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

Article history

The research reported in this issue of the journal was funded by the EME programme as project number 08/52/02. The contractual start date was in July 2009. The final report began editorial review in August 2014 and was accepted for publication in January 2015. The authors have been wholly responsible for all data collection, analysis and interpretation, and for writing up their work. The EME editors and production house have tried to ensure the accuracy of the authors’ report and would like to thank the reviewers for their constructive comments on the final report document. However, they do not accept liability for damages or losses arising from material published in this report.

Declared competing interests of authors

none

Copyright statement

© 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.

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. 6–8 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 (K_ATP) 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 K_ATP 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. 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 differed between the control group and the RIPC group (p < 0.001, two-way analysis of variance).

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.

Study design

The REPAIR trial was a multicentre, double-blind, European-based randomised controlled trial assessing the impact of RIPC on kidney function following renal transplantation. It used a 2 × 2 factorial design in which the recipients and their donors were randomised to RIPC or a sham procedure both 24 hours before surgery (late RIPC) and immediately pre surgery (early RIPC). Therefore, there were four arms in total: (1) a sham procedure both 24 hours before surgery and immediately pre surgery, (2) early RIPC and a sham procedure 24 hours before surgery, (3) late RIPC and a sham procedure immediately pre surgery and (4) late RIPC and early RIPC. Both donor and recipient were randomised to the same intervention group.

Aim

The REPAIR trial investigated whether RIPC improved kidney function and other clinical outcomes following renal transplantation. RIPC is a simple, non-invasive and virtually cost-free intervention, and any improvement in graft function might ultimately lead to prolonged allograft life in these patients, with resultant economic and quality of life benefits. The findings might also have implications for the use of RIPC in other clinical ischaemic syndromes.

Participants

The study intended to recruit 400 pairs of transplant recipients and their living donors from centres in the UK, the Netherlands, Belgium and France. Patients undergoing living-donor transplantation aged ≥ 18 years from 13 tertiary care hospitals in the UK, the Netherlands, Belgium and France were invited to take part in the study.

Recruitment

The 13 centres from which patients were recruited were Guy’s and St Thomas’ NHS Foundation Trust, London, UK; Leiden University Medical Centre, Leiden, the Netherlands; North Bristol NHS Trust, Bristol, UK; Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK; Royal Free London NHS Foundation Trust, London, UK; Queen Elizabeth Hospital, Birmingham, UK; Vrije Universiteit (VU) University Medical Centre, Amsterdam, the Netherlands; St George’s Hospital, London, UK; Royal London Hospital, London, UK; Western Infirmary, Glasgow, UK; Centre Hospitalier Universitaire (CHU) Erasme, Brussels, Belgium; CHU de Liège, Belgium; and Centre Hospitalier Régional Universitaire (CHRU) de Lille, France. The recruitment of patients was initiated in the outpatient setting. Both the recipient and the donor were given the information sheet prior to giving consent.

Randomisation, concealment and blinding

Patients were allocated at random in a 1 : 1 : 1 : 1 ratio to the control condition (sham RIPC), early RIPC alone (immediately pre surgery), late RIPC alone (24 hours pre surgery) and dual RIPC (RIPC 24 hours before surgery and immediately pre surgery). This was performed using a web-based service provided by Sealed Envelope^™ (Sealed Envelope Ltd, London, UK) through the Clinical Trials Unit (CTU) at the London School of Hygiene and Tropical Medicine. The method of randomisation was random permuted blocks of size four and eight stratified by recruiting centre. During the study only the unblinded statistician supporting the Data Monitoring Committee (DMC) had access to the randomisation codes.

The enrolment and preconditioning procedures were performed by an unblinded research nurse who was not involved in sample collection or data analysis. All other research personnel at each study site, including those responsible for assessing outcomes, remained blinded to the allocation of patients to either real or sham RIPC. The patients and donors were also blinded to the allocation of their randomised intervention by the use of sham procedures in those not allocated to RIPC. A limited number of staff at the CTU were unblinded to the allocations, in order to enter data onto the electronic case report forms (eCRFs), a web-based data capture system provided by Sealed Envelope. However, no member of staff at the CTU had involvement or influence in any outcome measures.

Treatment group allocation

The trial intervention was a physiological procedure and was performed on both the donor and recipient at two time points before transplantation (24 hours before surgery and immediately before surgery). At the time point immediately before surgery, the active or sham RIPC sequences were initiated before induction of anaesthesia and were completed in advance of the initiation of surgery. There were no other interventions. The interventions consisted of different combinations of the active RIPC intervention and sham RIPC intervention as described below. The active RIPC procedure consisted of four 5-minute inflations of a blood pressure cuff on the upper arm to 40 mmHg above systolic blood pressure separated by 5-minute periods when the cuff was deflated. The sham RIPC procedure consisted of four 5-minute inflations of a blood pressure cuff on the upper arm to a pressure that would not impede blood flow (40 mmHg) separated by 5-minute periods when the cuff was deflated. All patients and donors underwent either the sham or the active RIPC procedure at both time points and so were randomised to one of the following groups:

• control group: the control group underwent sham RIPC both 24 hours before surgery and immediately before surgery

• early RIPC group: the early RIPC group underwent sham RIPC 24 hours before surgery and active RIPC immediately before surgery

• late RIPC group: the late RIPC group underwent active RIPC 24 hours before surgery and sham RIPC immediately before surgery

• dual RIPC: the dual RIPC group underwent active RIPC both 24 hours before surgery and immediately before surgery.

Postintervention treatment regimens

Patients followed the same immunosuppressive protocol, which was agreed by all participating centres. Patients received methylprednisolone and/or prednisolone according to local practice and anti-CD25 antibody [basiliximab (Simulect^®, Novartis)] according to the manufacturer’s recommendations (20 mg intravenously pre transplant followed by 20 mg intravenously on day 4). Patients received mycophenolate or azathioprine according to local practice. Mycophenolate was administered as mycophenolate mofetil (CellCept^®; Roche), starting at a dose of at least 1 g/day, or as mycophenolate sodium (Myfortic^®; Novartis), starting at a dose of at least 720 mg/day. Azathioprine was administered at a starting dose of 2 mg/kg. Patients received tacrolimus with a target concentration according to local practice. Antimicrobial and antithrombotic prophylaxis was administered in accordance with local practice. It was anticipated that patients would receive prophylaxis against Pneumocystis carinii pneumonia, oral Candida albicans and cytomegalovirus (donor positive, recipient negative). There were no alterations to routine treatment and no changes from routine practice for anaesthesia.

Data collection and management

Baseline assessment

All recipients had a medical history taken and a clinical examination as part of usual care. The following information was recorded at baseline: weight, height, gender, ethnicity, systolic blood pressure, creatinine, urea and albumin levels, comorbidities, date of birth, date when started dialysis and type of dialysis at time of admission. All donors filled out either a UK Transplant or a Eurotransplant organ donor form, depending on location. The following information was collected from this form at baseline: weight, height, gender, ethnicity, systolic blood pressure, creatinine, urea and albumin levels, age and glomerular filtration rate (GFR).

Follow-up

Donors were followed up immediately after RIPC and at day 1 (the day of the transplant), day 2 and day 3. Recipients underwent follow-up immediately after RIPC, perioperatively and at day 1, day 2, day 3, day 5, month 3 and month 12. The 3-month and 1-year follow-ups were carried out primarily at clinic visits. If a patient was not able to attend, information was obtained directly from the patient or from the patient’s general practitioner. In addition, recipients are being followed up annually to 5 years using clinic visits, telephone calls and renal registry data to assess eGFR. Recipients at the Leiden University Medical Centre were also followed up at 6 months post transplant for renal fibrosis assessment.

Outcome measures

Laboratory techniques

Sample size

Statistical analysis

The primary analysis was conducted on an intention-to-treat (ITT) basis with all patients and donors, when information was available, considered in the groups to which they were randomised. A per-protocol (PP) analysis was undertaken including those who received the randomised intervention as specified [i.e. excluding those pairs in which the intervention was not undertaken or in which the intervention was incomplete (whether RIPC or sham)]. All p-values are two-sided.

Primary outcome

Secondary outcomes

Missing data

An analysis was undertaken by imputing the eGFR for those patients who did not have a GFR determined by iohexol clearance measured at 12 months. All other analyses were conducted on a complete case basis, excluding any participants who had missing data for the outcome or predictors of interest in that particular model.

Subgroup analyses

The main subgroup analysis was to assess whether any effect of RIPC differed according to a patient’s underlying risk of low GFR at 12 months. This was assessed using a linear regression model to predict GFR at 12 months from baseline explanatory variables. Potential predictors of GFR at 12 months were identified from the published literature. 51–54 For each potential predictor, a linear regression model was used to assess the independent association between the value at baseline and GFR measured by iohexol clearance at 12 months. Those factors that showed evidence of association with GFR were entered into a multiple linear regression model, starting with factors with the strongest association. The final model included all factors that were associated with GFR (p < 0.10) together with terms for early and late RIPC. From this model, a risk score was calculated for each patient as the predicted GFR at 12 months excluding the estimated effects of early and late RIPC (i.e. the prediction for that participant if they had received sham early and late RIPC). An interaction term was fitted between risk and treatment to establish if there was a difference in benefit according to the underlying risk. Patients were categorised into quartiles of baseline risk and the data were presented as the mean (SD) GFR by treatment group in each baseline risk group.

Two other specific subgroup analyses were conducted:

1. to compare patients for whom this was their first transplant with patients who had had a previous transplant

2. to compare patients with a ‘000’ mismatch (i.e. those with no mismatches at any of the major HLA loci) type with patients without a ‘000’ mismatch type.

These two subgroup analyses were chosen because they represent changes to the inclusion criteria of the study after it had begun. The mean difference in the treatment groups between each subgroup and 95% CIs were calculated and subgroups compared using an interaction test.

Ethical considerations

Ethical approval for the study in the UK was given by the Joint University College London (UCL)/University College London Hospital (UCLH) Committees on the Ethics of Human Research in June 2009 (reference number 09/H0715/48). Outside the UK, local research ethics committee approvals were gained at all recruiting sites and all hospitals that were involved in trial follow-up. The trial was registered with the International Standard Randomised Controlled Trial Register (reference number ISRCTN30083294). The trial had two committees overseeing its conduct: the Trial Steering Committee (TSC) and the Project Management Group (PMG). In addition, there was an independent DMC to ensure the safety of patients in the trial and to review operational issues such as recruitment. The DMC was the only group to review interim analyses broken down by treatment group during recruitment and follow-up of patients in the trial. The DMC performed interim safety analyses annually. The interim reports contained details of patient recruitment, demographic and baseline characteristics, transplant and intervention details, primary safety end points, the primary efficacy end point and other end points identified by the DMC, including adverse and serious adverse events. At the 18-month DMC meeting, a review of the assumptions on which the sample size calculations were based was carried out as requested by the Efficacy and Mechanism Evaluation (EME) programme board.

The TSC had overall responsibility for the scientific integrity and quality of the trial. This involved ensuring that the trial was conducted to the standards set out in the guidelines for GCP and that the protocol was adhered to as far as possible and having responsibility for overall patient safety as well as considering new relevant information arising throughout the duration of the trial. The TSC also had responsibility for considering any recommendations made by the DMC. The TSC met annually throughout the trial to monitor the progress and quality of the trial, to review the recruitment rate and consider protocol amendments. The PMG was responsible for the day-to-day running of the trial, meeting fortnightly during the setting up of the trial and the early stages of recruitment and then approximately monthly for the remainder of the trial.

Effect of remote ischaemic preconditioning on glomerular filtration rate

Table 9 and Figure 3 show GFR measured by iohexol clearance at 12 months and eGFR at 3 months and 12 months for each of the four groups in the 2 × 2 factorial design. Although there was substantial variability in GFR within each treatment group, mean GFR and eGFR were higher among those who were randomised to early or dual RIPC.

FIGURE 3.

Glomerular filtration rate at 3 and 12 months after transplantation by treatment group. (a) GFR by iohexol, 12 months; (b) GFR by iohexol and CKD-EPI equation, 12 months; (c) GFR by CKD-EPI equation, 12 months; (d) GFR by CKD-EPI equation, 3 months.

The results of the ITT analysis of the primary and secondary GFR outcomes are provided in Tables 10 and 11 respectively. In total, 331 patients were included in the analysis of the primary outcome, GFR measured by iohexol clearance at 12 months: 321 patients evaluated at 12 months’ follow-up (between 7 January 2011 and 22 May 2014) and 10 patients whose GFR was imputed as zero because they had died or experienced graft loss before 12 months. Reasons for missing data for this outcome are shown in the CONSORT diagram (see Figure 2).

In the primary ITT analysis, shown in Table 10, there was no strong evidence for a difference in mean GFR (ml/minute/1.73 m²) measured by iohexol clearance between those receiving early RIPC and those in the control group, although there was an observed increase in GFR after early RIPC (adjusted difference 3.08, 95% CI –0.89 to 7.04; p = 0.13). There was no evidence of a difference between those receiving late RIPC and those in the control group (adjusted difference 1.19, 95% CI –2.77 to 5.15; p = 0.56). In a formal assessment of whether any benefit of early or late RIPC depended on whether the other period of RIPC was given, there was no evidence of an interaction between early and late RIPC (p = 0.47). Of note are the higher than expected SDs in each group for the GFR at 12 months, which in turn impacted on the width of the CIs and hence the precision of the results.

When missing values of GFR by iohexol clearance were imputed using the eGFR at 12 months, the GFR (ml/minute/1.73 m²) was again numerically greater in those receiving early RIPC than in those in the control group (adjusted difference 3.41, 95% CI –0.21 to 7.04; p = 0.065). There was little evidence that GFR differed between those receiving late RIPC and those in the control group (adjusted difference 2.18, 95% CI –1.45 to 5.80; p = 0.24). At 3 and 12 months there was stronger evidence of a beneficial effect of early RIPC on eGFR compared with the control group, but there was little evidence that eGFR differed between the late RIPC group and the control group. There was no evidence of an interaction between early and late RIPC for any secondary GFR outcomes.

The secondary analysis, comparing the combined early and late RIPC groups with the control group, shown in Table 11, suggested a trend towards a higher GFR (ml/minute/1.73 m²) measured by iohexol clearance in the RIPC treatment groups than in the control group (adjusted difference 3.88, 95% CI –0.74 to 8.50; p = 0.099). There was evidence that, compared with the control group, the combined RIPC group had a higher GFR when missing values of GFR measured by iohexol clearance were imputed using the eGFR at 12 months (adjusted difference 4.56, 95% CI 0.34 to 8.77; p = 0.034); the combined RIPC group had a higher eGFR at 12 months (adjusted difference 5.51, 95% CI 1.04 to 9.98; p = 0.016); and there was a trend towards a higher eGFR at 3 months in the combined RIPC group (adjusted difference 3.68, 95% CI –0.22 to 7.58; p = 0.064).

The PP analyses for GFR outcomes are shown in Tables 12 and 13. These analyses includes outcomes for recipients when both donor and recipient received the full 16 cycles of sham or active RIPC according to protocol. The results were very similar to those of the ITT analysis. Compared with the control group, among those who received early RIPC there was a trend towards a higher GFR measured by iohexol clearance (p = 0.061), which was similar after missing values were imputed with the eGFR (p = 0.055). There was also evidence of a higher eGFR at 12 and 3 months among those who received early RIPC (p = 0.022 and p = 0.002, respectively). In contrast, there was no evidence that GFR measured by iohexol clearance at 12 months or eGFR at 12 or 3 months differed between the late RIPC group and the control group. There was no evidence of an interaction between early and late RIPC for any of the GFR or eGFR outcomes. When the early and late RIPC groups were combined (see Table 13), there was a trend towards a higher GFR measured by iohexol clearance in the RIPC groups than in the control group (p = 0.077), and evidence became stronger after missing values were imputed using eGFR (p = 0.029). Recipients who received RIPC had a higher eGFR at 12 months than those in the control group (p = 0.034) and there was a trend towards a higher eGFR at 3 months (p = 0.097).

Effect of remote ischaemic preconditioning on secondary outcomes

The results of the ITT analysis for the main secondary outcomes are shown in Table 18. There was no difference in the time for a fall in serum creatinine of 50% following surgery between the early RIPC group and the control group (p = 0.75) or between the late RIPC group and the control group (p = 0.64). As shown in Figure 4, the median time for a fall in serum creatinine of 50% was around 48 hours in all treatment groups. There was also little evidence of a difference in the rate of acute rejection between the early RIPC group and the control group (p = 0.86) or between the late RIPC group and the control group (p = 0.17). Just over 10% of participants experienced acute rejection during the trial (Figure 5). Only 12 patients experienced delayed graft function and so substantial uncertainty remains with regard to the effects of early and late RIPC on this outcome. There was little evidence that the incidence of delayed graft function differed between the early RIPC group and the control group (p = 0.61) but the incidence was lower among the late RIPC group than among the control group (1.0% vs. 5.2%, p = 0.031).

FIGURE 4.

Time for serum creatinine to fall by 50% by treatment group.

FIGURE 5.

Incidence of acute rejection, by treatment group.

Nine recipients experienced graft loss and only two recipients died during the initial 12 months following transplant. There was little evidence of any differences between those receiving RIPC and those in the control group but the small number of events means that there is substantial uncertainty around the effect of treatment on these outcomes.

There was little evidence of any differences between the control group and the combined early and late RIPC group for any of the secondary outcomes (Table 19).

The results of the PP analysis for the main secondary outcomes are shown in Table 20. This analysis includes outcomes for recipients when both donor and recipient received the full 16 cycles of sham or active RIPC according to protocol. The results were very similar to those of the ITT analysis for the secondary outcomes.

Adverse events

Table 21 shows the types and numbers of adverse events by randomised group. Adverse events were more commonly experienced by donors and recipients in the RIPC arms than in the control arm. In particular, and as expected, far more donors and recipients in the RIPC arms than in the control arm experienced pain/paraesthesia (33–45% RIPC vs. 2–7% control) or skin petechiae (8–20% RIPC vs. 1% control). Although pain/paraesthesia was experienced by at least one-third of donors and recipients in the RIPC groups, this was only severe enough to prevent completion of the RIPC intervention in four pairs in the early RIPC arm, two pairs in the late RIPC arm and three pairs in the dual RIPC arm. Similar proportions of recipients experienced adverse events during follow-up (between 52% and 60% depending on study arm), with very few adverse events reported in donors during follow-up.

There were two reported unexpected adverse events that occurred during or immediately after administration of the early RIPC intervention. The first event was an episode of hypotension in a recipient following administration of the early RIPC intervention, which was rated as a non-serious adverse event that was possibly related to the RIPC intervention. The second event was an anaphylactoid reaction to basiliximab in a recipient during administration of the early RIPC intervention. This event was rated as a serious adverse event that was unrelated to the RIPC intervention.

Mechanism of action of remote ischaemic preconditioning

Tables 22–24 present the analysis of TNF-α, IL-6, IL-1β and INF-γ levels in serum in donors and in serum and urine in recipients, respectively. There was little evidence of a difference in these outcomes between early RIPC and control and between late RIPC and control. Immunoblotting, immunohistochemistry and renal fibrosis analyses are in progress at the time of submission of this report.

Design of the REPAIR trial

Remote ischaemic preconditioning is a whole-body systemic reflex activated by localised transient ischaemia that is itself non-injurious. The discovery that limb ischaemia could activate this reflex, and limit tissue injury to vital organs in animals and humans, has stimulated a large number of clinical trials to detect protective effects in patients. Most of these have been too small to measure real clinical end points and the small sample sizes have doubtless contributed to heterogeneity and uncertainty in the trial findings. We chose living-donor kidney transplantation as a model for testing whether RIPC had more than a biological effect in patients. Kidney transplantation in this setting is carefully scheduled to facilitate the application of a preconditioning stimulus before surgery. It was possible to arrange for a preconditioning stimulus to be applied to the donor and recipient 24 hours before surgery and immediately before surgery to test for early and late effects of preconditioning. Lastly, the careful planning of surgery meant that kidney ischaemia times would be expected to be consistent, reducing the variability in ischaemia times, kidney injury and kidney function after surgery.

The RIPC stimulus that was used was based on that which had induced cardioprotection in a number of previous clinical trials, namely 5-minute cycles of arm ischaemia and reperfusion. Our previous work had indicated that at least three cycles were needed to induce systemic protection and, to build in a safety margin to ensure that the stimulus was sufficient, we designed the REPAIR trial to use a four-cycle preconditioning stimulus. Approximately 10% of patients did not receive the full preconditioning stimulus as planned, most commonly because arm ischaemia was poorly tolerated by a small number of donors or recipients. We planned a PP analysis of the effect of RIPC in those undergoing the full intervention cycle because it seemed unlikely that an inability to tolerate arm ischaemia would significantly bias the outcome of the trial.

We designed the REPAIR trial to have a large enough sample size to enable us to measure kidney function 12 months after transplantation. The usual measure of kidney function is the eGFR, calculated from the serum creatinine level, taking into account certain phenotypic features of the patient. In the REPAIR trial we elected to measure GFR directly from the rate of excretion of iohexol, taking the view that the increased precision of this assessment over the eGFR would be an advantage. We were mindful of the theoretical disadvantages of iohexol GFR, including its greater complexity requiring an additional outpatient visit and the greater potential for human error in administering the correct dose and in the timing of blood sampling. By setting iohexol GFR as our primary end point we attempted to optimise the possibility of capturing data on renal function using this direct measure.

The factorial design of the REPAIR trial was chosen to allow separate assessments of the immediate and delayed protection that RIPC might stimulate. In the early phase a window of protection of about 4 hours is provided immediately on completing a threshold preconditioning stimulus. Approximately 24 hours after preconditioning, a second more prolonged phase of protection is provided, mediated by a complex of anti-inflammatory mediators. The factorial design allowed us to investigate these two phases separately and, uniquely for a preconditioning study, to examine whether additional protection was provided by a combination of early and delayed protection stimulated by sequential application of preconditioning stimuli. To maximise the chances of inducing tissue protection during ischaemia and reperfusion, donors and recipients underwent the same preconditioning protocol. In this way the donor kidney underwent preconditioning in advance of its ischaemic insult, and reperfusion injury in the recipient might also be modulated by preconditioning.

Effects of remote ischaemic preconditioning on glomerular filtration rate

The major finding from the REPAIR trial was the effect of early RIPC on GFR. When measured by iohexol clearance the group randomised to early RIPC had a higher GFR at 12 months, although the evidence for an effect was weak. When measured by eGFR at 3 and 12 months, the evidence for a beneficial effect was stronger, with an increased eGFR in the early RIPC group, amounting to an approximate 5 ml/minute/1.73 m² difference at 12 months. When missing values of iohexol GFR were imputed using the eGFR, the strength of evidence for an effect of RIPC also increased. These conclusions were supported by PP analyses, which increased the size of the effect of early RIPC and increased confidence in the findings. Given a mean annual rate of decline of eGFR after living-donor transplantation of 1.5 ml/minute/1.73 m², a patient starting out after transplantation with a 5 ml/minute/1.73 m² advantage might reasonably expect a 2- to 3-year extension to the lifespan of the transplant. In contrast to these findings were the largely null effects of delayed preconditioning. In a pilot trial in paediatric transplantation, we had observed a protective effect of late RIPC on eGFR, but this was not apparent in the REPAIR trial. Indeed, using the iohexol GFR, eGFR at 3 and 12 months and iohexol GFR with imputed values for eGFR, and in the PP analyses, there was no evidence of a clinical effect of delayed preconditioning. The most likely explanation for the difference in the strength of evidence between the primary and secondary GFR end points is the greater variability in the more complex and less standardised measure of GFR and the attrition of data for this measure. This conclusion is supported by the consistency between the effects of early and delayed RIPC when assessed by either estimate of GFR.

Effects of remote ischaemic preconditioning on short-term secondary end points

There was no evidence of an effect of RIPC on the short-term secondary end points. The time taken for creatinine to fall by 50% following transplantation was similar between early RIPC and the control (p = 0.75) and between late RIPC and the control (p = 0.64), with a median time of 48 hours in all treatment groups. However, it is possible that there were insufficient sample analyses for creatinine in the first 24 hours after transplantation to detect very early effects of RIPC or to assess accurately the time taken for creatinine to fall by 50%. There was little evidence of a difference in the rate of acute rejection between early RIPC and the control (p = 0.86) or between late RIPC and the control (p = 0.17); however, only 10% of participants experienced acute rejection during the trial. There was little evidence that the incidence of delayed graft function differed between the early RIPC group and the control group (p = 0.61) but the incidence was lower among the late RIPC group than among the control group (1.0% vs. 5.3%, p = 0.031). However, only 12 patients experienced delayed graft function and so substantial uncertainty remains with regard to the effects of early and late RIPC on this outcome. The median length of hospital stay was 6 days in all groups. Nine recipients experienced graft loss and only two recipients died during the initial 12 months following transplantation. There was little evidence of any differences between those receiving RIPC and those in the control group. The results of the PP analysis for the main secondary outcomes were similar to those of the ITT analysis.

Remote ischaemic preconditioning had no effect on the systemic inflammatory response to surgery in the donor or the recipient, with similar profiles of TNF-α, IL-1β, INF-γ and IL-6. The lack of any anti-inflammatory effect of RIPC is consistent with the null effects of delayed RIPC. Other mechanistic analyses are ongoing.

Safety of remote ischaemic preconditioning

There were no major safety concerns around RIPC. As expected, RIPC caused some discomfort to approximately 40% of donors and recipients alike. However, RIPC was discontinued in only nine pairs because they were unable to tolerate the intervention. Direct pressure effects of the cuff used to occlude blood flow in the arm resulted in a small proportion experiencing asymptomatic petechiae. However, during follow-up there were no differences between the groups in the adverse events recorded, the majority of which were unscheduled hospital admissions unrelated to preconditioning.

Limitations of the REPAIR trial

The length of follow-up in the REPAIR trial was set at 12 months. Longer-term follow-up is needed to determine whether differences in GFR are maintained in subsequent years, to such an extent that one can have greater confidence that RIPC will alter the lifespan of the transplant. As only about one-third of screened patients were recruited, the generalisability of our findings can be legitimately questioned. RIPC might have exerted its action by inducing ischaemic protection in the donor or limiting reperfusion injury in the recipient; as the donor and the recipient underwent RIPC, our data do not inform on the phase of IR injury influenced by RIPC. The lack of any effect of late RIPC on kidney function contrasts with that seen in our pilot data in children. Possible explanations for this include small study bias, differences in preconditioning pathways between children and adults and loss of the precision of the intervention when moving from a single-centre to a multicentre study.

Research recommendations

Further studies will, of course, help define the role of RIPC in renal transplantation, as one trial rarely changes practice and it would be necessary to confirm the effects of early RIPC on kidney function after living-donor transplantation. The REPAIR trial also refines the optimal design for a trial of RIPC in deceased-donor transplantation, which should test the effects of early RIPC, applied to the recipient only, on eGFR.

Contributing sites

• Leiden University Medical Centre (65 patients, recruitment started 4 January 2010): Professor Hans de Fijter (principal investigator), J Dubbeld, Krista Glas, Ada Haasnoot, Sabrina Hendriksen, Clarisca Montero, Sonja van Berkel and Ruth van Dam.

• St George’s Hospital, London (83 patients, recruitment started 30 March 2010): Sarah Heap (principal investigator), Sharirose Abat, Rene Chang, Liz Cording, Mr Jiri Fronek, Helen Gregson, Mr Nicos Kessaris, Iain MacPhee, Joyce Popoola, Rajeshwar Ramkhelawon and Rhia Ventura.

• Royal Free Hospital, London (64 patients, recruitment started 21 February 2010): Dr Mark Harber (principal investigator), Obaayaa Buahin, Vash Deelchand, Peter Dupont, Dr Nadia Godigamuwe, Ruth Kinyanjui, Aisling O’Riordan, Alison Richardson, Alan Salama, David Wheele and Ruth Yang.

• Guy’s Hospital, London (23 patients, recruitment started 27 October 2010): Mr Jonathon Olsburgh (principal investigator), Golda-Grace Azanu, Karen Ignatian, Lisa Silas, Jane Watkins and all surgical, medical and nursing staff who helped with the patients.

• Southmead Hospital, Bristol (27 patients, recruitment started 30 September 2010): Mr Najib Kadi (principal investigator), Helen Andrew, Susan Dawson, Vicki Elnagar, Kate Humphries, Dominic Janssen, Dr Chris Johnson, Mr Paul Lear, Stacey McGary, Helen McNally, Ronelle Mouton, Freya Murch, Louise Pearse, Joana Vaz, Joao Vicente, Mr Andy Weale, Nicola Woollven and Dr Karine Zander.

• Queen Elizabeth Hospital, Birmingham (eight patients, recruitment started 13 October 2010): Mr Steve Mellor (principal investigator), Dr Chris Counsell, Mary Dutton, Jonathan Ellis, Melanie Field, Lesley B Fifer, Okdeep Kaur, Adnan Sharif and Chantelle Waite.

• Royal London Hospital (33 patients, recruitment started 14 October 2010): Dr Raj Thuraisingham (principal investigator), Belinda Englebright, Wancheung Li, Lilibeth Piso, Caroline Rolfe, Jamie Smith, Ray Trevitt, Clare Whittaker, Karen Williams and Magdi Yaqoob.

• Cambridge University Hospitals NHS Foundation Trust (36 patients, recruitment started 24 September 2010): Mr Chris Watson (principal investigator), Roberto Cayado-Lopez, Sylvia Cottee, Nirvana Croft, Julia Ertner, Faye Forsyth, Dorothee Koscielny-Lemaire, Sue Miles, Ann-Marie O’Sullivan, Lucy Randle, Annie Rivera and Myrna Udarbe.

• VU University Medical Centre, Amsterdam (42 patients, recruitment started 15 March 2010): Dr Azam Nurmohamed (principal investigator), Yvonne de Koter, Carla Schrauwers, Marjon van Vliet and Hiske Wellink.

• Western Infirmary, Glasgow (10 patients, recruitment started 28 September 2011): Marc Clancy (principal investigator), Emma Aitken, Laura Buist, Julie Glen, Cheryl Keenan, Donna Kelly, Louise Maxwell, Lorraine McGregor, Barbara McLaren and Maria Nicoletti.

• CHU Erasme, Brussels (seven patients, recruitment started 10 July 2012): Dr Annick Massart (principal investigator), Professor Daniel Abramowicz, Cherubine Addino, Brigitte Borre and Nicole Lietaer.

• CHU de Liège (three patients, recruitment started 18 September 2012): Laurent Weekers (principal investigator), Catherine Bonvoisin, Arnaud Borsu, Michèle Focan and Stephanie Grosch.

• CHRU de Lille (five patients, recruitment started 27 January 2013): Marc Hazzan (principal investigator), Celine Beaussart, Priscilla Couillet, Sophie Dessau, Christine Devlaminck, Laetitia Erichot, Valérie Fontaine, Marie Fruleux, Professor Gilles Lebuffe, Benedicte Mignot, Christian Noël, Ornella Savarino and Carole Zini.

• St Helier Hospital, Carshalton (1-year and longer-term follow-up for 49 patients from St George’s Hospital; 18 underwent iohexol clearance at St Helier): Shamshersingh Jeetun, Dr David Makanjuola, Aileen Moore, Dr Mysore Phanish and Gillian Thomas.

• Royal Sussex County Hospital, Brighton (1-year and longer-term follow-up for 10 patients from St George’s Hospital; all 10 underwent iohexol clearance at Brighton): Mary Flowerdew, Ed Kingdon and Richard Rye.

Contribution of authors

Raymond MacAllister (Clinical Pharmacologist) was the Chief Investigator on the REPAIR trial and was involved in the design and conduct of the trial, was a member of the TSC and PMG and provided input into the manuscript.

Tim Clayton (Medical Statistician) was a co-applicant on the REPAIR trial and was involved in the design and conduct of the trial (including the statistical analysis plan), was a member of the TSC and PMG and provided input into the manuscript.

Rosemary Knight (Senior Trial Manager) had overall responsibility for the REPAIR trial, ensuring that all relevant approvals were in place, organising the setting up and training of sites and ensuring that the project was delivered on time and to budget.

Steven Robertson (Senior Data Manager) had overall responsibility for all aspects of data management including involvement in the design and development of the CRF and eCRF. He was also responsible for data cleaning and preparation for the final analyses and was a member of the PMG.

Jennifer Nicholas (Medical Statistician) was the unblinded statistician supporting the DMC, was a member of the PMG and provided support in preparation of the statistical analysis plan. She conducted the statistical analysis and provided input into the manuscript.

Madhur Motwani (PhD Student) performed the ELISA experiments and organised the cytokine data for statistical analysis. He also co-ordinated the transfer of samples from the trial sites to UCL and was a member of the PMG.

Kristin Veighey (Clinical Research Fellow) was a member of the PMG and TSC. She carried out site induction and training and co-ordinated sample collection and transfer for storage and analysis at UCL. She also organised and performed the analysis of samples collected during the trial.

Disclaimers

This report presents independent research. The views and opinions expressed by authors in this publication are those of the authors and do not necessarily reflect those of the NHS, the NIHR, MRC, NETSCC, the EME programme or the Department of Health. If there are verbatim quotations included in this publication the views and opinions expressed by the interviewees are those of the interviewees and do not necessarily reflect those of the authors, those of the NHS, the NIHR, NETSCC, the EME programme or the Department of Health.

October 2009

Following approval further amendments were made to the trial protocol and information used for potential participants:

• The immunosuppression protocol was changed to reflect the standard protocols in use at each of the centres.

• RIPC/sham RIPC will be carried out before induction of anaesthesia to prevent any delays to transplantation.

• Patient follow-up was changed to reflect clinical practice.

• The timing of the collection of blood and urine samples from the donor was changed to 3 days postoperatively. Originally, samples were to be collected up to 5 days postoperatively but patients would likely have been discharged at this point.

• The blood sampling procedures were clarified.

• The arrangements for reporting adverse events were clarified.

• Changes were made to the consent form and participant information sheet and two versions (one for the recipient and one for the donor) were submitted. Treatment of the donor and recipient is different and having separate information sheets highlighted this fact.

December 2009

Patients who require HLA antibody removal therapy were excluded as they require a different immunosuppression regime that may have an effect on preconditioning.

May 2010

• Mycophenolate/azathiaprine were added to the immunosuppression regime as used by some of the recruiting hospitals. This would be beneficial in terms of increasing recruitment.

• All patients will receive tacrolimus, titrated according to local practice.

• Some of the centres wanted to send out an invitation letter to potential patients with a copy of the participant information sheet.

September 2010

Patients who have had a previous transplant, who were originally excluded from the trial, are now to be included. It had been considered that RIPC may not be as effective in patients who had undergone a previous transplant; however, there was a change of opinion and evidence and they could now be included.

December 2010

• Changes were made to the protocol to include more flexibility in the immunosuppression protocol and increase recruitment.

• Patients with a ‘000’ mismatch type are now to be included. These patients were originally excluded because their immunosuppression regime was different from that in other transplants. However, because of changes to the immunosuppression protocol since the original description, these patients can now be included.

January 2011

A letter was approved to remind patients to attend their 1-year appointment as this is the primary outcome. Patients were aware of this at the time of consent and it was included in the participant information sheet. Patients were followed up at 3 months and again at 1 year and, as there was a gap of 9 months between the two follow-ups, it was recommended that patients be reminded of the importance of attending for iohexol testing.

May 2011

• There was a change to the PI at St Georges’ Hospital.

• Further analysis was performed on the kidney tissue samples taken from the patients at the Royal Free Hospital. The patients were already aware that tissue samples were being gifted and had signed a consent form to agree to this. The analysis is in line with updated information.