The Effects of Oxygenation on Ex Vivo Kidneys Undergoing Hypothermic Machine Perfusion.

BACKGROUND: Supplemental oxygenation of the standard hypothermic machine perfusion (HMP) circuit has the potential to invoke favorable changes in metabolism, optimizing cadaveric organs before transplantation. METHODS: Eight pairs of porcine kidneys underwent 18 hours of either oxygenated (HMP/O2) or aerated (HMP/Air) HMP in a paired donation after circulatory death model of transplantation. Circulating perfusion fluid was supplemented with the metabolic tracer universally labeled glucose.Perfusate, end-point renal cortex, and medulla samples underwent metabolomic analysis using 1-dimension and 2-dimension nuclear magnetic resonance experiments in addition to gas chromatography-mass spectrometry. Analysis of C-labeled metabolic products was combined with adenosine nucleotide levels and differences in tissue architecture. RESULTS: Metabolomic analysis revealed significantly higher concentrations of universally labeled lactate in the cortex of HMP/Air versus HMP/O2 kidneys (0.056 mM vs 0.026 mM, P < 0.05). Conversely, newly synthesized [4,5-C] glutamate concentrations were higher in the cortex of HMP/O2 kidneys inferring relative increases in tricarboxylic acid cycle activity versus HMP/Air kidneys (0.013 mmol/L vs 0.003 mmol/L, P < 0.05). This was associated with greater amounts of adenoside triphosphate in the cortex HMP/O2 versus HMP/Air kidneys (19.8 mmol/mg protein vs 2.8 mmol/mg protein, P < 0.05). Improved flow dynamics and favorable ultrastructural features were also observed in HMP/O2 kidneys. There were no differences in thiobarbituric acid reactive substances and reduced glutathione levels, tissue markers of oxidative stress, between groups. CONCLUSIONS: The supplementation of perfusion fluid with high-concentration oxygen (95%) results in a greater degree of aerobic metabolism versus aeration (21%) in the nonphysiological environment of HMP, with reciprocal changes in adenoside triphosphate levels.

Recipient Outcomes From Nondirected Live Kidney Donors: A UK-based Cohort Study.

BACKGROUND: Increasing numbers of patients with end-stage renal failure are receiving kidneys from nondirected kidney donors (NKDs), also known as altruistic donors. Transplant outcomes for recipients of such kidneys are largely inferred from studies on specified kidney donors (SKDs), which may be inaccurate due to differences in donor, recipient and transplant specific factors. We report the outcomes for recipients of NKD in the United Kingdom. METHODS: Outcomes for 6861 patients receiving a living donor kidney transplant between January 2007 and December 2014 were analyzed using both the National Health Service Blood and Transplant and the UK Renal Registry datasets. Graft and patient outcomes were compared for patients receiving NKD and SKD organs using univariable and multivariable analyses. RESULTS: There was significant discordance between the NKD and SKD donors and recipients. These included increased donor age (median, 58 years vs 47 years; P < 0.001) and higher rates of hemodialysis and previous transplants in the NKD group (both P < 0.001). Despite such markers of increased risk among both donors and recipients of NKD kidneys, there was no difference in graft survival on univariable (hazard ratio, 1.20; 95% confidence interval, 0.77-1.86; P = 0.419) or multivariable analysis (hazard ratio, 1.13; 95% confidence interval, 0.65-1.95; P = 0.665). CONCLUSIONS: Despite some markers of transplant complexity, nondirected kidney donor organs are an excellent source of organs for transplantation.

The impact of donor body mass index on outcomes after deceased kidney transplantation - a national population-cohort study.

The aim of this study was to determine the effect of donor body mass index (BMI) on deceased donor kidney transplant outcomes. Data were collected from the UK Transplant Registry for all deceased donor kidney transplant recipients between January 2003 and January 2015. Univariable and multivariable analyses were undertaken to assess the impact of donor BMI on a range of outcomes. Donor BMI (kg/m2 ) was stratified as <18.5 (n = 380), 18.5-25.0 (n = 6890), 25.1-30.0 (n = 6669), 30.1-35.0 (n = 2503) and >35.0 (n = 1148). The prevalence of delayed graft function increased significantly with donor BMI (P < 0.001), with an adjusted odds ratio of 1.38 (95% CI: 1.16-1.63) for the >35.0 vs. 18.5-25.0 groups. However, there was no significant association between donor BMI and 12-month creatinine (P = 0.550), or patient (P = 0.109) or graft (P = 0.590) survival. In overweight patients, increasing donor BMI was associated with a significant increase in warm ischaemia time and functional warm ischaemia time, by an average of 4.6% (P = 0.043) and 5.2% (P = 0.013) per 10.0 kg/m2 . However, rising warm ischaemic time and functional warm ischaemic time was not significantly associated with delayed graft function, 12-month creatinine levels, graft loss or patient death. In this population cohort study, we identified no significant association between donor BMI and long-term clinical outcomes in deceased donor kidney transplantation.

Metabolic differences between cold stored and machine perfused porcine kidneys: A 1H NMR based study.

Hypothermic machine perfusion (HMP) and static cold storage (SCS) are the two methods used to preserve deceased donor kidneys prior to transplant. This study seeks to characterise the metabolic profile of HMP and SCS porcine kidneys in a cardiac death donor model. Twenty kidneys were cold flushed and stored for two hours following retrieval. Paired kidneys then underwent 24 h of HMP or SCS or served as time zero controls. Metabolite quantification in both storage fluid and kidney tissue was performed using one dimensional 1H NMR spectroscopy. For each metabolite, the net gain for each storage modality was determined by comparing the total amount in each closed system (i.e. total amount in storage fluid and kidney combined) compared with controls. 26 metabolites were included for analysis. Total system metabolite quantities following HMP or SCS were greater for 14 compared with controls (all p < 0.05). In addition to metabolic differences with control kidneys, the net metabolic gain during HMP was greater than SCS for 8 metabolites (all p < 0.05). These included metabolites related to central metabolism (lactate, glutamate, aspartate, fumarate and acetate). The metabolic environments of both perfusion fluid and the kidney tissue are strikingly different between SCS and HMP systems in this animal model. The total amount of central metabolites such as lactate and glutamate observed in the HMP kidney system suggests a greater degree of de novo metabolic activity than in the SCS system. Maintenance of central metabolic pathways may contribute to the clinical benefits of HMP.

(13)C glucose labelling studies using 2D NMR are a useful tool for determining ex vivo whole organ metabolism during hypothermic machine perfusion of kidneys.

BACKGROUND: The aim of this study is to determine the feasibility of using nuclear magnetic resonance (NMR) tracer studies ((13)C-enriched glucose) to detect ex vivo de novo metabolism in the perfusion fluid and cortical tissue of porcine kidneys during hypothermic machine perfusion (HMP). METHODS: Porcine kidneys (n = 6) were subjected to 24 h of HMP using the Organ Recovery Systems LifePort Kidney perfusion device. Glucose, uniformly enriched with the stable isotope (13)C ([U-(13)C] glucose), was incorporated into KPS-1-like perfusion fluid at a concentration of 10 mM. Analysis of perfusate was performed using both 1D (1)H and 2D (1)H,(13)C heteronuclear single quantum coherence (HSQC) NMR spectroscopy. The metabolic activity was then studied by quantifying the proportion of key metabolites containing (13)C in both perfusate and tissue samples. RESULTS: There was significant enrichment of (13)C in a number of central metabolites present in both the perfusate and tissue extracts and was most pronounced for lactate and alanine. The total amount of enriched lactate (per sample) in perfusion fluid increased during HMP (31.1 ± 12.2 nmol at 6 h vs 93.4 ± 25.6 nmol at 24 h p < 0.01). The total amount of enriched alanine increased in a similar fashion (1.73 ± 0.89 nmol at 6 h vs 6.80 ± 2.56 nmol at 24 h p < 0.05). In addition, small amounts of enriched acetate and glutamic acid were evident in some samples. CONCLUSIONS: This study conclusively demonstrates that de novo metabolism occurs during HMP and highlights active metabolic pathways in this hypothermic, hypoxic environment. Whilst the majority of the (13)C-enriched glucose is metabolised into glycolytic endpoint metabolites such as lactate, the presence of non-glycolytic pathway derivatives suggests that metabolism during HMP is more complex than previously thought. Isotopic labelled ex vivo organ perfusion studies using 2D NMR are feasible and informative.

Inhibition of Histone Deacetylase 6 Reveals a Potent Immunosuppressant Effect in Models of Transplantation.

BACKGROUND: Current transplant immunosuppression regimens have numerous limitations. Recent evidence suggests histone deacetylase inhibitors (HDACis) may represent a class of drug with immunosuppressive properties. This study compares cyclosporin A (CyA) with the pan-HDACi suberoylanilide hydroxamic acid (SAHA) and a novel HDAC6-specific inhibitor (KA1010) in models of alloreactivity. METHODS: Proliferation and mixed lymphocyte reaction (MLR)-based assays were used to determine the immunosuppressive effect of compounds, and a murine model of allogeneic skin transplantation was adopted to assess the in vivo effects of HDAC6 inhibition. RESULTS: KA1010 displayed superior inhibitory effects on the activation of peripheral mononuclear cells using in vitro models of transplantation. In a 1-way MLR, KA1010 (5 µΜ) reduced parent cell proliferation from 92% to 64% (P = 0.001). A 2-way MLR, adopting IFN-γ production as a marker of alloresponse, resulting in up to 91% reduction. Dose-response curves revealed dose-dependent profiles with greater potency of HDACis over CyA (IC50 values of 82.0 nM and 13.4 nM for KA1010 and SAHA).Mice treated with KA1010 displayed no significant features of skin allograft rejection upon histological analysis at 70 days and graft survival of 80% in subjects treated with 160 mg/kg. Immunological assessment, revealed a significant increase in CD4CD25forkhead box P3 regulatory T cells (from 18% to 25%, P = 0.0002) and a corresponding reduction in CD4 T cells (from 58% to 42%, P = 0.0009). CONCLUSIONS: HDAC6 may represent an optimal target for future immunosuppressant therapeutics with a particular role in transplantation. In this article, we have demonstrated a superior immunosuppressive effect of KA1010 over both CyA and SAHA, in the models of allotransplantation adopted.

Metabolomic analysis of perfusate during hypothermic machine perfusion of human cadaveric kidneys.

BACKGROUND: The metabolic processes occurring within the preserved kidney during hypothermic machine perfusion (HMP) are not well characterized. The aim of this study was to use nuclear magnetic resonance (NMR) spectroscopy to examine the metabolomic profile of HMP perfusate from human cadaveric kidneys awaiting transplantation and to identify possible discriminators between the profiles of kidneys with delayed graft function (DGF) and immediate graft function (IGF). METHODS: Perfusates from HMP kidneys were sampled at 45 min and 4 hr of preservation with the LifePort Kidney Transporter 1.0 (Organ Recovery Systems, Chicago, IL) using KPS-1. Prepared samples underwent 1-D Proton-NMR spectroscopy, and resultant spectra were analyzed. Clinical parameters were collected prospectively. RESULTS: Perfusate of 26 transplanted cadaveric kidneys was analyzed; 19(73%) with IGF and 7(27%) with DGF. Glucose concentrations were significantly lower in DGF kidneys compared to those with IGF at both 45 min (7.772 vs. 9.459 mM, P = 0.006) and 4 hr (8.202 vs. 10.235 mM, P = 0.003). Concentrations of inosine and leucine were significantly different between DGF and IGF kidneys at 45 min (0.002 vs. 0.013 mM, P = 0.009 and 0.011 vs. 0.006 mM, P = 0.036), and gluconate levels were also significantly different between DGF and IGF kidneys at 4 hr (49.099 vs. 59.513 mM, P = 0.009). CONCLUSION: Significant metabolic activity may be occurring in kidneys during HMP. The NMR spectroscopy of the perfusate can identify differences in the metabolomic profiles of DGF and IGF kidneys that might have a predictive role in viability assessment. Modification of harmful metabolic processes may improve outcomes for HMP kidneys.

Metabolomic perfusate analysis during kidney machine perfusion: the pig provides an appropriate model for human studies.

INTRODUCTION: Hypothermic machine perfusion offers great promise in kidney transplantation and experimental studies are needed to establish the optimal conditions for this to occur. Pig kidneys are considered to be a good model for this purpose and share many properties with human organs. However it is not established whether the metabolism of pig kidneys in such hypothermic hypoxic conditions is comparable to human organs. METHODS: Standard criteria human (n = 12) and porcine (n = 10) kidneys underwent HMP using the LifePort Kidney Transporter 1.0 (Organ Recovery Systems) using KPS-1 solution. Perfusate was sampled at 45 minutes and 4 hours of perfusion and metabolomic analysis performed using 1-D 1H-NMR spectroscopy. RESULTS: There was no inter-species difference in the number of metabolites identified. Of the 30 metabolites analysed, 16 (53.3%) were present in comparable concentrations in the pig and human kidney perfusates. The rate of change of concentration for 3-Hydroxybutyrate was greater for human kidneys (p<0.001). For the other 29 metabolites (96.7%), there was no difference in the rate of change of concentration between pig and human samples. CONCLUSIONS: Whilst there are some differences between pig and human kidneys during HMP they appear to be metabolically similar and the pig seems to be a valid model for human studies.

The use of NGAL and IP-10 in the prediction of early acute rejection in highly sensitized patients following HLA-incompatible renal transplantation.

Acute rejection is a significant problem for patients undergoing HLA-incompatible renal transplantation, affecting between 12 and 53% of patients. Any mechanism of detecting rejection in advance of current methods would offer significant benefit. This study aimed to evaluate whether serum biomarkers could predict rejection in HLAi transplants recipients. Sera from 94 HLAi transplant recipients from a single centre were analysed for a panel of biomarkers including: NGAL, KIM-1, IP-10, cystatin C, cathepsin L and VEGF. Biomarker levels pre-operatively, day 1 and at day 30 post-transplant were correlated with the development of early rejection. Significantly higher levels of IP-10 and NGAL were seen on day 1 following transplant in those patients who developed acute rejection (P < 0.001 and 0.005) and generated AUC of 0.73 and 0.67, respectively. No differences were seen for the other biomarkers or at the other time points. In this study cohort, IP-10 and NGAL have demonstrated good predictive ability for the development of acute rejection at a very early time point. They may have a role in identifying patients at higher risk for rejection and stratifying immunosuppression or surveillance.

Sickle cell and renal transplant: a national survey and literature review.

Sickle cell disease is an inherited, structural hemoglobin defect with multisystemic sequelae including renal failure. Patients with sickle cell disease are thought to benefit from renal transplant, but the long-term outcomes in such patients are unclear and have not been supported by any large prospective studies. Similarly, the renal morbidity and outcome after transplant in patients with sickle cell trait is also unclear. There is little evidence concerning living donation in donors with sickle cell disease or sickle cell trait, either for the donor health or for the graft outcome, and there are no United Kingdom guidelines. The aim of this study is to review the evidence surrounding renal transplant in recipients and donors with sickle syndromes and to determine the attitudes and current practices of United Kingdom transplant centers to performing such operations.

Hand-assisted laparoscopic donor nephrectomy: are hand port devices really necessary?

BACKGROUND: Hand port devices (HPD) are used routinely for hand-assisted laparoscopic surgery including hand-assisted laparoscopic donor nephrectomy (HALDN). However, the cost of such devices may prove prohibitive, particularly in centers with financial constraints. The authors aimed to identify any adverse effects of performing device-free HALDN. METHODS: A retrospective analysis was performed of patients undergoing HALDN at the authors' unit over a 3-year period (2007-2010). Eighty-four patients underwent device-free HALDN, whereas in 80 patients a HPD was used. The primary endpoint was duration of operation, with secondary endpoints including postoperative wound infections and incisional hernias. RESULTS: here was no difference in duration of operation for the device free (98 minutes; range = 43-215 minutes) compared with the HPD group (94 minutes; range = 36-180 minutes; P = .37). A device was required in 3 (3.6%) patients in which a device-free approach was attempted. There was no difference in either group in terms of rates of postoperative wound infections (0% vs 2.5%, respectively; P = .24) or incisional hernia incidence (1.5% vs 1.4%, respectively; P = .50). CONCLUSION: Device-free HALDN can be performed with no discernable compromise in operating time or patient outcome. This has implications in both cost benefit and translation of this technique to developing units.

Hand-assisted laparoscopic donor nephrectomy in patients with aberrant inferior vena caval anatomy.

OBJECTIVES: Hand-assisted laparoscopic donor nephrectomy has become an established technique for live-donor organ retrieval. In most cases, the left kidney is removed because of its more favorable anatomic relations, particularly with the major abdominal vessels. MATERIALS AND METHODS: We present 2 cases of live donation in which a hand-assisted laparoscopic approach was used to remove the right kidney as indicated by the presence of aberrant vascular anatomy, 1 being situs inversus totalis, the other a left-sided inferior vena cava. RESULTS: A 41-year-old woman and a 51-year-old man underwent assessment for live-kidney donation. During preoperative investigation, they underwent magnetic resonance imaging that demonstrated situs inversus totalis and a left-sided inferior vena cava. No contraindications to live donation were found during the investigation. In both cases, a right donor nephrectomy was performed owing to an anatomically longer right renal vein. Living donation proceeded without complication in both cases, and both patients had uneventful recoveries. CONCLUSIONS: Abnormalities in vascular anatomy should not be considered an absolute contraindication to donation, even by the hand-assisted laparoscopic donor approach. The use of magnetic resonance scanning preoperatively allows detailed planning of the approach required.

Mitochondrial complex activity in donor renal grafts, cold ischemia time, and recovery of graft function.

Indexed mitochondrial complex activities (MCAi) were determined in biopsies obtained from 52 donor kidneys at the end of cold ischemia (8-32 hr) to see if longer anoxia affected MCAi and accounted for the increased risk of delayed graft function (DGF) in recipients of grafts with longer cold ischemia time (CIT) or from non-heart-beating donors (NHBD). CITs were significantly different between those with and without DGF (P=0.02), being shorter in the latter, but MCAi were similar. CIT was correlated (r=0.43, P=0.003) with the time taken for creatinine concentration to fall to half the perioperative value (Crt(1/2)) but not with MCAi. Frequency of DGF, greater in NHBD, was significantly different from that of heart-beating donors (P=0.04), but CIT and MCAi were similar. However, Crt(1/2), was significantly different being longer in NHBD. Thus, the frequency of DGF increased and the speed of recovery diminished with longer CIT, whereas MCAi remained stable suggesting other factors determined tissue recovery.

Calciphylaxis following kidney transplantation: a case report.

INTRODUCTION: Calciphylaxis occurring after kidney transplantation is rare and rarely reported. It results in chronic non-healing wounds and is associated with a poor prognosis and is often fatal. We present a case of proximal lower limb calciphylaxis that occurred early after kidney transplantation. The patient had no classic associated risk factors. He had previously had a total parathyroidectomy but had normal serum calcium-phosphate product and parathyroid hormone levels. The clinical outcome of this case was favorable and highlights some fundamental issues relating to management. CASE PRESENTATION: A 70-year-old British Caucasian man with end-stage renal failure secondary to IgA nephropathy presented six months post kidney transplantation with cutaneous calciphylaxis lesions involving the medial aspect of the thigh bilaterally. CONCLUSION: To the best of our knowledge, this is the first reported case of rapid onset cutaneous calciphylaxis occurring soon after kidney transplantation that was associated with a favorable outcome. Cutaneous calciphylaxis lesions should be promptly managed with meticulous wound care, antimicrobial therapy and the correction of calcium-phosphate product where indicated.

Donor platelet and leukocyte load identify renal allografts at an increased risk of acute rejection.

BACKGROUND: A reduction in acute rejection may reduce graft losses from chronic rejection, benefiting the recipient and helping ease the huge donor organ shortfall in the UK. The prediction of recipients at greater risk of acute rejection might justify the administration to them of more potent immunosuppression, but defining this group on clinical parameters has been largely unsuccessful. Events impacting on the kidney by the time of donation may, if detectable histologically, predict those kidneys more likely to undergo rejection. METHODS: A prospective immunohistochemical analysis was undertaken to detect P-Selectin (PS), E-Selectin (ES), platelets, leukocyte common antigen, macrophages, T cells, and neutrophils in the pretransplant biopsies of 77 adult renal transplant recipients. Twenty-nine (38%) of this group rejected. RESULTS: A significantly increased number of recipients rejected if the donor biopsy was PS positive (63 vs. 24%, P=0.0007), contained five or more leukocytes/glomerulus (48 vs. 21%, P=0.03), contained >9.3 leukocytes/high power field (46.5 vs. 10.5%, P=0.006) or was both PS positive and contained >9.3 leukocytes/high power field (61.9 vs. 0.0%, P=0.0001). The PS was mostly of platelet origin and the majority of leukocytes were macrophages. Only previous CMV or any current infection in the donor correlated with the immunohistochemical changes. CONCLUSIONS: These immuno-histochemical changes are present before transplantation, which allows the prediction of recipients at both a higher and a lower risk of acute rejection, enabling the administration of recipient tailored immunosuppression, and supports the use of additional therapeutic intervention to reduce the injury response both within the recipient and the donor.