M Kaisar

Past, Present, and Future of Dynamic Kidney and Liver Preservation and Resuscitation

The increased demand for organs has led to the increased usage of “higher risk” kidney and liver grafts. These grafts from donation after circulatory death or expanded criteria donors are more susceptible to preservation injury and have a higher risk of unfavorable outcomes. Dynamic, instead of static, preservation could allow for organ optimization, offering a platform for viability assessment, active organ repair and resuscitation. Ex situ machine perfusion and in situ regional perfusion in the donor are emerging as potential tools to preserve and resuscitate vulnerable grafts. Preclinical findings have ignited clinical organ preservation research that investigates dynamic preservation, its various modes (continuous, preimplantation) and temperatures (hypo‐, sub, or normothermic). This review outlines the current status of dynamic preservation of kidney and liver grafts and describes ongoing research and emerging clinical trials.

Using an Integrated -Omics Approach to Identify Key Cellular Processes That Are Disturbed in the Kidney After Brain Death.

In an era where we are becoming more reliant on vulnerable kidneys for transplantation from older donors, there is an urgent need to understand how brain death leads to kidney dysfunction and, hence, how this can be prevented. Using a rodent model of hemorrhagic stroke and next‐generation proteomic and metabolomic technologies, we aimed to delineate which key cellular processes are perturbed in the kidney after brain death. Pathway analysis of the proteomic signature of kidneys from brain‐dead donors revealed large‐scale changes in mitochondrial proteins that were associated with altered mitochondrial activity and morphological evidence of mitochondrial injury. We identified an increase in a number of glycolytic proteins and lactate production, suggesting a shift toward anaerobic metabolism. Higher amounts of succinate were found in the brain death group, in conjunction with increased markers of oxidative stress. We characterized the responsiveness of hypoxia inducible factors and found this correlated with post–brain death mean arterial pressures. Brain death leads to metabolic disturbances in the kidney and alterations in mitochondrial function and reactive oxygen species generation. This metabolic disturbance and alteration in mitochondrial function may lead to further cellular injury. Conditioning the brain‐dead organ donor by altering metabolism could be a novel approach to ameliorate this brain death–induced kidney injury.

Plasma Biomarker Profile Alterations during Variable Blood Storage.

To the Editor: A major confounding factor in the discovery of disease-specific molecular signatures is the variability in handling clinical blood samples. Consequently, novel protein markers of diseases have failed the “iron test” of validation and implementation into clinical practice because, in addition to patient variability, a range of preanalytical parameters contributes to erroneous results. The temperature of storing, transporting, and processing of whole blood following collection is one factor that has not been standardized. Often, samples are transferred from the local laboratories to centralized biobanks in a cooled environment or even on dry ice. Recently, we established a UK biobank to conduct research into the quality of organs donated for transplantation [UK QUOD (Quality in Organ Donation)], collecting donor blood samples from 60 UK transplant centers (www.Quod.org.uk). Taking into consideration the logistical challenges in a clinical setting for sample collection and biobanking, we favored whole blood sample handling and processing at ambient temperature (22 ± 2 °C) before isolation of plasma by centrifugation and subsequent storage at −80 °C. We explored how the proteome and degradome (proteolytic processing of the proteome) may change when whole blood remains at ambient temperature for 30 min, 8 h, 24 h, and 48 h before plasma preparation. We …

Proteo-metabolomics reveals compensation between ischemic and non-injured contralateral kidneys after reperfusion

Ischaemia and reperfusion injury (IRI) is the leading cause of acute kidney injury (AKI), which contributes to high morbidity and mortality rates in a wide range of injuries as well as the development of chronic kidney disease. The cellular and molecular responses of the kidney to IRI are complex and not fully understood. Here, we used an integrated proteomic and metabolomic approach to investigate the effects of IRI on protein abundance and metabolite levels. Rat kidneys were subjected to 45 min of warm ischaemia followed by 4 h and 24 h reperfusion, with contralateral and separate healthy kidneys serving as controls. Kidney tissue proteomics after IRI revealed elevated proteins belonging to the acute phase response, coagulation and complement pathways, and fatty acid (FA) signalling. Metabolic changes were already evident after 4 h reperfusion and showed increased level of glycolysis, lipids and FAs, whilst mitochondrial function and ATP production was impaired after 24 h. This deficit was partially compensated for by the contralateral kidney. Such a metabolic balance counteracts for the developing energy deficit due to reduced mitochondrial function in the injured kidney.

Using “Omics” as a Novel Tool to Identify Mitochondrial Dysfunction and Metabolic Dysregulation as Critical Factors in Brain Death Induced Kidney Injury.: Abstract# 1475

1475 Using “Omics” as a Novel Tool to Identify Mitochondrial Dysfunction and Metabolic Dysregulation as Critical Factors in Brain Death Induced Kidney Injury. M. Akhtar, H. Huang, M. Kaisar, H. Leuvenink, B. Kessler, S. Fuggle, C. Pugh, R. Ploeg. Nuffi eld Derpartment of Surgery and Medicine, University of Oxford, Oxford, United Kingdom. Introduction: To gain better insight into patterns of injury and repair in kidneys from brain dead (BD) donors, we used next generation proteomics and metabolomics to identify the effect of BD on the protein signature of rodent renal samples. Methods: BD was induced in ventilated Fischer rats (250-300g) using a Fogarty balloon catheter infl ated in the epidural space (n=6) vs sham controls (n=6). Following BD, ventilation was continued for 4 h. Cortical proteins were extracted using in-solution trypsin digestion and subjected to proteomic analysis using liquid chromatography mass spectrometry (LC-MS/MS). Proteins were identifi ed based on having a >2 peptide sequence homology and analysed using Progenesis and Ingenuity Pathway Analysis (IPA). Samples were prepared for 1H-nuclear magnetic resonance (NMR) spectroscopy metabolomic analysis. Data was analysed as normalised intensity ratios and signifi cantly differentially expressed metabolites determined (p<0.05, Prism 6 Graphpad). Results: Over 1400 proteins were identifi ed, with 43 proteins being differentially expressed between BD and control samples (2 fold up or down regulated, p<0.05). Mitochondrial proteins and proteins concerning small molecule biochemistry and metabolism were the top dysregulated canonical pathways (IPA). Evaluation of the metabolome revealed signifi cantly increased amounts of lactate (p=0.04) in addition to alterations in other metabolites and intermediaries including increased isoleucine (p=0.002) and decreased AMP (p= 0.004), TMAO (p= 0.015) and aspartate (p=0.015). Creatinine, signifying renal dysfunction, was higher in the BD group in comparison to controls (p=0.009). Conclusion: This study underpins the importance of mitochondrial dysfunction and metabolic disturbances in the aetiology of BD induced kidney injury, suggesting a dependence on anaerobic metabolism of rodent kidneys in the BD state. To prevent kidney injury in the BD donor requires a multifaceted approach targeting protecting against mitochondrial damage and metabolic disturbance. Abstract# 1476 Brain Death Is Associated With Loss of TLR Signaling and Upregulation of Proinfl ammatory Proteins. J. Chen, S. Antipenko, R. Deierhoi, J. Locke, R. Mannon. Medicine and Surgery, University of Alabama at Birmingham, Birmingham. Background: Kidney transplants from brain dead (BD) donors have inferior outcomes than transplants from living donors. As such, BD initiates a systemic infl ammatory response, which may impact on recipient immunity. Prior studies in man have demonstrated an up-regulation of infl ammatory mediators following BD, but these may be affected by preservation and subsequent cold injury. We sought to determine the extent of BD on donor immune activation and impact on recipient function. Methods: In this pilot study, kidney biopsy, serum and urine were obtained from BD donors, just prior to organ retrieval and the start of cold preservation (N=8). Gene expression in biopsies was analyzed by customized Taqman real time low density arrays containing 96 molecules associated with innate and alloimmunity and ischemic injury. Serum and urine were analyzed by luminex assay and urine values normalized to urine creatinine. Donor PBMCs were cultured and stimulated with TLR ligands for 24h and cytokines produced in the media were measured by fl ow cytometry bead assay. Results: In BD donors, serum G-CSF was signifi cantly increased (170.9±38.5 vs. 64.8±9.8 pg/ml; p=0.045) and eotaxin was decreased (48.7±9.1 vs. 125.4±18.3 pg/ml; p=0.01) compared to normal individuals. Moreover, urine MCP-1 was signifi cantly increased (35.7±15.6 vs. 2.6±0.2 pg/mg creatinine; p=0.037). In this small dataset, there were no correlations to recipient eGFR at 1w or 1m except for urinary MCP1 where there was a trend towards signifi cance (Pearson’s r value 0.45, p=0.31). PCR analysis of BD kidney biopsies showed signifi cant upregulation of 22 genes and downregulation of 18 genes (A). In vitro stimulation of TLR1-9 in PMBCs showed a global reduction in cytokine production compared to normal PBMCs, with signifi cant reductions of IL-6 (B). 1476 Brain Death Is Associated With Loss of TLR Signaling and Upregulation of Proinfl ammatory Proteins. J. Chen, S. Antipenko, R. Deierhoi, J. Locke, R. Mannon. Medicine and Surgery, University of Alabama at Birmingham, Birmingham. Background: Kidney transplants from brain dead (BD) donors have inferior outcomes than transplants from living donors. As such, BD initiates a systemic infl ammatory response, which may impact on recipient immunity. Prior studies in man have demonstrated an up-regulation of infl ammatory mediators following BD, but these may be affected by preservation and subsequent cold injury. We sought to determine the extent of BD on donor immune activation and impact on recipient function. Methods: In this pilot study, kidney biopsy, serum and urine were obtained from BD donors, just prior to organ retrieval and the start of cold preservation (N=8). Gene expression in biopsies was analyzed by customized Taqman real time low density arrays containing 96 molecules associated with innate and alloimmunity and ischemic injury. Serum and urine were analyzed by luminex assay and urine values normalized to urine creatinine. Donor PBMCs were cultured and stimulated with TLR ligands for 24h and cytokines produced in the media were measured by fl ow cytometry bead assay. Results: In BD donors, serum G-CSF was signifi cantly increased (170.9±38.5 vs. 64.8±9.8 pg/ml; p=0.045) and eotaxin was decreased (48.7±9.1 vs. 125.4±18.3 pg/ml; p=0.01) compared to normal individuals. Moreover, urine MCP-1 was signifi cantly increased (35.7±15.6 vs. 2.6±0.2 pg/mg creatinine; p=0.037). In this small dataset, there were no correlations to recipient eGFR at 1w or 1m except for urinary MCP1 where there was a trend towards signifi cance (Pearson’s r value 0.45, p=0.31). PCR analysis of BD kidney biopsies showed signifi cant upregulation of 22 genes and downregulation of 18 genes (A). In vitro stimulation of TLR1-9 in PMBCs showed a global reduction in cytokine production compared to normal PBMCs, with signifi cant reductions of IL-6 (B). Conclusion: BD is an infl ammatory condition associated with systemic alterations that lead to recipient immune activation. However, TLR signaling in PBMCs appears to be downregulated, perhaps due to persistent activation in the BD donor. Further examination of the relationship between donor immune activation and transplant outcomes is needed. Abstract# 1477 Intermittent Subnormothermic Ex Vivo Liver Perfusion Reduces Endothelia Cell Death and Decreases Bile Duct Injury After Pig Liver Transplantation With DCD Grafts. V. Spetzler, M. Knaak, N. Goldaracena, K. Louis, D. Grant, M. Selzner. Multui Organ Transplant Program, Toronto General Hospital, Toronto, ON, Canada. 1477 Intermittent Subnormothermic Ex Vivo Liver Perfusion Reduces Endothelia Cell Death and Decreases Bile Duct Injury After Pig Liver Transplantation With DCD Grafts. V. Spetzler, M. Knaak, N. Goldaracena, K. Louis, D. Grant, M. Selzner. Multui Organ Transplant Program, Toronto General Hospital, Toronto, ON, Canada. Ischemic-type biliary lesions (ITBL) are the main obstacle for liver transplantation using organ donation after cardiac death (DCD). We developed a novel technique of subnormothermic ex vivo liver perfusion (SNEVLP) for the preservation of liver grafts suitable for a clinical setting. Methods: In a porcine DCD transplant model (45min warm ischemia time), liver grafts were either stored for 10hr at 4°C (CS, n=5) or preserved combining a total of 7hr cold storage and 3hr SNEVLP (33°C, n=5). Combination of cold storage and SNELP was used to simulate a clinical relevant scenario including graft transportation on ice and organ cooling for graft implantation. Parameters of hepatocyte (AST, INR), endothelial cell (Hyaluronic Acid, CD31 immunohistochemistry), Kupffer cell (β-Galactosidase), and biliary (alk. Phosphatase, Bilirubin) injury and function were determined. 5 day animal survival was assessed. Results: 5 day animal survival was similar between the CS and SNEVLP treated groups (40% vs 80%, p=0.8). No difference was observed between CS and SNEVLP groups regarding maximum INR (1.7 vs 2, p=0.9) or maximum AST within 48hr (2500±1100 U/L vs 3010±1530 U/L, p=0.3). In contrast, 7hr after reperfusion the CS vs SNEVLP group showed 5-fold higher Hyaluronic Acid levels (4195±2990 ng/ mL vs 737±450 ng/mL, p=0.01) as a marker of endothelial cell damage, and 2-fold increased β-Galactosidase levels (166±17 U/mL vs 95±25 U/ml, p<0.01) refl ecting increased Kupffer cell activation. CD31 staining of parenchymal biopsies at 8hr after reperfusion demonstrated massive endothelial cell injury in the CS group while endothelial cells were protected in SNEVLP livers. 3 days after transplantation CS vs SNEVLP groups had higher alk. Phosphatase (179±9 μmol/L vs 80±21 μmol/L, p≤0.05) and Bilirubin levels (20±22 μmol/L vs 6±2 μmol/L) as markers of biliary injury. Bile duct histology at the end of animal survival revealed severe bile duct necrosis in 3 out of 5 animals with CS, while no bile duct injury was observed in SNEVLP treated animals (p=0.026). Conclusion: SNEVLP preservation of DCD grafts reduces bile duct and endothelial cell injury in DCD grafts following liver transplantation. Intermitted SNEVLP preservation is a novel strategy to prevent ITBL in DCD liver grafts with extended warm ischemia times.