Warm Ischemia-reperfusion Injury Research Articles

#2027 Activation of histone deacetylase-1 in fibroblasts and pericytes induces renal interstitial fibrosis after ischemia-reperfusion injury

Abstract Background and Aims Following an acute kidney ischemic event the chromatin remodelling enzyme, histone deacetylase-1 (HDAC1), is activated in many cell types of the kidney including fibroblasts and pericytes. During kidney injury, the fibroblasts/pericytes differentiate into the myofibroblasts and produce extracellular matrix that can lead to a permanent decline in kidney function. Pharmacological inhibition of HDACs can attenuate ischemia-reperfusion-injury (IRI) mediated interstitial fibrosis. The hypothesis of this study was that fibroblast/pericyte HDAC1 activation promotes myofibroblast differentiation and interstitial fibrosis. Method Tamoxifen inducible, fibroblast/pericyte HDAC1 knockout (KO) mice (HDAC1Fl/Fl; Col1a2-CreEr) and littermate controls (HDAC1Fl/Fl) were used. Male and female mice were given tamoxifen by ip at 8-10 weeks of age and IRI or sham surgery completed after a 2-week tamoxifen washout period. A mild, 18 min, bilateral, warm IRI model was used, and samples collected over 4 weeks. Glomerular filtration rate (GFR) was assessed using transdermal FITC-Sinistrin clearance. In vitro experiments with kidney fibroblast cells (NRK49F) overexpressing empty vector or HDAC1 were used for RNA-sequencing to determine the HDAC1-dependent myofibroblast transcriptome. Results We confirmed that HDAC1 was knockdown in myofibroblast cells from KO mice with co-localization of HDAC1 and PDGFRβ or α-smooth muscle actin in the kidneys of IRI mice. Twenty-four hours post-surgery, all IRI mice exhibited 2-3 times increase in serum creatinine and a ∼50% reduction in GFR compared to sham mice (control IRI = 513 ± 129 μl /min/100 g body mass, control sham = 1010 ± 218, n = 4-5, p = 0.02; KO IRI = 529 ± 116, KO sham = 1187 ± 59 μl/min/100 g body mass, n = 4-5, p = 0.004). Subsequent GFR measurements over the next 4 weeks showed a gradual recovery of renal function of IRI mice starting at week 2 and fully restored to sham values by week 4. However, the male control IRI mice had significant focal interstitial fibrosis 2- and 4-weeks post injury (4-week data: sham = 0.3 ± 0.09 % of cortical area, IRI = 1.2 ± 0.2%, n = 6/group, p = 0.0011) but this was attenuated in the KO male IRI mice (sham = 0.4 ± 0.07% n = 4, IRI = 0.5 ± 0.1%, n = 7, p = 0.93). The female mice, regardless of genotype, had very little kidney damage or interstitial fibrosis at 4 weeks. Transcriptomes of NRK49F cells overexpressing HDAC1 had an upregulation of 55 genes related to pro-inflammatory cytokines and tumour necrosis factor signalling and downregulation of 367 genes related to cell junctions, cytoskeleton proteins, and cell adhesion compared to the empty vector control cells. Fibroblast proliferation rates were not statistically different among the groups, suggesting HDAC1 does not affect myofibroblast proliferation but rather pro-inflammatory gene expression. Conclusion In male mice, fibroblast/pericyte activation of HDAC1 leads to the production of pro-inflammatory cytokines that we predict will lead to immune cell infiltration and crosstalk that leads to the excessive production of interstitial fibrosis following IRI.

Effect of Subnormothermic Machine Perfusion on the Preservation of Vascularized Composite Allografts After Prolonged Warm Ischemia.

Warm ischemia time (WIT) and ischemia-reperfusion injury are limiting factors for vascularized composite allograft (VCA) transplantation. Subnormothermic machine perfusion (SNMP) has demonstrated the potential to extend WIT in organ transplantation. This study evaluates the effect of SNMP on VCA viability after prolonged WIT. Rat hindlimbs underwent WIT for 30, 45, 60, 120, 150, or 210 min, followed by 3-h SNMP. Monitoring of perfusion parameters and outflow determined the maximum WIT compatible with limb viability after SNMP. Thereafter, 2 groups were assessed: a control group with inbred transplantation (Txp) after 120 min of WIT and an experimental group that underwent WIT + SNMP + Txp. Graft appearance, blood gas, cytokine levels, and histology were assessed for 21 d. Based on potassium levels, the limit of WIT compatible with limb viability after SNMP is 120 min. Before this limit, SNMP reduces potassium and lactate levels of WIT grafts to the same level as fresh grafts. In vivo, the control group presented 80% graft necrosis, whereas the experimental group showed no necrosis, had better healing (P = 0.0004), and reduced histological muscle injury (P = 0.012). Results of blood analysis revealed lower lactate, potassium levels, and calcium levels (P = 0.048) in the experimental group. Both groups presented an increase in interleukin (IL)-10 and IL-1b/IL-1F2 with a return to baseline after 7 to 14 d. Our study establishes the limit of WIT compatible with VCA viability and demonstrates the effectiveness of SNMP in restoring a graft after WIT ex vivo and in vivo, locally and systemically.

Ceramides as Mediators of Mitochondrial Dysfunction Driving Kidney Injury

Objective. Perturbations in proximal tubule mitochondrial function and lipid metabolism potentiate acute kidney injury and failed repair. Sphingolipids such as ceramides are recognized as lipotoxic drivers of metabolic dysfunction and cell death. We hypothesized that ceramides drive mitochondrial dysfunction and tubular insult and that lowering ceramides would prevent acute kidney injury in a mouse model of bilateral ischemia reperfusion injury (IRI). Methods. We examined acute effects of ceramide treatment on primary mouse proximal tubule epithelial cell (PTEC) mitochondrial oxygen consumption and mitochondrial supercomplex assembly by Seahorse respirometry and blue native PAGE, respectively. In vivo, we profiled accumulation of ceramides via HPLC-MS/MS in kidney cortex tissue of mice challenged with warm bilateral IRI. We lowered whole-body ceramides via Degs1 inducible global knockout ( Degs1iGKO) in male C57Bl/6J mice prior to challenge with IRI. Clinical, transcriptional, and histopathological renal outcomes were compared to sham-operated and Degs1fl/fl controls 24 hours after kidney reperfusion. Results. Treatment of PTECs with 5-50uM C2-ceramide resulted in acute and dose-dependent decreases in mitochondrial ATP production rates (15-65% decrease from baseline, respectively, p<0.05), as well as disruption of electron transport chain supercomplex structures (i.e., III2+IV and I+III2+IV). In mice, IRI led to a 25% increase in total cortex ceramides (sham: 995±110 vs IRI: 1239±185 pmol lipid/mg protein, p<0.05). Lipid species within the de novo ceramide synthesis pathway were elevated as early as 5 minutes after IRI (e.g., 34% increase in Cer(d18:0/16:0), sham: 2.38±0.27 vs 5 minutes: 3.20±0.81 pmol lipid/mg protein, p<0.05) and continued to accumulate throughout the progression of the AKI up to 24 hours after reperfusion (e.g., 122% increase in Cer(d18:0/16:0), sham: 2.38±0.27 vs 24 hours: 5.29±1.71 pmol lipid/mg protein, p<0.001). Cortex ceramides, particularly deleterious long-chain (e.g., Cer(d18:1/16:0)) species, correlated positively with kidney injury biomarkers (plasma creatinine, Pearson r=0.57; BUN, r=0.74; and cortex expression of Lcn2, r=0.60 , and Havcr1, r=0.62 , p<0.05). Degs1 global knockout decreased cortex ceramide levels by 56% (fl/fl: 995±110 vs KO: 435±174 pmol lipid/mg protein, p<0.001). Degs1iGKO animals had improved cortex histopathology, blood urea nitrogen (fl/fl: 81.02±2.13 vs KO: 49.82±6.34 mg/dL, p <0.001), plasma creatinine (fl/fl: 1.28±0.4 vs KO: 0.32±0.11 mg/dL, p<0.001), and cortex Lcn2 expression (fl/fl: 36.36±21.50 vs KO: 5.31±4.61, p<0.01) following IRI. Conclusions. Renal ceramides are implicated as drivers of kidney injury and mitochondrial dysfunction. Global Degs1 knockout prevented AKI induced by ischemia reperfusion injury. Thus, ceramide-lowering interventions may be an effective strategy to prevent AKI. This work was supported by funding from the National Institutes of Health [R01KD130296 (SAS, WLH), R01DK131609 (SAS), U01CA272529 (SAS), R44DK116450 (SAS), R01DK112826 (WLH), T32DK091317 (RJN), F31DK134088 (RJN)]. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Differential Effects of HDAC6 Inhibition Versus Knockout During Hepatic Ischemia-reperfusion Injury Highlight Importance of HDAC6 C-terminal Zinc-finger Ubiquitin-binding Domain.

Ischemia-reperfusion injury (IRI) causes significant morbidity in liver transplantation among other medical conditions. IRI following liver transplantation contributes to poor outcomes and early graft loss. Histone/protein deacetylases (HDACs) regulate diverse cellular processes, play a role in mediating tissue responses to IRI, and may represent a novel therapeutic target in preventing IRI in liver transplantation. Using a previously described standardized model of murine liver warm IRI, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were assessed at 24 and 48 h after reperfusion to determine the effect of different HDAC inhibitors. Broad HDAC inhibition with trichostatin-A (TSA) was protective against hepatocellular damage (P < 0.01 for AST and P < 0.05 for ALT). Although HDAC class I inhibition with MS-275 provided statistically insignificant benefit, tubastatin-A (TubA), an HDAC6 inhibitor with additional activity against HDAC10, provided significant protection against liver IRI (P < 0.01 for AST and P < 0.001 for ALT). Surprisingly genetic deletion of HDAC6 or -10 did not replicate the protective effects of HDAC6 inhibition with TubA, whereas treatment with an HDAC6 BUZ-domain inhibitor, LakZnFD, eliminated the protective effect of TubA treatment in liver ischemia (P < 0.01 for AST and P < 0.01 for ALT). Our findings suggest TubA, a class IIb HDAC inhibitor, can mitigate hepatic IRI in a manner distinct from previously described class I HDAC inhibition and requires the HDAC6 BUZ-domain activity. Our data corroborate previous findings that HDAC targets for therapeutic intervention of IRI may be tissue-specific, and identify HDAC6 inhibition as a possible target in the treatment of liver IRI.

Protective effects of cyclosporine and its analog NIM-811 in a murine model of hepatic ischemia-reperfusion injury

Background and aimThe liver is susceptible to ischemia-reperfusion injury (IRI) during hepatic surgery, when the vessels are compressed to control bleeding, or liver transplantation, when there is an obligate period of ischemia. The hallmark of IRI comprises mitochondrial dysfunction, which generates reactive oxygen species, and cell death through necrosis or apoptosis. Cyclosporine (CsA), which is a well-known immunosuppressive agent that inhibits calcineurin, has the additional effect of inhibiting the mitochondrial permeability transition pore (mPTP), thereby, preventing mitochondrial swelling and injury. NIM-811, which is the nonimmunosuppressive analog of CsA, has a similar effect on the mPTP. In this study, we tested the effect of both agents on mitigating warm hepatic IRI in a murine model. Materials and methodsBefore ischemic insult, the mice were administered with intraperitoneal normal saline (control); CsA at 2.5, 10, or 25 mg/kg; or NIM-811 at 10 mg/kg. Thereafter, the mice were subjected to partial warm hepatic ischemia by selective pedicle clamping for 60 min, followed by 6 h of recovery after reperfusion. Serum alanine transaminase (ALT) was measured, and the liver tissue was examined histologically for the presence of apoptosis and the levels of inflammatory cytokines. ResultsCompared with the control mice, the mice treated with 10 and 25 mg/kg of CsA and NIM-811 had significantly lower ALT levels (P < 0.001, 0.007, and 0.031, respectively). Moreover, the liver tissue showed reduced histological injury scores after treatment with CsA at 2.5, 10, and 25 mg/kg and NIM-811 (P = 0.041, <0.001, 0.003, and 0.043, respectively) and significant decrease in apoptosis after treatment with CsA at all doses (P = 0.012, 0.007, and <0.001, respectively). Levels of the pro-inflammatory cytokines, particularly interleukin (IL)-1β, IL-2, IL-4, IL-10, and keratinocyte chemoattractant/human growth-regulated oncogene significantly decreased in the mice treated with the highest dose of CsA (25 mg/kg) than those in the control mice. ConclusionsPremedication with CsA or NIM-811 mitigated hepatic IRI in mice, as evidenced by the decreased ALT and reduced injury on histology. These results have potential implications on mitigating IRI during liver transplantation and resection.

Dynamic changes in the mouse hepatic lipidome following warm ischemia reperfusion injury

Liver failure secondary to metabolic dysfunction-associated steatotic liver disease (MASLD) has become the most common cause for liver transplantation in many parts of the world. Moreover, the prevalence of MASLD not only increases the demand for liver transplantation, but also limits the supply of suitable donor organs because steatosis predisposes grafts to ischemia–reperfusion injury (IRI). There are currently no pharmacological interventions to limit hepatic IRI because the mechanisms by which steatosis leads to increased injury are unclear. To identify potential novel mediators of IRI, we used liquid chromatography and mass spectrometry to assess temporal changes in the hepatic lipidome in steatotic and non-steatotic livers after warm IRI in mice. Our untargeted analyses revealed distinct differences between the steatotic and non-steatotic response to IRI and highlighted dynamic changes in lipid composition with marked changes in glycerophospholipids. These findings enhance our knowledge of the lipidomic changes that occur following IRI and provide a foundation for future mechanistic studies. A better understanding of the mechanisms underlying such changes will lead to novel therapeutic strategies to combat IRI.

Hepatoprotection of a Standardized Extract of Cultured Lentinula edodes Mycelia against Liver Injury Induced by Ischemia-Reperfusion and Partial Hepatectomy.

A standardized extract of cultured Lentinula edodes mycelia (ECLM, AHCC®) has been shown to have beneficial effects on organ metabolism. ECLM has been indicated to have liver protective properties by suppressing inflammatory responses. The pathogenesis of hepatic ischemia-reperfusion injury is thought to involve the induction of inflammatory mediators. However, whether ECLM affects inflammatory mediators caused by warm hepatic ischemia-reperfusion injury and partial hepatectomy (HIRI+PH) has not been clarified. In this study, we evaluated the protective effects of ECLM against liver damage caused by HIRI+PH. Rats were fed a normal diet (HIRI+PH) or a normal diet with 2% ECLM (HIRI+PH and ECLM) for ten days, then the liver and duodenal ligament were clamped and subjected to 15 min of hepatic ischemia. After 70% hepatectomy, the inflow occlusion was released, and liver and blood samples were collected at 3, 6, and 24 h. The effect of ECLM on mortality induced by 30 min of ischemia and hepatectomy was evaluated. The results showed that ECLM attenuated pathological liver damage, including apoptosis, in the rats treated with HIRI+PH, and decreased serum aminotransferase activity; ECLM decreased mRNA levels of the inflammation-related genes inducible nitric oxide synthase and C-X-C motif chemokine ligand 1, and increased mRNA levels of interleukin 10, an anti-inflammatory cytokine; ECLM increased hepatocyte growth factor mRNA levels and Ki-67 labeled nuclei in the liver at 24 h; ECLM significantly reduced HIRI+PH-induced mortality. In conclusion, ECLM may prevent HIRI+PH-induced liver injury in part by suppressing various inflammatory responses and promoting liver regeneration.

Cardioprotective effect of Interleukin-11 against warm ischemia-reperfusion injury in a rat heart donor model

Shortage of donor organs for heart transplantation is a worldwide problem. Donation after circulatory death (DCD) has been proposed to expand the donor pool. However, in contrast to the donation after brain death that undergoes immediate cold preservation, warm ischemia and subsequent reperfusion injury are inevitable in DCD. It has been reported that interleukin-11 (IL-11) mitigates ischemia-reperfusion injury in rodent models of myocardial infarction and donation after brain death heart transplantation. We hypothesized that IL-11 also offers benefit to warm ischemia in an experimental model of cardiac transplantation that resembles DCD. The hearts of naïve male Sprague Dawley rats (n = 15/group) were procured, subjected to 25-min warm ischemia, and reperfused for 60 min using Langendorff apparatus. IL-11 or saline was administered intravenously before the procurement, added to maintenance buffer, and infused via perfusion during reperfusion. IL-11 group exhibited significantly better cardiac function post-reperfusion. Severely damaged mitochondria was found in the electron microscopic analysis of control hearts whereas the mitochondrial structure was better preserved in the IL-11 treated hearts. Immunoblot analysis using neonatal rat cardiomyocytes revealed increased signal transducer and activator of transcription 3 (STAT3) phosphorylation at Ser727 after IL-11 treatment, suggesting its role in mitochondrial protection. Consistent with expected activation of mitochondrial respiration by mitochondrial STAT3, immunohistochemical staining demonstrated a higher mitochondrial cytochrome c oxidase subunit 2 expression. In summary, IL-11 protects the heart from warm ischemia reperfusion injury by alleviating mitochondrial injury and could be a viable therapeutic option for DCD heart transplantation.

Dynamics of necroptosis in kidney ischemia-reperfusion injury.

Necroptosis, a pathway of regulated necrosis, involves recruitment and activation of RIPK1, RIPK3 and MLKL, leading to cell membrane rupture, cell death and release of intracellular contents causing further injury and inflammation. Necroptosis is believed to play an important role in the pathogenesis of kidney ischemia-reperfusion injury (IRI). However, the dynamics of necroptosis in kidney IRI is poorly understood, in part due to difficulties in detecting phosphorylated MLKL (pMLKL), the executioner of the necroptosis pathway. Here, we investigated the temporal and spatial activation of necroptosis in a mouse model of unilateral warm kidney IRI, using a robust method to stain pMLKL. We identified the period 3-12 hrs after reperfusion as a critical phase for the activation of necroptosis in proximal tubular cells. After 12 hrs, the predominant pattern of pMLKL staining shifted from cytoplasmic to membrane, indicating progression to the terminal phase of necroptotic cell death. Mlkl-ko mice exhibited reduced kidney inflammation at 12 hrs and lower serum creatinine and tubular injury at 24 hrs compared to wild-type littermates. Interestingly, we observed increased apoptosis in the injured kidneys of Mlkl-ko mice, suggesting a relationship between necroptosis and apoptosis in kidney IRI. Together, our findings confirm the role of necroptosis and necroinflammation in kidney IRI, and identify the first 3 hrs following reperfusion as a potential window for targeted treatments.

MG53 mitigates warm ischemic lung injury in a murine model of transplantation

ObjectivesLung transplant warm ischemia–reperfusion injury (IRI) results in cellular injury, inflammation, and poor graft function. Mitsugumin 53 (MG53) is an endogenous protein with cell membrane repair properties and the ability to modulate the inflammasome. We hypothesize that the absence of circulating MG53 protein in the recipient increases IRI, and higher levels of circulating MG53 protein mitigate IRI associated with lung transplantation. MethodsTo demonstrate protection, wild-type (wt) lung donor allografts were transplanted into a wt background, a MG53 knockout (mg53−/−), or a constitutively overexpressed MG53 (tissue plasminogen activator-MG53) recipient mouse after 1 hour of warm ischemic injury. Mice survived for 5 days after transplantation. Bronchioalveolar lavage, serum, and tissue were collected at sacrifice. Bronchioalveolar lavage, serum, and tissue markers of apoptosis and a biometric profile of lung health were analyzed. Resultsmg53−/− mice had significantly greater levels of markers of overall cell lysis and endothelial cell injury. Overexpression of MG53 resulted in a signature similar to that of wt controls. At the time of explant, tissue plasminogen activator-MG53 recipient tissue expressed significantly greater levels of MG53, measured by immunohistochemistry, compared with mg53−/−, demonstrating uptake of endogenous overexpressed MG53 into donor tissue. ConclusionsIn a warm IRI model of lung transplantation, the absence of MG53 resulted in increased cell injury and inflammation. Endogenous overexpression of MG53 in the recipient results in protection in the wt donor. Together, these data suggest that MG53 is a potential therapeutic agent for use in lung transplantation to mitigate IRI.

Alternative splicing of CEACAM1 by hypoxia-inducible factor-1α enhances tolerance to hepatic ischemia in mice and humans.

Although alternative splicing (AS) drives transcriptional responses and cellular adaptation to environmental stresses, its contributions in organ transplantation have not been appreciated. We have shown that carcinoembryonic antigen-related cell adhesion molecule (Ceacam1; CD66a), a transmembrane biliary glycoprotein expressed in epithelial, endothelial, and immune cells, determines donor liver transplant quality. Here, we studied how AS of Ceacam1 affects ischemia-reperfusion injury (IRI) in mouse and human livers. We found that the short cytoplasmic isoform Ceacam1-S increased during early acute and late resolution phases of warm IRI injury in mice. Transfection of Ceacam1-deficient mouse hepatocytes with adenoviral Ceacam1-S mitigated hypoxia-induced loss of cellular adhesion by repressing the Ask1/p-p38 cell death pathway. Nucleic acid-blocking morpholinos, designed to selectively induce Ceacam1-S, protected hepatocyte cultures against temperature-induced stress in vitro. Luciferase and chromatin immunoprecipitation assays identified direct binding of hypoxia-inducible factor-1α (Hif-1α) to the mouse polypyrimidine tract binding protein 1 (Ptbp1) promoter region. Dimethyloxalylglycine protected mouse livers from warm IR stress and hepatocellular damage by inhibiting prolyl hydroxylase domain-containing protein 1 and promoting AS of Ceacam1-S. Last, analysis of 46 human donor liver grafts revealed that CEACAM1-S positively correlated with pretransplant HIF1A expression. This also correlated with better transplant outcomes, including reduced TIMP1, total bilirubin, proinflammatory MCP1, CXCL10 cytokines, immune activation markers IL17A, and incidence of delayed complications from biliary anastomosis. This translational study identified mouse Hif-1α-controlled AS of Ceacam1, through transcriptional regulation of Ptbp1 promoter region, as a functional underpinning of hepatoprotection against IR stress and tissue damage in liver transplantation.

Sodium thiosulfate refuels the hepatic antioxidant pool reducing ischemia-reperfusion-induced liver injury

Ischemia-reperfusion injury is a critical liver condition during hepatic transplantation, trauma, or shock. An ischemic deprivation of antioxidants and energy characterizes liver injury in such cases. In the face of increased reactive oxygen production, hepatocytes are vulnerable to the reperfusion driving ROS generation and multiple cell-death mechanisms. In this study, we investigate the importance of hydrogen sulfide as part of the liver's antioxidant pool and the therapeutic potency of the hydrogen sulfide donors sodium sulfide (Na2S, fast releasing) and sodium thiosulfate (STS, Na2S2O3, slow releasing). The mitoprotection and toxicity of STS and Na2S were investigated on isolated mitochondria and a liver perfusion oxidative stress model by adding text-butyl hydroperoxide and hydrogen sulfide donors. The respiratory capacity of mitochondria, hepatocellular released LDH, glutathione, and lipid-peroxide levels were quantified. In addition, wild-type and cystathionine-γ-lyase knockout mice were subjected to warm selective ischemia-reperfusion injury by clamping the main inflow for 1h followed by reperfusion of 1 or 24h. A subset of animals was treated with STS shortly before reperfusion. Glutathione, plasma ALT, and lipid-peroxide levels were investigated alongside mitochondrial changes in structure (electron microscopy) and function (intravital microscopy). Liver tissue necrosis quantified 24h after reperfusion indicates the net effects of the treatment on the organ. STS refuels and protects the endogenous antioxidant pool during liver ischemia-reperfusion injury. In addition, STS-mediated ROS scavenging significantly reduced lipid peroxidation and mitochondrial damage, resulting in better molecular and histopathological preservation of the liver tissue architecture. STS prevents tissue damage in liver ischemia-reperfusion injury by increasing the liver's antioxidant pool, thereby protecting mitochondrial integrity.

Warm Ischemia Induces Spatiotemporal Changes in Lysophosphatidylinositol That Affect Post-Reperfusion Injury in Normal and Steatotic Rat Livers.

Warm ischemia-reperfusion injury is a prognostic factor for hepatectomy and liver transplantation. However, its underlying molecular mechanisms are unknown. This study aimed to elucidate these mechanisms and identify the predictive markers of post-reperfusion injury. Rats with normal livers were subjected to 70% hepatic warm ischemia for 15, 30, or 90 min, while those with steatotic livers were subjected to 70% hepatic warm ischemia for only 30 min. The liver and blood were sampled at the end of ischemia and 1, 6, and 24 h after reperfusion. The serum alanine aminotransferase (ALT) activity, Suzuki injury scores, and lipid peroxidation (LPO) products were evaluated. The ALT activity and Suzuki scores increased with ischemic duration and peaked at 1 and 6 h after reperfusion, respectively. Steatotic livers subjected to 30 min ischemia and normal livers subjected to 90 min ischemia showed comparable injury. A similar trend was observed for LPO products. Imaging mass spectrometry of normal livers revealed an increase in lysophosphatidylinositol (LPI (18:0)) and a concomitant decrease in phosphatidylinositol (PI (18:0/20:4)) in Zone 1 (central venous region) with increasing ischemic duration; they returned to their basal values after reperfusion. Similar changes were observed in steatotic livers. Hepatic warm ischemia time-dependent acceleration of PI (18:0/20:4) to LPI (18:0) conversion occurs initially in Zone 1 and is more pronounced in fatty livers. Thus, the LPI (18:0)/PI (18:0/20:4) ratio is a potential predictor of post-reperfusion injury.

MP31-08 KIDNEY TRANSPLANTATION FROM DONATION AFTER BRAIN DEATH VERSUS DONATION AFTER CIRCULATORY DEATH USING ABDOMINAL NORMOTHERMIC OXYGENATED CIRCULATION IS THERE A DIFFERENCE IN SURGICAL COMPLICATIONS?

You have accessJournal of UrologyCME1 Apr 2023MP31-08 KIDNEY TRANSPLANTATION FROM DONATION AFTER BRAIN DEATH VERSUS DONATION AFTER CIRCULATORY DEATH USING ABDOMINAL NORMOTHERMIC OXYGENATED CIRCULATION IS THERE A DIFFERENCE IN SURGICAL COMPLICATIONS? Alberto Costa Silva, Teresa Pina-Vaz, Margarida Manso, Tiago Antunes-Lopes, Afonso Morgado, Carlos Martins-Silva, and João Alturas-Silva Alberto Costa SilvaAlberto Costa Silva More articles by this author , Teresa Pina-VazTeresa Pina-Vaz More articles by this author , Margarida MansoMargarida Manso More articles by this author , Tiago Antunes-LopesTiago Antunes-Lopes More articles by this author , Afonso MorgadoAfonso Morgado More articles by this author , Carlos Martins-SilvaCarlos Martins-Silva More articles by this author , and João Alturas-SilvaJoão Alturas-Silva More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003264.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Donation after circulatory death (DCD) is being considered a reliable source of kidneys given the scarcity of donation after brain death with standard- (DBD/SCD) and expanded-criteria (DBD/ECD). Our center has started a transplantation program for uncontrolled DCD donors after unsuccessful resuscitation (Maastricht II donors) using extracorporeal normothermic membrane oxygenation (ECMO). Our objective was to compare the surgical complications of kidney transplants from DCD/ECMO versus donation after DBD with SCD and ECD. METHODS: From January 2016 to October 2021, a prospective study was performed and three groups were considered – DCD/ECMO, DBD/SCD and DBD/ECD. Demographic data from donors and recipients were collected. Graft function evolution was assessed using creatine and glomerular filtration rate. Delayed graft function (DGF) was defined as the need for dialysis in the first week after transplantation. Surgical complications were classified as arterial, venous, ureteral, lymphatic, suture dehiscence, and/or need for early graft removal (defined as removal in the first 3 months after transplantation). The cause of early graft removal was noted and categorized as either a surgical or non-surgical complication. RESULTS: A total of 100 kidneys were implanted from DCD/ECMO, 108 from DBD/SCD and 115 from DBD/ECD.Despite a higher rate of DGF in DCD/ECMO groups vs DBD/SCD and DBD/EDC (65,9% vs 28,1% vs 30,8%; p<0,001), there were no differences in renal function between groups 12 months after transplantation. There were no statistically significant differences in surgical complications. Early graft removal was lower in DBD-SCD group. DCD/ECMO group had a significantly more early graft removals of non-surgical cause due to tubular necrosis, humoral rejection or infection, while DBD/ECD had a significantly more early graft removals of surgical cause due to arterial/venous thrombosis and hemorrhage. CONCLUSIONS: Kidney transplantation from DCD/ECMO can be a viable option. Despite higher rates of DGF in DCD/ECMO, those whose grafts survive have a similar renal function in the medium-to-long term. Early graft removal was mainly associated with a non-surgical cause, probably related to ischemia lesion caused by prolonged warm ischemia time and/or ischemia-reperfusion injury induced by the ECMO. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e432 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Alberto Costa Silva More articles by this author Teresa Pina-Vaz More articles by this author Margarida Manso More articles by this author Tiago Antunes-Lopes More articles by this author Afonso Morgado More articles by this author Carlos Martins-Silva More articles by this author João Alturas-Silva More articles by this author Expand All Advertisement PDF downloadLoading ...

Characteristics and management of thrombotic microangiopathy in kidney transplantation.

Thrombotic microangiopathy is not a rare complication of kidney transplantation and is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury with extensive thrombosis of the arterioles and capillaries. Various factors can cause thrombotic microangiopathy after kidney transplantation, including surgery, warm and cold ischemia-reperfusion injury, exposure to immunosuppressants, infection, and rejection. Many recent studies on atypical hemolytic uremic syndrome have described genetic abnormalities related to excessive activation of the alternative complement pathway. The affected patients' genetic backgrounds revealed significant genetic heterogeneity in several genes involved in complement regulation, including the complement factor H, complement factor H-related proteins, complement factor I, complement factor B, complement component 3, and CD46 genes in the alternative complement pathway. Although clinical studies have provided a better understanding of the pathogenesis of diseases, the diverse triggers present in the transplant environment can lead to thrombotic microangiopathy, along with various genetic predispositions, and it is difficult to identify the genetic background in various clinical conditions. Given the poor prognosis of posttransplant thrombotic microangiopathy, further research is necessary to improve the diagnosis and treatment protocols based on risk factors or genetic predisposition, and to develop new therapeutic agents.

Effect of the Non-steroidal Anti-inflammatory Drug Diclofenac on Ischemia-Reperfusion Injury in Rat Liver: A Nitric Oxide-Dependent Mechanism.

Ischemia/reperfusion injury (IRI) is an inevitable complication of liver surgery and transplantation. The purpose of this study was to examine the beneficial effects of diclofenac on hepatic IRI and the mechanism behind it. Wistar rats' livers were subjected to warm ischemia for 60min followed by 24h of reperfusion. Diclofenac was administered intravenously 15min before ischemia at 10, 20, and 40mg/kg body weight. To determine the mechanism of diclofenac protection, the NOS inhibitor L-Nitro-arginine methyl ester (L-NAME) was administered intravenously 10min after diclofenac injection (40mg/kg). Liver injury was evaluated by aminotransferases (ALT and AST) activities and histopathological analysis. Oxidative stress parameters (SOD, GPX, MPO, GSH, MDA, and PSH) were also determined. Then, eNOS gene transcription and p-eNOS and iNOS protein expressions were evaluated. The transcription factors PPAR-γ and NF-κB in addition to the regulatory protein IκBα were also investigated. Finally, the gene expression levels of inflammatory (COX-2, IL-6, IL-1β, IL-18, TNF-α, HMGB-1, and TLR-4) and apoptosis (Bcl-2 and Bax) markers were measured. Diclofenac, at the optimal dose of 40mg/kg, decreased liver injury and maintained histological integrity. It also reduced oxidative stress, inflammation, and apoptosis. Its mechanism of action essentially depended on eNOS activation rather than COX-2 inhibition, since pre-treatment with L-NAME abolished all the protective effects of diclofenac. To our knowledge, this is the first study demonstrating that diclofenac protects rat liver against warm IRI through the induction of NO-dependent pathway. Diclofenac reduced oxidative balance, attenuated the activation of the subsequent pro-inflammatory response and decreased cellular and tissue damage. Therefore, diclofenac could be a promising molecule for the prevention of liver IRI.

Pre-Treatment of Transplant Donors with Hydrogen Sulfide to Protect against Warm and Cold Ischemia-Reperfusion Injury in Kidney and Other Transplantable Solid Organs

Ischemia-reperfusion injury (IRI), a pathological condition resulting from prolonged cessation and subsequent restoration of blood flow to a tissue, is an inevitable consequence of solid organ transplantation. Current organ preservation strategies, such as static cold storage (SCS), are aimed at reducing IRI. However, prolonged SCS exacerbates IRI. Recent research has examined pre-treatment approaches to more effectively attenuate IRI. Hydrogen sulfide (H2S), the third established member of a family of gaseous signaling molecules, has been shown to target the pathophysiology of IRI and thus appears to be a viable candidate that can overcome the transplant surgeon's enemy. This review discusses pre-treatment of renal grafts and other transplantable organs with H2S to mitigate transplantation-induced IRI in animal models of transplantation. In addition, ethical principles of pre-treatment and potential applications of H2S pre-treatment in the prevention of other IRI-associated conditions are discussed.

The Novel MyD88 Inhibitor TJ-M2010-5 Protects Against Hepatic Ischemia-reperfusion Injury by Suppressing Pyroptosis in Mice.

. With the development of medical technology and increased surgical experience, the number of patients receiving liver transplants has increased. However, restoration of liver function in patients is limited by the occurrence of hepatic ischemia-reperfusion injury (IRI). Previous studies have reported that the Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88) signaling pathway and pyroptosis play critical roles in the development of hepatic IRI. . A mouse model of segmental (70%) warm hepatic IRI was established using BALB/c mice in vivo. The mechanism underlying inflammation in mouse models of hepatic IRI was explored in vitro using lipopolysaccharide- and ATP-treated bone marrow-derived macrophages. This in vitro inflammation model was used to simulate inflammation and pyroptosis in hepatic IRI. . We found that a MyD88 inhibitor conferred protection against partial warm hepatic IRI in mouse models by downregulating the TLR4/MyD88 signaling pathway. Moreover, TJ-M2010-5 (a novel MyD88 inhibitor, hereafter named TJ-5) reduced hepatic macrophage depletion and pyroptosis induction by hepatic IRI. TJ-5 treatment inhibited pyroptosis in bone marrow-derived macrophages by reducing the nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells, decreasing the release of high-mobility group box-1, and promoting endocytosis of lipopolysaccharide-high-mobility group box-1 complexes. . Inhibition of MyD88 may protect the liver from partial warm hepatic IRI by reducing pyroptosis in hepatic innate immune cells. These results reveal the mechanism underlying the development of inflammation in partially warm hepatic IRI and the induction of cell pyroptosis.

422.6: Effects of Argon Inhalation on Reducing Pulmonary Warm Ischemia-Reperfusion Injury in CLAWN Miniature Swine

Background: Ischemia-reperfusion injury (IRI) remains a major cause of early death in lung transplantation. In addition, warm ischemia of the donor organ is inevitable when transplanting from DCD donor, and it is important to develop new strategies for reducing warm IRI. The cytoprotective effects of chemically inert noble gases such as argon (Ar) have been demonstrated. Ar is particularly safe and cost-effective because it is non-anesthetic and the third most abundant gas in the atmosphere, suggesting higher safety levels and lower cost advantages. The inhibitory effect of IRI has been reported in small animal models of various organs, but there are conflicting reports on the effect in large animal models. In this study, we evaluated the cytoprotective effect and safety of Ar inhalation using large animal model of pulmonary warm IRI model. Methods: Ten CLAWN miniature swine were evenly divided into two groups (Ar-treated and control group). In both groups, warm ischemia was induced for 90 minutes by clamping the left bronchus, pulmonary artery and veins. Animals were inhaled with either 70% Ar (Ar-treated) or 70% nitrogen (control) in 30% oxygen for 360 minutes throughout the procedure. Lung function and structure were serially assessed via the ratio of partial pressure of oxygen to fractional inspired oxygen (pO2/FiO2) measured by both arterial blood and pulmonary vein (PV), chest X-ray (CXR) and lung biopsy, as well as the presence of side effects. Results: Ar inhalation dramatically decreased lung injury associated with ischemia and reperfusion without apparent adverse effects. In the control group, 90 minutes of warm ischemia resulted in a significant decrease in pO2/FiO2 by arterial blood, from 568 +/-12 mmHg before ischemia to 272 +/-39 mmHg 2 hours after reperfusion (p<0.05). In sharp contrast, animals in the Ar-treated group had no significant change in pO2/FiO2 by arterial blood despite 90-min ischemia (563 +/-18 mmHg before ischemia to 431 +/-49 mmHg 2 hours after reperfusion). Moreover, pO2/FiO2 by PV showed well-maintained lung function in the Ar-treated group (331 +/-40 vs. 186 +/-17 mmHg at 2 hours, p<0.05; 519 +/-19 vs. 292 +/-33 mmHg at 2 days after reperfusion, p<0.05). In addition, histological scores of lung biopsies (calculated based on four items: cellular infiltration, intralobular edema, fibrin exudation, and hemorrhage) were significantly better in the Ar-inhalation group for both 2-hour and 2-day biopsies after reperfusion. The evaluation of serum and biopsy samples suggested that anti-oxidant and anti-apoptosis were the main mechanisms of Ar inhalation. Conclusion: In this study, we demonstrated for the first time the inhibitory effect of perioperative Ar inhalation on pulmonary warm IRI in large animals. In order to apply this new therapy to clinical practice, further detailed analysis of the mechanism and evaluation using ischemic models including cold storage are required in the future.

Type 1 Innate Lymphoid Cells Are Proinflammatory Effector Cells in Ischemia-Reperfusion Injury of Steatotic Livers.

Innate lymphoid cells (ILCs), the most recently described family of lymphoid cells, play fundamental roles in tissue homeostasis through the production of key cytokine. Group 1 ILCs, comprised of conventional natural killer cells (cNKs) and type 1 ILCs (ILC1s), have been implicated in regulating immune-mediated inflammatory diseases. However, the role of ILC1s in nonalcoholic fatty liver disease (NAFLD) and ischemia-reperfusion injury (IRI) is unclear. Here, we investigated the role of ILC1 and cNK cells in a high-fat diet (HFD) murine model of partial warm IRI. We demonstrated that hepatic steatosis results in more severe IRI compared to non-steatotic livers. We further elicited that HFD-IRI mice show a significant increase in the ILC1 population, whereas the cNK population was unchanged. Since ILC1 and cNK are major sources of IFN-γ and TNF-α, we measured the level of ex vivo cytokine expression in normal diet (ND)-IRI and HFD-IRI conditions. We found that ILC1s in HFD-IRI mice produce significantly more IFN-γ and TNF-α when compared to ND-IRI. To further assess whether ILC1s are key proinflammatory effector cells in hepatic IRI of fatty livers, we studied both Rag1 −/− mice, which possess cNK cells, and a substantial population of ILC1s versus the newly generated Rag1 −/− Tbx21 −/− double knockout (Rag1-Tbet DKO) mice, which lack type 1 ILCs, under HFD IRI conditions. Importantly, HFD Rag1-Tbet DKO mice showed significant protection from hepatic injury upon IRI when compared to Rag1 −/− mice, suggesting that T-bet-expressing ILC1s play a role, at least in part, as proinflammatory effector cells in hepatic IRI under steatotic conditions.