Untargeted metabolomics of perfusate and their association with hypothermic machine perfusion and allograft failure

Abstract

Although hypothermic machine perfusion (HMP) is associated with improved kidney graft viability and function, the underlying biological mechanisms are unknown. Untargeted metabolomic profiling may identify potential metabolites and pathways that can help assess allograft viability and contribute to organ preservation. Therefore, in this multicenter study, we measured all detectable metabolites in perfusate collected at the beginning and end of deceased-donor kidney perfusion and evaluated their associations with graft failure. In our cohort of 190 kidney transplants, 33 (17%) had death-censored graft failure over a median follow-up of 5.0 years (IQR 3.0-6.1 years). We identified 553 known metabolites in perfusate and characterized their experimental and biological consistency through duplicate samples and unsupervised clustering. After perfusion-time adjustment and false discovery correction, six metabolites in post-HMP perfusate were significantly associated with death-censored graft failure, including alpha-ketoglutarate, 3-carboxy-4-methyl-5-propyl-2-furanpropanoate, 1-carboxyethylphenylalanine, and three glycerol-phosphatidylcholines. All six metabolites were associated with an increased risk of graft failure (Hazard Ratio per median absolute deviation range 1.04-1.45). Four of six metabolites also demonstrated significant interaction with donation after cardiac death with notably greater risk in the donation after cardiac death group (Hazard Ratios up to 1.69). Discarded kidneys did not have significantly different levels of any death-censored graft failure-associated metabolites. On interrogation of pathway analysis, production of reactive oxygen species and increased metabolism of fatty acids were upregulated in kidneys that subsequently developed death-censored graft failure. Thus, further understanding the role of these metabolites may inform the HMP process and help improve the objective evaluation of allograft offers, thereby reducing the discard of potentially viable organs. Although hypothermic machine perfusion (HMP) is associated with improved kidney graft viability and function, the underlying biological mechanisms are unknown. Untargeted metabolomic profiling may identify potential metabolites and pathways that can help assess allograft viability and contribute to organ preservation. Therefore, in this multicenter study, we measured all detectable metabolites in perfusate collected at the beginning and end of deceased-donor kidney perfusion and evaluated their associations with graft failure. In our cohort of 190 kidney transplants, 33 (17%) had death-censored graft failure over a median follow-up of 5.0 years (IQR 3.0-6.1 years). We identified 553 known metabolites in perfusate and characterized their experimental and biological consistency through duplicate samples and unsupervised clustering. After perfusion-time adjustment and false discovery correction, six metabolites in post-HMP perfusate were significantly associated with death-censored graft failure, including alpha-ketoglutarate, 3-carboxy-4-methyl-5-propyl-2-furanpropanoate, 1-carboxyethylphenylalanine, and three glycerol-phosphatidylcholines. All six metabolites were associated with an increased risk of graft failure (Hazard Ratio per median absolute deviation range 1.04-1.45). Four of six metabolites also demonstrated significant interaction with donation after cardiac death with notably greater risk in the donation after cardiac death group (Hazard Ratios up to 1.69). Discarded kidneys did not have significantly different levels of any death-censored graft failure-associated metabolites. On interrogation of pathway analysis, production of reactive oxygen species and increased metabolism of fatty acids were upregulated in kidneys that subsequently developed death-censored graft failure. Thus, further understanding the role of these metabolites may inform the HMP process and help improve the objective evaluation of allograft offers, thereby reducing the discard of potentially viable organs. Lay SummaryAlthough hypothermic machine perfusion (HMP) is associated with improved kidney transplant outcomes, the biological mechanisms are unknown. Metabolomic profiling of donor’s kidneys may illuminate metabolites that can aid in assessing organ viability and preservation. In this study, all detectable metabolites were measured from perfusate collected at the beginning and end of deceased-donor kidney HMP. Six metabolites were significantly associated with the risk of death-censored graft failure, including alpha-ketoglutarate, 3-carboxy-4-methyl-5-propyl-2-furanpropanoate, 1-carboxyethylphenylalanine, and 3 glycerol-phosphatidylcholines. On interrogation of pathway analysis, the production of reactive oxygen species and increased metabolism of fatty acids were upregulated in kidneys that subsequently developed death-censored graft failure. Thus, further understanding the role of these metabolites may inform the HMP process and help improve the objective evaluation of allograft offers, thereby reducing the discard of potentially viable organs. Although hypothermic machine perfusion (HMP) is associated with improved kidney transplant outcomes, the biological mechanisms are unknown. Metabolomic profiling of donor’s kidneys may illuminate metabolites that can aid in assessing organ viability and preservation. In this study, all detectable metabolites were measured from perfusate collected at the beginning and end of deceased-donor kidney HMP. Six metabolites were significantly associated with the risk of death-censored graft failure, including alpha-ketoglutarate, 3-carboxy-4-methyl-5-propyl-2-furanpropanoate, 1-carboxyethylphenylalanine, and 3 glycerol-phosphatidylcholines. On interrogation of pathway analysis, the production of reactive oxygen species and increased metabolism of fatty acids were upregulated in kidneys that subsequently developed death-censored graft failure. Thus, further understanding the role of these metabolites may inform the HMP process and help improve the objective evaluation of allograft offers, thereby reducing the discard of potentially viable organs. Hypothermic machine perfusion (HMP) is commonly used in the process of deceased donor kidney transplantation to preserve the organ during transport and help minimize cold ischemic injury to the allograft. HMP circulates cold perfusate solution through the graft kidney for several hours after removal from the donor and before transplantation into the recipient, and there is evidence that HMP reduces delayed graft function (DGF) and improves 3-year graft survival compared with static cold storage.1Peng P. Ding Z. He Y. et al.Hypothermic machine perfusion versus static cold storage in deceased donor kidney transplantation: a systematic review and meta-analysis of randomized controlled trials.Artif Organs. 2019; 43: 478-489Crossref PubMed Scopus (38) Google Scholar, 2Ravaioli M. De Pace V. Angeletti A. et al.Hypothermic oxygenated new machine perfusion system in liver and kidney transplantation of extended criteria donors: first Italian clinical trial.Sci Rep. 2020; 10: 6063Crossref PubMed Scopus (54) Google Scholar, 3Tingle S.J. Figueiredo R.S. Moir J.A. et al.Hypothermic machine perfusion is superior to static cold storage in deceased donor kidney transplantation: a meta-analysis.Clin Transplant. 2020; 34e13814Crossref PubMed Scopus (28) Google Scholar However, the biological mechanisms that underlie how HMP contributes to improved kidney graft health are unclear, with some studies suggesting possible roles for downregulation of inflammatory pathways and upregulation of cell survival pathways.4Fu Z. Ye Q. Zhang Y. et al.Hypothermic machine perfusion reduced inflammatory reaction by downregulating the expression of matrix metalloproteinase 9 in a reperfusion model of donation after cardiac death.Artif Organs. 2016; 40: E102-E111Crossref PubMed Scopus (17) Google Scholar,5Zhang Y. Fu Z. Zhong Z. et al.Hypothermic machine perfusion decreases renal cell apoptosis during ischemia/reperfusion injury via the Ezrin/AKT pathway.Artif Organs. 2016; 40: 129-135Crossref PubMed Scopus (23) Google Scholar Furthermore, although some perfusate proteins have demonstrated moderate predictive ability for DGF, no biomarkers have been associated with graft survival yet.6Guzzi F. Knight S.R. Ploeg R.J. Hunter J.P. A systematic review to identify whether perfusate biomarkers produced during hypothermic machine perfusion can predict graft outcomes in kidney transplantation.Transplant Int. 2020; 33: 590-602Crossref PubMed Scopus (25) Google Scholar The need for noninvasive biomarkers to assess allograft viability is greater than ever, as both the fraction of kidneys pumped and discarded increases.7Cooper M. Formica R. Friedewald J. et al.Report of National Kidney Foundation consensus conference to decrease kidney discards.Clin Transplant. 2019; 33e13419Crossref Scopus (57) Google Scholar,8Chang A. Schaubel D.E. Chen M. et al.Trends and outcomes of hypothermic machine perfusion preservation of kidney allografts in simultaneous liver and kidney transplantation in the United States.Transpl Int. 2022; 3510345Crossref Scopus (2) Google Scholar Even more recently, the change in the kidney allocation system to prioritize candidates within a distance of 250 nautical miles of the donor hospital has led to longer cold ischemia time and increased donor Kidney Donor Profile Indexes (KDPIs).9Adler J.T. Husain S.A. King K.L. Mohan S. Greater complexity and monitoring of the new Kidney Allocation System: implications and unintended consequences of concentric circle kidney allocation on network complexity.Am J Transplant. 2021; 21: 2007-2013Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar,10Rohan V.S. Pilch N. McGillicuddy J. et al.Early assessment of national kidney allocation policy change.J Am Coll Surg. 2022; 234: 565-570Crossref PubMed Scopus (6) Google Scholar Given that marginal kidneys are more likely to be pumped, HMP use for organ preservation will likely further increase.11Sharma N. Mahajan A. Qazi Y.A. Marginal kidney transplantation: the road less traveled.Curr Opin Organ Transplant. 2019; 24: 92-96Crossref PubMed Scopus (9) Google Scholar However, the lack of standardized guidelines for HMP use has led to significant variability among organ procurement organizations (OPOs) in the selection of organs for HMP and the use of biomarkers and other parameters to monitor perfusion. Following the logistical challenges associated with its implementation, ≈30% of perfused kidneys nationally are later discarded because of concerns about organ quality and viability.12Woodside K.J. Merion R.M. Leichtman A.B. et al.Utilization of kidneys with similar kidney donor risk index values from standard versus expanded criteria donors.Am J Transplant. 2012; 12: 2106-2114Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar Physical parameters, such as changes in kidney resistance and perfusate flow rates, are used to monitor the HMP process and infer the viability of kidneys on HMP.13Jochmans I. Moers C. Smits J.M. et al.The prognostic value of renal resistance during hypothermic machine perfusion of deceased donor kidneys.Am J Transplant. 2011; 11: 2214-2220Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar However, flow and resistance parameters are imperfect, nonstandardized measures that vary over time and do not appear to be linked to the biological health of the graft.14Parikh C.R. Hall I.E. Bhangoo R.S. et al.Associations of perfusate biomarkers and pump parameters with delayed graft function and deceased donor kidney allograft function.Am J Transplant. 2016; 16: 1526-1539Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar Developing our understanding of the active biological processes that occur during HMP and identifying key biomarkers directly linked to graft metabolism could improve the ability of transplant physicians to prognosticate allograft outcomes, design better perfusion solutions and strategies, and improve decision making around the use of HMP kidneys.15Arykbaeva A.S. de Vries D.K. Doppenberg J.B. et al.Metabolic needs of the kidney graft undergoing normothermic machine perfusion.Kidney Int. 2021; 100: 301-310Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar,16De Deken J. Kocabayoglu P. Moers C. Hypothermic machine perfusion in kidney transplantation.Curr Opin Organ Transplant. 2016; 21: 294-300Crossref PubMed Scopus (50) Google Scholar As such, there is growing interest regarding how metabolite levels in the perfusate may reflect ongoing graft metabolism and function, and prior exploratory studies have demonstrated the metabolic activity of the kidney during A.S. de Vries D.K. Doppenberg J.B. et al.Metabolic needs of the kidney graft undergoing normothermic machine perfusion.Kidney Int. 2021; 100: 301-310Abstract Full Text Full Text PDF PubMed Scopus (13) Google J. M. et of perfusate during hypothermic machine perfusion of PubMed Scopus Google Scholar Untargeted metabolomic profiling of are to in an to the active metabolic processes of kidney graft injury and in HMP. HMP perfusate an to the changes of the allograft biological as to ischemic there are changes in the of perfusate by the kidney of such as acids and to ongoing cell of of metabolism and of and in ischemic of perfusate biological pump parameters and clinical such as in the Kidney Donor Furthermore, the to ischemic during HMP may to its to within the after profiling into the of graft function and metabolomic profiling of kidney perfusate samples collected at the beginning and end of perfusion from we to metabolites that may reflect changes in the kidney during the of HMP. we the variability in the matrix of kidney perfusate solution in both duplicate samples and kidney we evaluated the changes in metabolites during HMP and death-censored graft failure and characterized active metabolic processes in kidneys that subsequently we on metabolites associated with for interaction with donation after cardiac death with DGF, and kidney is an to the Donor an ongoing cohort of deceased and their kidney and The Donor and have been in C.R. Hall I.E. Bhangoo R.S. et al.Associations of perfusate biomarkers and pump parameters with delayed graft function and deceased donor kidney allograft function.Am J Transplant. 2016; 16: 1526-1539Abstract Full Text Full Text PDF PubMed Scopus (67) Google I.E. et kidney injury is not associated with kidney allograft Int. 2019; Full Text Full Text PDF PubMed Scopus (57) Google Scholar, I.E. et al.Associations of deceased donor kidney injury with kidney discard and function after J Transplant. Full Text Full Text PDF PubMed Scopus Google Scholar, Hall I.E. et al.Associations deceased-donor injury biomarkers and kidney transplant Am 2016; PubMed Scopus Google Scholar The with Organ Donor and of that collected perfusate The cohort of kidneys that from at years of for were at kidney HMP. were no perfusate samples were for The by review and review for the All kidneys were pumped the Kidney the perfusion to the The about perfusion samples were collected from the perfusion machine at time within of to as the and a before the of the kidney to the to as the post-HMP The of by on and at at the to the were subsequently at the a controlled into and at the of metabolite All samples were measured by as et for the and of the of biological PubMed Scopus Google Scholar All and of to in samples were by Four samples of perfusate we to as were measured the kidney perfusate samples collected during HMP. We quality on the consistency of and post-HMP perfusate time and samples for the time of perfusate In we also of of duplicate samples to of for are in the donor and and outcomes, we linked to the United for Organ The United for Organ is to to the Organ and The Organ and system on all and transplant in the United by the of the Organ and and has been The and of and to the of the Organ and review of we donor with including and perfusion We review on at transplant in our network kidneys from and and as to after transplantation during the The of the kidney graft to death in this In a analysis, we also used the of DGF, by the need for in the first a within the first hours after I.E. et graft function and kidney transplant PubMed Scopus (34) Google Scholar were as median range for and as for We the on the donor of of as the of donation after cardiac of death and A to and the Kidney Donor Profile Scholar We the from the as per A to and the Kidney Donor Profile Scholar metabolites with of samples the in post-HMP perfusate we such that median absolute In metabolites with samples the of were metabolites are to as metabolites. the per median absolute we for perfusion and for We for the of kidneys from the donor by De metabolites were significantly associated with after on can be in the The by the review of all and from all their samples from all kidneys that HMP and were were for metabolomic samples from discarded by and were also to discarded versus and After quality samples from kidneys were and perfusate samples from 190 kidneys and discarded kidneys that HMP were The kidneys in the were by deceased are in and time kidneys hours of HMP before The cohort for the 190 HMP kidneys were a of were were and on The donor of discarded kidneys are in and donor and transplant of kidney of death to ischemia of kidneys discarded kidney donation after of Kidney Donor Profile Kidney Donor are in a new kidney donation after of Kidney Donor Profile Kidney Donor are metabolites from perfusate of were and were from the the 553 known were identified in the perfusate solution and are to as metabolites. We to the other metabolites as in perfusate the graft perfused The of metabolites in samples from of the with metabolites among samples from and metabolites among samples from consistency in the biological processes The 553 known metabolites and characterized from to in The median of of de and metabolites were and known metabolites identified in demonstrated significant associations changes during perfusion and perfusion time these (67) were in over time with perfusion We compared the of donor kidneys versus kidneys to the consistency of the perfusate through and clustering. the kidney from the post-HMP perfusate metabolites were with median kidneys were with median to After adjustment for perfusion time the metabolites with median the the median and kidneys suggesting a biological consistency of the perfusate and kidneys We unsupervised to and among metabolomic of kidney unsupervised The post-HMP perfusate of kidneys not also from other donor suggesting that perfusate metabolomic were to donor the 190 33 (17%) over a median follow-up of 5.0 The for per After for of we significant associations with in post-HMP perfusate metabolites for metabolites with false discovery and for metabolites with 3 and metabolites 1-carboxyethylphenylalanine, and significant with were for these metabolites. The with had a increased risk for per median absolute deviation Although no metabolites were associated with reduced risk of after false discovery correction, of the metabolites with were associated with reduced risk perfusate metabolite with in in per time in the and were the pump time and death-censored graft hypothermic machine Kidney Donor Profile median absolute metabolites were significantly associated with after for pump time and for false The after for and perfusate are also The in the and were the pump in a new death-censored graft hypothermic machine Kidney Donor Profile median absolute metabolites were significantly associated with after for pump time and for false The after for and perfusate are also the the 3 were with other and with and and were with other metabolites. We also evaluated whether risk donor demonstrated different associations for our by both donation after death and In for in we significant in including 1-carboxyethylphenylalanine, and In all of these risk associations were notably greater in the with up to although the of the in Although were in these metabolites in metabolites were significantly associated with in the group for all metabolites for significant were with active pathways in we used to metabolites the perfusate metabolites associated with the de were linked in the including all metabolites associated with The pathways in were cell of fatty production of reactive oxygen species and metabolism of in these pathways in production and fatty in kidneys with pathways among on the metabolites associated with at a value of pathway death and of fatty of of reactive oxygen of death-censored graft value of pathway in is the that the significant and a pathway is to in a new death-censored graft failure. value of pathway in is the that the significant and a pathway is to whether the associations for post-HMP perfusate metabolites were associated with production during as to their in perfusate measured at we a in we for the metabolites for perfusate levels and perfusion time We similar of for all suggesting the in metabolite levels perfusion were significantly associated with 1-carboxyethylphenylalanine, in this We also the of as a the 190 kidney in this study, Four of the metabolites were associated with DGF, including and from the discarded kidneys were compared perfusate from kidneys that did did not The kidneys were on HMP for a of hours before We no in the post-HMP of the metabolites discarded kidneys and all kidneys with and kidneys with no no in the metabolic of the kidneys at the time of In this study, we an metabolomic profiling of 190 kidneys from to identify 553 metabolites in allograft HMP We the de metabolites and in 190 kidneys with median follow-up of years to identify 1-carboxyethylphenylalanine, and 3 Given their significant interaction and greater risk with these metabolites may be in assessing viability of kidneys undergoing HMP during the allocation The of these metabolites with after for their at perfusion and their de production from the kidney during HMP is associated with graft failure. We characterized during HMP by identifying de metabolites significantly over that perfused kidneys are In we demonstrated that the metabolites did not significantly discarded and these in metabolites may further into for et solutions to discard of kidneys for Am 2016; PubMed Scopus Google discard not to transplantation and the of Kidney J. 2019; 12: PubMed Scopus Google Scholar biological may improve the of the discard process and help further the deceased-donor for organs. We also characterized the and consistency of the perfusate matrix for in this by the of of the 553 metabolites We demonstrated allograft perfusate are through duplicate and through the unsupervised of and kidneys from the we demonstrated that is to from perfusate over the of perfusion. In to the HMP a potential for prognostic and is as deceased-donor transplantation to with use of Although of all kidneys are pumped, and an greater fraction of and of of is discarded After these metabolites could help better organ by to an objective evaluation of allograft offers, thereby reducing the discard of potentially viable organs. of the post-HMP perfusate of kidneys that on to in production and in the of fatty acids compared with kidneys that did not has been that production during injury is a of injury during M. injury in kidney transplantation: mechanisms and 40: PubMed Scopus Google of ischemic kidney 2011; PubMed Scopus Google Scholar studies have that from kidney graft A. de et graft failure in renal transplant Am 2016; PubMed Scopus Google Scholar has also been as a risk for allograft a risk for allograft Int. Full Text Full Text PDF PubMed Google Scholar also that pathways in cell and transport were also We these pathways further HMP perfusate a matrix in to mechanisms in allograft processes that are during is an and can be from including directly from and from and fatty A.S. de Vries D.K. Doppenberg J.B. et al.Metabolic needs of the kidney graft undergoing normothermic machine perfusion.Kidney Int. 2021; 100: 301-310Abstract Full Text Full Text PDF PubMed Scopus (13) Google N. M. et and 2016; 24: PubMed Scopus Google Scholar of after may have by renal A. R. P. et of after Surg. Full Text Full Text PDF PubMed Scopus Google Scholar is also evidence that of reduces A. F. P. et of in a model of kidney J 2012; PubMed Scopus Google Scholar levels of in the perfusate may a greater from the kidney to and In this may be a of greater injury and graft of the metabolites associated with graft survival are are of cell and of may be of from ischemic V. et on the pathway in metabolism of 2019; PubMed Scopus (67) Google Y. S. C. et changes and of in kidney ischemia/reperfusion 2012; PubMed Scopus Google Scholar also renal from and are as and M. new about of 24: PubMed Scopus (54) Google Scholar has not been linked to S. A. et changes in renal allograft with PubMed Scopus (23) Google Scholar several can preserve function and thereby the of from ischemic the of and could the to to reperfusion A. et with 2016; Scopus Google Scholar the levels of perfusate and graft survival may that levels are of increased to ischemic may also be of underlying kidney injury that could to graft is a known that is significantly increased in kidney and cell via the of F. De R. et and of Am 2012; PubMed Scopus Google acids and the metabolite in the of renal 16: Google Scholar can also in the of by PubMed Scopus Google Scholar increased levels of 1-carboxyethylphenylalanine, a may also be a of kidney The of to is reduced in of kidney and metabolism in kidney Full Text Full Text PDF PubMed Google Scholar, Y. R. M. of to in renal Int. Full Text Full Text PDF PubMed Scopus Google Scholar, N. S. M. et al.The kidney is an for in in a metabolic role of the A. PubMed Scopus Google Scholar the of from and follow-up time for However, there are to As this is the first of new these candidates in In our associations were in perfusion challenges for how these inform of is the lack of a standardized time for perfusion and Although for perfusion time we have on potential metabolite likely more evaluated via collected we that graft outcomes are a of donor and to identify viability and our did not for In this multicenter de metabolites were associated with The associations were in are a growing of donor and are associated with challenges in the evaluation of graft quality and discard a better understanding of active pathways in transplant and may be to biological that reflect allograft to aid in and to improve and graft has from and to organs from He has for on the of with kidney is an of the of Kidney from and and is the of Kidney from and is of the and of All the other no for the Donor and the United for Organ by the Organ and for this are from the on is by the National of of and and Kidney and to and M. Kidney at to The have been by the United for Organ as the for the Organ and The and of these are the of the and in no be as an policy of by the the for the design and led all of the in with and and significantly to and of All to and of the for and of the with with metabolomic profiling in machine their and to clinical and have been the of in studies of kidney In this et the of to perfusate from donor kidneys to hypothermic machine perfusion. In to an model for kidney this the of allograft quality assessment and metabolites of interest in kidney PDF

Topics & Concepts