Results of an explorative clinical evaluation suggest immediate and persistent post-reperfusion metabolic paralysis drives kidney ischemia reperfusion injury
Authors: Jan H. Lindeman, Leonie G. Wijermars, Sarantos Kostidis, Oleg A. Mayboroda, Amy C. Harms, Thomas Hankemeier, Jörgen Bierau, Karthick B. Sai Sankar Gupta, Martin Giera, Marlies E. Reinders, Melissa C. Zuiderwijk, Sylvia E. Le Dévédec, Alexander F. Schaapherder, Jaap A. Bakker
Journal: Kidney International (2020)

Delayed graft function is the manifestation of ischemia reperfusion injury in the context of kidney transplantation. While hundreds of interventions successfully reduce ischemia reperfusion injury in experimental models, all clinical interventions have failed. This explorative clinical evaluation examined possible metabolic origins of clinical ischemia reperfusion injury combining data from 18 pre- and post-reperfusion tissue biopsies with 36 sequential arteriovenous blood samplings over the graft in three study groups. These groups included living and deceased donor grafts with and without delayed graft function. Group allocation was based on clinical outcome. Magic angle NMR was used for tissue analysis and mass spectrometry-based platforms were used for plasma analysis. All kidneys were functional at one-year. Integration of metabolomic data identified a discriminatory profile to recognize future delayed graft function. This profile was characterized by post-reperfusion ATP/GTP catabolism (significantly impaired phosphocreatine recovery and significant persistent (hypo)xanthine production) and significant ongoing tissue damage. Failing high-energy phosphate recovery occurred despite activated glycolysis, fatty-acid oxidation, glutaminolysis and autophagia, and related to a defect at the level of the oxoglutarate dehydrogenase complex in the Krebs cycle. Clinical delayed graft function due to ischemia reperfusion injury associated with a post-reperfusion metabolic collapse. Thus, efforts to quench delayed graft function due to ischemia reperfusion injury should focus on conserving metabolic competence, either by preserving the integrity of the Krebs cycle and/or by recruiting metabolic salvage pathways. Delayed graft function is the manifestation of ischemia reperfusion injury in the context of kidney transplantation. While hundreds of interventions successfully reduce ischemia reperfusion injury in experimental models, all clinical interventions have failed. This explorative clinical evaluation examined possible metabolic origins of clinical ischemia reperfusion injury combining data from 18 pre- and post-reperfusion tissue biopsies with 36 sequential arteriovenous blood samplings over the graft in three study groups. These groups included living and deceased donor grafts with and without delayed graft function. Group allocation was based on clinical outcome. Magic angle NMR was used for tissue analysis and mass spectrometry-based platforms were used for plasma analysis. All kidneys were functional at one-year. Integration of metabolomic data identified a discriminatory profile to recognize future delayed graft function. This profile was characterized by post-reperfusion ATP/GTP catabolism (significantly impaired phosphocreatine recovery and significant persistent (hypo)xanthine production) and significant ongoing tissue damage. Failing high-energy phosphate recovery occurred despite activated glycolysis, fatty-acid oxidation, glutaminolysis and autophagia, and related to a defect at the level of the oxoglutarate dehydrogenase complex in the Krebs cycle. Clinical delayed graft function due to ischemia reperfusion injury associated with a post-reperfusion metabolic collapse. Thus, efforts to quench delayed graft function due to ischemia reperfusion injury should focus on conserving metabolic competence, either by preserving the integrity of the Krebs cycle and/or by recruiting metabolic salvage pathways. Translational StatementThis study shows that delayed graft function (graft ischemia reperfusion injury) is preceded by a post-reperfusion metabolic collapse, which leads to an inability to sustain the organ's energy requirements. Efforts aimed at quenching delayed graft function (and ischemia reperfusion injury in general) should aim to preserve or restore metabolic competence. This study shows that delayed graft function (graft ischemia reperfusion injury) is preceded by a post-reperfusion metabolic collapse, which leads to an inability to sustain the organ's energy requirements. Efforts aimed at quenching delayed graft function (and ischemia reperfusion injury in general) should aim to preserve or restore metabolic competence. Ischemia reperfusion injury (IRI) is the phenomenon of increased tissue damage after reperfusion of previously ischemic tissue.1Gutteridge J.M.C. Halliwell B. Reactive species in disease: friends or foes?.in: Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. Oxford University Press, Oxford, UK2015: 511-638Google Scholar,2Davidson S.M. Ferdinandy P. Andreadou I. et al.Multitarget strategies to reduce myocardial ischemia/reperfusion injury: JACC review topic of the week.J Am Coll Cardiol. 2019; 73: 89-99Crossref PubMed Scopus (292) Google Scholar It is a main contributor to organ damage after myocardial or brain infarction3Lefer D.J. Bolli R. Development of an NIH consortium for preclinicAl AssESsment of CARdioprotective therapies (CAESAR): a paradigm shift in studies of infarct size limitation.J Cardiovasc Pharmacol Ther. 2011; 16: 332-339Crossref PubMed Scopus (62) Google Scholar and graft damage after organ transplantation.4Cavaillé-Coll M. Bala S. Velidedeoglu E. et al.Summary of FDA workshop on ischemia reperfusion injury in kidney transplantation.Am J Transplant. 2013; 13: 1134-1148Crossref PubMed Scopus (107) Google Scholar Although myriad interventions quench IRI in preclinical models, clinical success remains to be achieved.3Lefer D.J. Bolli R. Development of an NIH consortium for preclinicAl AssESsment of CARdioprotective therapies (CAESAR): a paradigm shift in studies of infarct size limitation.J Cardiovasc Pharmacol Ther. 2011; 16: 332-339Crossref PubMed Scopus (62) Google Scholar,4Cavaillé-Coll M. Bala S. Velidedeoglu E. et al.Summary of FDA workshop on ischemia reperfusion injury in kidney transplantation.Am J Transplant. 2013; 13: 1134-1148Crossref PubMed Scopus (107) Google Scholar Thus, there appears a translational gap between preclinical models and the clinical context. Delayed graft function (DGF) is the manifestation of IRI in the setting of kidney transplantation.5Schröppel B. Legendre C. Delayed kidney graft function: from mechanism to translation.Kidney Int. 2014; 86: 251-258Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar DGF is defined as the need for dialysis in the first week or weeks after transplantation.6Mallon D.H. Summers D.M. Bradley J.A. et al.Defining delayed graft function after renal transplantation: simplest is best.Transplantation. 2013; 96: 885-889Crossref PubMed Scopus (166) Google Scholar Although DGF is extremely rare in the context of living-donor graft procedures, it affects up to 90% of deceased donor graft transplantations.6Mallon D.H. Summers D.M. Bradley J.A. et al.Defining delayed graft function after renal transplantation: simplest is best.Transplantation. 2013; 96: 885-889Crossref PubMed Scopus (166) Google Scholar Previous work demonstrated an association between incident DGF and post-reperfusion normoxic glycolysis.7Wijermars L.G. Schaapherder A.F. de Vries D.K. et al.Defective postreperfusion metabolic recovery directly associates with incident delayed graft function.Kidney Int. 2016; 90: 181-191Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar This observation implies that DGF relates to a defect in graft energy homeostasis as result of mitochondrial dysfunction in the reperfusion phase.7Wijermars L.G. Schaapherder A.F. de Vries D.K. et al.Defective postreperfusion metabolic recovery directly associates with incident delayed graft function.Kidney Int. 2016; 90: 181-191Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar On this basis, we hypothesized that clinical DGF involves and may be driven by a metabolic defect (or defects). The objective of this study was to perform an in-depth analysis of metabolic responses to ischemia-reperfusion with and without IRI (DGF). This explorative metabolic evaluation is based on an integrated, time-resolved approach that involved sequential assessment of arteriovenous concentration (AV) differences over reperfused grafts and parallel profiling of graft (tissue) biopsies. Three study groups were included: grafts from deceased donor grafts with and without later IRI and living donor grafts. Group allocation of deceased donor grafts (+DGF and –DGF, respectively) was done retrospectively on basis of their clinical outcome. Living donor grafts were included as a reference because these grafts associate with an instantaneous functional recovery following reperfusion. To cover the primary aspects of metabolic homeostasis, the focus in this study was on the following gross metabolic clusters: nucleotide triphosphate metabolism, fatty acid (β)–oxidation, glycolysis/glutaminolysis, autophagy, Krebs cycle (defects), and cell damage. The data are presented accordingly. The study sample included 53 patients. Paired tissue biopsies were obtained from 18 patients, and sequential AV sampling was performed in 36 patients. One patient had both biopsies taken and underwent AV sampling. Clinical details for the 3 study groups are shown in Supplementary Table S1A (tissue biopsies) and S1B (AV sampling). All DGF cases required multiple dialyses over a time course of at least 7 days, and all showed adequate functional recovery. None of the deceased donors without DGF required dialysis after transplantation. One-year graft survival was 100%. We first explored putative differences in metabolic signatures for the 3 donor groups (the living [reference] donor grafts, the –DGF deceased donor grafts, and +DGF [IRI] deceased donor grafts) by mapping the plasma metabolome (AV differences) for the 30-minutes post-reperfusion time point (Figure 1a) and the tissue metabolome (tissue biopsies) for the 40-minutes post-reperfusion time point (Figure 1b). These time points were chosen to avoid interference from washout of metabolites that accumulated during ischemia or cold storage, or that were constituents of the preservation fluid (e.g., histidine washout from living donor grafts; Supplementary Figure S1 shows the selective use of H [Histidine] TK preservation fluid in these grafts).7Wijermars L.G. Schaapherder A.F. de Vries D.K. et al.Defective postreperfusion metabolic recovery directly associates with incident delayed graft function.Kidney Int. 2016; 90: 181-191Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar Results (z-scores) for these time points are summarized in the heat maps in Figure 1a (AV differences) and 1b (tissue). Grouping of the data was performed according to the 6 clusters that cover all metabolic data: (i) nucleoside triphosphate catabolism, (ii) β-oxidation, (iii) glycolysis/glutaminolysis, (iv) autophagy, (v) Krebs cycle defects, and (vi) cell damage.Figure 1Clustered heat maps for the arterial-venous metabolite concentration differences over the donor graft at 30 minutes and tissue metabolite contents 40 minutes after reperfusion. (a) Clustered heat map for the arterial-venous metabolite concentrations at t = 30 minutes after reperfusion. The columns represent the 3 donor groups (living donor grafts [reference group, n = 10]; deceased donor grafts without later delayed graft function [DGF {–DGF, n = 10}], and deceased donor grafts with later DGF [+DGF, n = 16]). Compounds are clustered according to the 5 metabolic clusters and, within each cluster, ranked based on the z-score of the living donors group. Green reflects net uptake by the graft and red reflects the net release from the graft. (b) Clustered heat map for tissue metabolites identified in the HR magic angle nuclear magnetic resonance analysis of graft biopsies taken 40 min after reperfusion. The columns represent the 3 donor groups (living donor group [reference group, n = 6], deceased donor grafts without later DGF [–DGF, n = 6), and deceased donor grafts with later DGF (+DGF, n = 6]). Red reflects a tissue content above and green below the geometric mean of the 3 groups.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Heat maps for the AV differences indicate parallel metabolic signatures for the living donor and –DGF grafts, and a clearly distinctive signature for the +DGF grafts (Figure 1a). A similar, although less pronounced, pattern was observed for the tissue metabolites (Figure 1b). Exclusive mapping of –DGF and +DGF grafts (without the living donor graft) resulted in similar conclusions (not shown), indicating that inclusion of the living donor (reference) data in the analysis did not interfere with the conclusions of the analysis. Collectively, the data provide a gross metabolic signature for renal IRI. For the sake of clarity, the data of the individual metabolites are presented in terms of the 6 metabolic clusters. To avoid interference from the initial washout of metabolites that have accumulated during cold storage within the first minutes of reperfusion, estimations for net post-perfusion release or uptake are based on integration of AV differences for the 10- to 30-minutes post-reperfusion time intervals (area between the curves). The first cluster of metabolites ("nucleoside triphosphate catabolism") signals a persistent post-reperfusion metabolic incompetence ("power shutdown") in grafts with later DGF (+DGF). This conclusion is based on impaired post-reperfusion recovery of the high-energy phosphate-buffer phosphocreatine in +DGF grafts (P < 0.001; Figure 2a), and by persistent post-reperfusion adenosine triphosphate/guanosine triphosphate (ATP/GTP) catabolism. The latter is reflected in the continued release (AV differences) of hypoxanthine and xanthine (Figure 2b and c, P < 0.0001 and 0.02, respectively), the terminal degradation products of ATP and GTP from these grafts. Data for the pre-reperfusion tissue biopsies showed graded degrees of inosine and hypoxanthine accumulation at the end ischemic storage period, with the lowest content found in living and the highest in deceased donor grafts (Figure 2d and e). Post-reperfusion (t = 40 min) hypoxanthine and inosine tissue contents were similar and low in all 3 donor groups (Figure 2d and e). Post-reperfusion ATP catabolism in +DGF grafts occurred despite an apparent post-reperfusion restoration of fatty acid β-oxidation (Supplementary Figure S2), activated glycolysis/glutaminolysis (Figure 3), and autophagy (Figure 4). All 3 graft types showed uniform restoration of tissue β-hydroxybutyrate content (Supplementary Figure S2A) and selective clearance (uptake) of medium-chain fatty acids (C8–C12) from the circulation (Supplementary Figures S1 and S2B–E), indicating uniform reinstatement of β-oxidation. However, tissue accumulation of acetyl-carnitine in the –DGF and +DGF deceased donor grafts (Supplementary Figure S2F), and washout (AV differences) of acetyl-carnitine from +DGF grafts (Supplementary Figure S2G, P < 0.03) imply graded defects in disposal of acetyl groups formed during β-oxidation in –DGF and +DGF deceased donor grafts.Figure 4Activated post-reperfusion autophagia in + delayed graft function (DGF) grafts. Curves for the arterial-venous differences (red curve is arterial, blue curve is venous). Tissue biopsies (bar graphs): white bars represent pre-reperfusion biopsies; gray bars represent post-reperfusion biopsies (t = 40 min after reperfusion). ∗P < 0.05. (a–c) Post-reperfusion release of the methionine, serine, and tyrosine (P < 0.0002, 2.10E-6, and 0.0005, respectively) from + DGF grafts (n = 10, 10, and 16 in the living, –DGF, and +DGF groups, respectively). (d,e) Release of butyryl carnitine (P < 0.003) and progressive isovaleryl-carnitine release (P < 0.006) from +DGF grafts showing oxidation of branched chain amino acids in the grafts.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Mapping of glycolysis/glutaminolysis networks (Figure 3) showed equal tissue glucose levels (Figure 4a) and confirmed persistent post-reperfusion normoxic glycolysis as an exclusive feature of +DGF donor grafts (viz. persistent lactate and pyruvate release (Figure 3b and c, P <0.0001 and <0.04, respectively) and release of the transamination products alanine and aspartate (Figure 3e and f, P < 0.02 and < 0.0001, respectively). Serine (Figure 4b) and phosphoserine (Supplementary Figure 3D) release from +DGF grafts may (partially) reflect transamination of the glycolysis intermediate phosphoglycerate. Persistent post-reperfusion glutamate release (Figure 3k, P < 0.002), selective release of the transamination products alanine and aspartate (Figure 3e and f), and exhaustion of the tissue asparagine pool (Figure 3j, P < 0.03) in +DGF grafts imply continued post-reperfusion glutaminolysis (alanine) and glutamine shuttling (asparagine-aspartate)8Zhang J. Fan J. Venneti S. et al.Asparagine plays a critical role in regulating cellular adaptation to glutamine depletion.Mol Cell. 2014; 56: 205-218Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar in the post-reperfusion phase of these grafts. Moreover, exclusive release of serine, methionine, and tyrosine (Figure 4a–c, all Ps < 0.0005), along with disposal of butyrylcarnitine and isovalerylcarnitine (Figure 4d and e, P <0.006 and <0.003, respectively), deamination products of the branched chain amino acids9Holecek M. Relation between glutamine, branched-chain amino acids, and protein metabolism.Nutrition. 2002; 18: 130-133Crossref PubMed Scopus (101) Google Scholar,10Newgard C.B. An J. Bain J.R. et al.A branched-chain amino acid–related metabolic signature that differentiates obese and lean humans and contributes to insulin resistance.Cell Metab. 2009; 9: 311-326Abstract Full Text Full Text PDF PubMed Scopus (1966) Google Scholar from +DGF grafts, but not from the other graft types (Figure 4a–e), implies post-reperfusion autophagy in these grafts.11Drake K.J. Sidorov V.Y. McGuinness O.P. et al.Amino acids as metabolic substrates during cardiac ischemia.Exp Biol Med (Maywood). 2012; 237: 1369-1378Crossref PubMed Scopus (69) Google Scholar Post-reperfusion acetyl-carnitine accumulation (tissue) in –DGF and +DGF grafts (Figure 2f) and initial (living donor and –DGF grafts) and continued (+DGF grafts) acetyl-carnitine release (P < 0.03) indicate a transient (–DGF grafts) or persistent (+DGF grafts) impaired acetyl-coenzyme A disposal after reperfusion (Supplementary Figure 2G). Although this accumulation may result from exaggerated glycolysis and β-oxidation, it may also indicate impaired acetyl disposal as result of a Krebs cycle defects. For +DGF grafts, the latter mechanism is supported by selective and persistent release of the Krebs cycle intermediate α-ketoglutarate (Figure 5c, P < 0.0005) as an exclusive feature in these grafts and by an impaired recovery of tissue succinate in the +DGF grafts (Figure 5e). A final cluster of discriminatory metabolites relates to ongoing cell damage. This cluster includes post-reperfusion release of uracil, an established marker of cell damage12De Jong J.W. Huizer T. Janssen M. et al.High-energy phosphates and their catabolites.in: Piper H.M. Preusse C.J. Ischemia-Reperfusion in Cardiac Surgery. Springer, Dordrecht, the Netherlands1993: 295-315Crossref Google Scholar,13van Os S. de Abreu R. Hopman J. et al.Purine and pyrimidine metabolism and electrocortical brain activity during hypoxemia in near-term lambs.Pediatr Res. 2004; 55: 1018-1025Crossref PubMed Scopus (9) Google Scholar (Supplementary Figure S3A, P < 0.0001) and of amino acid derivates that associate with hydrolysis of plasmalogens (viz. phospho-ethanolamine, ethanolamine, and phospho-serine; Supplementary Figure S3B–D, P < 0.001; Supplementary Figure S1). Although no AV differences were present for choline in the +DGF group (P = 0.60), this observation is in contrast to a net choline uptake in the living donor and –DGF group (P < 0.0001 and 0.02, respectively). Hence, in +DGF grafts, hydrolysis of choline plasmalogens may be masked by choline uptake. Such a mechanism is supported by the selective and progressive release of betaine, the oxidation product of choline14Blom H.J. De Vriese A.S. Why are homocysteine levels increased in kidney failure? A metabolic approach.J Lab Clin Med. 2002; 139: 262-268Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar in the +DGF group (Supplementary Figures S3G, P < 0.0001). The preceding observations associate incident IRI with persistent post-reperfusion ATP catabolism and ongoing cell damage in a context of mitochondrial failure and activation of glycolytic and lipolytic pathways (Figure 6). Considering the vital role of ATP in cellular homeostasis and survival, it was reasoned that recruitment of auxiliary ATP-regenerative pathways (viz. independent of mitochondrial respiration) would be beneficial. In this context, we considered inosine, a nucleoside that can generate ATP through nontraditional pathways. As shown in Figure 7, neither preventive nor rescue inosine delivery (in concentrations up to 10 mMol/l) rescued ATP exhaustion following chemically induced metabolic paralysis.Figure 7Both preventive and rescue inosine treatment fail to recover adenosine triphosphate (ATP) levels. The PK-1 renal cell line was stably transfected with the PercevalHR fluorescent biosensor of ATP-to–adenosine diphosphate (ADP) ratio. Chemically induced metabolic-paralysis was induced by adding rotenone/actinomycin/2-deoxyglucose, and the ATP-to-ADP ratio (relative fluorescence) was monitored. Brown, control; black, metabolic paralysis control; green, preventive inosine treatment (10 mMol/l); red, inosine rescue at t = 15 minutes after the induction of a metabolic paralysis.View Large Image Figure ViewerDownload Hi-res image Download (PPT) From this study, performed in the context of clinical kidney transplantation, the picture emerges of IRI (DGF) being a consequence of an almost instantaneous and persistent post-reperfusion failure of oxidative phosphorylation and an activated normoxic glycolysis that is unable to sustain energy homeostasis. In turn, high energy phosphate pools are progressively exhausted, and cellular integrity cannot be preserved, resulting in ongoing tissue damage. This clinical study is based on integration of metabolic data derived from tissue biopsies taken immediately before and 40 minutes after reperfusion and from sequential assessment of AV differences over the reperfused graft. These AV differences not only provide an indication for the pace and duration of metabolic (mal)adaptions but also allow for directing trends observed in the paired tissue biopsies and for appreciation of metabolite clearance of (e.g., lactate) or uptake from (e.g., medium-chain fatty acids) the circulation.15Ivanisevic J. Elias D. Deguchi H. et al.Arteriovenous blood metabolomics: a readout of intra-tissue metabostasis.Sci Rep. 2015; 5: 12757Crossref PubMed Scopus (47) Google Scholar,16Jang C. Hui S. Zeng X. et al.Metabolite exchange between mammalian organs quantified in pigs.Cell Metab. 2019; 30: 594-606Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar The resolution of the AV approach is clearly illustrated by the acylcarnitine data, which not only show selective uptake of medium-chain fatty acids but also suggest that the unsaturated C14 carnitine species tetradecenoyl- and tetradecadienyl carnitine behave similarly to medium-chain fatty acids (Supplementary Data S1) and may not rely on specific fatty acid transporters.17Bremer J. Carnitine-metabolism and functions.Physiol Rev. 1983; 63: 1420-1466Crossref PubMed Scopus (1352) Google Scholar In fact, in the process of data analysis, it was observed that sole reliance on tissue biopsies would have obscured most conclusions in this study because the majority of metabolites formed are efficiently cleared into the circulation. Stable arterial blood concentrations show that blood homeostasis is maintained, and consequently metabolites released or absorbed are effectively disposed of or replenished elsewhere.15Ivanisevic J. Elias D. Deguchi H. et al.Arteriovenous blood metabolomics: a readout of intra-tissue metabostasis.Sci Rep. 2015; 5: 12757Crossref PubMed Scopus (47) Google Scholar,16Jang C. Hui S. Zeng X. et al.Metabolite exchange between mammalian organs quantified in pigs.Cell Metab. 2019; 30: 594-606Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar Observed stable tissue contents, but clear AV differences challenge the validity of tissue-based metabolomic evaluations. Note that, in the context of deceased donor kidneys and the timeframe of the study, urinary clearance is not an interfering factor because all deceased donor grafts were anuric for the 40-minute measurement interval. Mapping of the data identifies a metabolic footprint that is fully discriminatory for IRI. Specifically, the reperfusion phase of grafts with future DGF is uniformly and distinctively characterized by severely impaired oxidative phosphorylation (histotoxic hypoxia)18Siggaard-Andersen O. Fogh-Andersen N. Gøthgen I.H. Larsen V.H. Oxygen status of arterial and mixed venous blood.Crit Care Med. 1995; 23: 1284-1293Crossref PubMed Scopus (53) Google Scholar and compensatory normoxic glycolysis that is unable to sustain ATP regeneration. The latter conclusion is based on the incomplete recovery of the high-energy phosphate buffer phosphocreatine19Stoica S.C. High-energy phosphates and the human donor heart.J Heart Lung Transplant. 2004; 23: S244-S246Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar and on persistent post-reperfusion ATP/GTP catabolism reflected by continued (hypo)xanthine release. In fact, approximation of adenosine losses for +DGF grafts based on hypoxanthine release (AV differences) in the 30 minutes after reperfusion (approximation based on reported post-reperfusion flow rates,20Lisik W. Gontarczyk G. Kosieradzki M. et al.Intraoperative blood flow measurements in organ allografts can predict postoperative function.Transplant Proc. 2007; 39: 371-372Crossref PubMed Scopus (25) Google Scholar average kidney tissue mass,21Molina D.K. DiMaio V.J. Normal organ weights in men: part II—the brain, lungs, liver, spleen, and kidneys.Am J Forensic Med Pathol. 2012; 33: 368-372Crossref PubMed Scopus (148) Google Scholar and renal ATP contents22Hems D.A. Brosnan J.T. Effects of ischaemia on content of metabolites in rat liver and kidney in vivo.Biochem J. 1970; 120: 105-111Crossref PubMed Scopus (164) Google Scholar) suggests near exhaustion of graft ATP pool 30 minutes post-reperfusion. Critical exhaustion of the ATP pool may result in a catabolic lockdown that renders the cell irresponsive to reestablishment of the proton-motive forces that drive ATP generation, rendering the cell unresponsive to rescue strategies. The post-reperfusion ATP deficit and histotoxic hypoxia in +DGF grafts may underlie the selective release of amino acids associated with hydrolysis of phospholipids (plasmalogens) in +DGF grafts. Experimental studies identified hydrolysis of plasmalogens and phospholipids as an early characteristic of tissue hypoxia,23De Medio G.E. Goracci G. Horrocks L.A. et al.The effect of transient ischemia on fatty acid and lipid metabolism in the gerbil brain.Ital J Biochem. 1980; 29: 412-432PubMed Google Scholar and hydrolysis of plasmalogens has been described in the context of ischemic kidney injury.24Rao S. Walters K.B. Wilson L. et al.Early lipid changes in acute kidney injury using SWATH lipidomics coupled with MALDI tissue imaging.Am J Physiol Renal Physiol. 2016; 310: F1136-F1147Crossref PubMed Scopus (40) Google Scholar Mechanistically, this phenomenon has been linked to membrane translocation and activation of a cytosolic calcium-independent phospholipase A2 resulting from hypoxia-driven complex formation between phospholipase and a phosphofructokinase regulatory element.25Portilla D. Shah S.V. Lehman P.A. et al.Role of cytosolic calcium-independent plasmalogen-selective phospholipase A2 in hypoxic injury to rabbit proximal tubules.J Clin Invest. 1994; 93: 1609-1615Crossref PubMed Scopus (77) Google Scholar,26Hazen S.L. Wolf M.J. Ford D.A. Gross R.W. The rapid and reversible association of phosphofructokinase with myocardial membranes during myocardial ischemia.FEBS Lett. 1994; 339: 213-216Crossref PubMed Scopus (27) Google Scholar Reversal of hypoxia or ATP treatment dissociates the phospholipase-phosphofructokinase complex and abolishes phospholipase activity. Note that the dynamics of post-reperfusion cessation of (phospho-)ethanolamine release from living donor and –DGF grafts, as well as persistent release in +DGF grafts, may reflect different degrees and rates of metabolic recovery. However, whereas earlier reports imply a role of a cytosolic calcium-independent phospholipase A2,25Portilla D. Shah S.V. Lehman P.A. et al.Role of cytosolic calcium-independent plasmalogen-selective phospholipase A2 in hypoxic injury to rabbit proximal tubules.J Clin Invest. 1994; 93: 1609-1615Crossref PubMed Scopus (77) Google Scholar,26Hazen S.L. Wolf M.J. Ford D.A. Gross R.W. The rapid and reversible association of phosphofructokinase with myocardial membranes during myocardial ischemia.FEBS Lett. 1994; 339: 213-216Crossref PubMed Scopus (27) Google Scholar observed AV differences for betaine and (phospho-)ethanolamine imply a more comprehensive activation of phospholipases that