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MacAllister R, Clayton T, Knight R, et al. REmote preconditioning for Protection Against Ischaemia–Reperfusion in renal transplantation (REPAIR): a multicentre, multinational, double-blind, factorial designed randomised controlled trial. Southampton (UK): NIHR Journals Library; 2015 May. (Efficacy and Mechanism Evaluation, No. 2.3.)

Cover of REmote preconditioning for Protection Against Ischaemia–Reperfusion in renal transplantation (REPAIR): a multicentre, multinational, double-blind, factorial designed randomised controlled trial

REmote preconditioning for Protection Against Ischaemia–Reperfusion in renal transplantation (REPAIR): a multicentre, multinational, double-blind, factorial designed randomised controlled trial.

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Chapter 1Introduction

Chronic kidney disease requiring renal replacement therapy affects approximately 50,000 adult patients in the UK.1 Approximately 20,000 of these patients have undergone transplantation, leaving about 30,000 requiring dialysis. Of these, approximately 6000 are deemed best treated by a kidney transplant and are on the active transplant list at any given time. After an average waiting time of about 3 years they undergo transplantation, with approximately 2000 such transplants performed in 2012–13.2 Increased transplant activity is just keeping pace with the increased demand for kidney transplants, not only from the increase in incident cases but also from those patients whose transplanted kidney has failed; this occurs in 3% of the transplant population per year, resulting in approximately 600 patients per year returning to the transplant list. The consequences for a patient deemed best treated by transplantation of remaining on or returning to the transplant list are substantial. Among these patients there is not only significant morbidity of dialysis but also appreciable annual mortality (approximately 3%) and a substantial impact on quality of life; dialysis is a time-consuming hospital-based treatment requiring three sessions per week, each occupying half a day, with substantial and permanent restrictions on lifestyle, including diet and fluid intake. This is in addition to the cost of dialysis (about £30,000 per patient per year, and greater than the costs of treating patients by transplantation), which consumes approximately 1–2% of the NHS budget.2 Therefore, approaches that maximise the lifespan of each transplanted kidney will benefit patients directly, contribute to a reduction in the transplant list and moderate the costs of renal replacement therapy.

Renal injury is caused by ischaemia and reperfusion during transplantation

When an organ or tissue is rendered ischaemic there is inevitable cell death and tissue injury, the extent of which can be limited by timely reperfusion. However, paradoxically, an additional injury occurs on reperfusion that limits the amount of tissue that can be salvaged. This composite injury is termed ‘ischaemia–reperfusion (IR) injury’. IR injury underlies much of the tissue damage that occurs in stroke and myocardial infarction, two of the most common clinical IR syndromes, but it also plays a part in damage to all organs when they become ischaemic. In many renal transplants the kidney is exposed to warm ischaemia before organ recovery, cold ischaemia caused by the delay in transplanting the recovered organ and a further period of warm ischaemia during the transplantation procedure.3 Cell death follows interruption of the blood supply to the kidney and successful reperfusion is mandatory for tissue salvage. Although reperfusion may be an integral part of the healing process, it may also contribute to tissue injury.4 The degree of IR injury determines the speed of recovery of organ function in the short term.5 In addition, it may modulate organ rejection in the longer term by priming the immune response early after transplantation.68 Reduction in IR injury has the potential to improve the outcome of kidney (and other organ) transplantation, in both the short term and the long term.9,10

Mechanisms of ischaemia–reperfusion injury

Ischaemia of the kidneys (like that of other tissues) deprives cells of adenosine triphosphate (ATP), as a result of which the cells are then unable to maintain essential homeostatic processes. This ultimately leads to cell death by apoptosis or necrosis if timely reperfusion does not occur.11 Reperfusion injury is multifactorial and is partly attributable to rapid reoxygenation of hypoxic tissues, resulting in oxidative damage, and calcium overload because of loss of ion pump homeostasis.12 Although any segment of the nephron may be affected, the cells most vulnerable to IR injury are in the renal proximal tubule and distal medullary thick ascending limb of the loop of Henle. This is because of the high metabolic rate required for ion transport in these cells and also because of a limited capacity for anaerobic metabolism. Additionally, there is marked microvascular congestion and hypoperfusion in this region that persists despite restoration of cortical blood flow, therefore contributing to prolonged ischaemic injury.13 Endothelial cell injury and endothelial dysfunction are primarily responsible for this phenomenon, known as the extension phase of acute kidney injury. Ischaemic injury causes loss of the apical brush border of proximal tubular cells. Disrupted microvilli detach from the apical surface forming membrane-bound blebs that are released into the tubular lumen. The detachment and loss of tubular cells, in combination with brush-border vesicle remnants and cellular debris, results in tubular casts that may cause obstruction.13

Strategies to limit clinical IR injury have mainly focused on timely reperfusion. These strategies include interventions such as primary coronary intervention, thrombolysis for stroke and reducing both warm and cold ischaemic times in transplantation. There has arguably been optimisation of therapeutic techniques and their timing (within the current framework of health-care delivery) to limit ischaemia times and attention has turned towards interventions that target IR injury, either to enhance resistance to ischaemia and/or to reduce reperfusion injury. One such strategy is ischaemic preconditioning (IPC).

Ischaemic preconditioning

Ischaemic preconditioning utilises sub-lethal ischaemia (preconditioning stimulus) to induce a state of protection against subsequent prolonged ischaemia.14 There are two phases of protection. The early phase of IPC occurs within minutes of the preconditioning stimulus and lasts for up to 4 hours.15 The mechanism of early IPC has been extensively studied in animals. It involves mediators that are generated during hypoxia (e.g. adenosine), which initiate the cascade of protection by activating G-protein-coupled receptors,15 promoting recruitment of protein kinase mediators [such as phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK), protein kinase C (PKC) and Janus kinase (JAK)/signal transducer and activator of transcription (STAT)].16 IPC activates at least three main salutatory pathways, the cyclic guanosine monophosphate (cGMP)-dependent protein kinase [cGMP/protein kinase G (PKG)] pathway,17 the reperfusion injury salvage kinase (RISK) pathway16 and the survivor activating factor enhancement (SAFE) pathway.18 There is a degree of overlap, in particular where the pathways converge in mitochondria.19 Here, although there is some uncertainty, the potassium-dependent ATP (KATP) channel is activated and leads to closure of the mitochondrial permeability transition pore (mPTP), preventing the influx of ions through this channel that would trigger mitochondrial rupture and cell death by apoptosis.

A late phase of IPC occurs 24 hours after the preconditioning stimulus and lasts for up to 72 hours; this is termed the ‘second window of protection’, distinguishing it from early IPC.15 The prolonged (24-hour) interval between the preconditioning event and its renewed protection 1 day later is consistent with new protein synthesis. IPC initiates a complex genomic and proteomic response that is thought to underpin the late phase of protection. This includes regulation of anti-apoptotic and anti-inflammatory gene transcription, likely to be responsible for the second window of protection.20 Later-phase protection requires synthesis of inducible nitric oxide synthase (iNOS), heat shock proteins (HSPs) or cyclo-oxygenase-2 (COX-2), secondary to induced upregulation of genes for these factors. These act locally through the mPTP or KATP channels to induce a state of protection.15 Although the majority of studies to date have demonstrated protection by IPC against IR injury to the myocardium of animals and humans, a smaller number of studies have investigated the potential of IPC to protect other organs, including the kidney. In animal models IPC attenuates injury and preserves function following renal IR and after renal transplantation.21

Remote ischaemic preconditioning

Nearly 30 years have elapsed since the first description of IPC but its therapeutic value in the clinical setting remains to be validated. This is largely because of the logistical difficulties of applying ischaemic stimuli to induce preconditioning in vital organs in humans. Nor has it yet been possible to induce IPC pharmacologically, a reflection of the incomplete understanding of the mechanisms and the likelihood that multiple biological targets need to be activated. Demonstrating that there is clinically relevant tissue protection would stimulate renewed interest in pharmacological approaches to modulate IPC. However, the realisation that IPC may protect tissues that are distant from those undergoing preconditioning has led to recent interest in the direct clinical application of IPC.22 This facet of preconditioning (termed remote ischaemic preconditioning; RIPC) has been shown to be protective against IR injury to the myocardium23,24 and the kidney.25 RIPC is mechanistically similar to IPC and causes a similar degree of tissue protection.

The mechanism by which the protective signal is transferred systemically from the area of index ischaemia has been the subject of some debate. Evidence for involvement of a humoral factor is supported by preclinical observations that protection can be transferred by the transfusion of serum from an animal that has undergone IPC to one that has not.26,27 This factor is believed to be heat stable, dialysable and < 15 kDa in size.28 In transplant studies in pigs, RIPC applied to the recipient animal conferred protection against IR injury to the denervated donor heart during transplantation, again supporting a humoral hypothesis.29 Attempts to identify this circulating factor have proved challenging. However, recently, stromal cell-derived factor-1 alpha (SDF-1α; also known as C–X–C motif chemokine 12 or CXCL12), a cardioprotective chemokine of 10 kDa that is induced by hypoxia, has been demonstrated to be upregulated following RIPC in rats. The resultant cardioprotection was blocked in rats treated with AMD3100, a highly specific inhibitor of C–X–C chemokine receptor type 4 (CXCR4), the target receptor for SDF-1α.30 Neurogenic mechanisms of signal transfer have also been suggested. In rats, Dong et al.31 demonstrated that femoral nerve section abolished the effects of limb IPC. In a rat myocardial infarction model,24 hexamethonium (an autonomic antagonist) abolished protection by RIPC achieved by mesenteric artery occlusion. The autonomic ganglionic blocker trimetaphan has been shown to inhibit RIPC in a human model.32 The humoral and neuronal pathways may work in series to spread protection systemically. Lim et al.33 demonstrated that, in mice, femoral vein occlusion or femoral and sciatic nerve resection abolished the protective effects of RIPC, implicating both humoral and neural pathways.

Clinical studies of remote ischaemic preconditioning

Most human studies have used limb ischaemia to activate RIPC because of the inaccessibility of vital organs for IPC. The first clinical study demonstrated an effect of limb ischaemia on experimental IR injury to the endothelium23 and was rapidly followed by the first clinical trial of RIPC. In this small study, eight patients undergoing coronary artery bypass grafting (CABG) were randomised to receive either RIPC or a control condition. The study demonstrated an increase in blood lactate dehydrogenase (collected from the coronary perfusion catheter) in the preconditioned group, which the investigators attributed to an ability to maintain anaerobic metabolism in preconditioned cells.

In 2007, Hausenloy et al.34 first demonstrated a reduction in troponin T levels in adults randomised to receive RIPC prior to CABG with cross-clamp fibrillation. In 2009, Venugopal et al.35 also demonstrated a reduction in troponin T following RIPC in patients undergoing cold blood cardioplegia. However, in 2010, Rahman et al.36 published the results of a larger single-centre, double-blind randomised controlled trial in which 162 patients undergoing CABG were randomised to receive either RIPC or placebo. In this study there was no difference in troponin release or any other clinical outcome between the two groups. Most recently, a larger single-centre study of 329 patients undergoing isolated CABG with cold blood cardioplegia and cardiopulmonary bypass who were randomised to RIPC or no RIPC demonstrated a reduction in postoperative troponin I in the preconditioned group.37 The authors also attempted to address the question of whether a reduction in troponin equated to a measurable longer-term clinical benefit. They reported a reduction in all-cause mortality in the preconditioned group, which was sustained over > 4 years of follow-up.

In 2006, Iliodromitis et al.38 first investigated whether RIPC would attenuate the inflammatory response in elective single-vessel percutaneous coronary intervention (PCI) for acute myocardial infarction with coronary stenting. They demonstrated an increase in cardiac enzymes and C-reactive protein in the preconditioned group (n = 41) and postulated that RIPC increased the inflammatory response. Subsequently, in 2009, Hoole et al.,39 in a study of 242 patients undergoing elective PCI, demonstrated that RIPC prior to PCI attenuated procedure-related troponin release. Botker et al.40 demonstrated that RIPC increased myocardial salvage in ST-segment elevation myocardial infarction. Increased interest in the clinical usefulness of RIPC in the setting of myocardial ischaemia (CABG or PCI) has led to the publication of many other small trials in recent years, all reporting differing outcomes. Other larger randomised controlled trials in cardioprotection are ongoing [Effect of Remote Ischaemic Preconditioning on Clinical Outcomes in CABG Surgery (ERICCA)41 and Remote Ischaemic Preconditioning for Heart Surgery (RIPHeart)42], recruiting over 3000 patients in total, and these are likely to eliminate some of the noise generated by the small studies and their attendant biases.

Evidence for a clinical benefit of remote ischaemic preconditioning in the kidney

Animal studies have demonstrated the therapeutic potential of RIPC in protecting against IR injury in the kidney;21 however, these benefits have yet to be established in clinical studies in humans. Although there have been several trials published, these have tended to be small, single-centre studies and they are likely to be affected by publication bias. Many report differing and short-term end points, making it difficult to compare outcomes or interpret the results. Additionally, the roles of coexistent comorbid states and polypharmacy in such patients are confounders, the degree to which cannot easily be ascertained.

A recent meta-analysis of randomised studies in cardiac/abdominal aortic aneurysm surgery suggested that there was a benefit of RIPC in reducing renal injury post surgery.43 However, only five trials had absolute creatinine values documented and could be included and, of these, differing measures were reported and so the results were adjusted and reported as standardised mean values. Additionally, these trials were not powered individually for renal end points.

One other potential application that has been investigated in a clinical trial is the use of RIPC to protect against contrast-induced acute kidney injury. Patients with pre-existing renal dysfunction [serum creatinine > 1.4 mg/dl or estimated glomerular filtration rate (eGFR) < 60 ml/minute/1.73 m2] were randomised to receive RIPC (four times 5-minute arm cuff inflations) or sham treatment prior to elective coronary angioplasty. The authors reported a reduction in the rate of contrast-induced acute kidney injury, from 40% in the control group to 12% in the RIPC group (n = 100, p = 0.002).44

The use of direct IPC in transplantation (preconditioning of the donor organ at retrieval by repeated clamping/unclamping of the arterial supply) has been investigated in clinical trials in liver transplantation;45 however, no similar studies have yet been published in kidney transplantation. A pilot clinical trial carried out by our group in the setting of paediatric living-donor renal transplantation demonstrated the protective effects of late (‘second window’) RIPC.46 A blood pressure cuff was used to produce 5-minute periods of limb ischaemia (three cycles; applied to the donor and recipient) 24 hours in advance of surgery. A prospective cohort of patients (n = 20) was randomised in a blinded fashion to sham RIPC or RIPC (n = 10 in each group). There was a beneficial effect of RIPC on long-term renal function (Figure 1).

FIGURE 1. Effect of RIPC on long-term graft function following transplantation.

FIGURE 1

Effect of RIPC on long-term graft function following transplantation. The graph shows eGFR against time (1–60 months post transplantation) in the control and RIPC groups (mean ± standard error). The eGFR against time curves (more...)

A second randomised controlled study of RIPC in renal transplantation was published (as a letter to the editor) in 2013.47 In this study of 40 patients, live-donor kidney transplant recipients and their donors were randomised in pairs to receive either donor RIPC, recipient RIPC or no RIPC. In this small study the authors did not observe any differences between the three groups in urine volume, plasma creatinine level, acute kidney injury biomarkers, length of hospital stay or cost.

Rationale for the REmote preconditioning for Protection Against Ischaemia–Reperfusion in renal transplantation trial

The REmote preconditioning for Protection Against Ischaemia–Reperfusion in renal transplantation (REPAIR) trial was designed to provide definitive evidence for a benefit of RIPC in renal IR injury. The REPAIR trial measured the effects of early and late RIPC on renal IR injury using a 2 × 2 factorial design. Early RIPC activates an immediate but non-sustained protective effect. Identifying a protective effect of the early phase of RIPC has implications for future studies in deceased-donor transplantation, in which the unpredictable availability of organs precludes the scheduling of a preconditioning protocol 24 hours in advance of surgery. In this clinical setting (and currently the majority of transplants) the only feasible preconditioning stimulus will be early RIPC. Late RIPC had demonstrable benefits in a pilot study of renal transplantation,46 which we hypothesised were secondary to its prolonged and sustained phenotype. This profile might reduce IR injury and blunt the secondary inflammatory response to tissue injury. Applying the RIPC stimulus 24 hours before surgery enabled the late and sustained effects of RIPC on renal function (primary end point) to be assessed and was considered to establish aspects of the mechanism in humans. Moreover, the factorial design of the REPAIR trial allowed an assessment of the combination of early and late RIPC. Lastly, we sought to investigate the mechanism of RIPC in human tissue samples, recovered perioperatively from donor and recipient. These were sections of renal graft artery and vein that are trimmed from vessels to facilitate anastomosis and redundant biopsy material from protocol biopsies.

Copyright © Queen’s Printer and Controller of HMSO 2015. This work was produced by MacAllister et al. under the terms of a commissioning contract issued by the Secretary of State for Health. This issue may be freely reproduced for the purposes of private research and study and extracts (or indeed, the full report) may be included in professional journals provided that suitable acknowledgement is made and the reproduction is not associated with any form of advertising. Applications for commercial reproduction should be addressed to: NIHR Journals Library, National Institute for Health Research, Evaluation, Trials and Studies Coordinating Centre, Alpha House, University of Southampton Science Park, Southampton SO16 7NS, UK.

Included under terms of UK Non-commercial Government License.

Bookshelf ID: NBK294372

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