under an overhead lamp in order to avoid anaesthetic hypothermia. Their temperature was monitored continually by using an electronic thermometer.
After the induction of anaesthesia, the peritoneal cavity was opened, the right renal vascular pedicle was exposed and ligated using 3–0 silk sutures twice, and the kidney was removed according to a modified method proposed by Dobashi [6]. To avoid vessel wall trauma and peri-vessels haematoma the left kidney and its pedicle were dissected off the surrounding perirenal fat along the renal surface; then both renal artery and vein were clamped using a Schwartz clip (RS-5452, Roboz).
In the non-protected I/R group, 90 min after ischaemia, the clamp was removed and the renal blood flow restored. In the postconditioning group (P-IR), 90 min after ischaemia, postconditioning was performed treating the rats by 3 min, 6 min and 12 min of reperfusion consecutively, separated by 5 min of reocclusion (Figure 1). The abdomen was closed and the wound sterilized by applying 10% povidoneiodin. Animals were killed at the end of Ischaemia (EI), at 36 min of reperfusion (corresponding to the end of postconditioning), and at 24 (24h) and 48 (48h) of reperfusion; blood was collected and the left kidney rapidly removed for functional and morphological analysis. In addition, two sets of eight animals were killed after right nephrectomy and before left renal ischaemia and the kidneys were considered as controls (BI). To prevent the risk of dehydration all the animals were pretreated with 3 ml of subcutaneous physiologic saline solution (PSS).
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Renal function and histological analysis
Serum creatinine and blood urea nitrogen (BUN) were measured as renal functional parameters using standard diagnostic kits. The kidneys were preserved in phosphate-buffered 10% formalin, embedded in paraffin and used for histopathological examination. Then, 2-µm-thick sections were cut, deparaffinized, hydrated and stained with haematoxylin and eosin and with periodic acid-Shiff (PAS) reagents. Renal sections were examined in blind fashion for tubular cell swelling, cellular vacuolization, pyknotic nuclei, medullary congestion and the degree of necrosis. A minimum of 10 fields for each slide were examined and assigned for severity of changes using scores on a scale of 0 = no change, 1 = patchy isolated damage, 2 = damage less than 25%, 3 = moderate, damage between 25% and 50%, 4 = severe, damage between 50% and 75% and 5 = very severe, complete destruction of epithelial cells [7].
Mitochondrial isolation, oxygraphic measurements and western blot analysis
Fresh kidney tissue was chilled on ice and washed with mitochondria buffer (0.25 M Sucrose, 5 mM K-EDTA, 10 mM-Tris HCl pH 7.4) containing 0.5% BSA, to remove blood and connective tissue. Subsequently, the tissue was minced with scissors and washed three times with the same buffer in a prechilled glass beaker, and centrifuged to extract mitochondria according to Kun [8]. Total protein concentration was determined using the Lowry micromethod kit (Sigma-Aldrich, St. Louis, MO, USA). Freshly prepared mitochondria were assayed for oxygen consumption at 30°C by an oxygraph apparatus equipped with a Clark's electrode and a rapid mixing device (Hansatech Instruments Ltd and Oxygraph Plus 1.01 Software).
State 4 respiration was started by the addition of 10 mM glutamate/5 mM malate or 10 mM succinate in the presence of 1 µM rotenone. State 3 respiration was induced by addition of 100 µM ADP. Rates of oxygen consumption were determined after sequential addition of the following substrates and inhibitors: 0.5 µM carbonylcyanide M-chlorophenylhydrazone (CCCP), 0.02 µM antimycin A, 10 mM ascorbate plus 0.25 N',N'-tetramethylbenzene-1,4-diamine (TPMD), 0.3 µM potassium cyanide (KCN). The ascorbate/TMPD respiration rate was measured as that which was KCN sensitive. Respiration control index (RCI: ratio of state 3 to state 4 respiration), ADP/O (ratio of ADP added and oxygen consumed) and respiratory activity of Complex I, III and IV were calculated as previously reported [9].
In addition, qualitative analysis of mitochondrial oxidized proteins, as expression of oxidative protein damage, was performed by western blot analysis using the Oxyblot kit (Chemicon International). Briefly, approximately 15 µg of mitocondrial proteins was reacted with dinitrophenylhydrazine (DNPH) for 20 min, followed by neutralization with a solution containing glycerol and 2-mercaptoethanol, resolved in 12.5% SDS-PAGE, transferred to a nitrocellulose membrane, blocked with non-fat milk and then incubated with a rabbit anti-DNPH antibody as the primary antibody (1:150) at 4°C overnight. After washing, the membrane was incubated with the secondary antibody (1:300) conjugated to horseradish peroxidase (HRP) and detected by a chemiluminescence detection kit (Cell Signaling Technology, Beverly, MA, USA, #7071). Reactive bands were visualized by the enhanced chemiluminescence method on VersaDoc Image System (Biorad).
Measurement of peroxide production in kidney mitochondria
The rate of peroxide production over time in kidney mitochondria was determined by a modification of the method described by Barja as previously reported [10]. Mitochondria were incubated at 37°C with 5 mM pyruvate plus 2.5 mM malate or with 10 mM succinate in 2 mL of 5 mM phosphate buffer, pH 7.4, containing 0.1 mM EGTA, 3 mM MgCl2, 145 mM KCl, 30 mM HEPES, 0.1 mM homovanilic acid and 6 U/mL HRP. The incubation was stopped at 5, 10 and 15 min with 1 mL of cold 2 M glycine buffer containing 50 mM EDTA and 2.2 M NaOH. The fluorescence of supernatants at these times was measured using 312 nm as excitation wavelength and 420 nm as emission wavelength. The rate of peroxide production was calculated using a standard curve of H2O2.
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Data were expressed as mean ± standard deviation (SD). All the variables were analysed for normal Gaussian distribution by the Kolgomorov–Smirnov test. Actual data were analysed by the two-way ANOVA and Bonferroni correction test as the post-hoc test. Non-parametric histological scores were analysed by the Mann–Whitney test. In all instances P < 0.05 was taken as significant. The package GraphPad Prism 4 for Windows (GraphPad Software Inc., San Diego, CA, USA) was used to perform all the statistical analyses.
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Effect of postconditioning on the survival rate of the rats subjected to kidney ischaemia/reperfusion
Ten rats of each group of experimental protocol were used for the survival study. Rats that lived more than 12 days after kidney I/R were considered survivors.
As shown in Figure 2 no mortality was observed in the animals of the control group, whereas rats undergoing I/R injury without protection presented more than 60% of mortality compared to 25% of the postconditioned group.
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Protective effect of postconditioning on renal function
Blood urea nitrogen (BUN) and serum creatinine were evaluated as indices of renal function and their profiles are reported in Figure 3A–B. Rats from the non-protected ischaemic group (I/R) presented severe renal failure 24 h after reperfusion with a peak of BUN on Day 2. Twenty-four hours after postconditioning reperfusion (P-IR) BUN and serum creatinine levels were significantly lower compared to the I/R group and, more relevantly, plasma levels did not increase on Day 2 (P < 0.05 for BUN and P < 0.005 for creatinine).
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Effect of postconditioning on kidney morphology after I/R injury
Renal sections were stained with haematoxylin and eosin and with periodic acid-Schiff (PAS) reagents. Histological sections of control (BI) kidneys showed normal renal architecture with well-preserved glomeruli and normal thickness of the brush border of the tubules (score = 0, Figure 4A). I/R rat kidneys showed almost complete tissue destruction at 24 and 48 h of reperfusion (Figure 4B and C). Most significant changes were seen in the proximal tubules with shedding of the lining cells into the lumen together with increased amounts of protein in the lumen. Semi-quantitative analysis of tubular necrosis showed a severely increased necrosis score from 0.5 (range 0.5–1) at end-ischaemia to a median of 4.5 (range 3.5–5) and 4 (range 3–5) on Day 1 and 2, respectively. Kidney from the P-IR group showed a significantly lower necrosis score of 2 (range 0.5–2.5) and 3 (range 0.5–4) on Day 1 and 2, demonstrating in some cases no notable difference from before ischaemia (Figure 4D and E).
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Effect of ischaemia and long-term reperfusion on the mitochondria respiratory chain
The respiratory control index (RCI), expression of the coupling respiration/phosphorylation, dramatically decreased at the end of ischaemia in renal mitochondria from the I/R group as shown in Figure 5. Twenty-four hours after reperfusion, RCI remained lower, and no recovery was observed after 48 h. The pattern of the changes was similarly independent of the oxidative substrate used (Figure 5A and B). In sharp contrast, mitochondria of P-IR rats showed a significant improvement in RCI at 24 and 48 h with both substrates over that in the I/R group, suggesting a rapid restoration of mitochondrial function in postconditioned rats (P < 0.01 for Complex I and P < 0.0001 for Complex II).
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Respiration activity measurements suggested a differential sensitivity of the components of the mitochondrial electron transport chain to ischaemia and reperfusion. In fact, at the end of ischaemia the respiratory activity of Complex I appeared to be decreased and no recovery was observed 24 and 48 h after reperfusion (Figure 6A, white squares). In contrast, Complex II appeared significantly impaired even at the end of ischaemia and its activity remained lower during reperfusion (Figure 6B, white squares).
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Ischaemic postconditioning reduces mitochondria eroxide production and oxidative protein damage during reperfusion
Peroxide production by kidney mitochondria was measured using pyruvate and malate as Complex I-linked substrates and succinate as a Complex II-linked substrate at the end of postconditioning reperfusion (36 min) and 24 h later in I/R and P-IR rats.
The rate of peroxide production in kidney mitochondria from I/R rats was significantly higher in I/R kidneys than in the P-IR group at the end of 36 min of reperfusion (P < 0.05 for the Complex I- and P < 0.01 for Complex II-linked substrate respectively). The rate of hydroperoxide synthesis returned to the basal level in both groups 24 h later (Figure 7).
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To better characterize the protection exerted by postconditioning against oxidative damage, carbonyl groups of oxidized proteins were derivatized with DNPH on Oxyblot and detected using an anti-DNP antibody by western blot, as described in the Materials and Methods section. Representative analysis of Oxyblot are shown in Figure 8. An appreciable decrease in oxidized proteins was detected in postconditioned rats when compared to non-protected rats.
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Discussion |
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Transient ischaemia leads to a heterogeneous pattern of tissue damage in the kidney. Short period of ischaemia (<45 min) irreversibly damages distal segments of the proximal tubules, whereas more proximal segments suffer reversible injury [11]. The same pattern of damage can be induced in the proximal segments after longer periods of ischaemia (90 min) [12]. In the present study we applied a severe model of ischaemia (90 min) after right nephrectomy, firstly proposed by Dobashi et al. [6], that induced about a 10-fold increase in creatinine and BUN level 24 and 48 h after reperfusion, related to large necrosis of renal tubules. This experimental model induces, in male Wistar rats, a mortality rate of about 70%.
The gradually increased blood flow has been shown to have beneficial effects of reducing the damage of reperfusion injury on an ischaemic rat muscle model [13] and on an ischaemic rat kidney [5]. In addition, Vinten-Johansen et al. have reported that gradual restoration of perfusion pressure during early reperfusion was associated with a reduction in microvascular injury, infarct size and tissue oedema in the heart [2].
According to these observations, in the present study, the application of 3, 6 and 12 min of reperfusion alternated to 5 min of ischaemia before definitive reperfusion (postconditioning) was chosen arbitrarily and demonstrated to be able to reduce renal failure of about 50% and in some cases, almost completely abolished the tubular damage. Moreover, the mortality rate was less than 25% as compared to 60% of non-protected rats.
We observed that the protection exerted by postconditioning acts on the mitochondria respiratory chain function and hydroperoxide synthesis. In fact, to our knowledge, we firstly demonstrated that at the end of the application of postconditioning, the rate of mitochondrial H2O2 synthesis is significantly lower than that in non-treated rats and the mitochondria chain improved after ischaemia, suggesting a novel role of postconditioning in the protection exerted against renal reperfusion injury.
Ischaemia/Reperfusion impairs the mitochondrial respiratory chain and induces mitochondrial protein oxidation
Complete or partial cessation followed by restoration of the blood flow is a serious event that affects many organs, such as the heart, brain, liver and kidney. Ischaemia–reperfusion contributes to abnormal signal transduction or cellular dysfunction and initiates the cascade of apoptosis/necrosis, with subsequent inflammatory infiltration. Reperfusion remains the definitive treatment to salvage tissue [14]. However, reperfusion is associated with injury that was not apparent at the time of reflow. Reperfusion injury is an integrated response to the restoration of the blood flow after ischaemia, and is initiated at the very early moments of reperfusion, lasting potentially for days [15]. Reperfusion injury has been a strategic target of therapy for many years in renal surgery. It has been reported that ischaemia substantially inhibits electron transport without affecting the activity complex. The impairment of the electron transport chain could be due to ultrastructural derangement related to conformational changes and protein modification [16]. Other studies supposed that this impairment might be ascribed to generalized protein oxidation [17], but no data are available on long-term reperfusion in the renal model of I/R injury. In the present study, the oxymetric tests performed in mitochondria isolated from non-protected I/R rats confirm that the coupling respiration/phosphorylation is severely impaired at the end of ischaemia, according to previous observations [18]. In addition, our results show that ischaemia inhibited electron transport throughout Complexes II and IV, without affecting the respiratory activity of Complex I at the end of ischaemia. This could be related to the regulatory role of NADH dehydrogenase (Complex I). Upon re-entry of oxygen, the redistribution of the electron flow increases the percentage of electrons that leak to form ROS [16] at the ubisemiquinone level, causing inactivation of Complex II through generalized protein oxidation [17]. In a recent work [3], we have found that ischaemia–reperfusion causes an increase in peroxide production through mitochondrial Complexes I and III, suggesting a role played by mitochondria in oxidative stress during ischaemia–reperfusion. Here we report a five-fold increase in the rate of superoxide anion/hydroperoxide production after reperfusion in the rat kidney [16], which, together with the reported two- to five-fold increases in the intracellular steady state concentration of hydrogen peroxide in the rat kidney [18], supports the idea of mitochondria as a major source of oxygen free radicals in some oxidative stress situations. The increase of ROS production is only partially counteracted by the increase of the activity of the intramitochondrial superoxide dismutase [18]. Nevertheless, the increase in the rate of mitochondrial production of oxygen free radicals is considered enough to damage some of the respiratory chain components and to cause cell damage [16,18].
Our data support these observations, since 24 h after reperfusion all three complexes are impaired in the non-protected I/R rat group. Immunoblotting analysis of mito- chondria protein reveals a significant increase in protein oxidation accountable for the respiratory chain impairment. More experimental studies are necessary to properly discern whether and which of mitochondria respiratory chain complexes are oxidated.
In addition, our data show that long-term reperfusion (24 h) after prolonged ischaemia (90 min) also affects the regulatory activity of the Complex I and dramatically alters the cell bioenergetics. The recovery of the oxidative phosphorylation after reperfusion is essential for restoring ATP stores depleted during ischaemia, and is a prerequisite for organ viability and survival following organ transplantation.
Postconditioning prevents mitochondria impairment and reduces protein oxidation
A well-recognized and effective surgical approach to limit reperfusion injury is ischaemic preconditioning, first reported by Murry in 1986 [1], who reported the increased tolerance to prolonged ischaemia by pre-exposing the organ to one or more cycles of brief ischaemia and reperfusion (I/R). The mechanisms of preconditioning include adenosine receptor mediated activation of adenosine triphosphate (ATP)-gated potassium channels, nitric oxide synthesis, free radical generation and upregulation of molecular chaperones. It is well recognized that mitochondria play a critical part in I/R injury and in preconditioning protection, by their pivotal role in energy production, by the generation of ROS and the initiation of apoptosis.
Recently, Zhao et al. have reported that ‘postconditioning’ was as effective as preconditioning in protecting cardiac tissue against reperfusion injury [2]. We have recently shown, in an isolated and perfused heart model, that postconditioning protects the heart against I/R injury by preventing mitochondria peroxide production [3]. The mechanical procedure of postconditioning seems to induce a multiplicity of events that together attenuate reperfusion injury at many cellular and intracellular sites, as opposed to inhibiting individual biochemical or molecular targets [19]. Postconditioning attenuates oxidants and oxidant-mediated injury, but may also preserve the signalling function of both oxidants and nitric oxide [19]. It is under debate whether postconditioning may be applied directly to other organs and in other procedures such as organ transplantation. So far no data are available, in vivo, on the possible application of postconditioning in the renal model of I/R injury. Therefore, we designed a rat model of kidney I/R injury where short episodes of I/R were performed during the first minutes of reperfusion.
The present study suggests involvement of mitochondria in the protection exerted by postconditioning. There is a wide consensus that the protective mechanism of postconditioning involves prevention of the burst or prolonged elevation of free radical generation. Data from the present study are consistent with this reduction in oxygen radical species, and further suggest that those oxidants derived from mitochondria were reduced by postconditioning. This is the first report demonstrating that postconditioning is associated with protection of mitochondria respiratory chain in kidneys. In the postconditioning group, an almost complete recovery of the respiration rate is observed. In particular, the recovery of the maximum respiratory rate is observed when succinate was added into the medium. In contrast, when glutamate–malate was used as oxidative substrate, the recovery was partially obtained, suggesting the loss of functional integrity at the level of Complex I as target of oxidative reperfusion injury.
Oxidant generation in the kidney may lead to apoptosis [4] particularly in tubular epithelium. The in situ study showed that ROS are produced in significant amounts in tubule epithelium under reperfusion conditions and therefore may be responsible for the apoptotic death of these cells [4]. It has been clearly established that mitochondrial oxidative stress makes the tubular cells much more susceptible to ischaemia-induced apoptosis [2,4]. Our results show that while reperfusion causes mitochondrial dysfunction, postconditioning would protect the kidney against cell death by preventing mitochondrial oxidative stress.
Recently, Argaud et al. has shown in the rabbit heart that postconditioning can modify mitochondrial permeability transition pore (mPTP) opening [20]; mPTP opening results in uncoupling the respiratory chain, efflux of cytochrome c and other proapoptotic factors, leading to either apoptosis or necrosis. Ischaemia–reperfusion combines several conditions that can trigger mPTP opening, including matrix Ca2+ overload, overproduction of reactive oxygen species, depletion of adenine nucleotides and accumulation of inorganic phosphates. In particular postconditioning seems to delay Ca2+-induced mPTP opening. To date, no data are available on the role of mPTP in an ischaemia-reperfused rat kidney. It is conceivable that postconditioning might affect the degree of mPTP also in renal ischaemia. In the present study, however, we did not determine whether tubular injury was reduced by necrosis or apoptosis, and which mechanism of cell death was influenced by postconditioning.
In conclusion, the present study shows, for the first time, that postconditioning improves in vivo renal function after prolonged ischaemia, which is associated with attenuation of mitochondria bioenergetics dysfunction, ROS synthesis and protein damage. Postconditioning, in contrast to preconditioning, which requires a foreknowledge of the ischaemic event, can be clinically applicable at the onset of reperfusion at the point of clinical service, i.e. renal artery and kidney surgery or transplantation, where reperfusion injury is expressed.
Future studies are needed to define the ‘gold algorithm’ of postconditioning sequences, as well as other mechanisms involved in the protective effect.
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Acknowledgments |
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The authors are grateful to Professor N. Capitanio and Dr F. Bellanti for valuable suggestions and to Dr A. Petrella for animals care. This study was partially supported by the Ministero dell’Università e della Ricerca (MIUR)—Progetto di Ricerca di interesse nazionale 2007 and by a grant from Cassa di Risparmio di Puglia Foundation.
Conflict of interest statement. None declared.
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[Abstract/Free Full Text]
Accepted in revised form: 5.10.07
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