Michael L. Nicholson

Non-Heart Beating Donor Kidneys with Delayed Graft Function Have Superior Graft Survival Compared with Conventional Heart-Beating Donor Kidneys That Develop Delayed Graft Function

Delayed graft function may have an association with reduced graft survival, and nonheart‐beating donor (NHBD) kidneys have higher rates of delayed graft function (DGF) than heart‐beating donor (HBD) kidneys. This study compared outcome of renal transplants from HBDs who developed DGF, with NHBDs who developed DGF.

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.

Sotrastaurin, a novel small molecule inhibiting protein-kinase C: Randomized phase II study in renal transplant recipients

Sotrastaurin, a selective protein‐kinase‐C inhibitor, blocks early T‐cell activation through a calcineurin‐independent mechanism. In this study, de novo renal transplant recipients with immediate graft function were randomized 1:2 to tacrolimus (control, n = 44) or sotrastaurin (300 mg b.i.d.; n = 81). All patients received basiliximab, mycophenolic acid (MPA) and steroids. The primary endpoint was the composite of treated biopsy‐proven acute rejection (BPAR), graft loss, death or lost to follow‐up at month 3. The main safety assessment was estimated glomerular filtration rate (eGFR); modification of diet in renal disease (MDRD) at month 3. Composite efficacy failure at month 3 was higher for the sotrastaurin versus control regimen (25.7% vs. 4.5%, p = 0.001), driven by higher BPAR rates (23.6% vs. 4.5%, p = 0.003), which led to early study termination. Median (± standard deviation [SD]) eGFR was higher for sotrastaurin versus control at all timepoints from day 7 (month 3: 59.0 ± 22.3 vs. 49.5 ± 17.7 mL/min/1.73 m2, p = 0.006). The most common adverse events were gastrointestinal disorders (control: 63.6%; sotrastaurin: 88.9%) which led to study‐medication discontinuation in two sotrastaurin patients. This study demonstrated a lower degree of efficacy but better renal function with the calcineurin‐inhibitor‐free regimen of sotrastaurin+MPA versus the tacrolimus‐based control. Ongoing studies are evaluating alternative sotrastaurin regimens.

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.

A prospective study of the predictive power of spiral computed tomographic angiography for defining renal vascular anatomy before live-donor nephrectomy.

The advent of laparoscopic donor nephrectomy for renal transplantation has focused minds to an even greater degree on the vascular anatomy of the donor kidney. The authors from Leicester determined the accuracy of spiral CT imaging for donor venous anatomy and comparing it to the operative findings, either at open or laparoscopic donor nephrectomy. They found the technique to be an excellent way of assessing potential renal donors in terms of the gonadal and adrenal veins, but less so for predicting lumbar veins.

Comparison of right and left laparoscopic live donor nephrectomy

To compare the anatomy and function of right and left kidneys retrieved by laparoscopic live donor nephrectomy (LDN).

Biomarkers of oxidative damage to predict ischaemia-reperfusion injury in an isolated organ perfusion model of the transplanted kidney.

Ischaemia-reperfusion (IR) injury is known to be a risk factor influencing both short and long-term graft function following transplantation. The pathophysiology of IR injury is suggested to involve elevated reactive oxygen species production resulting in oxidative damaged cellular macromolecules. The objective of this study was to evaluate oxidative damage following IR using an isolated organ perfusion model of the transplanted kidney, in order to determine a simple, preferably non-invasive biomarker for IR injury. Porcine kidneys were retrieved with 10 or 40 min warm ischaemic (WI) time and haemoperfused for 6 h on an isolated organ perfusion machine. ELISA was used to detect carbonyls, 8-isporostane and 8-hydroxy-2′-deoxyguanosine, representing protein, lipid and DNA damage respectively in pre and post reperfusion samples of plasma, urine and biopsy material. Plasma carbonyl and 8-isporostane and were significantly increased in the 40 min group compared to pre-perfusion (0.96 ± 0.10 vs. 0.62 ± 0.06, P < 0.001 and 1.57(1.28–4.9) vs. 0.36(0.09–0.59), P < 0.05). The levels also correlated with creatinine clearance used to determine renal function (r = − 0.6150, P < 0.01 and r = − 0.7727, P < 0.01). The results of this study suggest both plasma carbonyl and 8-isporostane to be reliable biomarkers to predict the level IR injury.

Differential expression of cytoprotective and apoptotic genes in an ischaemia‐reperfusion isolated organ perfusion model of the transplanted kidney

The optimal kidney preservation system and methods to ameliorate reperfusion injury are major factors in accomplishing successful graft function following transplantation. Ischaemia and reperfusion lead to cellular stress and the adaptive response may include the activation of genes involved in cellular protection and/or cell death by apoptosis. We investigated the expression of cytoprotective heme oxygenase‐1 (HO‐1), anti‐apoptotic Bcl‐2 and pro‐apoptotic Bax after 6u2003h isolated organ perfusion in porcine kidneys that had been given 10 and 40u2003min warm ischaemic time. The level of HO‐1 was shown to be significantly higher in the 10‐min warm ischaemic group compared with 40‐min group (0.90u2003±u20030.03 vs. 0.83u2003±u20030.03; Pu2003=u20030.002). The levels of HO‐1 showed a significant positive correlated with parameters of renal function, creatinine clearance, and renal blood flow and urine output (AUC; ru2003=u20030.8042, Pu2003=u20030.03; ru2003=u20030.6028, Pu2003=u20030.04; ru2003=u20030.6055, Pu2003=u20030.04), demonstrating a possible protective role of this gene in this model of renal transplantation.

Static normothermic preservation of renal allografts using a novel nonphosphate buffered preservation solution

The aim of this study was to assess the viability and function of renal allografts under normothermic conditions using a novel nonphosphate buffered preservation solution AQIX®RS‐I. Porcine kidneys were flushed at 30u2003°C with AQIX®RS‐I at 100u2003mmHg pressure after 5–10u2003min warm ischaemic time and stored statically at either 4u2003°C or 30u2003°C for 2u2003h (nu2003=u20036 per group). Assessment of renal function by physiological and biochemical parameters was performed by perfusing the organs with autologous blood at 37u2003°C, with an initial circulating serum creatinine concentration of 1000u2003μmol/l on an isolated organ perfusion system for 6u2003h. Although the hypothermic group demonstrated overall superior renal function, the normothermic stored kidneys displayed a statistically comparable acid‐base balance (7.37u2003±u20030.15 vs. 7.3u2003±u20030.09, Pu2003=u20030.24). Furthermore, renal function was still evident after 6u2003h perfusion with increasing oxygen consumption, renal blood flow and reduced renal vascular resistance. The effectiveness and versatility of AQIX®RS‐I as a preservation solution under both normothermic and hypothermic conditions has been demonstrated. Renal viability was maintained after 2u2003h static normothermic storage. This study provides a foundation for further analysis utilizing normothermic preservation.