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Hydrogen sulphide-induced hypometabolism in human-sized porcine kidneys.

ABSTRACT: BACKGROUND:Since the start of organ transplantation, hypothermia-forced hypometabolism has been the cornerstone in organ preservation. Cold preservation showed to protect against ischemia, although post-transplant injury still occurs and further improvement in preservation techniques is needed. We hypothesize that hydrogen sulphide can be used as such a new preservation method, by inducing a reversible hypometabolic state in human sized kidneys during normothermic machine perfusion. METHODS:Porcine kidneys were connected to an ex-vivo isolated, oxygen supplemented, normothermic blood perfusion set-up. Experimental kidneys (n = 5) received a 85mg NaHS infusion of 100 ppm and were compared to controls (n = 5). As a reflection of the cellular metabolism, oxygen consumption, mitochondrial activity and tissue ATP levels were measured. Kidney function was assessed by creatinine clearance and fractional excretion of sodium. To rule out potential structural and functional deterioration, kidneys were studied for biochemical markers and histology. RESULTS:Hydrogen sulphide strongly decreased oxygen consumption by 61%, which was associated with a marked decrease in mitochondrial activity/function, without directly affecting ATP levels. Renal biological markers, renal function and histology did not change after hydrogen sulphide treatment. CONCLUSION:In conclusion, we showed that hydrogen sulphide can induce a controllable hypometabolic state in a human sized organ, without damaging the organ itself and could thereby be a promising therapeutic alternative for cold preservation under normothermic conditions in renal transplantation.

SUBMITTER: Maassen H

PROVIDER: S-EPMC6863563 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Hydrogen sulphide-induced hypometabolism in human-sized porcine kidneys.

Maassen Hanno H Hendriks Koen D W KDW Venema Leonie H LH Henning Rob H RH Hofker Sijbrand H SH van Goor Harry H Leuvenink Henri G D HGD Coester Annemieke M AM

PloS one 20191119 11

<h4>Background</h4>Since the start of organ transplantation, hypothermia-forced hypometabolism has been the cornerstone in organ preservation. Cold preservation showed to protect against ischemia, although post-transplant injury still occurs and further improvement in preservation techniques is needed. We hypothesize that hydrogen sulphide can be used as such a new preservation method, by inducing a reversible hypometabolic state in human sized kidneys during normothermic machine perfusion.<h4>M ...[more]

PMID: 31743376

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Project description:IntroductionHydrogen sulphide (H2S) has been established as a key member of the gasotransmitters family that recently showed a pivotal role in various pathological conditions including cancer.ObjectivesThis study investigated the role of H2S in breast cancer (BC) pathogenesis, on BC immune recognition capacity and the consequence of targeting H2S using non-coding RNAs.MethodsEighty BC patients have been recruited for the study. BC cell lines were cultured and transfected using validated oligonucleotide delivery system. Gene and protein expression analysis was performed using qRT-PCR, western blot and flow-cytometry. In-vitro analysis for BC hallmarks was performed using MTT, BrdU, Modified Boyden chamber, migration and colony forming assays. H2S and nitric oxide (NO) levels were measured spectrophotometrically. Primary natural killer cells (NK cells) and T cell isolation and chimeric antigen receptor transduction (CAR T cells) were performed using appropriate kits. NK and T cells cytotoxicity was measured. Finally, computational target prediction analysis and binding confirmation analyses were performed using different software and dual luciferase assay kit, respectively.ResultsThe H2S synthesizing enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), exhibited elevated levels in the clinical samples that correlated with tumor proliferation index. Knock-down of CBS and CSE in the HER2+ BC and triple negative BC (TNBC) cells resulted in significant attenuation of BC malignancy. In addition to increased susceptibility of HER2+ BC and TNBC to the cytotoxic activity of HER2 targeting CAR T cells and NK cells, respectively. Transcriptomic and phosphoprotein analysis revealed that H2S signaling is mediated through Akt in MCF7, STAT3 in MDA-MB-231 and miR-155/ NOS2/NO signaling in both cell lines. Lastly, miR-4317 was found to function as an upstream regulator of CBS and CSE synergistically abrogates the malignancy of BC cells.ConclusionThese findings demonstrate the potential role of H2S signaling in BC pathogenesis and the potential of its targeting for disease mitigation.

Project description:In porcine kidney auto-transplant models, red blood cells (RBCs) are required for ex-vivo normothermic machine perfusion (NMP). As large quantities of RBCs are needed for NMP, utilising autologous RBCs would imply lethal exsanguination of the pig that is donor and recipient-to-be in the same experiment. The purpose of this study was to determine if an isolated porcine kidney can also be perfused with allogeneic porcine or human RBCs instead. Porcine kidneys, autologous and allogeneic blood were obtained from a local slaughterhouse. Human RBCs (O-pos), were provided by our transfusion laboratory. Warm ischaemia time was standardised at 20 minutes and subsequent hypothermic machine perfusion lasted 1.5-2.5 hours. Next, kidneys underwent NMP at 37°C during 7 hours with Williams' Medium E and washed, leukocyte depleted RBCs of either autologous, allogeneic, or human origin (n = 5 per group). During perfusion all kidneys were functional and produced urine. No macroscopic adverse reactions were observed. Creatinine clearance during NMP was significantly higher in the human RBC group in comparison with the allogeneic group (P = 0.049) but not compared to the autologous group. The concentration of albumin in the urine was significantly higher in the human RBC group (P <0.001) compared to the autologous and allogeneic RBC group. Injury marker aspartate aminotransferase was significantly higher in the human RBC group in comparison with the allogeneic group (P = 0.040) but not in comparison with the autologous group. Renal histology revealed glomerular and tubular damage in all groups. Signs of pathological hyperfiltration and microvascular injury were only observed in the human RBC group. In conclusion, perfusion of porcine kidneys with RBCs of different origin proved technically feasible. However, laboratory analysis and histology revealed more damage in the human RBC group compared to the other two groups. These results indicate that the use of allogeneic RBCs is preferable to human RBCs in a situation where autologous RBCs cannot be used for NMP.

Project description:Background The gap between demand and supply of kidneys for transplantation necessitates the use of kidneys from extended criteria donors. Transplantation of these donor kidneys is associated with inferior results, reflected by an increased risk of delayed graft function. Inferior results might be explained by the higher immunogenicity of extended criteria donor kidneys. Normothermic machine perfusion (NMP) could be used as a platform to assess the quality and function of donor kidneys. In addition, it could be useful to evaluate and possibly alter the immunological response of donor kidneys. In this study, we first evaluated whether complement was activated during NMP of porcine and human discarded kidneys. Second, we examined the relationship between complement activation and pro-inflammatory cytokines during NMP. Third, we assessed the effect of complement activation on renal function and injury during NMP of porcine kidneys. Lastly, we examined local complement C3d deposition in human renal biopsies after NMP. Methods NMP with a blood-based perfusion was performed with both porcine and discarded human kidneys for 4 and 6 h, respectively. Perfusate samples were taken every hour to assess complement activation, pro-inflammatory cytokines and renal function. Biopsies were taken to assess histological injury and complement deposition. Results Complement activation products C3a, C3d, and soluble C5b-9 (sC5b-9) were found in perfusate samples taken during NMP of both porcine and human kidneys. In addition, complement perfusate levels positively correlated with the cytokine perfusate levels of IL-6, IL-8, and TNF during NMP of porcine kidneys. Porcine kidneys with high sC5b-9 perfusate levels had significantly lower creatinine clearance after 4 h of NMP. In line with these findings, high complement perfusate levels were seen during NMP of human discarded kidneys. In addition, kidneys retrieved from brain-dead donors had significantly higher complement perfusate levels during NMP than kidneys retrieved from donors after circulatory death. Conclusion Normothermic kidney machine perfusion induces complement activation in porcine and human kidneys, which is associated with the release of pro-inflammatory cytokines and in porcine kidneys with lower creatinine clearance. Complement inhibition during NMP might be a promising strategy to reduce renal graft injury and improve graft function prior to transplantation.

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Hydrogen sulphide-induced hypometabolism in human-sized porcine kidneys.

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Hydrogen sulphide-induced hypometabolism in human-sized porcine kidneys.

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