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The Effects of Free Heme on Functional and Molecular Changes During Ex Vivo Normothermic Machine Perfusion of Human Kidneys

Front Immunol. 2022 May 2;13:849742 doi: 10.3389/fimmu.2022.849742.

Normothermic machine perfusion (NMP) is a technique of kidney preservation designed to restore cellular metabolism after cold ischemia. Kidneys are perfused with an oxygenated banked red blood cell (RBC) based solution for 1h at 36°C. During NMP, RBCs can become damaged, releasing free heme into the perfusate. This can act as a damage-associated molecular pattern (DAMP) activating inflammatory signalling pathways. The aim of this study was to measure the levels of free heme during NMP, assess the effect on kidney function and determine any association with inflammatory and stress related gene expression. Levels of free heme were measured in perfusate samples from a series of donation after circulatory death (DCD) kidneys undergoing NMP as part of a randomised controlled trial (RCT). The age of RBCs and levels of free heme were correlated with perfusion parameters. Changes in gene expression were analysed in a series of kidneys declined for transplantation using the NanoString nCounter Organ Transplant Panel and qRT-PCR. Older units of RBCs were associated with higher levels of free heme and levels increased significantly during NMP (Pre 8.56 ± 7.19µM vs 26.29 ± 15.18µM, P<0.0001). There was no association with levels of free heme and perfusion parameters during NMP (P > 0.05). Transcriptional and qPCR analysis demonstrated the upregulation of differentially expressed genes associated with apoptosis (FOS and JUN), inflammatory cytokines (IL-6, SOCS3, ATF3), chemokines (CXCL8, CXCL2, CC3/L1) and oxidative stress (KLF4) after NMP. However, these did not correlate with levels of free heme (P >0.05). A significant amount of free heme can be detected in the perfusate before and after NMP particularly when older units of red cells are used. Although transcriptional analysis demonstrated significant upregulation of genes involved with apoptotic, inflammatory and oxidative pathways these were not associated with high levels of free heme.

CET Conclusion
Reviewer: Mr Simon Knight, Centre for Evidence in Transplantation, Nuffield Department of Surgical Sciences University of Oxford
Conclusion: This paper reports a secondary analysis of an RCT of normothermic machine perfusion of the kidney prior to transplantation. The authors measured the level of free heme in the perfusate during 1 hour of NMP in a series of 42 DCD kidneys, and related this to perfusion parameters or gene expression. They found that significant levels of free heme are present in the perfusate even during short periods of NMP, and these correlate with the age of the RBC used for perfusion. However, there was no association between levels of free heme and gene expression. As this is a secondary analysis and the number are small with a short perfusion duration, it is unclear whether these findings truly reflect the clinical effects of high levels of free heme in the perfusate, or whether differences in outcome could be detected in a larger sample. Certainly, the age of red cells is likely to be important, and will also effect oxygen carrying capacity and delivery during NMP. Whilst this does not appear to have a major impact during short periods of NMP, it is possible that it will be more significant during longer duration perfusions.
Study Details
Aims: This secondary analysis of a randomised controlled trial aimed to measure free heme levels during normothermic machine perfusion (NMP) and assess their influence on kidney function, and also to determine any correlation with inflammatory and stress related gene expression.
Interventions: Kidneys in the original trial were randomly assigned receive either ex vivo normothermic perfusion (EVNP) or remain in static cold storage.
Participants: 42 kidneys that underwent 1 hour normothermic machine perfusion (NMP).
Outcomes: Levels of free heme, correlation of free heme levels and pRBC age with perfusion parameters and with inflammatory and stress related gene expression.
Follow Up: 1 hour
Funding: Non-industry funding
Publication type: Randomized Controlled Trial, Randomised Controlled Trial
Trial registration: ISRCTN15821205
Organ: Kidney
Language: English
Author email:
MeSH terms: Cold Ischemia; Heme; Humans; Kidney; Organ Preservation; Perfusion; 42VZT0U6YR (Heme); Kidney Transplantation