Ischemic Conditioning in Kidney Transplantation

Introduction

When an organ or tissue is rendered ischemic, there is inevitable injury, the extent of which can be limited by timely reperfusion. However, paradoxically, an additional injury occurs upon reperfusion, akin to a disordered biological reboot, that limits the amount of tissue that can be salvaged. This composite injury is termed “ischemia–reperfusion (IR) injury” and complicates many clinical syndromes, including kidney transplantation.

Despite an improvement in 1-year kidney transplant survival, long-term graft survival has not altered significantly in recent years. ^{1 ∓3} Ischemia–reperfusion injury at the time of transplantation is associated with an increased risk of acute rejection, delayed graft function (DGF), and poor overall graft function. ⁴ Even in the setting of living donor transplantation, the deleterious impact of small increases in warm ischemic time (WIT) is becoming more and more apparent. ^5,6 Therefore, strategies to reduce IR injury at the time of organ harvest and transplantation may be the best therapeutic intervention to increase graft longevity.

Ischemic preconditioning (IPC) is an intervention targeted against IR injury whereby brief, nonlethal periods of ischemia activate an innate response that confers protection against a more prolonged and thus potentially lethal period of ischemia. The therapeutic potential of this simple and cost-effective protective strategy has attracted much attention in recent years, with 2 notable clinical trials in the setting of renal transplantation having recently published their results.

Ischemia–Reperfusion Injury in Kidney Transplantation

Ischemia–reperfusion injury is an inevitable consequence of kidney transplantation. Strategies to limit injury have mainly focused on the development of organ allocation systems that minimize the duration of cold ischemia, minimizing warm ischemia at the time of surgery (such as by the use of parallel theatres in living donor transplantation), and strategies such as cooling of the organ on ice and modification of the perfusate to prevent tissue damage and cell death. However, there has arguably been maximal optimization of measures to reduce the ischemic insult within the current framework of health-care delivery, and additionally, a significant proportion of tissue dysfunction and cell death is attributable to the injury that occurs on reperfusion. Therefore, attention has turned toward interventions that target IR injury either to enhance resistance to ischemia and/or to reduce reperfusion injury. One such strategy is IPC.

Ischemic Preconditioning and Remote Ischemic Preconditioning

Ischemic preconditioning is a whole-body innate reflex that protects against subsequent IR injury and is activated by brief, nonlethal periods of tissue or organ ischemia. The phenomenon was first described by Murray et al in 1986, who demonstrated that a series of four 5-minute periods of circumflex coronary artery occlusion, separated by 5 minutes of reperfusion, could significantly reduce myocardial infarct size in dogs following subsequent prolonged ischemia. ⁷ The magnitude of effect size observed was much greater than with any pharmacological agent, and the phenomenon was demonstrated to be widely reproducible, and was observed in many in different animal models and species, including chicken, pig, rat, dog, mouse, and sheep. ⁸

Following on from Murray’s discovery, Przyklenk et al demonstrated that ischemic conditioning could be applied remotely; brief periods of ischemia applied to 1 vascular bed could remotely protect another—in this case, circumflex artery preconditioning protected the anterior descending coronary artery territory from injury following a subsequent prolonged occlusion. ⁹ Subsequent studies established that the preconditioning stimulus could be applied to a different organ, with protection spreading to remote organs, including heart, brain, and kidney. Now termed remote ischemic preconditioning (RIPC), this intervention gained potential clinical applicability with the discovery that the IPC stimulus could be applied noninvasively in humans using a blood pressure cuff placed on a limb and inflated above systolic blood pressure (SBP) to induce limb ischemia. ¹⁰ The discovery that preconditioning could be activated both noninvasively and remotely led directly to potential clinical applications of this therapy.

The protective effects of IPC have been demonstrated to occur in 2 “windows,” the initial period of protection occurring immediately following the preconditioning stimulus, lasting for between 1 and 4 hours, ^{11 ∓13} and a delayed or “second window of protection” with an onset of 24 hours following preconditioning and lasting for between 24 and 72 hours. ^8,12,14 However, it should be noted that timely reperfusion remains a requirement because IPC⁷ delays rather than abrogates the onset of cellular death.

Ischemic Perconditioning and Postconditioning

Although IPC refers to direct preconditioning of the tissue or organ at risk, and RIPC refers to the activation of the protective reflex remotely and prior to the IR insult, the remote preconditioning stimulus may also be applied during ischemia, termed remote ischemic perconditioning (RIPerC), or by a process of staged reperfusion to the organ at risk, termed ischemic postconditioning (IPostC). Some studies have also utilized a remote conditioning protocol analogous to RIPC, but applied during reperfusion, a form of remote ischemic postconditioning (RIPostC).

Pathophysiology of Renal Transplant IR Injury

Ischemia–reperfusion injury in the transplanted kidney is underpinned by similar pathophysiological events as in other tissues—oxygen depletion leads to a switch toward anaerobic metabolism with depletion of ATP, leading to lysosomal destabilization and ultimately mitochondrial permeability transition, and cellular death. ¹⁵ Reperfusion carries an additional insult with the creation of toxic oxygen and nitrogen-free radicals causing further tissue damage and inflammation. There is migration of neutrophils into tissues with subsequent release of pro-inflammatory cytokines. Dendritic cells are induced by damage-associated molecular proteins (DAMPs) or pathogen-associated molecular proteins (PAMPs) and represent the connecting bridge between the innate and the adaptive immune systems. Toll-like receptors (TLRs) recognize PAMPs or DAMPs and, once activated, recruit adaptor molecules within the cytoplasm, activating kinases and transcription factors such as Nuclear Factor Kappa-Light-Chain-Enhancer of activated B cells (NF-κB), and induce an inflammatory response. Damage-associated molecular proteins may also activate all 3 complement pathways, and there is cross talk between the TLRs and complement. ¹⁶ Activation of C3 and upregulation of TLRs in the donor kidney prior to transplantation due to oxidative stress in the donor have been demonstrated to be detrimental to allograft outcomes.

Although it is well established in deceased donor transplantation that a prolonged cold ischemic time predisposes to DGF and its sequelae, a prolonged WIT in living donor transplantation should not be expected to be a benign phenomenon. A US study of 946 consecutive living donor/recipient pairs demonstrated that increased mean WITs (defined as the time from clamping of renal artery supply in the donor to submersion in iced saline) of the order of only 1.5 minutes predisposed to poorer early graft function (EGF, defined as delayed or slow graft function), which in turn predisposed to increased risk of acute rejection and poorer long-term graft function. ⁶ In a similar study in the Netherlands, the authors also documented a significant influence of increased WIT (first WIT defined as the period between clamping of the renal artery and start of cold perfusion, second WIT as the time between ending of cold storage and recirculation in the recipient) in predisposing to poorer EGF in 472 living donor transplant recipients, and again, this led to poorer rejection-free and long-term graft survival. ^17,18 Another study examined the influence of WIT in both living and deceased donor transplantation, demonstrating in a cohort of 131 677 patients in whom WIT was documented that a prolonged WIT was associated with increased rates of death/graft failure. This observation persisted when the living donor cohort was examined in isolation. ⁵

Mechanisms of Protection of Ischemic Conditioning in Renal Transplantation

Local trigger factors released at the time of preconditioning initiate the process of propagating a protective signal to the organ or tissue of subsequent injury. The precise mechanism for this signal transduction is unclear; however, there is evidence for both humoral and neurological components, which may work in series or parallel. ¹⁹ The protection exerts its effect on stabilizing the mitochondrial permeability transition pore (mPTP) via prosurvival kinases such as the reperfusion injury salvage kinase (RISK) and survivor activating factor enhancement (SAFE) pathways. Reduction in the inflammatory and therefore immunological sequelae of IR injury as detailed could be expected to translate into better graft outcomes.

Evidence for a Clinical Benefit of IPC in Kidney Transplantation

Animal models have suggested a potential benefit of IPC in protecting against IR injury in the setting of kidney transplantation. ^{20 ∓22} However, arguably, these models do not represent the complexity of the clinical picture and the degree to which increasing age, coexistent comorbid states, and polypharmacy in such patients are confounders. The fact that the mechanism of IPC is still incompletely understood may also impede or reduce our ability to transfer these effects to the human clinical setting in such patients without inadvertently confounding the mechanism. One example of this is the application of RIPC during propofol anesthesia, which is known to inhibit vagal pathways that may be integral to signal transduction. ¹⁹

Our group was the first to investigate the potential of IPC in kidney transplantation. A prospective cohort of pediatric living donor renal transplant recipients and their donors (n = 20) were randomized in a blinded fashion to sham RIPC or RIPC (n = 10 in each group). A blood pressure cuff was used to cause 5-minute periods of limb ischemia (3 cycles, applied to the donor and recipient) 24 hours in advance of surgery. Remote ischemic preconditioning resulted in significant improvement in long-term renal function, as illustrated in Figure 1. Postoperative excretion of retinol-binding protein (RBP; area under the curve for RBP 72 hours posttransplantation) in RIPC patients was significantly reduced compared to controls (1.2 × 10⁵ vs 1.5 × 10⁵, respectively; P  =  .02), and the time for the creatinine to halve was shorter in the RIPC group than in the controls (5.5 ± 2.3 vs 9.4 ± 3.5, respectively; P  =  .007). ²³

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Figure 1.

Effect of remote ischemic preconditioning (RIPC) on long-term graft function following transplantation. Estimated glomerular filtration rate (eGFR) against time (1-60 months posttransplantation) in control and remote ischemic preconditioning (RIPC) groups (mean ± standard error of the mean [SEM]). The eGFR against time curves differed between control and RIPC groups (P < .001, 2-way analysis of variance [ANOVA]). ²³

A second randomized controlled study of RIPC in living donor renal transplantation has been published as a letter to the editor. In this study, 60 living donor kidney transplant recipients and their donors were randomized in pairs to receive donor RIPC, recipient RIPC, or none (20 in each group). The RIPC stimulus was 3- × 5-minute leg cuff inflations to 300 mm Hg, separated by 5 minutes of reperfusion. The timing of the RIPC stimulus prior to surgery was not specified; however, it could be assumed that this intervention was performed during anesthesia, as this magnitude of leg cuff inflation would otherwise be difficult to tolerate. In this small study, the authors did not observe any differences in terms of urine volumes, plasma creatinine, acute kidney injury biomarkers, length of hospital stay, or cost between the 3 groups. ²⁴

The largest study of RIPC in living donor renal transplantation, Renal Protection Against Ischemia Reperfusion in transplantation (REPAIR), was published as a monograph for the UK National Institution for Health Research Efficiency and Mechanism Programme in 2015. ²⁵ In this study, 406 donors and their recipients were randomized in pairs in a factorial design to receive early RIPC (immediately prior to surgery), late RIPC (24 hours prior to surgery), or both or neither (sham procedure). Remote ischemic preconditioning was applied as 4 cycles of upper arm cuff inflation to SBP plus 40 mm Hg for 5 minutes, followed by 5 minutes of cuff deflation. This study demonstrated a trend toward an improvement in formal measured iohexol glomerular filtration rate (GFR) at 12 months following transplantation in those who received early RIPC; however, this fell short of statistical significance (GFR: 55.9 control vs 58.3 early RIPC, adjusted difference: 3.08, 95% CI: −0.89 to 7.04, P  =  .13). However, there was a significant benefit of early RIPC in improving estimated glomerular filtration rate (eGFR) at both 3 and 12 months (3 months eGFR: 54.2 control vs 58.5 early RIPC, adjusted difference: 4.99, CI: 1.69-8.29, P = .003; 12 months eGFR: 60.7 control vs 64.8 early RIPC, adjusted difference: 4.98, 95% CI: 1.13-8.29; P  =  .011). There was no effect of late RIPC at any time point.

The Remote Ischemic Preconditioning in Neurological Death Organ Donors (RIPNOD) study (clinicaltrials.gov reference NCT01515072) has closed to recruitment, and the results are awaited. This study aimed to recruit 320 neurological death donors to assess the efficacy of four 5-minute cycles of RIPC applied immediately following confirmation of brain death and again at harvesting to investigate donor stability, organ quality, organ yield, and early posttransplant clinical outcomes. An abstract from this study has been presented, ²⁶ which suggested a reduction in markers of acute liver injury Aspartate transaminase (AST)/Alanine transaminase (ALT) in the preconditioned group; however, no markers of kidney injury were presented. The associated Remote Ischemic PreConditioning in abdominal Organ Transplantation (RIPCOT) study (clinicaltrials.gov reference NCT00975702) has also closed to recruitment, again awaiting results. This study aimed to recruit 580 deceased organ donors and recipients of kidneys, livers, and pancreas. Organ donors were randomized to receive either RIPC (leg cuff inflation on each side for 10 minutes) or no RIPC before organ recovery, performed in the operating room after commencement of surgery. Early postoperative outcomes would then be assessed using markers of organ function and cell injury parameters. Long-term outcomes will be assessed by graft and recipient survival.

Evidence for a Clinical Benefit of RIPerC and IPostC in Kidney Transplantation

Studies have also investigated the potential benefits of IPerC and IPostC in kidney transplantation. A randomized controlled trial by Nicholson et al, in which 80 patients undergoing living donor kidney transplantation were randomized to either RIPerC (4 cycles of leg cuff inflation to 200 mm Hg or SBP + 25 mm Hg for 5 minutes, followed by reperfusion) or a sham procedure (cuff inflation to 25 mm Hg) performed during ischemia, demonstrated no effect of IPerC on kidney function (Modification of Diet in Renal Disease [MDRD] eGFR) at 1 or 3 months. ²⁷ A study of RIPerC in deceased donor transplants, Remote ischemic conditioning in renal transplantation - effect on immediate and extended kidney graft function (CONTEXT), reported in late 2016. ²⁸ This study recruited 225 patients undergoing cadaveric kidney transplantation, who were randomized to receive either early RIPC delivered as cycles of 4- × 5-minute leg cuff inflations followed by 5-minute reperfusion or a sham procedure. The intervention was performed during surgery but prior to reperfusion. The primary end point of this study was the time for the baseline creatinine to fall by half following transplantation. An effect on this, or other early outcomes, was not observed in this study. Long-term outcomes, such as kidney function at 1 year, are awaited. One conclusion from these studies is that for preconditioning to have a measurable effect, it has to be applied in a timely fashion in advance of the ischemic insult.

A pilot human study in donation after circulatory death (DCD) kidney transplantation recruited recipients to receive IPostC as 3 cycles of clamp release for 1 minute followed by reclamping for 1 minute. ²⁹ The control groups consisted of 40 historical controls and also a paired kidney analysis on the contralateral kidney (n = 11). The primary outcome was the rate of adverse events, with secondary outcomes of DGF and kidney function at 3 months. Of note, donor age and serum creatinine were higher in the IPostC group, and this group experienced more DGF. Also of note, the historical control group was also younger and had better kidney function than the IPostC group. There was no difference in serum creatinine/MDRD eGFR at 3 months between the IPostC and either control group. Also of note, 1 patient had a venous tear that was attributed to the intervention. The authors still concluded that the intervention was safe on the basis that no serious adverse events were observed; however, clearly the incidence of venous damage gives some cause for concern.

Another study randomized 60 recipients of living donor kidney transplants to receive either RIPostC (3 cycles of upper limb cuff inflation for 5 minutes, followed by 5-minute reperfusion) carried out at the time of graft reperfusion or none. Kidney function (creatinine and eGFR) was assessed 2 hours after surgery and at 12-hour intervals for 96 hours. Urine output and urine creatinine were assessed until postoperative day 7, and hospital stay and complication rates were compared. The time for the creatinine to reach 50% of its preoperative level was significantly shorter in the preconditioned group (12 [12-24] hours vs 24 [21-36] hours, P = .005), and the number of patients whose creatinine fell by 50% within 24 hours was also significantly greater in the preconditioned group (n = 26 [87%] vs n = 18 [60%] P = .020). However, there were no differences in creatinine or eGFR thereafter, the incidence of graft dysfunction, or complication rates between groups. ³⁰ A summary of published studies of ischemic conditioning in renal transplantation is shown in Table 1.

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Table 1.

Clinical Trials of Ischemic Conditioning in Renal Transplantation.

Year	n	Author	Patient Group	Conditioning Protocol	Treated Group	Conditioned During Anesthetic?	Results
2005	20	Loukogeorgakis et al 23	Pediatric living donor	RIPC 24 hours pretransplant Arm 4 × 5 minutes	Donor and recipient (same allocation)	No	Improved MDRD eGFR over 60-month follow-up. Reduced urinary biomarkers (RBP) of AKI in RIPC group. Improvement in time for creatinine to fall by half in RIPC group
2013	60	Chen et al 24	Living donor	RIPC Leg 3 × 5 minutes	Donor/recipient/both/neither	Yes	No difference in urine volumes, urinary biomarkers (MDA, NAG, NGAL, RBP, SOD), or creatinine
2015	406 pairs	MacAllister et al 25	Living donor	Early (immediately pretransplant)/late (24 hours pretransplant)/dual RIPC Arm 4 × 5 minutes	Donor and recipient (same allocation)	No	No effect on iohexol GFR at 1 year. Significant effect on CKD-EPI eGFR at 3 and 12 months
2016	225	Krogstrup et al 27	Deceased donor	Early RIPC Leg 4 × 5 minutes	Recipient only	Yes	No effect on time for creatinine to fall by half
2013	20 (plus 40 historical controls)	van den Akker et al 29	DCD	IPostC 2 cycles external iliac artery cross clamping 1 minute, reperfusion 1 minute	Recipient only	Yes	No effect on creatinine or DGF at 3 months
2015	80	Nicholson et al 28	Living donor	IPerC Leg cuff 10 minutes to 200 mm Hg/SBP plus 25 mm Hg	Recipient only	Yes	No effect on MDRD eGFR at 1 or 3 months
2014	60	Kim et al 30	Living donor	RIPostC Arm 3 × 5 minutes	Recipient only	Yes	Reduction in time for creatinine to fall by half, no effect on graft function thereafter or complication rates

Abbreviations: AKI, acute kidney injury; CKD-EPI, Chronic Kidney Disease Epidemiology Collaboration; DCD, donation after circulatory death; DGF, delayed graft function; eGFR, estimated glomerular filtration rate; GFR, glomerular filtration rate; IPerC, ischemic preconditioning; IPostC, ischemic postconditioning; MACCE, major adverse cardiac and cerebral event; MDRD, Modification of Diet in Renal Disease; MDA, malondialdehyde; NAG, N-acetyl-d-glucosaminidase; NGAL, neutrophil gelatinase-associated lipocalin; RBP, retinol-binding protein; RIPC, remote ischemic preconditioning; RIPostC, remote ischemic postconditioning.

Discussion

Ischemic conditioning is a simple and low-cost intervention which has demonstrated great promise in the animal experimental setting yet proved difficult to translate into beneficial clinical outcomes in humans. There are several small published studies that report varying end points, and the absence of a complete understanding regarding the mechanism of signal transduction adds to the potential for inadvertent confounding, such as by anesthetic agents.

The only published randomized controlled study to have reported promising outcomes has been the REPAIR study. ²⁵ Although the primary end point was negative, there was significant dropout of patients not attending for the 4-hour formalized iohexol clearance at 1 year; additionally, there was significant variability in this “gold-standard” measure when performed outside experienced centers, which exceeded the variability of eGFR. The REPAIR is the largest clinical study in this setting, and there may be several plausible reasons as to why this study reported a benefit on kidney function (eGFR), despite other studies being negative. Firstly, in this study, RIPC was delivered by applying the intervention to both donor and recipient, thus in advance of both ischemia and reperfusion. Secondly, the potential interaction of anesthetic agents, which may impede transfer of the preconditioning signal, was avoided in that the intervention was applied prior to these agents being given. Of note, Gourine et al demonstrated in a rat model of IR injury that RIPC was abolished following subdiaphragmatic vagotomy, gastric vagotomy, or sectioning of the posterior gastric branch, suggesting that the circulating factor (or factors) of RIPC is produced and released into the systemic circulation by the visceral organ(s) innervated by the posterior gastric branch of the vagus nerve. ¹⁹ The authors therefore postulated that the explanation for the recent negative cardiac RIPC studies (Remote Ischemic Preconditioning for heart surgery (RIPHeart) ³¹ and Effect of Remote Ischemic preconditioning on Clinical outcomes in patients undergoing Coronary Artery bypass graft surgery ³² ) could be attributed to the effects of propofol, an anesthetic agent known to suppress the activity of vagal preganglionic neurons and inhibit autonomic reflex pathways.

The heterogeneous nature of the currently published studies (and the RIPCOT/RIPNOD studies, for which results are awaited) in terms of patient population and end points, and the often small size of exploratory studies, means that these do not definitively answer the question of the utility of this intervention in each individual clinical setting. This is further compounded by the fact that the mechanism of IPC remains elusive, and therefore, it is not yet clear how to best design studies to maximize any potential benefits. Further adequately powered studies incorporating long-term clinical outcome measures are required in each patient group (ie, living donor, DCD, and donation after brain death (DBD) transplantation).

Summary

Ischemic preconditioning is a safe, inexpensive, and well-tolerated intervention that might have significant clinical benefits in reducing tissue and organ damage following IR injury. Further elucidation of the underlying mechanisms is required to enable more targeted investigation in the clinical setting; however, this intervention may show promise in the setting of living donor kidney transplantation.