In organ transplantation, the paradigm is to initiate reperfusion as soon as possible. Although reperfusion relieves ischemia, it concurrently initiates a cascade of harmful events, leading to so-called ischemia-reperfusion injury (IRI). Multiple processes are involved in IRI, including ion accumulation, dissipation of mitochondrial membrane potential, free reactive oxygen species (ROS) formation, endothelial dysfunction, platelet aggregation with microembolization, no-reflow phenomenon, and immune activation (reviewed in (1)).
Delayed graft function (DGF) frequently complicates kidney transplantation (KT). IRI is considered to be an important cause of DGF, which is associated with prolonged hospital stay, acute rejection, and, possibly, chronic transplant loss (2–5). Finding new strategies to prevent DGF has therefore become a major issue in transplantation. Attempts to reduce IRI have hitherto focused on improving hypothermic organ preservation and reducing the nephrotoxic effects of calcineurin inhibitors (5–7).
The possibility to create resistance against IRI through “organ conditioning” has become an area of increasing interest. In 1986, Murry et al. demonstrated that four cycles of 5-min occlusions of the coronary artery, interspersed with 5-min cycles of reperfusion applied immediately before prolonged ischemia, reduced myocardial infarct size by 75% in a canine heart (8). This phenomenon was named ischemic preconditioning (IPC). Subsequent research has shown that the mechanisms involved in the protective effects of IPC confer a complex set of signal transduction pathways (9). However, IPC is an intervention that has to be applied before the onset of ischemia and is thus not suitable for clinical implementation, because the onset of an ischemic insult is often difficult to predict. This has restricted its use to elective (cardiac) surgery (10).
A clinically more suitable approach would be to perform the “conditioning” intervention at the onset of reperfusion, because it is a more controllable time point. In fact, repeated sequences of ischemia and reperfusion after a prolonged ischemic episode, so-called ischemic postconditioning (IPoC), also reduce myocardial infarct size by approximately 40% in animals (11–13) and in humans (14). In addition, IPoC has demonstrated its clinical potential in several animal models of noncardiac IRI, including the kidney (15). Many of the protective pathways involved in IPoC are similar to those of IPC, although differences do exist (16). Thus far, no human studies have been performed to limit IRI through IPoC in transplantation. Here, we provide an overview of animal experiments on IPoC in relation to renal IRI and assess whether there is a rationale to perform IPoC to improve the outcomes of human KT.
RENAL ISCHEMIA-REPERFUSION INJURY MODELS AND ISCHEMIC POSTCONDITIONING ALGORITHMS
In animal models, KT is most frequently mimicked by inducing warm ischemia of the native kidneys by clamping the renal pedicle for a certain period of time. The major disadvantage of this method is the absence of cold ischemia. Unfortunately, in small animals, (auto)transplantation is technically very challenging. As a result, most reports on IPoC are restricted to the warm ischemia renal IRI model.
In Table 1, the various animal studies investigating IPoC are summarized. Interpretation of these data is complicated by the fact that different animal species were studied (rats (17–26), mice (27), and mongrel dogs (28, 29)), the sex of the animals was different, and the duration of ischemia and the particular IPoC algorithm used were not the same (Table 1). In general, larger animals need to be exposed to a longer duration of ischemia to induce the same effect on an organ, although conclusive evidence for this is lacking. Furthermore, females appear to be more resistant to ischemia than males; therefore, longer ischemia times are required to reach the same effect (30). In the studied reports, most experiments involved unilateral nephrectomy with contralateral clamping of the renal pedicle to induce renal IRI. Two groups performed bilateral renal clamping (24, 25), and in one study, autotransplantation was performed in a canine model, in which the kidney was exposed to 24-hr cold ischemia (29).
Duration of warm ischemia varied from 30 to 90 min, where 45-min ischemia was most common (all in rats). Only Wang et al. (23) and Serviddio et al. (19) included longer ischemia times of 60 and 90 min, respectively. The expected damage would be more extensive in these groups, probably resulting in a higher mortality, but as the animals were sacrificed at 24 or 48 hr, this could not be observed. As mentioned, there were also differences in the IPoC algorithm, the duration of reperfusion and ischemia in IPoC. Most groups used an algorithm of 6×10 sec (all in rats). It is unclear whether the different algorithms used have different effects, which complicates the comparison of the reports. Nevertheless, previous reports mention no difference between different algorithms in healthy animals (31, 32), although Jiang et al. (28) compared three different algorithms in mongrel dogs: six cycles of 15 sec, six cycles of 30 sec, and three cycles of 60 sec and did find significant differences between the three algorithms. The effect of the six cycles of 15-sec algorithm was most effective. The absence of consensus on the IPoC algorithm complicates translation to the clinic.
EFFECTS OF ISCHEMIC POSTCONDITIONING ON KIDNEY FUNCTION
Kidney function markers serum creatinine and blood urea nitrogen are the most frequently used parameters to evaluate the severity of IRI. To date, in all studies, IPoC seems to reduce the severity of renal IRI (Table 1) irrespective of index ischemia, algorithm of IPoC, and animal species studied. In the experiment of Eldaif et al. (20), who used a unique IPoC algorithm (Table 1), rats with IRI+IPoC had a kidney function (24 hr after reperfusion), which was comparable with that of sham-operated animals. This indicates that IPoC abolished the detrimental effects of renal IRI on kidney function, which was significantly higher in the IRI (without IPoC) group.
In myocardial IRI, the duration of index ischemia dictates the outcome of IPoC (33); a detrimental effect of IPoC on myocardial infarct size after shorter periods of index ischemia was seen (i.e., 15–30 min), whereas a protective effect was observed with longer durations of ischemia (i.e., 45–60 min). This protective effect waned when index ischemia was increased even further (i.e., 90–120 min). In the kidney, no detrimental effect of IPoC on kidney function has been demonstrated.
Another way to investigate renal damage after ischemia is by histopathologic analysis, where damage is studied at the cellular level, mostly by observing morphologic changes and measuring necrosis. Damage can be scored objectively with a renal damage scale like Jablonski et al.’s (34). This sort of scale enables an objective measurement of the damage after IRI and comparison between IPoC and IRI. Substantially less histologic damage was observed between IPoC and IRI alone (Table 1). When damage was scored only 2 hr after reperfusion, no differences were found (25). Serviddio et al. (19) used a semiquantitative scale and showed observable differences between kidneys after IRI with or without IPoC. In the IRI group, increased necrosis after ischemia was found, whereas, in the IRI+IPoC group, hardly any notable differences with preischemia samples were seen (19). Zhang et al. (24) showed a significantly lower percentage of apoptosis in IPoC compared with IRI. Finally, IPoC led to less tubulointerstitial fibrosis at 12 weeks after IRI probably due to less inflammation in the acute phase of IRI (26). Thus, IPoC ameliorates cellular damage caused by renal IRI, resulting in less kidney dysfunction.
MECHANISM OF ISCHEMIC POSTCONDITIONING
The underlying pathophysiologic mechanisms of IRI are very complex. The reports on IPoC in the kidney have focused on different aspects of IRI, such as ROS formation and scavenging, apoptosis, and inflammation. Figure 1 represents an integrated view of the most important mechanisms of IPoC, as reported in the reviewed publications.
Reactive Oxygen Species and Reactive Oxygen Species Scavengers
Formation of ROS plays a critical role in the balance between cell survival and apoptosis (35–37) and is also considered a major factor in IRI-induced damage (38–40). There are different ways to measure the formation of ROS or oxidative stress, because multiple mechanisms are involved. Superoxide dismutase (SOD) and malondialdehyde (MDA) are markers of ROS and oxidative stress; an up-regulation of these markers is likely after inducing renal IRI. In the two renal IRI models studying these markers, the expression of MDA was found to be significantly lower in IPoC compared with IRI (21, 28). SOD was significantly higher in both reports, which means less ROS are formed and less SOD is needed to reduce ROS levels. Jiang et al. (29) repeated their experiment but replaced the simple clamping of the kidney by autotransplantation. In this way, they added 24-hr cold ischemia to the ischemic attack. Results were similar to the warm ischemia model, with an increase in SOD and a decrease of MDA in IPoC compared with IRI (29).
These findings suggest an attenuation of ROS formation due to IPoC. During reperfusion, the sudden influx of oxygen will cause ROS formation. Furthermore, antioxidants, induced by IPoC, can scavenge ROS, mostly through converting superoxide in water. Glutathione (GSH) peroxidase is an example of an enzyme with antioxidant activity. Catalase can catalyze this mechanism. An up-regulation of GSH, GSH peroxidase, and catalase is seen after IPoC compared with IRI alone (21). Thus, besides the fact that IPoC attenuates the formation of ROS, it is suggested to stimulate the production of ROS-neutralizing scavengers more quickly.
Mitochondria and Apoptosis
Mitochondria are important ROS producers after IRI. The formation of mitochondrial permeability transition pores (MPTPs) at reperfusion can lead to apoptosis (41). Zhang et al. (24) measured mitochondrial membrane potential to examine MPTPs and demonstrated a higher membrane potential after IPoC compared with IRI. Decreased membrane potential after IRI correlates with MPTP formation and can thus be used as parameter for mitochondrial damage. Furthermore, mitochondrial KATP channels (mitoKATP) provide protection of cell survival (42). It is shown that mitoKATP are involved in the protective effect of IPoC (43–45). Zhang et al. (24) showed the involvement of mitoKATP in IPoC in the kidney and that its effect was diminished when a selective inhibitor of mitoKATP (5-hydroxydecanoate) was administered.
Apoptosis is considered a major contributor to IRI and can induce an inflammatory response (36, 37). Caspase-3, Bax, cytochrome c, extracellular signal-regulated kinase, Akt, and bcl-2 are proteins involved in apoptotic pathways. After kidney IRI, up-regulation of proapoptotic proteins caspase-3, Bax, and cytochrome c has been observed (18). This up-regulation is significantly decreased after IPoC. Furthermore, bcl-2 and Akt are both upregulated after IPoC, resulting in inhibition of apoptosis (18).
Eldaif et al. confirmed that IPoC in kidney IRI, just as in IPC (41), acts through adenosine, which activates protein kinase C to affect mitochondrial permeability, which results in cell survival (20). The effect of IPoC at nitric oxide (NO) and renal NO synthase (NOS; endothelial NOS and inducible NOS) was examined by the same group (17). In IRI, NO could have a dual role, because it attenuates neutrophil events but reacts with ROS as well, forming the cytotoxic oxidant peroxynitrite. This complicates the judgment whether NO is protective or not. Possibly, its effect is dependent on the concentration and the site of release of NO. IPoC increases NO, endothelial NOS, and inducible NOS expression and is associated with improved renal function. When a NOS inhibitor was administered before IPoC, the effect of IPoC was abolished. This suggests that NO actually has a protective effect in renal IRI, which is invigorated by IPoC (17). A suggested pathway is shown in Figure 1.
Cellular damage caused by renal IRI induces an inflammatory cascade, resulting in further damage. Tumor necrosis factor-α (TNF-α) is one of the key mediators of the inflammatory response after IRI (46). During ischemia, necrosis develops in the kidney, accompanied by cytokine formation. Cytokines induce an inflammatory response augmenting damage (47), activating pathways such as mitogen-activated protein kinase, nuclear factor-κB, and apoptotic cell death. Miklos et al. (25) examined TNF-α serum levels and TNF-α infiltration in the kidney 2 hr after reperfusion. Serum TNF-α was significantly increased after IRI compared with preoperative levels in both control and IPoC. The rise in serum TNF-α and TNF-α infiltration in the kidney was significantly less after IPoC compared with control (P<0.05).
Inflammation due to cellular damage also encompasses neutrophil infiltration in the kidney; neutrophils produce cytokines, proteases, and myeloperoxidase (MPO). MPO activity was less in IPoC compared with controls, suggesting less neutrophil infiltration after IPoC (21, 28). Cyclooxygenase (COX)-1 and -2 are involved in the arachnoid acid metabolism. COX-1 is normally expressed in most tissues, whereas COX-2 is associated with inflammatory responses in various tissues, including the kidney, induced by cytokines (interleukin-1, TNF-α, and interferon-gamma). An up-regulation of COX-2 is seen after renal IRI (48). Inhibition of COX-2 is again associated with amelioration of renal IRI (49). Yun et al. (22) showed that COX-1 expression fluctuated in a similar manner between groups (sham, IRI, and IPoC). A significant up-regulation of COX-2 was found compared with sham-operated rats. No difference was observed between IPoC and IRI. However, the COX-2/COX-1 ratio was found to be significantly different between IRI and IPoC in favor of IPoC.
The complement system is involved in IRI (50, 51). C3a and C5a are formed during the complement cascade and are markers of complement involvement. Wang et al. (23) found a significant down-regulation of DAF and up-regulation of C5a after IRI. IPoC prevented this complement activation, resulting in significantly higher DAF and significantly lower C5a expression compared with IRI.
Since 2003, when IPoC was shown to limit myocardial IRI (13), many studies have investigated this endogenous protective mechanism, followed by the translation to other organ systems, with the first publication of IPoC in the kidney in 2007 (27). Since then, several experimental studies with IPoC in renal IRI models have been published (17–28). The general conclusion of these experiments is that IPoC improves renal function after IRI. Renal damage parameters are significantly attenuated in IPoC compared with IRI alone. IPoC leads to a reduction of ROS production and an increase in ROS scavengers. This would suggest that IPoC not only prevents ROS formation but also stimulates antioxidant activity, preventing cellular damage. Furthermore, mitochondria seem to be more resistant to damage through IPoC, leading to less apoptosis by up-regulation of antiapoptotic proteins as well as attenuation of proapoptotic gene expression. Renal IRI has a very complex pathophysiologic mechanism, which is exemplified in the reports reviewed here. Nonetheless, all the studied pathways involved are stimulated or inhibited in a protective manner (Fig. 1). Thus, ROS, apoptosis, and inflammation are all reduced. They are all causative factors of DGF, which can lead to chronic kidney failure.
Unfortunately, the optimal IPoC algorithm remains unknown. Generally, in smaller animals with a more rapid heartbeat, a shorter period of time can be used (5–10 versus 30–60 sec in larger animals). For humans, an algorithm of 1 min may be sufficient considering these speculations. These differences are thought to be correlated with the metabolic rate of a certain species (9, 14), although conclusive evidence is lacking. Nonetheless, IPoC is a simple intervention that may easily be incorporated in everyday clinical transplant practice to prevent the consequences of IRI.
Because transplantation is ordinarily not an elective procedure, IPC cannot always be performed. However, the acceptor could be preconditioned. IPoC can be performed in both elective and primary procedures because the surgeon is in control of reperfusion. Despite the rising amount of proof, clinicians remain critical about the concept of IPoC. One explanation is that, in most animal experiments, healthy young animals were used. The population in which IPoC would be used is older and obviously has some kind of (co)morbidity. To mimic the clinical situation, experiments have been performed in animal models with comorbidities, which do show a reduction of both myocardial (31, 32, 52) and renal IRI (25).
Another argument is the used IRI model in animal experiments, which only investigated warm ischemia. Hitherto, only one report was published with an autologous KT model in mongrel dogs, which showed favorable effects of IPoC (29). Furthermore, several small clinical trials with IPoC have already been performed, especially in cardiology and cardiothoracic surgery (13, 14). More structural collaboration between different medical specialties is needed to benefit from translational experience obtained in another but related field.
Based on the lessons learned in cardiology, where both IPC and IPoC have been discovered, it seems sensible to first explore possible beneficial effects of IPoC in KT using deceased donors. The effect of IPoC on myocardial infarct size has been shown to be dependent on the duration of myocardial index ischemia and may even increase infarct size after shorter durations of index ischemia (33). Therefore, applying IPoC in live donor KT is probably not the most suitable approach, because ischemic damage is usually significantly less.
Another reason why IPoC has not yet been studied clinically may be the fear that repetitive clamping of a human artery increases the risk for postoperative complications, such as releasing atherosclerotic plaques into the vascular system or causing iatrogenic injury to the clamped vessel. This may be especially relevant to patients with end-stage renal disease who often have extensive atherosclerosis. However, the available clinical trials in cardiothoracic surgery do not report such serious adverse events, although these trials contained small numbers of patients (53, 54). Furthermore, in clinical studies on IPC in both kidney and liver transplant recipients, no increased risk of vascular complications was observed (1, 55).
Remote IPC is controlled organ or tissue ischemia other than the target organ. By inducing one or more defined ischemic periods in a distant tissue, it is attempted to protect the target organ from IRI. Initial successes have been achieved, particularly in patients undergoing cardiothoracic surgery (56). However, negative reports begin to emerge now that more clinical trials are being performed applying variations in remote conditioning protocols, different intervals between the remote stimulus and the onset of myocardial ischemia, and differences in baseline patient characteristics and choice of anesthetic agents (because pharmacologic conditioning can be applied as well). One clinical study is currently ongoing, which aims to test the effect of remote ischemic conditioning in KT from deceased donors (ClinicalTrials.gov NCT01395719).
Interestingly, it has recently been shown that the application of IPoC can be delayed showing that IRI is not only an acute process on the onset of reperfusion but also later on. Roubille et al. (57) showed in mice that myocardial infarct size could be reduced by delaying IPoC up to 45 min after the onset of reperfusion. Basalay et al. (58) confirmed these observations and showed in rats that remote ischemic IPoC applied to the femoral arteries was effective up to 10 min after the onset of myocardial reperfusion but was ineffective when this period was extended up to 30 min. Furthermore, they showed that remote IPC is critically dependent on afferent innervation of the remote organ as well as intact parasympathetic activity because denervation of peripheral tissue or vagotomy abolished this protection (58). These observations have yet to be extended to renal IRI.
CONCLUSIONS AND FUTURE PERSPECTIVES
IPoC is a simple method to provide protection against IRI in different organs. Although IPoC has shown positive effects on renal IRI in animal experiments, no attempts have yet been made to investigate the efficacy and safety of IPoC in human KT. In KT, IRI and DGF remain an important clinical problem and much benefit may be gained from protective interventions such as IPoC. It would be of great interest to know if IPoC can reduce IRI, which in ultimo might lead to the more widespread acceptance of organs from extended criteria donors or of organs that would otherwise be discarded. If IPoC decreases the incidence and severity of DGF and chronic kidney allograft loss, it may also have great socioeconomic implications.
Attempts to reduce IRI in clinical KT have mostly focused on improving hypothermic organ preservation (aiming for short ischemia times with optimum procurement, preservation, and transport measures). Interventions to improve kidney recovery after KT can be done in donors, recipients, and during graft preservation. Donor resuscitation may involve therapies aimed at improving (renal) hemodynamics and the inflammatory response, whereas graft preservation may improve by using different preservation solutions or hypothermic or normothermic machine preservation (1, 5–7, 59). If benefits are gained by such interventions, they could all be implemented in clinical transplantation, from procurement to transplantation.
It remains to be determined whether IPoC is universally applicable in solid organ transplantation. Finally, all manners of conditioning (i.e., ischemic, pharmacologic, remote, or delayed) should be tested to see if these methods have clinical potential. To answer some of these questions, we are currently performing a proof-of-principle trial to investigate the feasibility, safety, and effectiveness of IPoC in human deceased donor KT (Dutch trial registry number NTR 3117).
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