Association of Impaired Reactive Aldehyde Metabolism with Delayed Graft Function in Human Kidney Transplantation

Delayed graft function is an early complication following kidney transplantation with an unclear molecular mechanism. Here we determined whether impaired reactive aldehyde metabolism is associated with delayed graft function. Human kidney biopsies from grafts with delayed graft function were compared with grafts that did not develop delayed graft function by Ingenuity gene pathway analysis. A second series of grafts with delayed graft function (n=10) were compared to grafts that did not develop delayed graft function (n=10) by measuring reactive aldehyde metabolism, reactive aldehyde-induced protein adduct formation, and aldehyde dehydrogenase (ALDH) gene and protein expression. In the first series of kidney biopsies, several gene families known for metabolizing reactive aldehydes, such as aldehyde dehydrogenase (ALDH), aldo-keto reductase (AKR), and glutathione-S transferase (GSTA), were upregulated in kidneys that did not develop delayed graft function versus those that did. In the second series of kidney grafts, we focused on measuring aldehyde-induced protein adducts and ALDH enzymatic activity. The reactive aldehyde metabolism by ALDH enzymes was reduced in kidneys with delayed graft function compared to those that did not (37 ± 12∗ vs. 79 ± 5 μg/min/mg tissue, ∗P<0.005, respectively). ALDH enzymatic activity was also negatively correlated with length of hospital stay after a kidney transplant. Together, our study identifies a reduced ALDH enzymatic activity with kidneys developing delayed graft function compared to those that did not. Measuring ALDH enzymatic activity and reactive aldehyde-induced protein adducts can potentially be further developed as a biomarker to assess for delayed graft function and recovery from a kidney transplant

Succinate Accumulation and Ischemia-Reperfusion Injury: Of Mice but Not Men, a Study in Renal Ischemia-Reperfusion

A recent seminal paper implicated ischemia-related succinate accumulation followed by succinate-driven reactive oxygen species formation as a key driver of ischemia-reperfusion injury. Although the data show that the mechanism is universal for all organs tested (kidney, liver, heart, and brain), a remaining question is to what extent these observations in mice translate to humans. We showed in this study that succinate accumulation is not a universal event during ischemia and does not occur during renal graft procurement; in fact, tissue succinate content progressively decreased with increasing graft ischemia time (p <0.007). Contrasting responses were also found with respect to mitochondrial susceptibility toward ischemia and reperfusion, with rodent mitochondria robustly resistant toward warm ischemia but human and pig mitochondria highly susceptible to warm ischemia (p <0.05). These observations suggest that succinate-driven reactive oxygen formation does not occur in the context of kidney transplantation. Moreover, absent allantoin release from the reperfused grafts suggests minimal oxidative stress during clinical reperfusio

Oxidative damage in clinical ischemia/reperfusion injury: a reappraisal

Ischemia/reperfusion (I/R) injury is a common clinical problem. Although the pathophysiological mechanisms underlying I/R injury are unclear, oxidative damage is considered a key factor in the initiation of I/R injury. Findings from preclinical studies consistently show that quenching reactive oxygen and nitrogen species (RONS), thus limiting oxidative damage, alleviates I/R injury. Results from clinical intervention studies on the other hand are largely inconclusive. In this study, we systematically evaluated the release of established biomarkers of oxidative and nitrosative damage during planned I/R of the kidney and heart in a wide range of clinical conditions. Sequential arteriovenous concentration differences allowed specific measurements over the reperfused organ in time. None of the biomarkers of oxidative and nitrosative damage (i.e., malondialdehyde, 15(S)-8-iso-prostaglandin F2α, nitrite, nitrate, and nitrotyrosine) were released upon reperfusion. Cumulative urinary measurements confirmed plasma findings. As of these negative findings, we tested for oxidative stress during I/R and found activation of the nuclear factor erythroid 2-related factor 2 (Nrf2), the master regulator of oxidative stress signaling. This comprehensive, clinical study evaluates the role of RONS in I/R injury in two different human organs (kidney and heart). Results show oxidative stress, but do not provide evidence for oxidative damage during early reperfusion, thereby challenging the prevailing paradigm on RONS-mediated I/R injury. Findings from this study suggest that the contribution of oxidative damage to human I/R may be less than commonly thought and propose a re-evaluation of the mechanism of I/

Results of an explorative clinical evaluation suggest immediate and persistent post-reperfusion metabolic paralysis drives kidney ischemia reperfusion injury

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.Analytical BioScience

The neglectable impact of delayed graft function on long-term graft survival in kidneys donated after circulatory death associates with superior organ resilience

OBJECTIVE:To explore putative different impacts of delayed graft function (DGF) on long-term graft survival in kidneys donated after brain death (DBD) and circulatory death (DCD). BACKGROUND:Despite a 3-fold higher incidence of DGF in DCD grafts, large studies show equivalent long-term graft survival for DBD and DCD grafts. This observation implies a differential impact of DGF on DBD and DCD graft survival. The contrasting impact is remarkable and yet unexplained. METHODS:The impact of DGF on DBD and DCD graft survival was evaluated in 6635 kidney transplants performed in The Netherlands. DGF severity and functional recovery dynamics were assessed for 599 kidney transplants performed at the Leiden Transplant Center. Immunohistochemical staining, gene expression profiling, and Ingenuity Pathway Analysis were used to identify differentially activated pathways in DBD and DCD grafts. RESULTS:While DGF severely impacted 10-year graft survival in DBD grafts (HR 1.67; P &lt; 0.001), DGF did not impact graft survival in DCD grafts (HR 1.08; P = 0.63). Shorter dialysis periods and superior posttransplant eGFRs in DBD grafts show that the differential impact was not caused by a more severe DGF phenotype in DBD grafts. Immunohistochemical evaluation indicates that pathways associated with tissue resilience are present in kidney grafts. Molecular evaluation showed selective activation of resilience-associated pathways in DCD grafts. CONCLUSIONS:This study shows an absent impact of DGF on long-term graft survival in DCD kidneys. Molecular evaluation suggests that the differential impact of DGF between DBD and DCD grafts relates to donor-type specific activation of resilience pathways in DCD grafts

Defective postreperfusion metabolic recovery directly associates with incident delayed graft function

Delayed graft function (DGF) following kidney transplantation affects long-term graft function and survival and is considered a manifestation of ischemia reperfusion injury. Preclinical studies characterize metabolic defects resulting from mitochondrial damage as primary driver of ischemia reperfusion injury. In a comprehensive approach that included sequential establishment of postreperfusion arteriovenous concentration differences over the human graft, metabolomic and genomic analysis in tissue biopsies taken before and after reperfusion, we tested whether the preclinical observations translate to the context of clinical DGF. This report is based on sequential studies of 66 eligible patients of which 22 experienced DGF. Grafts with no DGF immediately recovered aerobic respiration as indicated by prompt cessation of lactate release following reperfusion. In contrast, grafts with DGF failed to recover aerobic respiration and showed persistent adenosine triphosphate catabolism indicated by a significant persistently low post reperfusion tissue glucose-lactate ratio and continued significant post-reperfusion lactate and hypoxanthine release (net arteriovenous difference for lactate and hypoxanthine at 30 minutes). The metabolic data for the group with DGF point to a persistent post reperfusion mitochondrial defect, confirmed by functional (respirometry) and morphological analyses. The archetypical mitochondrial stabilizing peptide SS-31 significantly preserved mitochondrial function in human kidney biopsies following simulated ischemia reperfusion. Thus, development of DGF is preceded by a profound post-reperfusion metabolic deficit resulting from severe mitochondrial damage. Strategies aimed at preventing DGF should be focused on safeguarding a minimally required post-reperfusion metabolic competenc