Severe Ischemia-reperfusion Injury Research Articles

Canagliflozin Mitigates Acute Kidney Injury Secondary to Resuscitative Endovascular Balloon Occlusion of the Aorta in a Porcine Model of Hemorrhagic Shock.

We investigated the potential of acute canagliflozin administration to mitigate acute kidney injury (AKI) and attenuate deleterious pro-inflammatory cytokine release in a clinically relevant swine model of severe renal ischemia reperfusion injury (IRI) induced by hemorrhage and aortic occlusion. Long-term canagliflozin use attenuates renal function decline and reduces AKI in diabetes mellitus and heart failure patients. Whilst several reports indicate prophylactic SGLT2 inhibition prevents AKI in IRI, the efficacy of acute administration on IRI and inflammation is not known. Female swine (n=16) underwent controlled hemorrhage of 25% blood volume, followed by 90 min of aortic occlusion at the level of the renal ostia (via Resuscitative Endovascular Balloon Occlusion of the Aorta). A single 300mg dose of oral canagliflozin or vehicle (saline) was delivered 5 mins into aortic occlusion. Hemodynamic monitoring, markers of renal function (serum creatinine, blood urea nitrogen, proteinuria and urinary neutrophil gelatinase-associated lipocalin) and serum cytokine concentrations (including interleukins: IL-1RA, IL-6, IL-8, IL-10, IL-18; and Tumor necrosis factor alpha) were analyzed after IRI, and during a 6h critical care phase. Compared to controls, animals receiving canagliflozin had less severe AKI, improved creatinine clearance, reduced proteinuria, and significantly lower tubular damage as evidenced by histopathology and urinary NGAL. Furthermore, the pro-inflammatory cytokine IL-6 was markedly attenuated without reduction in anti-inflammatory cytokines (IL-1RA and IL-10). A single dose of canagliflozin administered shortly into ischemic insult mitigates AKI and attenuates harmful pro-inflammatory cytokine release following trauma or surgery. These findings suggest a potential novel therapeutic role for canagliflozin in mitigating the effects of renal IRI worthy of further investigation.

MSR1-dependent efferocytosis improved ischemia-reperfusion injury following aged-donor liver transplantation in mice by regulating the pro-resolving polarisation of macrophages

Compared with young liver donors, aged liver donors are more susceptible to ischemia-reperfusion injury (IRI) following transplantation, which may be related to excessive inflammatory response and macrophage dysfunction, but the specific mechanism is unclear. Macrophage scavenger receptor 1 (MSR1) is a member of the scavenger receptor family, and plays an important regulatory role in inflammation response and macrophage function regulation. But its role in IRI following aged-donor liver transplantation is still unclear. This study demonstrates that MSR1 expression is decreased in macrophages from aged donor livers, inhibiting their efferocytosis and pro-resolving polarisation. Decreased MSR1 is responsible for the more severe IRI suffered by aged donor livers. Overexpression of MSR1 using F4/80-labelled AAV9 improved intrahepatic macrophage efferocytosis and promoted pro-resolving polarisation, ultimately ameliorating IRI following aged-donor liver transplantation. In vitro co-culture experiments further showed that overexpression of MSR1 promoted an increase in calcium concentration, which further activated the PI3K-AKT-GSK3β pathway, and induced the upregulation of β-catenin. Overall, MSR1-dependent efferocytosis promoted the pro-resolving polarisation of macrophages through the PI3K-AKT-GSK3β pathway-induced up-regulating of β-catenin leading to improved IRI following aged-donor liver transplantation.

#2385 Inhibition of histone lysine demethylases 5 attenuates acute kidney injury but not acute kidney disease or chronic kidney disease

Abstract Background and Aims The histone lysine demethylases 5 (KDM5) are members of the family of Jumonji C domain-containing histone demethylases and catalyze the removal of di- and tri-methyl moieties from the fourth lysine of histone 3 (H3K4me2/3). It has been reported that the KDM5 family mediates a range of physiological and pathological events including cell differentiation, motility, senescence and epithelial-mesenchymal transition (EMT) via activating or repressing transcription in demethylase-dependent or independent manners in both homeostasis and disease. This study investigated the potential role of KDM5 in the development and progression of renal fibrosis in acute kidney injury (AKI), chronic kidney disease (CKD) or AKI to CKD transition (acute kidney disease, AKD). Method We evaluated therapeutic effects of KDM5 inhibition in AKI model of unilateral nephrectomy plus contralateral ischemia-reperfusion (IR) injury, AKI to CKD transition model of unilateral IR injury plus removal of contralateral uninjured kidney after 8 days of IR injury, and CKD model of unilateral ureteral obstruction (UUO) in mice. Results KDM5 C70 inhibitor prevented the increase of blood urea nitrogen and positive areas of trichrome, Sirius red, F4/80 and α-smooth muscle actin in injured kidneys 7 days after IR injury. Significant reductions in renal mRNA levels of pro-inflammatory cytokine/chemokine, EMT and pro-fibrotic markers (IL-1β, IL-6, TNF-α, CCL2, CCL3, GM-CSF, MMP9, fibronectin, vimentin, TGF-β1, COL4A1) were also observed with attenuated mRNA levels of KDM5a-c. In addition, renal protein levels of antioxidants such as catalase, SOD1 and SOD2 were increased in injured kidneys of KDM5 inhibitor-treated mice. However, parameters of renal fibrosis and inflammation were similar between mice without and with KDM5 inhibitor in both AKD and CKD models. Conclusion These data demonstrate that inhibition of KDM5 could prevent renal inflammation and fibrosis after acute and severe IR injury. The activation of KDM5 might be an early step in the development of renal fibrosis after AKI insult.

End-ischemic hypothermic oxygenated machine perfusion does not improve renal outcome following liver transplantation from aged donors: A single-center retrospective report.

Organ transplantation using grafts from elderly donors entails a higher risk for severe ischemia-reperfusion injury (IRI). Advanced IRI after liver transplantation (LT) seems to be associated with the development of acute kidney injury (AKI). We studied if end-ischemic hypothermic oxygenated machine perfusion (HOPE) of liver grafts, aimed at mitigating liver IRI, impacts on the frequency and severity of AKI after LT. LTs performed at our center between January 2017 and December 2022 using organs from deceased brain-dead donors aged 70 or older were reviewed. From November 2020 on, HOPE was performed routinely in this donor category. The frequency and severity of AKI (KDIGO criteria) within 48 hours of graft reperfusion and the model of early allograft function (MEAF) were compared between HOPE-LTs (n = 30) and control LTs (n = 71). AKI developed in 23/30 (77%) HOPE-LTs and in 40/71 (56%) control LTs (p = n.s.), with no difference in severity and timing between groups. Renal replacement therapy was required in 3/30 (10%) HOPE-LTs and 6/71 (8%) control LTs. In addition, transaminase leak during the first week (marker of IRI) and MEAF were similar between groups. These findings persisted after propensity matching. Histology showed more hepatocyte vacuolization and higher Suzuki score in HOPE-LTs. Although this analysis could have been underpowered, no trends supporting the benefit of HOPE on liver and renal injury after LT were ever identified. In conclusion, HOPE in this group of older donors does not seem to improve either graft IRI, or the incidence of early AKI after LT.

Resolvin D1 prevents injurious neutrophil swarming in transplanted lungs

Neutrophils are the primary cell type involved in lung ischemia-reperfusion injury (IRI), which remains a frequent and morbid complication after organ transplantation. Endogenous lipid mediators that become activated during acute inflammation-resolution have gained increasing recognition for their protective role(s) in promoting the restoration of homeostasis, but their influence on early immune responses following transplantation remains to be uncovered. Resolvin D1, 7S,8R,17S-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid (RvD1), is a potent stereoselective mediator that exhibits proresolving and anti-inflammatory actions in the setting of tissue injury. Here, using metabololipidomics, we demonstrate that endogenous proresolving mediators including RvD1 are increased in human and murine lung grafts immediately following transplantation. In mouse grafts, we observe lipid mediator class switching early after reperfusion. We use intravital two-photon microscopy to reveal that RvD1 treatment significantly limits early neutrophil infiltration and swarming, thereby ameliorating early graft dysfunction in transplanted syngeneic lungs subjected to severe IRI. Through integrated analysis of single-cell RNA sequencing data of donor and recipient immune cells from lung grafts, we identify transcriptomic changes induced by RvD1. These results support a role for RvD1 as a potent modality for preventing early neutrophil-mediated tissue damage after lung IRI that may be therapeutic in the clinics.

Small extracellular vesicles from HO-1-modified bone marrow-derived mesenchymal stem cells attenuate ischemia-reperfusion injury after steatotic liver transplantation by suppressing ferroptosis via miR-214-3p

Donor shortage is a major problem that limits liver transplantation availability. Steatotic donor liver presents a feasible strategy to solve this problem. However, severe ischemia-reperfusion injury (IRI) is an obstacle to the adoption of steatotic transplanted livers. Evidence from our prior studies indicated that bone marrow mesenchymal stem cells modified with heme oxygenase-1 (HMSCs) can attenuate non-steatotic liver IRI. However, the contribution of HMSCs in transplanted steatotic liver IRI is unclear. Here, HMSCs and their derived small extracellular vesicles (HM-sEVs) alleviated IRI in transplanted steatotic livers. After liver transplantation, there was significant enrichment of the differentially expressed genes in the glutathione metabolism and ferroptosis pathways, accompanied by ferroptosis marker upregulation. The HMSCs and HM-sEVs suppressed ferroptosis and attenuated IRI in the transplanted steatotic livers. MicroRNA (miRNA) microarray and validation experiments indicated that miR-214-3p, which was abundant in the HM-sEVs, suppressed ferroptosis by targeting cyclooxygenase 2 (COX2). In contrast, COX2 overexpression reversed this effect. Knockdown of miR-214-3p in the HM-sEVs diminished its ability to suppress ferroptosis and protect liver tissues/cells. The findings suggested that HM-sEVs suppressed ferroptosis to attenuate transplanted steatotic liver IRI via the miR-214-3p–COX2 axis.

#3665 DCR2 PROMOTES TUBULAR MALADAPTIVE REPAIR BY INHIBITING KETONE BODY–INDUCED FOXO3 SIGNALING AFTER ACUTE KIDNEY INJURY

Abstract Background and Aims The proximal tubule has a capacity for repair after acute kidney injury (AKI), but this capacity is limited, especially in severe AKI. Tubular maladaptive repair after AKI leads to chronic kidney disease (CKD), even to end-stage renal disease (ESRD). However, the mechanism of maladaptive repair is not fully clarified, and there is a lack of effective treatment for AKI in clinic. Our previous study found that decoy receptor 2 (DcR2), a trans-membrane receptor of tumor necrosis factor-related apoptosis inducing ligand (TRAIL), was specifically expressed renal tubules and did not have the ability of proliferation in AKI, suggesting DcR2 may be associated with maladaptive repair of tubular cells. This study aims to investigate the role and mechanism of DcR2-positive tubular cells in the repair of AKI. Method The DcR2-GFP lineage trace mice,KSP-creDcR2f/f mice (Distal tubular DcR2 Conditional Knockout CKO) mice and GGT1-creDcR2f/f (proximal tubular DcR2 Conditional Knockout CKO) mice were constructed, and three AKI mouse models (moderate and severe ischemia-reperfusion injury and cisplatin-induced AKI) were conducted. Light microscopic examination of paraffin-embedded sections stained with haematoxylin and eosin, periodic acid–Schiff stain and Masson stain. Confocal analysis the co-expression of DcR2-GFP and proximal tubular markers(AQP1, Villin), distal markers (AQP2), failed repair markers (Vcam1, Dcdc2), proliferative markers (Ki-67, Edu, PCNA), Kim1, differentiated markers (pax2, sox9, six2), senescent markers (P16, P21, SA-β-gal), senescent phenotype (IL-6, TGF-β1) and fibrotic markers (a-SMA, collagen I, Fibronectin). And wild type (WT) mice and DcR2 CKO mice were used to compare the degree of kidney injury, renal function and tubular repair after AKI. Furthermore, quantitative proteomics and bioinformatics analyzed the downstream molecules of DcR2 in renal tissues from WT-AKI and CKO-AKI, and validated studied were done. Results In this study, we found decoy receptor DcR2 was highly expressed in renal tubules and associated with kidney damage in AKI patients and mouse models. DcR2-GFP transgenic mice verified that DcR2 was specifically expressed in proximal tubular epithelial cells (PTECs) after AKI. The levels of Scr, BUN and urinary DcR2 and renal acute and chronic injury scores were significantly lower inGGT1-creDcR2f/f -AKI than that of WT-AKI. Meanwhile, the proliferative markers, senescent markers and area of renal fibrosis and fibrotic markers expression was decreased inGGT1-creDcR2f/f mice compared with WT. However, the above effects were not obviously improved in KSP-creDcR2f/f mice after AKI. These results suggested that proximal tubular DcR2 knockout alleviated kidney injury and promote tubular repair after AKI. Consistent with findings in vivo, DcR2 promoted cell senescence and inhibited cell proliferation in hypoxia-reoxygenation and cisplatin-treated primary PTECs. Further quantitative proteomics and validated studies showed Hmgcs2, a key enzyme for ketone synthesis, was increased in GGT1-creDcR2f/f mice compared with WT. And the levels of urinary and renal β-hydroxybutyrate (β-OHB) were higher inGGT1-creDcR2f/f mice, suggesting DcR2 affects the synthesis of β-hydroxybutyrate through regulating the expression of Hmgcs2. Inhibition or deletion of Hmgcs2 aggravated kidney damage and repressed renal repair, but administration with β-OHB rescued these phenomena. Subsequently, PTEC-specific DcR2/Hmgcs2 double deletion decreased β-OHB levels, which inhibited FOXO3 signaling by regulating histone acetylation, thereby boosting tubular maladaptive repair after AKI. Conclusion DcR2 promotes maladaptive repair of tubular cells by regulating Hmgcs2-induced β-OHB production, and β-OHB affects FOXO3 signaling by regulating histone acetylation. The findings suggests that DcR2/Hmgcs2/ketogenesis/FOXO3 signaling mediates tubular maladaptive repair, and DcR2 could serve as a promising target for improving renal repair and AKI prognosis.

#5790 IMPAIRED FATTY ACID METABOLISM PERPETUATES LIPOTOXICITY ALONG THE TRANSITION TO CHRONIC KIDNEY INJURY

Abstract Background and Aims Understanding the cell-intrinsic mechanisms contributing to the maintenance of a dysfunctional cellular state in chronic kidney disease (CKD) and identifying therapeutic targets are research priorities in renal medicine. A key contributor to chronic kidney histological damage is acute kidney injury, especially ischemia-reperfusion injury (IRI). Persistent cell-extrinsic perturbations generated upon IRI, for example hypoxia, impair the energetic metabolism of Proximal Tubular Cells (PTC) participating in the process of transition from acute to chronic kidney injury. Here, we propose to investigate the PTC intrinsic factors involved in the perpetuation of an impaired cellular state which contribute to CKD progression Method We combined single nucleus transcriptomic, metabolomic and lipidomic approaches in experimental models and patient cohorts to investigate the molecular bases of the progression to chronic kidney allograft injury initiated by IRI. Results The urinary metabolome of kidney transplant recipients with chronic allograft injury and who experienced severe IRI was significantly enriched with long chain fatty acids (FA). We identified a renal FA-related gene signature with low levels of Cpt2 and Acsm5 and high levels of Acsl4 and Acsm5 associated with IRI, transition to chronic injury, and established CKD in mouse models and kidney transplant recipients. The findings were consistent with the presence of Cpt2-, Acsl4+, Acsl5+, Acsm5- PTC failing to recover from IRI as identified by single nucleus RNA sequencing. In vitro experiments indicated that endoplasmic reticulum (ER) stress contributes to CPT2 repression, which, in turn, promotes lipids accumulation, drives profibrogenic epithelial phenotypic changes, and activates the unfolded protein response. Conclusion ER stress through CPT2 inhibition and lipid accumulation, engages an auto-amplification loop leading to lipotoxicity and self-sustained cellular stress. Thus, IRI imprints a persistent FA metabolism disturbance in the proximal tubule sustaining the progression to chronic kidney allograft injury.

Can Hypothermic Oxygenated Perfusion Mitigate Ischemia-Reperfusion Injury in Ex-Situ Split Grafts? – A Comparative Study with Living Donation in Pediatric Liver Transplantation

Abstract Background The gold standard in pediatric split liver transplantation (SLT) is living donation (LD) providing high-quality grafts with short static cold storage (SCS). Aims This study investigates the protective effect of hypothermic oxygenated perfusion (HOPE) on ex-situ partial grafts from deceased donors in comparison to standard ex-situ Static-Split and LD SLT. Methods We included all consecutive HOPE-Split, Static-Split and LD SLT performed from 2018-2022 at a single center. The primary endpoint was early ischemia-reperfusion injury (IRI) based on reperfusion biopsy (graded from none/0 to severe/4), the occurrence of post reperfusion syndrome (PRS, drop≥30% of systolic arterial pressure) and post-LT transaminase release. Results A total of 46 SLT (14 HOPE-Split, 17 Static-Split, and 15 LD) were included. With a median perfusion duration of 100min, HOPE-Split had a significant decrease of SCS compared to Static-Split (473 min vs 538 min; p=0.02) with similar total preservation time (p=0.13). This translated into lower rates of mild to severe IRI (grade≥2; p=0.03) and significantly reduced neutrophilic infiltrate than Static-Split (p=0.04; Fig.1). The PRS was also reduced in HOPE-Split (0% vs 35%, p=0.02) with less transaminase release. Despite prolonged SCS (473 vs 117min, p<0.001), HOPE-Split was comparable to LD regarding grade≥2 IRI (64 vs 40%; p=0.17) and PRS (0 vs 6.7% p=0.34), with however higher transaminase release (571 vs 244 UI/L/100g; p=0.004). Overall, 3-months surgical complications, graft and recipient survival did not differ among groups. Conclusions HOPE allowed improved preservation of ex-situ split grafts compared to static cold storage, resulting in similar early IRI profiles then LD grafts. Preservation with HOPE may offer a promising strategy to improve outcomes and expand selection criteria in split LT for pediatric recipients.

Urinary Kidney Injury Biomarkers Are Associated with Ischemia-Reperfusion Injury Severity in Kidney Allograft Recipients.

We explored the potential of emerging and conventional urinary kidney injury biomarkers in recipients of living donor (LD) or donation after circulatory death (DCD) kidney transplantation, patients with chronic kidney disease (CKD), and individuals from the general population. Urine samples from kidney allograft recipients with mild (LD; n = 199) or severe (DCD; n = 71) ischemia-reperfusion injury (IRI) were analyzed for neutrophil gelatinase-associated lipocalin (NGAL), insulin-like growth factor-binding protein 7 (IGFBP7), tissue inhibitor of metalloproteinases 2 (TIMP2), kidney injury molecule-1 (KIM-1), chemokine C-X-C motif (CXCL9), solute carrier family 22 member 2 (SLC22A2), nephrin, and uromodulin (UMOD) by quantitative multiplex LC-MS/MS analysis. The fold-change in biomarker levels was determined in mild and severe IRI and in patients with CKD stage 1-2 (n = 127) or stage ≥3 (n = 132) in comparison to the general population (n = 1438). Relationships between the biomarkers and total protein, β2-microglobulin (B2M), creatinine, and osmolality were assessed. NGAL, IGFBP7, TIMP2, KIM-1, CXCL9, and UMOD were quantifiable, whereas nephrin and SLC22A2 were below the limit of detection. Kidney injury biomarkers were increased up to 6.2-fold in allograft recipients with mild IRI and 8.3-fold in recipients with severe IRI, compared to the reference population, with the strongest response observed for NGAL and B2M. In CKD stage 1-2, B2M, NGAL, IGFBP7, TIMP2, KIM-1, UMOD, and CXCL9 were not altered, but in individuals with CKD stage ≥3, B2M, NGAL, and KIM-1 were increased up to 1.3-fold. IGFBP7, TIMP2, NGAL, and CXCL9 were strongly correlated (all r ≥ 0.8); correlations with B2M and TP were smaller (all r ≤ 0.6). IRI, but not stable CKD, was associated with increased urinary levels of kidney injury biomarkers determined by LC-MS/MS. Absolute and multiplexed protein quantitation by LC-MS/MS is an effective strategy for biomarker panel evaluation for translation toward the clinical laboratory.

Targeted VEGFA therapy in regulating early acute kidney injury and late fibrosis.

Damage to peritubular capillaries is a key process that contributes to acute kidney injury (AKI) progression. Vascular endothelial growth factor A (VEGFA) plays a critical role in maintaining the renal microvasculature. However, the physiological role of VEGFA in various AKI durations remains unclear. A severe unilateral ischemia‒reperfusion injury model was established to provide an overview of VEGFA expression and the peritubular microvascular density from acute to chronic injury in mouse kidneys. Therapeutic strategies involving early VEGFA supplementation protecting against acute injury and late anti-VEGFA treatment for fibrosis alleviation were analyzed. A proteomic analysis was conducted to determine the potential mechanism of renal fibrosis alleviation by anti-VEGFA. The results showed that two peaks of extraglomerular VEGFA expression were observed during AKI progression: one occurred at the early phase of AKI, and the other occurred during the transition to chronic kidney disease (CKD). Capillary rarefaction progressed despite the high expression of VEGFA at the CKD stage, and VEGFA was associated with interstitial fibrosis. Early VEGFA supplementation protected against renal injury by preserving microvessel structures and counteracting secondary tubular hypoxic insults, whereas late anti-VEGFA treatment attenuated renal fibrosis progression. The proteomic analysis highlighted an array of biological processes related to fibrosis alleviation by anti-VEGFA, which included regulation of supramolecular fiber organization, cell-matrix adhesion, fibroblast migration, and vasculogenesis. These findings establish the landscape of VEGFA expression and its dual roles during AKI progression, which provides the possibility for the orderly regulation of VEGFA to alleviate early acute injury and late fibrosis.

Effect of a Combined Drug Approach on the Severity of Ischemia-Reperfusion Injury During Liver Transplant

In a porcine model of liver transplant, a combined drug approach that targeted the donor graft and graft recipient reduced ischemia-reperfusion injury, a major hurdle to the success of liver transplant. To assess the effect of a clinical form of a perioperative combined drug approach delivered immediately before implantation to the procured liver and to the liver recipient on the degree of ischemia-reperfusion injury. This unicentric, investigator-driven, open-label randomized clinical trial with 2 parallel arms was conducted in Belgium from September 2013 through February 2018, with 1-year follow-up. Adults wait-listed for a first solitary full-size liver transplant were screened for eligibility. Exclusion criteria were acute liver failure, kidney failure, contraindication to treatment, participation in another trial, refusal, technical issues, and death while awaiting transplant. Included patients were enrolled and randomized at the time of liver offer. Data were analyzed from May 20, 2019, to May 27, 2020. Participants were randomized to a combined drug approach with standard of care (static cold storage) or standard of care only (control group). In the combined drug approach group, following static cold preservation, donor livers were infused with epoprostenol (ex situ, portal vein); recipients were given oral α-tocopherol and melatonin prior to anesthesia and intravenous antithrombin III, infliximab, apotransferrin, recombinant erythropoietin-β, C1-inhibitor, and glutathione during the anhepatic and reperfusion phase. The primary outcome was the posttransplant peak serum aspartate aminotransferase (AST) level within the first 72 hours. Secondary end points were the frequencies of postreperfusion syndrome, ischemia-reperfusion injury score, early allograft dysfunction, surgical complications, ischemic cholangiopathy, acute kidney injury, acute cellular rejection, and graft and patient survival. Of 93 randomized patients, 21 were excluded, resulting in 72 patients (36 per study arm) in the per protocol analysis (median recipient age, 60 years [IQR, 51.7-66.2 years]; 52 [72.2%] men). Peak AST serum levels were not different in the combined drug approach and control groups (geometric mean, 1262.9 U/L [95% CI, 946.3-1685.4 U/L] vs 1451.2 U/L [95% CI, 1087.4-1936.7 U/L]; geometric mean ratio, 0.87 [95% CI, 0.58-1.31]; P = .49) (to convert AST to μkat/L, multiply by 0.0167). There also were no significant differences in the secondary end points between the groups. In this randomized clinical trial, the combined drug approach targeting the post-cold storage graft and the recipient did not decrease ischemic-reperfusion injury. The findings suggest that in addition to a downstream strategy that targets the preimplantation liver graft and the graft recipient, a clinically effective combined drug approach may need to include an upstream strategy that targets the donor graft during preservation. Dynamic preservation strategies may provide an appropriate delivery platform. ClinicalTrials.gov Identifier: NCT02251041.

Hypothermic Perfusion Modifies the Association Between Anti-LG3 Antibodies and Delayed Graft Function in Kidney Recipients.

We previously reported associations between autoantibodies to the LG3 fragment of perlecan, anti-LG3, and a higher risk of delayed graft function (DGF) in kidney transplant recipients. Here, we aimed to determine whether some factors that modulate ischemia-reperfusion injury (IRI) can modify this association. We performed a retrospective cohort study in kidney transplant recipients in 2 university-affiliated centers. In 687 patients, we show that high pre-transplant anti-LG3 are associated with DGF when the kidney is transported on ice (odds ratio (OR): 1.75, 95% confidence interval 1.02-3.00), but not when placed on hypothermic perfusion pump (OR: 0.78, 95% CI 0.43-1.37). In patients with DGF, high pre-transplant anti-LG3 are associated with a higher risk of graft failure (subdistribution hazard ratio (SHR): 4.07, 95% CI: 1.80, 9.22), while this was not the case in patients with immediate graft function (SHR: 0.50, 95% CI 0.19, 1.29). High anti-LG3 levels are associated with a higher risk of DGF in kidneys exposed to cold storage, but not when hypothermic pump perfusion is used. High anti-LG3 are also associated with a higher risk of graft failure in patients who experience DGF, a clinical manifestation of severe IRI.

Alterations of long noncoding RNAs and mRNAs in extracellular vesicles derived from the murine heart post-ischemia-reperfusion injury.

Extracellular vesicles (EVs) play important roles in cardiovascular diseases by delivering their RNA cargos. However, the features and possible role of the lncRNAs and mRNAs in cardiac EVs during ischemia-reperfusion (IR) remain unclear. Therefore, we performed RNA sequencing analysis to profile the features of lncRNAs and mRNAs and predicted their potential functions. Here, we demonstrated that the severity of IR injury was significantly correlated with cardiac EV production. RNA sequencing identified 73 significantly differentially expressed (DE) lncRNAs (39 upregulated and 34 downregulated) and 720 DE-mRNAs (317 upregulated and 403 downregulated). Gene Ontology (GO) and pathway analysis were performed to predict the potential functions of the DE-lncRNAs and mRNAs. The lncRNA-miRNA-mRNA ceRNA network showed the possible functions of DE-lncRNAs with DE-mRNAs which are enriched in the pathways of T cell receptor signalling pathway and cell adhesion molecules. Moreover, the expressions of ENSMUST00000146010 and ENSMUST00000180630 were negatively correlated with the severity of IR injury. A significant positive correlation was revealed between TCONS_00010866 expression and the severity of the cardiac injury. These findings revealed the lncRNA and mRNA profiles in the heart derived EVs and provided potential targets and pathways involved in cardiac IR injury.

Partial Resuscitative Endovascular Balloon Occlusion of the Aorta Limits Ischemia-Reperfusion Injury After Simulated Aeromedical Evacuation

IntroductionOne of the advantages of partial Resuscitative Endovascular Balloon Occlusion of the Aorta (pREBOA) compared to the original model is the mitigation of reperfusion injury. The safety and efficacy of pREBOA have not been demonstrated in the setting of aeromedical evacuation. We hypothesized that the pREBOA would result in less ischemia-reperfusion injury after altitude exposure. MethodsTwenty-four swine underwent femur fracture with hemorrhage for 20 min, followed by resuscitative endovascular balloon occlusion of the aorta (REBOA) deployment to Zone 1 and were randomized to pREBOA-PRO (Prytime Medical Devices Inc) full inflation, partial inflation, or sham inflation and then an altitude exposure of ground level or 8000 ft for 15 min. The primary endpoint was to examine if the balloon functioned at altitude. Our secondary endpoint was investigating evidence of ischemia-reperfusion by hemodynamic instability, electrolyte derangements, and acidosis. Comparisons were made by ANOVA. ResultsAfter deflation, the partially inflated group maintained a higher mean arterial pressure (MAP) compared to fully inflated group (P = 0.026). Full REBOA pigs were more tachycardic compared to sham pREBOA at ground (P < 0.001) and this was exacerbated at altitude (P < 0.001). Full REBOA pigs were more acidotic than sham and pREBOA at ground pigs (P = 0.0006 and P = 0.0002, respectively). Altitude increased the acidosis in full REBOA pigs, resulting in a greater base deficit (P < 0.0001), lactate (P < 0.0001), and IL-6 (P = 0.006). ConclusionsPREBOA resulted in less severe ischemia-reperfusion injury at both altitude and ground, while full balloon inflation at altitude exacerbated acidosis and ischemia-reperfusion injury. Efforts should therefore be made to utilize partial balloon occlusion when employing the REBOA catheter.

Postreperfusion Biopsy as a Predictor of Biliary Complication After Deceased Donor Liver Transplantation. A Retrospective Cohort Study

Ischemia reperfusion injury (IRI) on postreperfusion biopsies is associated with worse outcomes after liver transplantation, although the influence on biliary complications (BC) remains poorly studied. Therefore, the primary aim of our study was to assess the influence of IRI on the incidence of BC. A secondary aim was to assess the influence of steatosis on biliary complications and determine factors that predictor BC. We report a retrospective cohort study including patients with liver transplantation and postreperfusion injury. Biopsies were classified as relevant and nonrelevant ischemia reperfusion injury for assessment of BC. BC included anastomotic stricture, ischemic cholangiopathy, leaks, and bilomas. Independent predictive factors of biliary complications were assessed using univariate and multivariate analyses. 302 patients were included, and 125 patients fulfilled the criteria for relevant IRI (41.4%). Worse IRI was not associated with biliary complications (42.5% vs 40.1%; P=.68), nor was liver graft steatosis associated with BC (40.5% vs 41.5%, P=.95). The median time until biliary complications did not differ between the 2 groups (2 months; interquartile range=1-15 vs 3 months; interquartile range=1-12.5; P=.18). Hepatic artery thrombosis (odds ratio [OR]=3.4; 95% confidence interval [CI], 1.4-8.2; P=.004), older donor age (OR=2.1; 95% CI, 1.1-4.1; P=.024), and prolonged cold ischemia time (OR=1.9; 95% CI, 1.1-3.2) were independent factors of biliary complications. Severe IRI on the postreperfusion injury does not predict development of biliary complications.

S3200 Unexpected Outcome of Ischemia-Reperfusion Injury Following Liver Transplantation: A Case Series

Introduction: Severe ischemia-reperfusion injury is a potential deadly complication after liver transplantation that is characterized by a significant elevation of liver enzymes. It is usually complicated by severe organ dysfunction, rejection, and re-transplantation. In this case series, we report three cases of severe ischemia-reperfusion injury following liver transplantation with unexpected clinical course. Case Description/Methods: Three cases of patients with autoimmune hepatitis, alcoholic cirrhosis, and idiopathic liver cirrhosis were selected. All of them underwent liver transplantation. Few hours after the surgery, their liver enzymes started to rise to the thousands raising concern for ischemia-reperfusion injury (Image 1). Patients remained clinically stable with reduced but preserved urine output and state of consciousness. Doppler-ultrasound of the graft revealed patent hepatic artery, hepatic vein, and portal vein. Therefore, the patients received supportive care. After few days of observation, liver enzymes started to decrease unexpectedly reaching a normal level after a week. The patients gradually improved and their liver function remained stable upon follow-up after discharge from the hospital. Discussion: Severe ischemia-reperfusion injury is a potential deadly complication of liver transplantation. Liver function test abnormalities following liver transplantation are a hallmark of the clinical presentation. Upon diagnosis, the outcome is usually poor without re-transplantation. In this study, we presented three cases of patients with increased LFTs following liver transplantation, who improved with supportive measures alone. Although LFTs are commonly accepted as indicators of liver injury post-transplant, these cases highlight the importance of taking into account other parameters such as the clinical picture and radiological findings before determining the outcome of the ischemia-reperfusion injury following liver transplant; therefore, avoiding unnecessary and invasive measures.Figure 1.: Post-transplant AST and ALT levels in patient with autoimmune hepatitis (a), alcoholic cirrhosis (b), and liver cirrhosis of unknown origin (c).

Impaired fatty acid metabolism perpetuates lipotoxicity along the transition to chronic kidney injury.

Energy metabolism failure in proximal tubule cells (PTCs) is a hallmark of chronic kidney injury. We combined transcriptomic, metabolomic, and lipidomic approaches in experimental models and patient cohorts to investigate the molecular basis of the progression to chronic kidney allograft injury initiated by ischemia/reperfusion injury (IRI). The urinary metabolome of kidney transplant recipients with chronic allograft injury and who experienced severe IRI was substantially enriched with long chain fatty acids (FAs). We identified a renal FA-related gene signature with low levels of carnitine palmitoyltransferase 2 (Cpt2) and acyl-CoA synthetase medium chain family member 5 (Acsm5) and high levels of acyl-CoA synthetase long chain family member 4 and 5 (Acsl4 and Acsl5) associated with IRI, transition to chronic injury, and established chronic kidney disease in mouse models and kidney transplant recipients. The findings were consistent with the presence of Cpt2-Acsl4+Acsl5+Acsm5- PTCs failing to recover from IRI as identified by single-nucleus RNA-Seq. In vitro experiments indicated that ER stress contributed to CPT2 repression, which, in turn, promoted lipids' accumulation, drove profibrogenic epithelial phenotypic changes, and activated the unfolded protein response. ER stress through CPT2 inhibition and lipid accumulation engaged an auto-amplification loop leading to lipotoxicity and self-sustained cellular stress. Thus, IRI imprints a persistent FA metabolism disturbance in the proximal tubule, sustaining the progression to chronic kidney allograft injury.

Suppression of NRF2 Activity by HIF-1α Promotes Fibrosis after Ischemic Acute Kidney Injury.

Acute kidney injury (AKI) is a rapid decline in renal function and can occur after ischemia/reperfusion injury (IRI) to the tubular epithelia. The nuclear factor erythroid-2-related factor 2 (NRF2) pathway protects against AKI and AKI-to-chronic kidney disease (CKD) progression, but we previously demonstrated that severe IRI maladaptively reduced NRF2 activity in mice. To understand the mechanism of this response, we subjected C57BL/6J mice to unilateral kidney IRI with ischemia times that were titrated to induce mild to severe injury. Mild IRI increased NRF2 activity and was associated with renal recovery, whereas severe IRI decreased NRF2 activity and led to progressive CKD. Due to these effects of ischemia, we tested the hypothesis that hypoxia-inducible factor-1α (HIF-1α) mediates NRF2 activity. To mimic mild and severe ischemia, we activated HIF-1α in HK-2 cells in nutrient-replete or nutrient-deficient conditions. HIF-1α activation in nutrient-replete conditions enhanced NRF2 nuclear localization and activity. However, in nutrient-deficient conditions, HIF-1α activation suppressed NRF2 nuclear localization and activity. Nuclear localization was rescued with HIF-1α siRNA knockdown. Our results suggest that severe ischemic AKI leads to HIF-1α-mediated suppression of NRF2, leading to AKI-to-CKD progression.

422.7: Necroptosis Plays a Role in Acute Kidney Injury (AKI) and the Progression to Renal Fibrosis Following Ischemia Reperfusion Injury (IRI)

Introduction: In kidney transplantation, ischemia reperfusion injury (IRI), a type of acute kidney injury (AKI), is associated with poorer outcomes including delayed graft function, rejection and chronic kidney disease (CKD) with fibrosis. Therapies to reduce AKI and CKD following transplantation have been investigated but none has been translated into clinical practice. Here we examined necroptosis, a pathway of regulated necrosis that has been implicated in kidney IRI. Necroptosis is triggered by recruitment of the intracellular kinases RIPK1 and RIPK3 and activation of the pseudokinase MLKL. Active MLKL causes cell death by plasma membrane rupture, driving “necroinflammation”. RIPK1 and RIPK3 may also have a cell death-independent role in the development of kidney fibrosis. The aims of this study were to (1) investigate the role of necroptosis in a mouse IRI model of AKI, using knockout mice and small molecule inhibitors, and (2) determine whether inhibition of RIPK1 or RIPK3 after AKI interferes with the progression to CKD. Methods: AKI model: 10-12 week old male MLKL KO mice and wild type (WT) littermates underwent right nephrectomy followed by 18, 20, 22 or 24 min left kidney ischemia. Samples were collected at 24 h to assess renal injury, inflammation and necroptosis. In a separate experiment using the 18 min model, WT mice were treated either before or after IR with a RIPK1 inhibitor Nec-1s, a RIPK3 inhibitor GSK872, or vehicle.CKD-after-AKI model: This model was adapted from the AKI 18 min model by (i) omitting right nephrectomy, (ii) delaying the endpoint to 28 days, and (iii) extending the analyses to include renal fibrosis (Masson’s trichrome staining) and relevant gene expression. WT mice were treated with Nec-1s, GSK872 or vehicle daily on days 3-9. Results: In the AKI model, expression of necroptotic (RIPK1, RIPK3 and MLKL), kidney injury (KIM-1, NGAL) and inflammatory (IL-6, IL-1β, IL-33, TNFα) genes was significantly upregulated following moderate (18 min) and severe (20-24 min) ischemia. MLKL KO mice were protected from moderate but not severe IRI, with lower creatinine (µmol/L) (47.13±6.28 vs 80.78±10.45, p=0.007) and less tubular injury (%) (16.79±2.80 vs 26.95±3.68, p=0.048) compared to WT littermates. WT mice receiving Nec-1s before or after IR showed reduced creatinine and tubular injury compared to vehicle controls, whereas GSK872 was not protective.In the CKD-after-AKI model, vehicle-treated WT mice showed significant renal fibrosis at 28 days compared to sham (fibrosis score 28.68±1.25 vs 2.33±0.11, p=0.0025), with increased expression of RIPK1 (p=0.0079), RIPK3 (p=0.0025), and the pro-fibrotic genes TGFβ (p=0.0025), HIF1α (p=0.0025) and Col-I (p=0.0025). Inhibiting RIPK1 or RIPK3 on days 3-9 resulted in reduced fibrosis.Conclusion: Our data support a role for necroptosis in AKI and fibrosis following IR, and identify inhibition of the necroptosis pathway as a potential therapeutic strategy. NHMRC scholarship GNT1168307.