Adam D. Barlow

Evidence for Rapamycin Toxicity in Pancreatic β-Cells and a Review of the Underlying Molecular Mechanisms

Rapamycin is used frequently in both transplantation and oncology. Although historically thought to have little diabetogenic effect, there is growing evidence of β-cell toxicity. This Review draws evidence for rapamycin toxicity from clinical studies of islet and renal transplantation, and of rapamycin as an anticancer agent, as well as from experimental studies. Together, these studies provide evidence that rapamycin has significant detrimental effects on β-cell function and survival and peripheral insulin resistance. The mechanism of action of rapamycin is via inhibition of mammalian target of rapamycin (mTOR). This Review describes the complex mTOR signaling pathways, which control vital cellular functions including mRNA translation, cell proliferation, cell growth, differentiation, angiogenesis, and apoptosis, and examines molecular mechanisms for rapamycin toxicity in β-cells. These mechanisms include reductions in β-cell size, mass, proliferation and insulin secretion alongside increases in apoptosis, autophagy, and peripheral insulin resistance. These data bring into question the use of rapamycin as an immunosuppressant in islet transplantation and as a second-line agent in other transplant recipients developing new-onset diabetes after transplantation with calcineurin inhibitors. It also highlights the importance of close monitoring of blood glucose levels in patients taking rapamycin as an anticancer treatment, particularly those with preexisting glucose intolerance.

Rapamycin toxicity in MIN6 cells and rat and human islets is mediated by the inhibition of mTOR complex 2 (mTORC2).

Aims/hypothesisRapamycin (sirolimus) is one of the primary immunosuppressants for islet transplantation. Yet there is evidence that the long-term treatment of islet-transplant patients with rapamycin may be responsible for subsequent loss of islet graft function and viability. Therefore, the primary objective of this study was to elucidate the molecular mechanism of rapamycin toxicity in beta cells.MethodsExperiments were performed on isolated rat and human islets of Langerhans and MIN6 cells. The effects of rapamycin and the roles of mammalian target of rapamycin complex 2 (mTORC2)/protein kinase B (PKB) on beta cell signalling, function and viability were investigated using cell viability assays, insulin ELISA assays, kinase assays, western blotting, pharmacological inhibitors, small interfering (si)RNA and through the overproduction of a constitutively active mutant of PKB.ResultsRapamycin treatment of MIN6 cells and islets of Langerhans resulted in a loss of cell function and viability. Although rapamycin acutely inhibited mTOR complex 1 (mTORC1), the toxic effects of rapamycin were more closely correlated to the dissociation and inactivation of mTORC2 and the inhibition of PKB. Indeed, the overproduction of constitutively active PKB protected islets from rapamycin toxicity whereas the inhibition of PKB led to a loss of cell viability. Moreover, the selective inactivation of mTORC2 using siRNA directed towards rapamycin-insensitive companion of target of rapamycin (RICTOR), mimicked the toxic effects of chronic rapamycin treatment.Conclusions/interpretationThis report provides evidence that rapamycin toxicity is mediated by the inactivation of mTORC2 and the inhibition of PKB and thus reveals the molecular basis of rapamycin toxicity and the essential role of mTORC2 in maintaining beta cell function and survival.

A Pilot Study Assessing the Feasibility of a Short Period of Normothermic Preservation in an Experimental Model of Non Heart Beating Donor Kidneys

BACKGROUNDnRestoring metabolism to an organ after hypothermic storage and before transplantation could reverse some of the detrimental effects of ischemic injury. This may be particularly beneficial for kidneys from non-heart-beating (NHBD) donors that sustain significant periods of warm and cold ischemic injury. This pilot study assessed the feasibility of a short period of normothermic preservation (NP) in a porcine autotransplant model.nnnMETHODSnKidneys were subjected to 30 min of warm ischemia, then preserved by hypothermic machine perfusion (HMP) for 22 h or 20 h HMP followed by 2 h of NP using autologous blood. Kidneys were then re-implanted, a contralateral nephrectomy performed, and renal function measured over 10 d.nnnRESULTSnPost-transplant, 4/6 animals survived in the NP group compared with 5/6 in the HMP group (P = 1.00). Creatinine levels fell below 250 μmol/L in all four of the surviving animals in the NP group compared with 2/5 in the HMP group (P = 0.608). There was no difference in levels of renal function (peak creatinine, HMP = 1736 ± 866 versus NP = 1553 ± 516 μmol/L; P ≥ 0.990). Levels of lipid peroxidation were significantly lower 60 min post-transplant in the NP group (NP = 477 ± 118.0 versus HMP = 671 ± 99.4 pg/mL; P = 0.026).nnnCONCLUSIONnA period of NP at the end of the renal preservation period in NHB kidneys is a feasible method of kidney preservation. NP could prove to be a useful technique to predetermine graft function and allow pre-transplant modification of organs.

Current state of pancreas preservation and implications for DCD pancreas transplantation.

One of the main factors limiting potential uptake of pancreas transplantation, particularly in the United Kingdom, is the shortage of grafts. There has therefore been a recent expansion, particularly in the United Kingdom, in the utilization of grafts from donation after cardiac death (DCD) donors. These grafts are subjected to a greater ischemic insult and are arguably at higher risk of poor functional outcome. Although conventional preservation techniques may be adequate for donation after brain death (DBD) and low-risk DCD pancreases, as the number of DCD pancreas transplants increase and the threshold for rejecting organs decreases, the importance of optimal preservation techniques is going to increase. Over recent years, there have been significant advances in preservation techniques for DCD kidneys, improving the outcome of these marginal grafts. However, the use of such techniques for pancreas preservation is extremely limited and mainly historical. This overview describes the background and results of the established method of pancreas preservation for DBD, namely, cold static storage, and describes the use of the two-layer method. It also reviews pulsatile machine perfusion and normothermic perfusion for pancreas preservation techniques, which have shown promise in the preservation of DCD kidney grafts. The use of these techniques in pancreas preservation is predominantly historical but warrants reevaluation as to the feasibility of applying these techniques to DCD pancreas grafts not only for preservation but also for viability assessment. Further areas for development of pancreas preservation are discussed.

A Double Blind Randomized Clinical Trial of Remote Ischemic Conditioning in Live Donor Renal Transplantation.

Abstract Ischemic conditioning involves the delivery of short cycles of reversible ischemic injury in order to induce protection against subsequent more prolonged ischemia. This randomized controlled trial was designed to determine the safety and efficacy of remote ischemic conditioning (RC) in live donor kidney transplantation. This prospective randomized clinical trial, 80 patients undergoing live donor kidney transplantation were randomly assigned in a 1:1 ratio to either RC or to a control group. RC consisted of cycles of lower limb ischemia induced by an arterial tourniquet cuff placed around the patients thigh. In the RC treatment group, the cuff was inflated to 200 mm Hg or systolic pressure +25 mm Hg for 4 cycles of 5 min ischemia followed by 5 min reperfusion. In the control group, the blood pressure cuff was inflated to 25 mm Hg. Patients and medical staff were blinded to treatment allocation. The primary end-point was renal function measured by estimated glomerular filtration rate (eGFR) at 1 and 3 months posttransplant. Donor and recipient demographics were similar in both groups (Pu200a<u200a0.05). There were no significant differences in eGFR at 1 month (control 52u200a±u200a14 vs RC 54u200a±u200a17u200amL/min; Pu200a=u200a0.686) or 3 months (control 50u200a±u200a14 vs RC 49u200a±u200a18u200amL/min; Pu200a=u200a0.678) between the control and RC treatment groups. The RC technique did not cause any serious adverse effects. RC, using the protocol described here, did not improve renal function after live donor kidney transplantation.

External iliac artery dissection causing early renal transplant dysfunction

External iliac artery dissection after kidney transplantation is a rare, catastrophic but potentially reversible complication. Treatment which may save both the transplant and the patient requires clinical suspicion, timely imaging, and prompt intervention. This case report describes successful diagnosis of this complication and surgical intervention which saved the kidney and safeguarded blood supply to the patients leg.

Surgery for renal replacement therapy

Surgical intervention is required for long-term access for both haemodialysis and peritoneal dialysis. As the number and complexity of patients on dialysis increases, this presents an increasing challenge. Successful renal access surgery requires both careful planning and technical skill. This article describes the evaluation of patients for surgery, explores the surgical options for both dialysis modalities and discusses the common complications of these.

Transplantation/ImmunologyA Pilot Study Assessing the Feasibility of a Short Period of Normothermic Preservation in an Experimental Model of Non Heart Beating Donor Kidneys

BACKGROUNDnRestoring metabolism to an organ after hypothermic storage and before transplantation could reverse some of the detrimental effects of ischemic injury. This may be particularly beneficial for kidneys from non-heart-beating (NHBD) donors that sustain significant periods of warm and cold ischemic injury. This pilot study assessed the feasibility of a short period of normothermic preservation (NP) in a porcine autotransplant model.nnnMETHODSnKidneys were subjected to 30 min of warm ischemia, then preserved by hypothermic machine perfusion (HMP) for 22 h or 20 h HMP followed by 2 h of NP using autologous blood. Kidneys were then re-implanted, a contralateral nephrectomy performed, and renal function measured over 10 d.nnnRESULTSnPost-transplant, 4/6 animals survived in the NP group compared with 5/6 in the HMP group (P = 1.00). Creatinine levels fell below 250 μmol/L in all four of the surviving animals in the NP group compared with 2/5 in the HMP group (P = 0.608). There was no difference in levels of renal function (peak creatinine, HMP = 1736 ± 866 versus NP = 1553 ± 516 μmol/L; P ≥ 0.990). Levels of lipid peroxidation were significantly lower 60 min post-transplant in the NP group (NP = 477 ± 118.0 versus HMP = 671 ± 99.4 pg/mL; P = 0.026).nnnCONCLUSIONnA period of NP at the end of the renal preservation period in NHB kidneys is a feasible method of kidney preservation. NP could prove to be a useful technique to predetermine graft function and allow pre-transplant modification of organs.