Prolonged Cold Ischemia Research Articles

Donor Preconditioning with Inhaled Sevoflurane Mitigates the Effects of Ischemia-Reperfusion Injury in a Swine Model of Lung Transplantation.

Primary graft dysfunction (PGD) and ischemia-reperfusion injury (IRI) occur in up to 30% of patients undergoing lung transplantation and may impact on the clinical outcome. Several strategies for the prevention and treatment of PGD have been proposed, but with limited use in clinical practice. In this study, we investigate the potential application of sevoflurane (SEV) preconditioning to mitigate IRI after lung transplantation. The study included two groups of swines (preconditioned and not preconditioned with SEV) undergoing left lung transplantation after 24-hour of cold ischemia. Recipients' data was collected for 6 hours after reperfusion. Outcome analysis included assessment of ventilatory, hemodynamic, and hemogasanalytic parameters, evaluation of cellularity and cytokines in BAL samples, and histological analysis of tissue samples. Hemogasanalytic, hemodynamic, and respiratory parameters were significantly favorable, and the histological score showed less inflammatory and fibrotic injury in animals receiving SEV treatment. BAL cellular and cytokine profiling showed an anti-inflammatory pattern in animals receiving SEV compared to controls. In a swine model of lung transplantation after prolonged cold ischemia, SEV showed to mitigate the adverse effects of ischemia/reperfusion and to improve animal survival. Given the low cost and easy applicability, the administration of SEV in lung donors may be more extensively explored in clinical practice.

Hypothermic machine perfusion can safely prolong cold ischemia time in deceased donor kidney transplantation. A retrospective analysis on postoperative morbidity and graft function.

In deceased donor kidney transplantation (KT), a prolonged cold ischemia time (CIT) is a negative prognostic factor for KT outcome, and the efficacy of hypothermic machine perfusion (HMP) in prolonging CIT without any additional hazard is highly debated. We conducted a retrospective study on a cohort of 154 single graft deceased donor KTs, in which a delayed HMP, after a preliminary period of static cold storage (SCS), was used to prolong CIT for logistic reasons. Primary outcomes were postoperative complications as well as 1year graft survival and function. 73 cases (47.4%) were managed with HMP and planned KT, while 81 (52.6%) with SCS and urgent KT. The median CIT in HMP group and SCS group was 29hour:57minutes [27-31hour:45minutes] and 11hour:25minutes [9-14hour:30minutes], respectively (P<.001). The period of SCS in the HMP group was significantly shorter than in the SCS group (10 vs. 11hour:25minutes, P=.02) as well as the prevalence of expanded criteria donors was significantly higher (43.8% vs. 18.5%, P<.01). After propensity score matching for these two baseline characteristics, the HMP and SCS groups showed comparable outcomes in terms of delayed graft function, vascular, and urologic complications, infections, and episodes of graft rejection. At 1 year follow-up, serum creatinine levels were comparable between the groups. Therefore, the use of HMP to prolong the CIT and convert KT into a planned procedure seemed to have an adequate safety profile, with outcomes comparable to KT managed as an urgent procedure and a CIT nearly three time shorter.

Donor-Recipient Matching to Optimize the Utility of High Kidney Donor Profile Index Kidneys

BackgroundIn December 2014, the Kidney Donor Profile Index (KDPI) was developed to give more precise information on donor kidney quality. Kidneys with KDPI scores ≥ 85 (K ≥ 85) have been reported to have inferior outcomes to kidneys with KDPI scores < 85. MethodsAfter the implementation of the new Kidney Allocation System, we developed a protocol to evaluate K ≥ 85 use. We analyzed the safety and efficacy of our institutional criteria and evaluated post-transplant outcomes. K ≥ 85 recipients were stratified based on their 1-year creatinine and estimated glomerular filtration rates to elucidate characteristics associated with serum creatinine < 1.7 mg/dL or estimated glomerular filtration rates ≤ 45 mL/min/1.73 m2. ResultsFrom December 2014 to December 2019, 304 deceased donor kidney transplants were performed at Hartford Hospital; 58 (19%) were K ≥ 85 with an average KDPI of 91%. There were 4 graft losses; 2 were death censored. Prolonged cold ischemia time and black recipient race were associated with inferior recipient graft function at 1 year. ConclusionsHigh KDPI kidney use requires a multifaceted evaluation that takes into account donor and recipient characteristics for an ideal match. We have identified several characteristics that may predict optimal post-transplant kidney function.

Effects of prolonged cold-ischemia on autophagy in the graft lung in a rat orthotopic lung transplantation model

IntroductionIschemia-reperfusion (I/R) injury causes present challenges in the field of graft transplantation which is also a major contributor to early graft dysfunction or failure after organ transplantation. The study focuses on the effects of prolonged cold-ischemia (CI) on the autophagic activity in the graft lung in a rat orthotopic lung transplantation model. Material and methodsDonor lungs were preserved under CI conditions for different periods. An orthotopic lung transplantation model was developed, and the lung tissues from donor lungs subjected to CI preservation and reperfusion were harvested. We evaluated the effects of different CI periods on autophagy, reactive oxygen species (ROS) and glucose consumption. Additionally, the mechanism by which prolonged CI affected autophagy was investigated through determination of the molecules related to the mTOR pathway after treatment with 3-Methyladenine (3-MA), rapamycin and an adenosine triphosphate (ATP) synthase inhibitor oligomycin (OM). ResultsProlonged CI led to increased activities of key glycolytic enzymes, glucose consumption and lactic acid production. Autophagy, ROS and glucose consumption were induced in the graft lung after I/R, which reached peak levels after 6 h and was gradually decreased. Most importantly, the perfusion treatment of 3-MA or OM decreased ROS level and autophagy, but increased the extent of mTOR phosphorylation, while the perfusion treatment of rapamycin induced ROS and autophagy. ConclusionTaken together, autophagy mediated by a prolonged CI preservation affects the glucose consumption and ROS production in the graft lung via the mTOR signaling pathway.

Impact of donor and prolonged cold ischemia time of neonatal pig pancreas on neonatal pig islet transplant outcome.

Genetically modified pigs (GMP) have been developed to alleviate the shortage of donors in human islet transplantation and rejection. In this study, we characterized and compared the islets from GalTKO, GalTKO/hCD46, GalTKO/hCD46/hCD39, and wild-type (WT) neonatal pigs. Islets were isolated from GMP and WT pig pancreases that have been packaged with ice pack for at least 24hours. The difference in gene expression and function of islets were evaluated by microarray analysis and transplantation of islets under the kidney capsule of streptozotocin-induced diabetic immune-deficient mice, respectively. Blood glucose levels of these mice were monitored weekly post-transplantation for >100days, and islet grafts were collected and evaluated for the presence of endocrine cells. The genes involved in extracellular components, cell adhesion, glucose metabolism, and inflammatory response are differentially expressed between GMP and WT pig islets. Variation in the ability of pig islets in correcting the diabetic state of the mouse recipients appears to be dependent on the pig donor. In addition, prolonged cold ischemia time had a negative effect on the transplant outcome. All normoglycemic mice were able to respond well to glucose challenge despite the initial differences in the ability of islet transplants to reverse their diabetic state. Islet xenografts of normoglycemic mice contained abundant insulin- and glucagon-positive cells. The effect of GMP and WT neonatal pig islet transplants on hyperglycemia in mice appears to be dependent on the pig donor, and prolonged cold ischemia time negatively affects the neonatal pig islet transplant outcome.

Functional state of the cardiorespiratory system after orthotopic heart transplantation with prolonged cold ischemia time

Objective: to assess the functional state of the cardiorespiratory system in the long term after orthotopic heart transplantation (HT) with prolonged cold ischemia time. Materials and methods. The results of 60 orthotopic HTs performed at Meshalkin National Medical Research Center were analyzed. A comparison was made of the immediate and long-term outcomes of HTs in the group with cold ischemia time lasting for less than 240 minutes and in those with farther distance between donor and recipient sites with cold ischemia time of 240 minutes or more. In the long-term follow-up after HT, all patients underwent cardiopulmonary exercise testing, body plethysmography, assessment of the diffusing capacity of the lungs, and quality of life assessment. Results. Prolonged cold ischemia showed a negative effect on the early postoperative period – decreased myocardial contractility on postoperative day 1 and longer duration of inotropic support. At the same time, the survival rate and incidence of graft rejection reactions in the early and late post-HT periods in the studied groups did not differ significantly. Peak oxygen consumption in the general group in the long term after HT was 17 (14.7–21.0) mL/kg/min, VE/ VCO2 slope was 30 (29–36) at 100 (90–120) W threshold load power. All the parameters of pulmonary function tests did not differ significantly depending on cold ischemia duration. Quality of life also did not show significant differences depending on the duration of graft ischemia in terms of both physical and psycho-emotional health components of the SF-36 questionnaire. Conclusion. Long-term cold ischemia of the graft did not show any negative impact on the functional state of the cardiorespiratory system and quality of life in the long term after HT. The studied group of recipients was characterized by high efficiency of pulmonary ventilation and gas exchange, as well as high tolerance to physical activity in the long-term post-HT period.

Ex vivo delivery of Mirococept: A dose-finding study in pig kidney after showing a low dose is insufficient to reduce delayed graft function in human kidney.

The complement system plays a pivotal role in the pathogenesis of ischemia-reperfusion injury in solid organ transplantation. Mirococept is a potent membrane-localizing complement inhibitor that can be administered ex vivo to the donor kidney prior to transplantation. To evaluate the efficacy of Mirococept in reducing delayed graft function (DGF) in deceased donor renal transplantation, we undertook the efficacy of mirococept (APT070) for preventing ischaemia-reperfusion injury in the kidney allograft (EMPIRIKAL) trial (ISRCTN49958194). A dose range of 5-25mg would be tested, starting with 10mg in cohort 1. No significant difference between Mirococept at 10mg and control was detected; hence the study was stopped to enable a further dose saturation study in a porcine kidney model. The optimal dose of Mirococept in pig kidney was 80mg. This dose did not induce any additional histological damage compared to controls or after a subsequent 3hours of normothermic machine perfusion. The amount of unbound Mirococept postperfusion was found to be within the systemic dose range considered safe in the Phase I trial. The ex vivo administration of Mirococept is a safe and feasible approach to treat DGF in deceased donor kidney transplantation. The porcine kidney study identified an optimal dose of 80mg (equivalent to 120mg in human kidney) that provides a basis for further clinical development.

DOES THE COLD STORAGE REALLY «COLD»? LIVER THERMOGRAPHY AND MICRODIALYSIS FINDINGS

Background: We suggest that in clinical practice organ temperature (Temp) during static cold storage is an underestimated factor affecting both the organ metabolic state before transplantation and the initial graft function. To verify this hypothesis we conducted a pilot clinical study involving living (LD) and deceased donors (DD) liver grafts. Material and Methods: The study included 4 LD right lobe liver grafts (cases L1 – L4) and 7 grafts from standard criteria DD with brain death (cases D1 – D7). Two DD grafts were not transplanted due to prolonged cold ischemia time (CIT) exceeding 13 hours (cases D6 and D7). Histidine-tryptophan-ketoglutorate solution was used for all grafts. Thermography (Guide D160) and microdialysis (M Dialysis AB, Sweden) were performed in each case at the end of the CIT. Microdialysis catheter was placed in SIV (for DD) or in SVIII (for LD). Samples of interstitial fluid were collected within 30-45 minutes before thermography and analysed for glucose (Glu), lactate (Lac), pyruvate (Pyr) and glycerol (Glyc). All transplantations were primary, non-urgent, performed in patients with cirrhosis and MELD value of 12-24 points.Early allograft dysfunction (EAD) was diagnosed with Olthoff criteria. There were no primary non-functioning grafts. Results and Discussion: CITs for LD grafts were: 0,5 – 1,0 h (median–0,5); for all DD grafts: 8,0 – 14,0 h (median–11,0) and for DD transplanted grafts: 8,0 – 12,5 (median–9). The medians of Temp [T(med)] were significantly higher than 4°C and varied widely. For LD: 13.4°C - 16.8°C, for DD: 7.7°C - 12.4°C. Moreover, the Temp of each individual graft also varied widely: the difference between the hottest and coldest points was in the range of 3.5°C – 7.3°C.At the end of CIT the Temp of DD grafts with normal initial function (D1, D2 and D3) were significantly lower than in grafts with EAD (D3 and D4): 8,5 [7,8 – 10,1](6,6 – 12,0) vs. 12,1 [11,5 – 12,7](9,2 – 15,7), p = 0.005. In addition to the level of Glyc, there were no other significant differences in microdialysis parameters between DD and LD grafts.This was probably due to a strong correlation between Glyc level and CIT: Spearman R = 0,96; p <0,001. Based on the suggesting that not only CIT but also T(med) affects the graft metabolic state immediately before transplantation, a new variable was introduced: [CIT * T(med)] (hours * °C). Apparently, the relationship of this parameter with interstitial levels of Glu, Lac, and Glyc was nonlinear. The best results of dynamic fitting in all cases were achieved using the equation: f = y0+a*exp(b*x). The R2 values for Glu, Lac and Glyc were 0,80, 0,60 and 0,81, respectively.Conclusion: Our results confirm the strong influence of Temp on the state of metabolism in the graft and its initial function. The development of devices for continuous assessment of metabolic state and Temp control during cold storage is promising. Russian Science Foundation (grant # 19-75-10040).

Characterization of Diabetic and Non-Diabetic Foot Ulcers Using Single-Cell RNA-Sequencing.

Background: Recent advances in high-throughput single-cell sequencing technologies have led to their increasingly widespread adoption for clinical applications. However, challenges associated with tissue viability, cell yield, and delayed time-to-capture have created unique obstacles for data processing. Chronic wounds, in particular, represent some of the most difficult target specimens, due to the significant amount of fibrinous debris, extracellular matrix components, and non-viable cells inherent in tissue routinely obtained from debridement. Methods: Here, we examined the feasibility of single cell RNA sequencing (scRNA-seq) analysis to evaluate human chronic wound samples acquired in the clinic, subjected to prolonged cold ischemia time, and processed without FACS sorting. Wound tissue from human diabetic and non-diabetic plantar foot ulcers were evaluated using an optimized 10X Genomics scRNA-seq platform and analyzed using a modified data pipeline designed for low-yield specimens. Cell subtypes were identified informatically and their distributions and transcriptional programs were compared between diabetic and non-diabetic tissue. Results: 139,000 diabetic and non-diabetic wound cells were delivered for 10X capture after either 90 or 180 min of cold ischemia time. cDNA library concentrations were 858.7 and 364.7 pg/µL, respectively, prior to sequencing. Among all barcoded fragments, we found that 83.5% successfully aligned to the human transcriptome and 68% met the minimum cell viability threshold. The average mitochondrial mRNA fraction was 8.5% for diabetic cells and 6.6% for non-diabetic cells, correlating with differences in cold ischemia time. A total of 384 individual cells were of sufficient quality for subsequent analyses; from this cell pool, we identified transcriptionally-distinct cell clusters whose gene expression profiles corresponded to fibroblasts, keratinocytes, neutrophils, monocytes, and endothelial cells. Fibroblast subpopulations with differing fibrotic potentials were identified, and their distributions were found to be altered in diabetic vs. non-diabetic cells. Conclusions: scRNA-seq of clinical wound samples can be achieved using minor modifications to standard processing protocols and data analysis methods. This simple approach can capture widespread transcriptional differences between diabetic and non-diabetic tissue obtained from matched wound locations.

Impact of warm ischemia time on outcomes for kidneys donated after cardiac death Post-KAS.

Prolonged warm (WIT) and cold (CIT) ischemia times are often important considerations in the discard of DCD kidneys, but their impact on post-transplant outcomes in the post-KAS era is unclear. We examined the association of ischemia time on delayed graft function (DGF) and death-censored graft failure for DCD kidneys. The 2018 SRTR SAF was utilized to identify post-KAS DCD kidney transplants occurring from 2015 to 2018. Relative risk and Cox regression were used to calculate risk of delayed graft function and hazard of death-censored graft failure, respectively. We identified 4,680 kidneys from DCD donors transplanted from 2015 to 2018 with recorded WIT and CIT times. Median WIT was 21.0minutes (IQR 14.0-28.0), and CIT was 18.5hours (IQR 13.9-23.5). The overall incidence of DGF was 42.7%. In a univariable relative risk regression model, extended CIT (24-30hours:RR 1.37, 95% CI 1.15-1.77; >30hours:RR 1.47, 95% CI 1.22-1.77) and WIT (20-40minutes:RR 1.10, 95% CI 1.03-1.17) were associated with increased risk of DGF. When included in a multivariable model, neither prolonged CIT nor WIT were significantly associated with death-censored graft failure. Prolonged WIT and CIT are associated with increased DGF but not death-censored graft failure in recipients of DCD kidney transplants in the post-KAS era. Extended ischemia alone should not be used as a basis for discard or non-utilization of these organs.

Update on the Management of BK Virus Infection.

The BK polyomavirus was isolated in 1971; it has been a significant risk factor for both graft dysfunction and failure in renal transplant recipients. So far, no specific treatment option has been available for effective treatment or prophylaxis for BK virus infections. Although the use of heavy immunosuppression has been the main risk factor for BK virus infection, other risk factors are equally important, including elderly recipients, prior rejection episodes, male sex, human leukocyte antigen mismatching, prolonged cold ischemia time, pretransplant BK virus serostatus, and ureteral stenting. Regular follow-up for BK virus infections according to each institution's policy has been, so far, effective in detecting patients with BK virus viremia and consequently preventing allograft loss. The mainstay of management continues to be reduction of immunosuppression. However, newer options are providing new insights, such as cellular immunotherapy. In this review, we will address the diagnosis, screening, new diagnostic tools, and updated management of BK virus infections.

The Differential Influence of Cold Ischemia Time on Outcome After Liver Transplantation for Different Indications-Who Is at Risk? A Collaborative Transplant Study Report.

Introduction: Despite increasing awareness of the negative impact of cold ischemia time (CIT) in liver transplantation, its precise influence in different subgroups of liver transplant recipients has not been analyzed in detail. This study aimed to identify liver transplant recipients with an unfavorable outcome due to prolonged cold ischemia.Methods: 40,288 adult liver transplantations, performed between 1998 and 2017 and reported to the Collaborative Transplant Study were analyzed.Results: Prolonged CIT significantly reduced graft and patient survival only during the first post-transplant year. On average, each hour added to the cold ischemia was associated with a 3.4% increase in the risk of graft loss (hazard ratio (HR) 1.034, P < 0.001). The impact of CIT was strongest in patients with hepatitis C-related (HCV) cirrhosis with a 24% higher risk of graft loss already at 8–9 h (HR 1.24, 95% CI 1.05–1.47, P = 0.011) and 64% higher risk at ≥14 h (HR 1.64, 95% CI 1.30–2.09, P < 0.001). In contrast, patients with hepatocellular cancer (HCC) and alcoholic cirrhosis tolerated longer ischemia times up to <10 and <12 h, respectively, without significant impact on graft survival (P = 0.47 and 0.42). In HCC patients with model of end-stage liver disease scores (MELD) <20, graft survival was not significantly impaired in the cases of CIT up to 13 h.Conclusion: The negative influence of CIT on liver transplant outcome depends on the underlying disease, patients with HCV-related cirrhosis being at the highest risk of graft loss due to prolonged cold ischemia. Grafts with longer cold preservation times should preferentially be allocated to recipients with alcoholic cirrhosis and HCC patients with MELD <20, in whom the effect of cold ischemia is less pronounced.

Ultrastructural Characterization of Prolonged Normothermic and Cold Cerebral Ischemia in the Adult Rat.

The ultrastructural effects of prolonged normothermic and cold ischemia on the cerebral cortex of the adult rat were investigated. Complete cerebral ischemia was produced by cardiac arrest and the animals' temperature was maintained at 37°C for periods ranging from 0 to 81 hours before electron microscopy preparation. Electron micrographs of cold cerebral ischemia were generated after stabilizing the rat's temperature at 0°C after cardiac arrest for periods ranging from 0 hours to 6 months. A qualitative examination of the electron micrographs shows structural signatures of energy depletion such as vessel leaking and chromatin clumping after 1 hour at 37°C and after 24 hours at 0°C, followed by synapse degradation after 6 hours at 37°C and 1 week at 0°C. Evidence of advanced necrosis was observed after 36 hours at 37°C and 2 months at 0°C. A deep learning algorithm was introduced that demonstrates the temperature dependence of ischemia-induced ultrastructural changes and that can also successfully distinguish between early ischemic changes and advanced necrosis.

Mitochondrial DNA Stimulates TLR9-Dependent Neutrophil Extracellular Trap Formation in Primary Graft Dysfunction.

The immune system is designed to robustly respond to pathogenic stimuli but to be tolerant to endogenous ligands to not trigger autoimmunity. Here, we studied an endogenous damage-associated molecular pattern, mitochondrial DNA (mtDNA), during primary graft dysfunction (PGD) after lung transplantation. We hypothesized that cell-free mtDNA released during lung ischemia-reperfusion triggers neutrophil extracellular trap (NET) formation via TLR9 signaling. We found that mtDNA increases in the BAL fluid of experimental PGD (prolonged cold ischemia followed by orthotopic lung transplantation) and not in control transplants with minimal warm ischemia. The adoptive transfer of mtDNA into the minimal warm ischemia graft immediately before lung anastomosis induces NET formation and lung injury. TLR9 deficiency in neutrophils prevents mtDNA-induced NETs, and TLR9 deficiency in either the lung donor or recipient decreases NET formation and lung injury in the PGD model. Compared with human lung transplant recipients without PGD, severe PGD was associated with high levels of BAL mtDNA and NETs, with evidence of relative deficiency in DNaseI. We conclude that mtDNA released during lung ischemia-reperfusion triggers TLR9-dependent NET formation and drives lung injury. In PGD, DNaseI therapy has a potential dual benefit of neutralizing a major NET trigger (mtDNA) in addition to dismantling pathogenic NETs.

Effects of Delayed Hypothermic Machine Perfusion on Kidney Grafts with a Preliminary Period of Static Cold Storage and a Total Cold Ischemia Time of Over 24 Hours.

BackgroundHypothermic machine perfusion (HMP) appears to exert a reconditioning effect on the ischemic damage of kidney grafts. However, some concerns still remain about its real effectiveness when it is delayed after a preliminary period of static cold storage (SCS) or with prolonged overall cold ischemia time (CIT).Material/MethodsThe effect of HMP on hemodynamic, metabolic, histological and ultrastructural features of grafts was investigated in 21 single-kidney grafts treated with a delayed HMP after SCS and with a total CIT of over 24 h.ResultsThe mean CIT, SCS, and HMP times were 29 h, 12 h, and 18 h, respectively. Longer SCS was associated with higher vascular resistance and lower arterial flow. In the pre- vs. post-HMP comparison, a significant decrease in arterial resistances and increase of flow were recorded. The hemodynamic improvement was independent of HMP duration. The perfused grafts retained some metabolic activity, with a statistically significant decrease of pH, pO2, and glucose levels, and increase of lactates in the perfusion liquid, by the end of HMP. Longer SCS was associated with higher pH and greater pO2 decrease during HMP. Light microscopy and transmission electronic microscopy revealed no significant variations in nuclear, cytoplasmic, or ultrastructural damage. SCS, HMP, and CIT were not identified as risk factor for delayed graft function or rejection.ConclusionsA delayed and extended HMP can recover the graft hemodynamic function, maintain some metabolic activity, and stabilize the accumulated ischemic damage due to a preliminary SCS.

Targeting of intragraft reactive oxygen species by APP-103, a novel polymer product, mitigates ischemia/reperfusion injury and promotes the survival of renal transplants.

Inflammatory responses associated with ischemia/reperfusion injury (IRI) play a central role in alloimmunity and transplant outcomes. A key event driving these inflammatory responses is the burst of reactive oxygen species (ROS), with hydrogen peroxide (H2 O2 ) as the most abundant form that occurs as a result of surgical implantation of the donor organ. Here, we used a syngeneic rat renal transplant and IRI model to evaluate the therapeutic properties of APP-103, a polyoxalate-based copolymer molecule containing vanillyl alcohol (VA) that exhibits high sensitivity and specificity toward the production of H2 O2 . We show that APP-103 is safe, and that it effectively promotes kidney function following IRI and survival of renal transplants. APP-103 reduces tissue injury and IRI-associated inflammatory responses in models of both warm ischemia (kidney clamping) and prolonged cold ischemia (syngeneic renal transplant). Mechanistically, we demonstrate that APP-103 exerts protective effects by specifically targeting the production of ROS. Our data introduce APP-103 as a novel, nontoxic, and site-activating therapeutic approach that effectively ameliorates the consequences of IRI in solid organ transplantation.

Prolonged Cold Ischemia Time Offsets the Benefit of Human Leukocyte Antigen Matching in Deceased Donor Kidney Transplant

BackgroundThe consequences of prolonging cold ischemia time (CIT) to facilitate HLA matching in kidney transplantation are not known. MethodsPatients with a history of kidney transplant in the United States (2000-2016) with 0 HLA mismatch (MM) were categorized based on CIT (< 10; 10 to < 15; 15 to < 20; 20 to < 25; 25 to < 30; and ≥ 30 hours). Time to graft loss was compared for each CIT category to a reference group of individuals with > 0 HLA MM and short CIT (< 10 hours) using a multivariable Cox proportional hazards model. ResultsThe adjusted risk of graft failure was significantly lower for 0 HLA MM with the shortest CIT compared to the reference group (hazard ratio, 0.82; 95% confidence interval, 0.72-0.94), and this survival advantage persisted to a threshold of < 20 hours of CIT. No survival advantage was observed for the 0 HLA MM group once CIT was > 20 hours. This trend persisted after excluding highly sensitized recipients (panel reactive antibody > 98%) where shipping of organs occurs to achieve more equitable access to organs rather than optimize HLA match. ConclusionsCIT > 20 hours offsets the benefit of 0 HLA MM in kidney transplantation. This may have implications in organ shipping to facilitate immunologic match.

Long non-coding RNA X-inactive specific transcript silencing ameliorates primary graft dysfunction following lung transplantation through microRNA-21-dependent mechanism.

BackgroundPrimary graft dysfunction (PGD) is a known acute lung injury (ALI) and a major cause of fatality post-lung transplantation. Though some long non-coding RNAs (lncRNAs) have been studied in ALI through regulation of microRNAs (miRNAs), their effects on PGD remain undefined. The present study aims to explore the underlying mechanism of lncRNA X-inactive specific transcript (XIST) in PGD after lung transplantation.MethodsInitially, the expression of miR-21, IL-12A and XIST was determined by RT-qPCR and western blot analysis. The dual luciferase reporter assay, RNA pull-down and RIP assay were performed to identify the targeting relationship between miR-21 and IL-12A and the binding relationship between miR-21 and XIST. Loss- and gain-of-function investigations were conducted in rats treated with prolonged cold ischemia and polymorphonuclear neutrophils (PMNs).FindingsmiR-21 was decreased, whilst XIST and IL-12A were increased in the bronchoalveolar lavage fluid of PGD patients after lung transplantation. Enhanced miR-21 expression in rats and PMNs resulted in downregulated expression of pro-inflammatory factors and chemokines, and enhanced the apoptosis of PMNs. XIST was found to upregulate IL-12A expression in a miR-21-dependent manner. Additionally, XIST silencing enhanced the apoptosis of PMNs and inhibited the neutrophil extracellular trap (NET) formation through upregulation of miR-21 but downregulation of IL-12A in vivo.InterpretationIn summary, lncRNA XIST upregulates IL-12A by binding to miR-21, thereby inducing NET formation and accelerating PGD after lung transplantation. This suggests that inhibition of XIST and NET may be beneficial for the treatment of PGD.

Injury Pathways That Lead to AKI in a Mouse Kidney Transplant Model.

Prolonged cold ischemia (CI) is a risk factor for acute kidney injury after kidney transplantation. We endeavored to determine the pathways involved in the development of tubular cell injury and death before and after transplantation. We hypothesized that ex vivo cold storage before transplant would produce a different injury phenotype to that seen after engraftment in kidney transplants with or without CI. Four groups of mouse donor kidneys were studied: (1) nontransplanted control kidneys; (2) donor kidneys subjected to ex vivo cold ischemia (CI); (3) donor kidneys subjected to kidney transplant without CI (Txp); and (4) donor kidneys subjected to CI followed by transplantation (CI+Txp). Acute kidney injury only occurred in the CI+Txp group, which had significantly increased sCr versus the Txp group and the control mice. Histologically, the CI group demonstrated significantly increased tubular cell apoptosis and caspase-9 expression, whereas the Txp group demonstrated only mild brush border injury without apoptosis or necrosis. In contrast, the CI+Txp group had tubular cell apoptosis associated with expression of caspase-8, TNFR1, and increased serum TNF-α. CI+Txp also led to significantly higher ATN scores in association with increased RIP1, RIP3, pMLKL, and TLR4 expression. Our results suggest distinct therapies are needed at different times during organ preservation and transplantation. Prevention of apoptosis during cold storage is best achieved by inhibiting intrinsic pathways. In contrast, prevention of cell death and innate immunity after CI+Txp requires inhibition of both the extrinsic death receptor pathway via TNFR1 and caspase-8 and inhibition of programmed necrosis via TLR4 and TNFR1.

The UK Experience of Renal Transplantation in AL Amyloidosis

Systemic AL amyloidosis, caused by monoclonal light chain depositing as amyloid fibril protein, commonly presents with renal involvement. A third of patients with renal involvement have or develop significant renal dysfunction leading to end-stage renal failure (ESRF). In an era of improving life expectancy for patients with AL amyloidosis, renal transplantation, with its marked impact on improving quality of life and reducing the cost burden, is increasingly considered. We report the outcomes of fifty patients who had received a renal allograft between 2004-2019, seen at the UK National Amyloidosis Centre Patient characteristics were: 60% male; median age at diagnosis was 53.5 years (38-69 years); median ECOG performance status 1 (0-3); median presenting creatinine, eGFR and proteinuria were 199μmol/L (69-756 μmol/L), 27ml/min and 8.95g/24h (0.4-19.7g/24h) respectively. Involved light chains were - lambda - 36 (72%) and kappa - 14 (28%). 40 patients were evaluable (5 transplanted for non-amyloid conditions prior to diagnosis, 4 lost to follow up, 1 transplanted for amyloidosis overseas prior to referral). Thirteen (32.5%) had isolated renal involvement whilst 35 (87.5%) patients had other multi-organ involvement including 13/40 (32.5%) with cardiac involvement. Median time from diagnosis to ESRF was 15 months (0-115 months) and from renal replacement therapy to renal allograft was 28 months (3-83 months). Four (10%) received a pre-emptive renal allograft. At time of renal transplantation, patients had a median of 2 prior lines of chemotherapy (range 1-4) whilst 10/40 patients (25%) had a prior autologous stem cell transplant. At transplant, haematological responses (HR) were: complete response (CR) - 22 (55%), very good partial response (VGPR) - 12 (30%), partial response (PR) - 4 (10%) and stable disease - 2 (5%). Median time from HR to renal transplant was 29 months (0-93 months). From renal transplant, haematological PFS was 82.7 months (95% CI 61.2-104.2). Patients achieving a CR achieved a markedly higher PFS of 102.5 months (95% CI 68.8-136.3 months) (p=0.023) as did those with a dFLC &lt;10mg/L at renal transplantation, 121.1 months (95% CI 91.5-150.6 months) (p=0.0001). At a median follow up of 107 months (26-231 months), 14 (35%) patients died, of whom only one had graft failure at the time of death. One patient (with stage 3 cardiac AL) died within 2 days of a renal allograft after a post-operative hypotensive episode. Nine (22.5%) patients had post-transplant chemotherapy for relapse. Median OS from renal transplantation was 106.6 months (95% CI 85.0-128.3 months); significantly better in patients achieving a CR (133.0 months, p=0.005) or dFLC &lt;10mg/L (137.8 months, p=0.0001) prior to transplant. Stage of chronic kidney disease at presentation, plasma cell burden, age at renal transplantation and multi-organ involvement did not impact overall survival. Of the patients experiencing graft failure, 1 graft failed within a week of implantation due to primary non-function after a prolonged cold ischaemia time, 1 functioned poorly from implantation and failed within 4 months and a third failed after 45 months with evidence of amyloid recurrence in the graft. In conclusion, the outcomes of carefully selected patients with AL amyloidosis undergoing renal transplantation is promising and justifies careful consideration. There was only 1 peri-operative death. Patients in a complete response or who have a dFLC &lt;10mg/L prior to renal allograft implantation have significantly better outcomes. At a median follow ~9 years, graft failure is rare (only 7.5%). Disclosures No relevant conflicts of interest to declare.