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Through improved insights into the increasing incidence and detrimental effects of acute kidney injury (AKI), its clinical relevance has become more and more apparent. Although treatment strategies for AKI have also somewhat improved, an adequate remedy still does not exist. Finding one is complicated by a multifactorial pathophysiology and by heterogeneity in the patient population. Alkaline phosphatase (ALP) has been suggested as a therapy for sepsis-associated AKI because of its protective effects against lipopolysaccharide (LPS)-induced inflammation and kidney injury in animals. However, its effectiveness as an AKI treatment has not been demonstrated definitively. Because the anti-inflammatory properties of ALP are likely not reliant on a direct effect on LPS itself, we postulate that other pathways are much more important in explaining the renoprotective properties ascribed to ALP. The re-evaluation of which properties of the ALP enzyme are responsible for the benefit seen in the lab is an important step in determining where the true potential of ALP as a treatment strategy for AKI in the clinic lies. In this review we will discuss how ALP can prevent activation of harmful pro-inflammatory receptors, redirect cell–cell signalling and protect barrier tissues, which together form the basis for current knowledge of the role of ALP in the kidney. With this knowledge in mind and by analysing currently available clinical evidence, we propose directions for new research that can determine whether ALP as a treatment strategy for AKI has a future in the clinical field. [ABSTRACT FROM AUTHOR]

Background: Ischaemia–reperfusion injury (IRI) is a critical complication post-limb replantation. The oxidative stress and cellular apoptosis due to IRI considerably hinder the healing process. This study aimed to investigate the modulatory effects of pre-perfusion with hydrogen-rich heparin sodium on the nuclear factor erythroid 2–related factor 2 (NRF2)/haeme oxygenase-1 (HO-1) pathway and its potential mechanisms in mitigating skeletal muscle IRI post-limb replantation. Methods: Forty healthy Sprague–Dawley rats (250–300 g) were classified into five groups (n = 8 each): normal control, IRI + heparin sodium pre-perfusion (heparin group), IRI + hydrogen-rich heparin sodium pre-perfusion (hydrogen-rich heparin group), IRI + hydrogen-rich heparin sodium pre-perfusion + NRF2 inhibitor (hydrogen-rich heparin + all-trans retinoic acid [ATRA] group), and IRI + heparin sodium pre-perfusion + NRF2 inhibitor (heparin + ATRA group). The activation of the NRF2/HO-1 pathway in skeletal muscle IRI was evaluated based on HO-1 expression using western blotting and immunofluorescence. Furthermore, haematoxylin and eosin staining and transmission electron microscopy were employed to determine the histopathological characteristics. Additionally, superoxide dismutase and malondialdehyde levels in skeletal muscle tissue were measured to assess antioxidant capacity and the degree of oxidative stress damage. Tissue hypoxia was assessed based on hypoxia-inducible factor 1-alpha expression, whereas apoptosis markers BCL-2-associated X protein (BAX) and Caspase-3 in skeletal muscle tissues were analysed using western blotting with terminal deoxynucleotidyl transferase dUTP nick end labelling staining to quantify cell apoptosis. Results: Compared with the control group, the heparin group exhibited significant pathological changes, including inflammatory infiltration and cellular hypertrophy, with increased apoptosis and oxidative stress. Notably, NRF2 suppression aggravated these effects. However, hydrogen-rich heparin sodium prominently activated the NRF2/HO-1 pathway, enhancing antioxidant defence and reducing BAX/Caspase-3-mediated apoptosis, thereby mitigating IRI-induced damage. The use of an NRF2 inhibitor to inhibit NRF2 excitation by hydrogen-rich heparin sodium notably weakened NRF2 activation and the antioxidant response, resulting in a substantial increase in cellular apoptosis. Conclusion: Pre-perfusion with hydrogen-rich heparin sodium markedly diminishes the BAX/Caspase-3-mediated apoptotic pathway in skeletal muscle tissues with IRI through the excitation of the NRF2/HO-1 pathway.

Ischaemia-reperfusion (IR) injury is the paradoxical consequence of the rapid restoration of blood flow to an ischaemic organ. Although reperfusion is essential for tissue survival in conditions such as myocardial infarction and stroke, the excessive production of mitochondrial reactive oxygen species (ROS) upon reperfusion initiates the oxidative damage that underlies IR injury, by causing cell death and inflammation. This ROS production is caused by an accumulation of the mitochondrial metabolite succinate during ischaemia, followed by its rapid oxidation upon reperfusion by succinate dehydrogenase (SDH), driving superoxide production at complex I by reverse electron transport. Inhibitors of SDH, such as malonate, show therapeutic potential by decreasing succinate oxidation and superoxide production upon reperfusion. To better understand the mechanism of mitochondrial ROS production upon reperfusion and to assess potential therapies, we set up an in vitro model of IR injury. For this, isolated mitochondria were incubated anoxically with succinate to mimic ischaemia and then rapidly reoxygenated to replicate reperfusion, driving a burst of ROS formation. Using this system, we assess the factors that contribute to the magnitude of mitochondrial ROS production in heart, brain, and kidney mitochondria, as well as screening for inhibitors of succinate oxidation with therapeutic potential.

Background and Purpose: Fibrosis in kidney allografts is a major post‐transplant complication that contributes to graft failure. Lately, multiple potent inhibitors of fibrosis‐related pathways have been developed such as galunisertib, an inhibitor of the transforming growth factor‐beta (TGF‐β/TGFβ1) signalling pathway. This drug, however, poses risks for adverse effects when administered systemically. Therefore, we devised a new repurposing strategy in which galunisertib is administered ex vivo. We combined machine perfusion and tissue slices to explore the antifibrotic effects of galunisertib in renal grafts. Experimental Approach: Porcine kidneys were subjected to 30 min of warm ischaemia, 24 h of oxygenated hypothermic machine perfusion and 6 h of normothermic machine perfusion with various treatments (i.e. untreated control, TGFβ1, galunisertib or TGFβ1 + galunisertib; n = 8 kidneys per group). To determine whether effects persisted upon ceasing treatment, kidney slices were prepared from respective kidneys and incubated for 48 h. Key Results: Galunisertib treatment improved general viability without negatively affecting renal function or elevating levels of injury markers or by‐products of oxidative stress during perfusion. Galunisertib also reduced inflammation and, more importantly, reduced the onset of fibrosis after 48 h of incubation. Conclusions and Implications: Our findings demonstrate the value of using machine perfusion for administering antifibrotic drugs such as galunisertib, proving it to be an effective example of repurposing. [ABSTRACT FROM AUTHOR]

Background: Ischaemia-reperfusion injury (IRI) is the damage that occurs when blood flow is restored to a tissue or organ after a period of ischaemia. Postconditioning is a therapeutic strategy aimed at reducing the tissue damage caused by IRI. Postconditioning in rodents is a useful tool to investigate the potential mechanisms of postconditioning. Currently, there is no convenient approach for postconditioning rodents. Methods: Rats were subjected to a balloon postconditioning procedure. A balloon was used to control the flow in the vessel. This allowed for easy and precise manipulation of perfusion. Evans blue and triphenyltetrazolium chloride (TTC) double staining were used to determine the infarct size. Apoptosis in the myocardium was visualised and quantified by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Western blotting was performed to assess the expression of key apoptotic proteins, i.e., B-cell lymphoma 2 (Bcl-2), Bcl-2 Associated X (Bax), and cleaved caspase-3. Results: The balloon control approach to postconditioning provided accurate control of coronary blood flow and simplified the postconditioning manipulation. Infarct size reduction was observed in IRI rats after post-conditioning. There was a decrease in cardiac apoptosis in IRI rats after conditioning, as detected by TUNEL staining. IRI rats showed increased Bcl-2 levels and decreased Bax and cleaved caspase-3 levels in the myocardium. Conclusions: Postconditioning was successfully applied in rats using this novel approach. Postconditioning with this approach reduced infarct size and apoptosis in the area at risk. [ABSTRACT FROM AUTHOR]

Aims: Ischemia‐reperfusion injury (IRI) during kidney transplant procedures is associated with adverse outcome. Alkaline phosphatase (AP) is an enzyme that has the potential to dampen IRI. Prior to this study, it had not been tested in the setting of kidney transplantation. This study aimed to evaluate the safety and feasibility of peri‐procedural AP administration in living donor kidney transplantation. Methods: In this double blind, randomized, placebo‐controlled, single‐center pilot study, all eligible recipients of living donor kidneys were asked to give informed consent. AP (bRESCAP) or a placebo was administered intravenously over 24 hours after the transplantation procedure. The primary outcome—graft function at 1 year—was represented by iohexol measured glomerular filtration rate (mGFR). Serum and urine biomarkers within seven days after surgery were used as surrogate markers of kidney function and injury. Results: Eleven patients were enrolled of whom five were treated with bRESCAP and six with placebo. After 1 year, mGFR was not different between groups. No specific adverse events were observed in the bRESCAP group. Urine expression of injury biomarkers CCL14, NGAL and Cystatin C was lower in the bRESCAP group at day seven. This was statistically significant. Conclusion: This study illustrates that bRESCAP treatment is feasible in kidney transplantation, might have a dampening effect on IRI induced renal inflammation, and raises no safety concerns. Future research will evaluate the effects of bRESCAP treatment in donation after circulatory death kidney transplantation where IRI is more pronounced. [ABSTRACT FROM AUTHOR]

Open in new tab Download slide OBJECTIVES In myocardial infarction, the addition of mineralocorticoid receptor blockers to standard therapies, such as angiotensin-converting enzyme inhibitors or beta-blockers, reportedly reduces mortality and cardiac events. We investigated whether the non-steroidal mineralocorticoid receptor blocker esaxerenone has cardioprotective effects and its protective mechanisms. METHODS Isolated rat hearts were Langendorff-perfused (constant pressure, 80 mmHg) with oxygenated Krebs–Henseleit bicarbonate buffer and reperfused for 60 min; afterwards, recovery of function (left ventricular pressure, measured with an intraventricular balloon) and myocardial injury were measured. In a preliminary study, we determined the optimal concentration of esaxerenone required for myocardial protection. Next, esaxerenone was administered in the pre- and post-ischaemic phases to determine the optimal timing of administration. In addition, we assessed coronary flow response to acetylcholine with and without esaxerenone. We examined whether esaxerenone-induced cardioprotection was prevented by targeting putative components in the preconditioning manner (the mitochondrial ATP-sensitive potassium [KATP] channel). RESULTS Myocardial protection by esaxerenone was observed when esaxerenone was administered before ischaemia but not after ischaemia. The coronary flow response to acetylcholine was significantly better in the esaxerenone group than in the control group. The cardioprotective effect of esaxerenone was eliminated by the mitochondrial KATP channel blocker, 5-hydroxy decanoate. CONCLUSIONS This study confirmed the myocardial protective effect of the pre-ischaemic administration of esaxerenone. Esaxerenone may contribute to coronary endothelial protection and exert pharmacological preconditioning via the mitochondrial KATP channel. [ABSTRACT FROM AUTHOR]

Abstract Tetrahydrobiopterin (BH4) is an essential cofactor for nitric oxide (NO) synthases in which its production of NO is crucial for cardiac function. However, non‐canonical roles of BH4 have been discovered recently and the cell‐specific role of cardiomyocyte BH4 in cardiac function and metabolism remains to be elucidated. Therefore, we developed a novel mouse model of cardiomyocyte BH4 deficiency, by cardiomyocyte‐specific deletion of Gch1, which encodes guanosine triphosphate cyclohydrolase I, a required enzyme for de novo BH4 synthesis. Cardiomyocyte (cm)Gch1 mRNA expression and BH4 levels from cmGch1 KO mice were significantly reduced compared to Gch1flox/flox (WT) littermates. Transcriptomic analyses and protein assays revealed downregulation of genes involved in fatty acid oxidation in cmGch1 KO hearts compared with WT, accompanied by increased triacylglycerol concentration within the myocardium. Deletion of cardiomyocyte BH4 did not alter basal cardiac function. However, the recovery of left ventricle function was improved in cmGch1 KO hearts when subjected to ex vivo ischaemia–reperfusion (IR) injury, with reduced infarct size compared to WT hearts. Metabolomic analyses of cardiac tissue after IR revealed that long‐chain fatty acids were increased in cmGch1 KO hearts compared to WT, whereas at 5 min reperfusion (post‐35 min ischaemia) fatty acid metabolite levels were higher in WT compared to cmGch1 KO hearts. These results indicate a new role for BH4 in cardiomyocyte fatty acid metabolism, such that reduction of cardiomyocyte BH4 confers a protective effect in response to cardiac IR injury. Manipulating cardiac metabolism via BH4 could play a therapeutic role in limiting IR injury.

IntroductionInvestigating coronary microvascular perfusion responses after myocardial infarction (MI) would aid in the development of flow preserving therapies. Laser speckle contrast imaging (LSCI) is a powerful tool used for real-time, non-contact, full-field imaging of blood flow in various tissues/organs. However, its use in the beating heart has been limited due to motion artifacts.MethodsIn this paper, we report the novel use of LSCI, combined with custom speckle analysis software (SpAn), to visualise and quantitate changes in ventricular perfusion in adult and aged mice undergoing ischaemia-reperfusion (IR) injury. The therapeutic benefit of inhibiting the actions of the pro-inflammatory cytokine interleukin-36 (IL-36) was also investigated using an IL-36 receptor antagonist (IL-36Ra).ResultsImaging from uncovered and covered regions of the left ventricle demonstrated that whilst part of the LSCI flux signal was derived from beating motion, a significant contributor to the flux signal came from ventricular microcirculatory blood flow. We show that a biphasic flux profile corresponding to diastolic and systolic phases of the cardiac cycle can be detected without mathematically processing the total flux data to denoise motion artifacts. Furthermore, perfusion responses to ischaemia and postischaemia were strong, reproducible and could easily be detected without the need to subtract motion-related flux signals. LSCI also identified significantly poorer ventricular perfusion in injured aged mice following IR injury which markedly improved with IL-36Ra.DiscussionWe therefore propose that LSCI of the heart is possible despite motion artifacts and may facilitate future investigations into the role of the coronary microcirculation in cardiovascular diseases and development of novel therapies.

AimsRisks and outcomes of myocardial infarction (MI) are different between men and women and some studies have demonstrated that the latter have a higher risk of mortality. Whilst there are many reasons for this, it may also partially be linked to stronger innate and adaptive immune responses mounted by females compared to males. However, little is known about how sex impacts the coronary microvessels, the site where inflammatory processes take place, after an MI. Intravital and laser speckle microscopy was used to image coronary microvessels and ventricular perfusion in vivo in response to myocardial ischaemia-reperfusion (IR) injury in male and female mice. Interleukin-36 (IL-36) is the latest addition to the IL-1 superfamily of pro-inflammatory cytokines and has recently been shown to mediate inflammation in a number of non-cardiovascular diseases. Its role in mediating potential sex-related microcirculatiory pertubations in the heart are unknown. Therefore, the vasculoprotective efficacy of an IL-36 receptor antagonist (IL-36Ra) was also investigated.Methods and resultsImmunostaining and flow cytometry demonstrated higher expression of IL-36 and its receptor in female hearts, an observation confirmed in human samples. Intravital imaging of the anaesthetised mouse beating heart identified significantly greater neutrophil recruitment in female hearts, but a greater burden of thrombotic disease in male hearts. Male mice had reduced functional capillary density and were unable to restore perfusion to baseline values as effectively as females. However, female mice had significantly larger infarcts. Interestingly, IL-36Ra decreased inflammation, improved perfusion, and reduced infarct size in both sexes despite increasing platelet presence in male hearts. Mechanistically, this was explained by IL-36Ra attenuating endothelial oxidative damage and VCAM-1 expression. Importantly, IL-36Ra administration during ischaemia was critical for vasculoprotection to be realised.ConclusionThis novel study identified notable sex-related differences in the coronary microcirculatory response to myocardial IR injury which may explain why some studies have noted poorer outcomes in women after MI. Whilst contemporary MI treatment focuses on anti-platelet strategies, the heightened presence of neutrophils in female IR injured coronary microvessels necessitates the development of an effective anti-inflammatory approach for treating female patients. We also emphasise the importance of early intervention during the ischaemic period in order to maximise therapeutic effectiveness.

Phagocytosis plays vital roles in injury and repair, while its regulation by properdin and innate repair receptor, a heterodimer receptor of erythropoietin receptor (EPOR)/p common receptor (βcR), in renal ischaemia-reperfusion (IR) remains unclear. Properdin, a pattern recognition molecule, facilitates phagocytosis by opsonizing damaged cells. Our previous study showed that the phagocytic function of tubular epithelial cells isolated from properdin knockout (PKO) mouse kidneys was compromised, with upregulated EPOR in IR kidneys that was further raised by PKO at repair phase. Here, helix B surface peptide (HBSP), derived from EPO only recognizing EPOR/βcR, ameliorated IR-induced functional and structural damage in both PKO and wild-type (WT) mice. In particular, HBSP treatment led to less cell apoptosis and F4/80+ macrophage infiltration in the interstitium of PKO IR kidneys compared to the WT control. In addition, the expression of EPOR/βcR was increased by IR in WT kidneys, and furthered increased in IR PKO kidneys, but greatly reduced by HBSP in the IR kidneys of PKO mice. HBSP also increased PCNA expression in IR kidneys of both genotypes. Moreover, iridium-labelled HBSP (HBSP-Ir) was localized mainly in the tubular epithelia after 17-h renal IR in WT mice. HBSP-Ir also anchored to mouse kidney epithelial (TCMK-1) cells treated by H2O2. Both EPOR and EPOR/ pcR were significantly increased by H2O2 treatment, while further increased EPOR was showed in cells transfected with small interfering RNA (siRNA) targeting properdin, but a lower level of EPOR was seen in EPOR siRNA and HBSP-treated cells. The number of early apoptotic cells was increased by EPOR siRNA in H2O2-treated TCMK-1, but markedly reversed by HBSP. The phagocytic function of TCMK-1 cells assessed by uptake fluorescence-labelled E.coli was enhanced by HBSP dose-dependently. Our data demonstrate for the first time that HBSP improves the phagocytic function of tubular epithelial cells and kidney repair post IR injury, via upregulated EPOR/βcR triggered by both IR and properdin deficiency. [ABSTRACT FROM AUTHOR]

Acute myocardial infarction (AMI) is the leading cause of morbidity and mortality worldwide and the primary underlying risk factor for heart failure. Despite decades of research and clinical trials, there are no drugs currently available to prevent organ damage from acute ischaemic injuries of the heart. In order to address the increasing global burden of heart failure, drug, gene, and cell-based regeneration technologies are advancing into clinical testing. In this review we highlight the burden of disease associated with AMI and the therapeutic landscape based on market analyses. New studies revealing the role of acid-sensitive cardiac ion channels and other proton-gated ion channels in cardiac ischaemia are providing renewed interest in pre- and post-conditioning agents with novel mechanisms of action that may also have implications for gene- and cell-based therapeutics. Furthermore, we present guidelines that couple new cell technologies and data resources with traditional animal modelling pipelines to help de-risk drug candidates aimed at treating AMI. We propose that improved preclinical pipelines and increased investment in drug target identification for AMI is critical to stem the increasing global health burden of heart failure. [ABSTRACT FROM AUTHOR]

Sphingosine 1-phosphate (S1P) and S1P receptors (S1PR) regulate many cellular processes, including lymphocyte migration and endothelial barrier function. As neutrophils are major mediators of inflammation, their transendothelial migration may be the target of therapeutic approaches to inflammatory conditions such as ischaemia–reperfusion injury (IRI). The aim of this project was to assess whether these therapeutic effects are mediated by S1P acting on neutrophils directly or indirectly through the endothelial cells. First, our murine model of peritoneum cell recruitment demonstrated the ability of S1P to reduce CXCL8-mediated neutrophil recruitment. Mechanistic in vitro studies revealed that S1P signals in neutrophils mainly through the S1PR1 and 4 receptors and induces phosphorylation of ERK1/2; however, this had no effect on neutrophil transmigration and adhesion. S1P treatment of endothelial cells significantly reduced TNF-α-induced neutrophil adhesion under flow (p < 0.01) and transendothelial migration towards CXCL8 during in vitro chemotaxis assays (p < 0.05). S1PR1 agonist CYM5442 treatment of endothelial cells also reduced neutrophil transmigration (p < 0.01) and endothelial permeability (p < 0.005), as shown using in vitro permeability assays. S1PR3 agonist had no effects on chemotaxis or permeability. In an in vivo mouse model of renal IRI, S1PR agonism with CYM5442 reduced endothelial permeability as shown by reduced Evan's Blue dye extravasation. Western blot was used to assess phosphorylation at different sites on vascular endothelial (VE)–cadherin and showed that CYM5442 reduced VEGF-mediated phosphorylation. Taken together, the results of this study suggest that reductions in neutrophil infiltration during IRI in response to S1P are mediated primarily by S1PR1 signalling on endothelial cells, possibly by altering phosphorylation of VE–cadherin. The results also demonstrate the therapeutic potential of S1PR1 agonist during IRI. [ABSTRACT FROM AUTHOR]

New Findings: What is the central question of this study?What are the physiological roles of cardiomyocyte‐derived tetrahydrobiopterin (BH4) in cardiac metabolism and stress response?What is the main finding and its importance?Cardiomyocyte BH4 has a physiological role in cardiac metabolism. There was a shift of substrate preference from fatty acid to glucose in hearts with targeted deletion of BH4 synthesis. The changes in fatty‐acid metabolic profile were associated with a protective effect in response to ischaemia–reperfusion (IR) injury, and reduced infarct size. Manipulating fatty acid metabolism via BH4 availability could play a therapeutic role in limiting IR injury. Tetrahydrobiopterin (BH4) is an essential cofactor for nitric oxide (NO) synthases in which its production of NO is crucial for cardiac function. However, non‐canonical roles of BH4 have been discovered recently and the cell‐specific role of cardiomyocyte BH4 in cardiac function and metabolism remains to be elucidated. Therefore, we developed a novel mouse model of cardiomyocyte BH4 deficiency, by cardiomyocyte‐specific deletion of Gch1, which encodes guanosine triphosphate cyclohydrolase I, a required enzyme for de novo BH4 synthesis. Cardiomyocyte (cm)Gch1 mRNA expression and BH4 levels from cmGch1 KO mice were significantly reduced compared to Gch1flox/flox (WT) littermates. Transcriptomic analyses and protein assays revealed downregulation of genes involved in fatty acid oxidation in cmGch1 KO hearts compared with WT, accompanied by increased triacylglycerol concentration within the myocardium. Deletion of cardiomyocyte BH4 did not alter basal cardiac function. However, the recovery of left ventricle function was improved in cmGch1 KO hearts when subjected to ex vivo ischaemia–reperfusion (IR) injury, with reduced infarct size compared to WT hearts. Metabolomic analyses of cardiac tissue after IR revealed that long‐chain fatty acids were increased in cmGch1 KO hearts compared to WT, whereas at 5 min reperfusion (post‐35 min ischaemia) fatty acid metabolite levels were higher in WT compared to cmGch1 KO hearts. These results indicate a new role for BH4 in cardiomyocyte fatty acid metabolism, such that reduction of cardiomyocyte BH4 confers a protective effect in response to cardiac IR injury. Manipulating cardiac metabolism via BH4 could play a therapeutic role in limiting IR injury. [ABSTRACT FROM AUTHOR]

The present study was designed to investigate the efficacy of post-conditioning (POC) in the diabetic heart with myopathy (DCM) against ischaemia-reperfusion (I/R) injury in an isolated rat heart model. Present work includes three groups of male Wistar rat viz., (i) normal, (ii) diabetes mellitus (DM) and (iii) DCM and each group was subdivided into normal perfusion, I/R, and POC. Isolated heart from the rats was analysed for tissue injury, contractile function, mitochondrial function, and oxidative stress. Results demonstrated that unlike in DM heart and normal heart, POC procedure failed to recover the DCM heart from I/R induced cardiac dysfunction (measured via cardiac hemodynamics and infarct size. POC was unsuccessful in preserving mitochondrial subsarcolemmal fraction during I/R when compared with DM and normal heart. To conclude, the development of myopathy in diabetic heart abolished the cardioprotective efficacy of POC and the underlying pathology was linked with the mitochondrial dysfunction. Early studies reported contradicting response of diabetic heart towards post-conditioning mediated cardioprotection. Deteriorated mitochondrial function underlines the failure of post-conditioning in DCM. Efficacy of cardioprotection depends on the varying pathology of different diabetes stages. [ABSTRACT FROM AUTHOR]

Objectives: Acute kidney injury (AKI) is a sudden impairment in kidney function that is associated with high morbidity and mortality. Inflammation, oxidative stress, mitochondrial impairment and energy depletion, along with organ dysfunction are hallmarks of AKI. This study aimed to evaluate the effects of Eplerenone, an aldosterone receptor antagonist, on the kidney injury caused by ischaemia/reperfusion (I/R). Methods: Male Wistar rats (n = 24) were randomly allocated into four groups: sham, IR, Eplerenone and Eplerenone+IR. Rats in the two last groups 1 h before I/R induction, were treated with Eplerenone (100 mg/kg) via intraperitoneal injection. Protein levels of Klotho, heat shock protein 70 (HSP70), sirtuin1 (SIRT1), SIRT3 and peroxisome proliferator-activated receptor-gamma coactivator 1-α (PGC-1α) along with antioxidant, apoptotic (caspase 3, Bax and Bcl2) and inflammatory [nuclear factor kappa-B (NF-κB) p65, Interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α) and cyclooxygenase-2 (COX-2)] factors were evaluated in the kidney tissues of the experimental groups. Key findings: Eplerenone pre-treatment significantly could improve IR-induced pathological changes and kidney function and increase the renal antioxidant factors compared to the IR group (P < 0.05). Furthermore, in the Eplerenone + IR group, significant elevation of the Klotho, SIRT1, SIRT3 and PGC-1α at the protein level was identified compared to the IR group. Eplerenone pretreatment could not only downregulate NF-κB signalling and its downstream inflammatory factors (IL-6, COX-2 and TNF-α) but also could decrease apoptotic factors (P ≤ 0.01). Conclusions: The results recommended that Eplerenone exerts a protective effect against kidney IR injury by up-regulating Klotho, HSP70, sirtuins and PGC-1α to preserve mitochondrial function and cell survival. Moreover, it hinders renal inflammation by suppressing NF-κB signalling. These results offer insight into the prevention or treatment of AKI in the future. [ABSTRACT FROM AUTHOR]

Abstract Ischaemia–reperfusion injury (IRI) encompasses the deleterious effects on cellular function and survival that result from the restoration of organ perfusion. Despite their unique tolerance to ischaemia and hypoxia, afforded by their dual (pulmonary and bronchial) circulation as well as direct oxygen diffusion from the airways, lungs are particularly susceptible to IRI (LIRI). LIRI may be observed in a variety of clinical settings, including lung transplantation, lung resections, cardiopulmonary bypass during cardiac surgery, aortic cross-clamping for abdominal aortic aneurysm repair, as well as tourniquet application for orthopaedic operations. It is a diagnosis of exclusion, manifesting clinically as acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). Ischaemic conditioning (IC) signifies the original paradigm of treating IRI. It entails the application of short, non-lethal ischemia and reperfusion manoeuvres to an organ, tissue, or arterial territory, which activates mechanisms that reduce IRI. Interestingly, there is accumulating experimental and preliminary clinical evidence that IC may ameliorate LIRI in various pathophysiological contexts. Considering the detrimental effects of LIRI, ranging from ALI following lung resections to primary graft dysfunction (PGD) after lung transplantation, the association of these entities with adverse outcomes, as well as the paucity of protective or therapeutic interventions, IC holds promise as a safe and effective strategy to protect the lung. This article aims to provide a narrative review of the existing experimental and clinical evidence regarding the effects of IC on LIRI and prompt further investigation to refine its clinical application.

Ischaemic heart disease, which often manifests clinically as myocardial infarction (MI), remains a major cause of mortality worldwide. Despite the development of effective pre-clinical cardioprotective therapies, clinical translation has been disappointing. Nevertheless, the 'reperfusion injury salvage kinase' (RISK) pathway appears to be a promising target for cardioprotection. This pathway is crucial for the induction of cardioprotection by numerous pharmacological and non-pharmacological interventions, such as ischaemic conditioning. An important component of the cardioprotective effects of the RISK pathway involves the prevention of mitochondrial permeability transition pore (MPTP) opening and subsequent cardiac cell death. Here, we will review the historical perspective of the RISK pathway and focus on its interaction with mitochondria in the setting of cardioprotection. [ABSTRACT FROM AUTHOR]

Earlier studies revealed the presence of cysteine persulfide (CysSSH) and related polysulfide species in various mammalian tissues. CysSSH has both antioxidant and oxidant properties, modulates redox-dependent signal transduction and has been shown to mitigate oxidative stress. However, its functional relevance in the setting of myocardial ischaemia-reperfusion injury (IRI) remains unknown. The present study was undertaken to (1) study the dynamics of production and consumption of persulfides under normoxic and hypoxic conditions in the heart, and (2) determine whether exogenous administration of the CysSSH donor, cysteine trisulfide (Cys-SSS-Cys) at the onset of reperfusion rescues functional impairment and myocardial damage by interfering with lipid peroxidation. Utilising a well-established ex vivo Langendorff murine model, we here demonstrate that endogenous tissue concentrations of CysSSH are upregulated when oxygen supply is compromised (global myocardial ischaemia) and rapidly restored to baseline levels upon reperfusion, suggestive of active regulation. In a separate set of experiments, exogenous administration of Cys-SSS-Cys for 10 min at the onset of reperfusion was found to decrease malondialdehyde (MDA) concentrations, formation of 4-hydroxynonenal (4-HNE) protein adducts and rescue the heart from injury. Cys-SSS-Cys also restored post-ischaemic cardiac function, improving both coronary flow and left ventricular developed pressure (LVDP). Taken together, these results support the notion that endogenous CysSSH plays an important role as a “redox preconditioning” agent to combat the oxidative insult in myocardial IRI.

Vascular surgery patients are exposed to the risk of secondary trauma, which arises on the basis of pathological changes caused first by ischemia and then by reperfusion. Ischemia-reperfusion syndrome due to oxidative stress and increased cell death contributes significantly to the high perioperative morbidity and mortality of these patients. In an attempt to reduce the negative impact of these processes, a number of measures have been developed, including the concept of remote ischemic preconditioning. Based on the analogy between the pathophysiology of ischemic preconditioning and sleep disordered breathing, we can hypothesize a smaller impact of ischemia-reperfusion changes in patients with obstructive sleep apnea. [ABSTRACT FROM AUTHOR]

Following a myocardial infarction (MI), the prognosis of patients is highly dependent upon the re‐establishment of perfusion not only in the occluded coronary artery, but also within the coronary microcirculation. However, our fundamental understanding of the pathophysiology of the tiniest blood vessels of the heart is limited primarily because no current clinical imaging tools can directly visualise them. Moreover, in vivo experimental studies of the beating heart using intravital imaging have also been hampered due to obvious difficulties related to significant inherent contractile motion, movement of the heart brought about by nearby lungs and its location in an anatomically challenging position for microscopy. However, recent advances in microscopy techniques, and the development of fluorescent reporter mice and fluorescently conjugated antibodies allowing visualisation of vascular structures, thromboinflammatory cells and blood flow, have allowed us to overcome some of these challenges and increase our basic understanding of cardiac microvascular pathophysiology. In this review, the elegant attempts of the pioneers in intravital imaging of the beating heart will be discussed, which focussed on providing new insights into the anatomy and physiology of the healthy heart microvessels. The reviews end with the more recent studies that focussed on disease pathology and increasing our understanding of myocardial thromboinflammatory cell recruitment and flow disturbances, particularly in the setting of diseases such as MI. [ABSTRACT FROM AUTHOR]

Ischaemia–reperfusion injury (IRI) encompasses the deleterious effects on cellular function and survival that result from the restoration of organ perfusion. Despite their unique tolerance to ischaemia and hypoxia, afforded by their dual (pulmonary and bronchial) circulation as well as direct oxygen diffusion from the airways, lungs are particularly susceptible to IRI (LIRI). LIRI may be observed in a variety of clinical settings, including lung transplantation, lung resections, cardiopulmonary bypass during cardiac surgery, aortic cross-clamping for abdominal aortic aneurysm repair, as well as tourniquet application for orthopaedic operations. It is a diagnosis of exclusion, manifesting clinically as acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). Ischaemic conditioning (IC) signifies the original paradigm of treating IRI. It entails the application of short, non-lethal ischemia and reperfusion manoeuvres to an organ, tissue, or arterial territory, which activates mechanisms that reduce IRI. Interestingly, there is accumulating experimental and preliminary clinical evidence that IC may ameliorate LIRI in various pathophysiological contexts. Considering the detrimental effects of LIRI, ranging from ALI following lung resections to primary graft dysfunction (PGD) after lung transplantation, the association of these entities with adverse outcomes, as well as the paucity of protective or therapeutic interventions, IC holds promise as a safe and effective strategy to protect the lung. This article aims to provide a narrative review of the existing experimental and clinical evidence regarding the effects of IC on LIRI and prompt further investigation to refine its clinical application. [ABSTRACT FROM AUTHOR]

Acute ST-elevation myocardial infarction (STEMI) remains a globally significant health challenge in spite of improvement in management strategy. Being aware that mitochondrial dysfunction plays a crucial role in ischaemia-reperfusion injury (IRI) modulation, empirical evidence suggests functional mitochondrial transplantation strikes as a reliable therapeutic approach for patients with acute myocardial infarction. We conducted a prospective, triple-blinded, parallel-group, blocked randomised clinical trial to investigate the therapeutic effects and clinical outcomes of platelet-derived mitochondrial transplantation in 30 patients with acute STEMI, such that the 15 subjects in the control group were given standard of care treatment, whereas the subjects in the intervention group received autologous platelet-derived mitochondria through the intracoronary injection. We observed that within 40 days, the intervention group had a slightly greater improvement in the left ventricular ejection fraction (LVEF) compared to the control group and experienced a significant enhancement in the exercise capacity (p < 0.001). Moreover, major adverse cardiac events (MACE), arrhythmia, fever, and tachycardia were compared between the groups and lack of significant difference marks the safety of mitochondrial transplantation (p > 0.05). Furthermore, the two groups were not significantly distinct as regards the average length of stay for a hospitalisation (p > 0.05). We suggest platelet-derived mitochondrial transplantation appears as a beneficial and highly promising therapeutic option for patients of ischaemic heart disease (IHD); however, we are aware that further in-depth studies with larger sample sizes along with longer follow-up periods are necessary for validating the clinical implications of our findings. • Platelet- derived mitochondrial transplantation in ischaemic heart disease is safe. • Platelet- derived mitochondrial transplantation mitochondrial transplantation increases the ejection fraction. • Platelet- derived mitochondrial transplantation emerges as a novel therapeutic approach for ischemic heart disease. [ABSTRACT FROM AUTHOR]

Background & aimsGlycogen synthase kinase 3β (Gsk3β [Gsk3b]) is a ubiquitously expressed kinase with distinctive functions in different types of cells. Although its roles in regulating innate immune activation and ischaemia and reperfusion injuries (IRIs) have been well documented, the underlying mechanisms remain ambiguous, in part because of the lack of cell-specific tools in vivo.MethodsWe created a myeloid-specific Gsk3b knockout (KO) strain to study the function of Gsk3β in macrophages in a murine liver partial warm ischaemia model.ResultsCompared with controls, myeloid Gsk3b KO mice were protected from IRI, with diminished proinflammatory but enhanced anti-inflammatory immune responses in livers. In bone marrow-derived macrophages, Gsk3β deficiency resulted in an early reduction of Tnf gene transcription but sustained increase of Il10 gene transcription on Toll-like receptor 4 stimulation in vitro. These effects were associated with enhanced AMP-activated protein kinase (AMPK) activation, which led to an accelerated and higher level of induction of the novel innate immune negative regulator small heterodimer partner (SHP [Nr0b2]). The regulatory function of Gsk3β on AMPK activation and SHP induction was confirmed in wild-type bone marrow-derived macrophages with a Gsk3 inhibitor. Furthermore, we found that this immune regulatory mechanism was independent of Gsk3β Ser9 phosphorylation and the phosphoinositide 3-kinase-Akt signalling pathway. In vivo, myeloid Gsk3β deficiency facilitated SHP upregulation by ischaemia-reperfusion in liver macrophages. Treatment of Gsk3b KO mice with either AMPK inhibitor or SHP small interfering RNA before the onset of liver ischaemia restored liver proinflammatory immune activation and IRI in these otherwise protected hosts. Additionally, pharmacological activation of AMPK protected wild-type mice from liver IRI, with reduced proinflammatory immune activation. Inhibition of the AMPK-SHP pathway by liver ischaemia was demonstrated in tumour resection patients.ConclusionsGsk3β promotes innate proinflammatory immune activation by restraining AMPK activation.Lay summaryGlycogen synthase kinase 3β promotes macrophage inflammatory activation by inhibiting the immune regulatory signalling of AMP-activated protein kinase and the induction of small heterodimer partner. Therefore, therapeutic targeting of glycogen synthase kinase 3β enhances innate immune regulation and protects liver from ischaemia and reperfusion injury.

Aims Prolyl hydroxylation is a post-translational modification that regulates protein stability, turnover, and activity. The proteins that catalyze prolyl hydroxylation belong to the 2-oxoglutarate- and iron-dependent oxygenase family of proteins. 2-oxoglutarate- and iron-dependent oxygenase domain-containing protein 1 (Ogfod1), which hydroxylates a proline in ribosomal protein s23 is a newly described member of this family. The aims of this study were to investigate roles for Ogfod1 in the heart, and in the heart's response to stress. Methods and results We isolated hearts from wild-type (WT) and Ogfod1 knockout (KO) mice and performed quantitative proteomics using tandem mass Tag labelling coupled to liquid chromatography and tandem mass spectrometry (LC-MS/MS) to identify protein changes. Ingenuity pathway analysis identified 'Urate Biosynthesis/Inosine 5′-phosphate Degradation' and 'Purine Nucleotides Degradation II (Aerobic)' as the most significantly enriched pathways. We performed metabolomics analysis and found that both purine and pyrimidine pathways were altered with the purine nucleotide inosine 5′-monophosphate showing a 3.5-fold enrichment in KO hearts (P = 0.011) and the pyrimidine catabolism product beta-alanine showing a 1.7-fold enrichment in KO hearts (P = 0.014). As changes in these pathways have been shown to contribute to cardioprotection, we subjected isolated perfused hearts to ischaemia and reperfusion (I/R). KO hearts showed a 41.4% decrease in infarct size and a 34% improvement in cardiac function compared to WT hearts. This protection was also evident in an in vivo I/R model. Additionally, our data show that treating isolated perfused WT hearts with carnosine, a metabolite of beta-alanine, improved protection in the context of I/R injury, whereas treating KO hearts with carnosine had no impact on recovery of function or infarct size. Conclusions Taken together, these data show that Ogfod1 deletion alters the myocardial proteome and metabolome to confer protection against I/R injury. [ABSTRACT FROM AUTHOR]

Background: Ischaemia-reperfusion injury (IRI) is a critical complication post-limb replantation. The oxidative stress and cellular apoptosis due to IRI considerably hinder the healing process. This study aimed to investigate the modulatory effects of pre-perfusion with hydrogen-rich heparin sodium on the nuclear factor erythroid 2-related factor 2 (NRF2)/haeme oxygenase-1 (HO-1) pathway and its potential mechanisms in mitigating skeletal muscle IRI post-limb replantation., Methods: Forty healthy Sprague-Dawley rats (250-300 g) were classified into five groups (n = 8 each): normal control, IRI + heparin sodium pre-perfusion (heparin group), IRI + hydrogen-rich heparin sodium pre-perfusion (hydrogen-rich heparin group), IRI + hydrogen-rich heparin sodium pre-perfusion + NRF2 inhibitor (hydrogen-rich heparin + all-trans retinoic acid [ATRA] group), and IRI + heparin sodium pre-perfusion + NRF2 inhibitor (heparin + ATRA group). The activation of the NRF2/HO-1 pathway in skeletal muscle IRI was evaluated based on HO-1 expression using western blotting and immunofluorescence. Furthermore, haematoxylin and eosin staining and transmission electron microscopy were employed to determine the histopathological characteristics. Additionally, superoxide dismutase and malondialdehyde levels in skeletal muscle tissue were measured to assess antioxidant capacity and the degree of oxidative stress damage. Tissue hypoxia was assessed based on hypoxia-inducible factor 1-alpha expression, whereas apoptosis markers BCL-2-associated X protein (BAX) and Caspase-3 in skeletal muscle tissues were analysed using western blotting with terminal deoxynucleotidyl transferase dUTP nick end labelling staining to quantify cell apoptosis., Results: Compared with the control group, the heparin group exhibited significant pathological changes, including inflammatory infiltration and cellular hypertrophy, with increased apoptosis and oxidative stress. Notably, NRF2 suppression aggravated these effects. However, hydrogen-rich heparin sodium prominently activated the NRF2/HO-1 pathway, enhancing antioxidant defence and reducing BAX/Caspase-3-mediated apoptosis, thereby mitigating IRI-induced damage. The use of an NRF2 inhibitor to inhibit NRF2 excitation by hydrogen-rich heparin sodium notably weakened NRF2 activation and the antioxidant response, resulting in a substantial increase in cellular apoptosis., Conclusion: Pre-perfusion with hydrogen-rich heparin sodium markedly diminishes the BAX/Caspase-3-mediated apoptotic pathway in skeletal muscle tissues with IRI through the excitation of the NRF2/HO-1 pathway., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Background: A minimal decrease in renal function in partial nephrectomy is an important clinical and experimental research objective. We compared the effect of intermittent hilar occlusion (IHO) and continuous hilar occlusion (CHO) on kidney injury and oxidative damage. Method: Using CHO or IHO for 24, 30, and 45 min, intraoperative ischaemia, as well as a sham group, were set up in an ischaemia-reperfusion long-term survival mouse model. Renal function, pathological injury, subcellular structure injury, and cell apoptosis were evaluated at 1, 30, and 90 d postoperatively to study the protective effect of IHO. To do so, oxidative damage analysis, antioxidant activities, and ischaemia time dose-response curves on relative acute renal function injury were analysed. Results: All parameters indicated that kidney injury was dramatically attenuated in the IHO groups compared to those in the corresponding CHO groups. Particularly, mice in the IHO 24-min group incurred no tubular injury or renal functional injury; mice in the CHO 30-min group incurred severe acute and chronic kidney injury, but mice in the IHO 30-min group only experienced transient mild renal functional injury. IHO prolonged tolerable ischaemia duration. Conclusions: IHO can prolong the duration of tolerable ischaemia, at least partly through protecting the kidney from oxidative stress, consequently attenuating or preventing renal ischaemia-reperfusion injury. Reduced kidney injury after ischaemia-reperfusion is linked to a greater maintenance of antioxidant activity and less oxidative damage. The protective effect of IHO can be extremely efficient if properly applied; IHO may avoid renal ischaemia-reperfusion injury during partial nephrectomy.

Sphingosine 1-phosphate (S1P) and S1P receptors (S1PR) regulate many cellular processes, including lymphocyte migration and endothelial barrier function. As neutrophils are major mediators of inflammation, their transendothelial migration may be the target of therapeutic approaches to inflammatory conditions such as ischaemia–reperfusion injury (IRI). The aim of this project was to assess whether these therapeutic effects are mediated by S1P acting on neutrophils directly or indirectly through the endothelial cells. First, our murine model of peritoneum cell recruitment demonstrated the ability of S1P to reduce CXCL8-mediated neutrophil recruitment. Mechanistic in vitro studies revealed that S1P signals in neutrophils mainly through the S1PR1 and 4 receptors and induces phosphorylation of ERK1/2; however, this had no effect on neutrophil transmigration and adhesion. S1P treatment of endothelial cells significantly reduced TNF-α-induced neutrophil adhesion under flow (p < 0.01) and transendothelial migration towards CXCL8 during in vitro chemotaxis assays (p < 0.05). S1PR1 agonist CYM5442 treatment of endothelial cells also reduced neutrophil transmigration (p < 0.01) and endothelial permeability (p < 0.005), as shown using in vitro permeability assays. S1PR3 agonist had no effects on chemotaxis or permeability. In an in vivo mouse model of renal IRI, S1PR agonism with CYM5442 reduced endothelial permeability as shown by reduced Evan’s Blue dye extravasation. Western blot was used to assess phosphorylation at different sites on vascular endothelial (VE)–cadherin and showed that CYM5442 reduced VEGF-mediated phosphorylation. Taken together, the results of this study suggest that reductions in neutrophil infiltration during IRI in response to S1P are mediated primarily by S1PR1 signalling on endothelial cells, possibly by altering phosphorylation of VE–cadherin. The results also demonstrate the therapeutic potential of S1PR1 agonist during IRI.

Acute kidney injury (AKI) is a common clinical condition, results in patient morbidity and mortality, and incurs considerable health care costs. Sepsis, ischaemia-reperfusion injury (IRI) and drug nephrotoxicity are the leading causes. Mounting evidence suggests that perturbations in circular RNAs (circRNAs) are observed in AKI of various aetiologies, and have pathogenic significance. Aberrant circRNA expressions can cause altered intracellular signalling, exaggerated oxidative stress, increased cellular apoptosis, excess inflammation, and tissue injury in AKI due to sepsis or IRI. While circRNAs are dysregulated in drug-induced AKI, their roles in pathogenesis are less well-characterised. CircRNAs also show potential for clinical application in diagnosis, prognostication, monitoring, and treatment. Prospective observational studies are needed to investigate the role of circRNAs in the clinical management of AKI, with special focus on the safety of therapeutic interventions targeting circRNAs and the avoidance of untoward off-target effects. [ABSTRACT FROM AUTHOR]

Remote ischaemic preconditioning (RIPC), induced by intermittent periods of limb ischaemia and reperfusion, confers cardiac and vascular protection from subsequent ischaemia–reperfusion (IR) injury. Early animal studies reliably demonstrate that RIPC attenuated infarct size and preserved cardiac tissue. However, translating these adaptations to clinical practice in humans has been challenging. Large clinical studies have found inconsistent results with respect to RIPC eliciting IR injury protection or improving clinical outcomes. Follow‐up studies have implicated several factors that potentially affect the efficacy of RIPC in humans such as age, fitness, frequency, disease state and interactions with medications. Thus, realizing the clinical potential for RIPC may require a human experimental model where confounding factors are more effectively controlled and underlying mechanisms can be further elucidated. In this review, we highlight recent experimental findings in the peripheral circulation that have added valuable insight on the mechanisms and clinical benefit of RIPC in humans. Central to this discussion is the critical role of timing (i.e. immediate vs. delayed effects following a single bout of RIPC) and the frequency of RIPC. Limited evidence in humans has demonstrated that repeated bouts of RIPC over several days uniquely improves vascular function beyond that observed with a single bout alone. Since changes in resistance vessel and microvascular function often precede symptoms and diagnosis of cardiovascular disease, repeated bouts of RIPC may be promising as a preclinical intervention to prevent or delay cardiovascular disease progression. [ABSTRACT FROM AUTHOR]

To document changes in urinary biomarker concentration and conventional diagnostic tests of acute kidney injury (AKI) following hypotension and fluid resuscitation in anaesthetized dogs. Experimental, repeated measures, prospective study. A group of six male adult Greyhound dogs. Following general anaesthesia, severe hypotension was induced by phlebotomy, maintaining mean arterial blood pressure (MAP) < 40 mmHg for 60 minutes, followed by resuscitation with intravenous gelatine solution to maintain MAP > 60 mmHg for 3 hours. Following euthanasia, renal tissue was examined by light microscopy (LM) and transmission electron microscopy (TEM). Urinary and serum concentrations of neutrophil gelatinase-associated lipocalin (NGAL), cystatin C (CysC), and gamma-glutamyl transpeptidase (GGT), serum creatinine and urine output were measured at baseline and hourly until euthanasia. Data are presented as mean and 95% confidence interval and analysed using repeated measures analysis of variance with Dunnett's adjustment, p < 0.05. Structural damage to proximal renal tubular cells was evident on LM and TEM. Urinary biomarker concentrations were significantly elevated from baseline, peaking 2 hours after haemorrhage at 19.8 (15.1–25.9) ng mL–1 NGAL (p = 0.002), 2.54 (1.64–3.43) mg mL–1 CysC (p = 0.009) and 2043 (790–5458) U L–1 GGT (p < 0.001). Serum creatinine remained within a breed-specific reference interval in all dogs. Urinary protein–creatinine ratio (UPC) was significantly elevated in all dogs from 1 hour following haemorrhage. Urinary NGAL, CysC and GGT concentrations, and UPC were consistently elevated within 1 hour of severe hypotension, suggesting that proximal renal tubules are damaged in the earliest stage of ischaemia-reperfusion AKI. Measurement of urinary biomarkers may allow early diagnosis of AKI in anaesthetized dogs. Urinary GGT concentration and UPC are particularly useful as they can be measured on standard biochemistry analysers. [ABSTRACT FROM AUTHOR]

Purpose: The purpose of this study was to evaluate the effects of increasing zone II resuscitative endovascular balloon occlusion of the aorta (REBOA) occlusion times on physiological, end-organ and inflammatory responses in rabbits to assess the safe aortic occlusion time in a normovolemic rabbit model.Methods: The zone ll aorta was occluded with a balloon in 32 rabbits (8 animals each for 15, 30, 60, and 90 min). 8 rabbits served as a control. ELISAs were used to examine the serum levels of ALT, AST, Cr, BUN, MDA, SOD, IL-8, IL-6, and TNF-α; HE staining was used to identify the morphological changes in the kidney; RT-PCR was used to detect the mRNA levels of IL-6, IL-8, TNF-α and NF-κB in the kidney and uterus; and Western blotting was used to measure the protein expression levels of IL-6, IL-8, TNF-α and NF-κB in the kidney and uterus.Results: Plasma concentrations of liver markers, kidney markers, inflammatory factors and oxidative stress indicators were significantly increased at the end of reperfusion in the 30 min, 60 min and 90 min groups. Damage to the kidney occurred in the 30 min, 60 min and 90 min groups. The mRNA and protein expression levels of IL-6, IL-8, TNF-α and NF-κB in the kidney and uterus were significantly increased at the end of reperfusion in the 30 min group, and as the time of occlusion extended, these levels continued to increase.Conclusion: Activation of systemic inflammation and ischaemia-reperfusion injury of end-organs occurred when the occlusion time reached 30 min. Therefore, 15 min should be regarded as a safe period of REBOA in zone II. [ABSTRACT FROM AUTHOR]

Ischaemia–reperfusion injury (IRI) is an inevitable process in transplantation and results in inflammation and immune system activation. Alpha-1 antitrypsin (AAT) has anti-inflammatory properties. Normothermic machine perfusion (NMP) can be used to deliver therapies and may help in assessing the effects of IRI and immunity. This study investigated the effects of AAT on IRI and inflammation in pig kidneys when administered during preservation, followed by normothermic reperfusion (NR) with autologous whole blood, as a surrogate for transplant. Two different models were used to deliver AAT or placebo to paired slaughterhouse pig kidneys: Model 1: 7-h static cold storage (SCS) + 3-h NR (n = 5 pairs), where either AAT (10 mg/ml) or placebo was delivered in the flush following retrieval; Model 2: 4-h SCS + 3-h NMP + 3-h NR (n = 5 pairs), where either AAT or placebo was delivered during NMP. Injury markers and cytokines levels were analysed in the perfusate, and heat shock protein 70 KDa (HSP-70) was analysed in biopsies. AAT delivered to kidneys showed no adverse effects on perfusion parameters. HSP-70 fold changes were significantly lower in the AAT group during NMP (P < 0.01, paired t-test) but not during NR. Interleukin-1 receptor antagonist (IL-1ra) fold changes were significantly higher in the AAT group during NR model 1 (p < 0.05, two-way ANOVA). In contrast to the AAT group, significant upregulation of interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) between t = 90 min and t = 180 min and interleukin-8 (IL-8) between baseline and t = 90 min was observed in the control group in NR model 2 (p < 0.05, Tukey's multiple comparison test). However, overall inflammatory cytokines and injury markers showed similar levels between groups. Delivery of AAT to pig kidneys was safe without any detrimental effects. NMP and NR provided excellent methods for comparison of inflammation and immune activation in the delivery of a novel therapy. [ABSTRACT FROM AUTHOR]

Ischaemia–reperfusion injury (IRI) is an inevitable process in transplantation and results in inflammation and immune system activation. Alpha-1 antitrypsin (AAT) has anti-inflammatory properties. Normothermic machine perfusion (NMP) can be used to deliver therapies and may help in assessing the effects of IRI and immunity. This study investigated the effects of AAT on IRI and inflammation in pig kidneys when administered during preservation, followed by normothermic reperfusion (NR) with autologous whole blood, as a surrogate for transplant. Two different models were used to deliver AAT or placebo to paired slaughterhouse pig kidneys: Model 1: 7-h static cold storage (SCS) + 3-h NR (n = 5 pairs), where either AAT (10 mg/ml) or placebo was delivered in the flush following retrieval; Model 2: 4-h SCS + 3-h NMP + 3-h NR (n = 5 pairs), where either AAT or placebo was delivered during NMP. Injury markers and cytokines levels were analysed in the perfusate, and heat shock protein 70 KDa (HSP-70) was analysed in biopsies. AAT delivered to kidneys showed no adverse effects on perfusion parameters. HSP-70 fold changes were significantly lower in the AAT group during NMP (P < 0.01, paired t-test) but not during NR. Interleukin-1 receptor antagonist (IL-1ra) fold changes were significantly higher in the AAT group during NR model 1 (p < 0.05, two-way ANOVA). In contrast to the AAT group, significant upregulation of interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) between t = 90 min and t = 180 min and interleukin-8 (IL-8) between baseline and t = 90 min was observed in the control group in NR model 2 (p < 0.05, Tukey’s multiple comparison test). However, overall inflammatory cytokines and injury markers showed similar levels between groups. Delivery of AAT to pig kidneys was safe without any detrimental effects. NMP and NR provided excellent methods for comparison of inflammation and immune activation in the delivery of a novel therapy.

There is a dearth of evidence regarding detorsion as a surgical management of testicular torsion in studies of experimental testicular damage in rabbits. We investigated histopathological changes, oxidative stress and antioxidant response to testicular ischaemia-reperfusion injury following torsion. Ten (10) New Zealand male peripubertal rabbits (Mean Age: 4.6 ± 1.2 months; Mean Weight: 4.3 ± 0.6 kg) were randomly handpicked (with five (5) in each) into two groups-A and B, equally. Group A was the control while Group B underwent left testicular torsion for 1 hour and detorsion for surgical correction. Testicular tissue samples of the affected testicles were analysed for histopathological changes, Malondialdehyde (MDA), Superoxide Dismutase (SOD) and Glutathione Peroxidase (GPx). Oedema and loss of interstitial spaces with coagulative necrosis, lobular segmentation and complete loss of architecture of seminiferous tubules were evident. There was a significant (P < 0.0001) decrease in SOD but a significant increase in MDA and GPx when compared to the control group. Surgical detorsion as correction for testicular torsion did not produce satisfactory outcome. [ABSTRACT FROM AUTHOR]

Amifostine is a cytoprotective compound that is beneficial in ischaemic stroke cases. However, the neuroprotective effect of amifostine on ischaemia/reperfusion (I/R)-induced brain injury and its underlying mechanism are still poorly understood. Herein, we constructed an animal model of middle cerebral artery occlusion and reperfusion (MCAO/R) injury and an in vitro model of oxygen and glucose deprivation and reperfusion (OGD/R) injury. After administration of amifostine, we found significant improvements in neurological deficits, infarct size, and cerebral oedema. Moreover, amifostine alleviated histopathological alteration and increased the number of surviving neurons. Biochemical analysis showed that treatment with amifostine obviously improved the brain damage of MCAO/R mice, as manifested by a decrease in reactive oxygen species (ROS) and malondialdehyde (MDA) generation, and an increase in superoxide dismutase (SOD) activity. Moreover, amifostine decreased the mitochondrial membrane potential (m) loss, and cytochrome c escaping to cytoplasm, but increased the ATP level. In vitro, amifostine also showed an antioxidant effect, which was reflected by the reduced ROS generation, decreased mitochondrial superoxide generation, increased total SOD, SOD1 (Cu/Zn SOD, cytoplasmic SOD), and SOD2 (mitochondrial SOD) activities, and decreased m loss. Furthermore, amifostine suppressed neuronal apoptosis, accompanied by the reduction of Bax, cleaved caspase-9, cleaved caspase-3, and Bcl-2 upregulation. Amifostine also reduced the expression of p-p38 (Thr 180/Tyr 182) in vivo and in vitro. In short, amifostine exhibits a protective effect on cerebral I/R damage through modulating p38-related oxidative stress, mitochondrial dysfunction, and apoptosis.

β-adrenoceptor antagonists are commonly used in ischaemic heart disease (IHD) patients, yet may impair signalling and efficacy of 'cardioprotective' interventions. We assessed effects of chronic β1-adrenoceptor antagonism on myocardial resistance to ischaemia-reperfusion (IR) injury and the ability of cardioprotective interventions [classic ischaemic preconditioning (IPC); novel sustained ligand-activated preconditioning (SLP)] to reduce IR injury in murine hearts. Young male C57Bl/6 mice were untreated or received atenolol (0.5g/l in drinking water) for 4 weeks. Subsequently, two cardioprotective stimuli were evaluated: morphine pellets implanted (to induce SLP, controls received placebo) 5 days prior to Langendorff heart perfusion, and IPC in perfused hearts (3×1.5min ischaemia/2min reperfusion). Atenolol significantly reduced in vivo heart rate. Untreated control hearts exhibited substantial left ventricular dysfunction (~50% pressure development recovery, ~20mmHg diastolic pressure rise) with significant release of lactate dehydrogenase (LDH, tissue injury indicator) after 25min ischaemia/45min reperfusion. Contractile dysfunction and elevated LDH were reduced >50% with IPC and SLP. While atenolol treatment did not modify baseline contractile function, post-ischaemic function was significantly depressed compared to untreated hearts. Atenolol pre-treatment abolished beneficial effects of IPC, whereas SLP protection was preserved. These data indicate that chronic β1-adrenoceptor blockade can exert negative effects on functional IR tolerance and negate conventional IPC (implicating β1-adrenoceptors in IR injury and IPC signalling). However, novel morphine-induced SLP is resistant to inhibition by β1-adrenoceptor antagonism.

Ischaemia–reperfusion (I/R) is one of the main factors of acute kidney injury (AKI). mitochondrial damage pathway are important features of I/R induced‐acute kidney injury (IRI‐AKI). Hypoxia‐inducible factor (HIF) expression in renal tubule segments is up‐regulated during AKI. Herein, we investigated the role of FG‐4592 in a mouse model of IRI‐AKI to confirm whether FG‐4592 is beneficial in AKI. We found that pretreatment with FG‐4592 significantly ameliorated renal function and renal histological damage in mice after IRI. Furthermore, these results suggest that pretreatment with FG‐4592 significantly reduced the tubular cells apoptosis (decreased TUNEL‐positive cells, Bax, caspase12 levels), attenuated mitochondrial damage (increased ATPβ, PPARγ, mitochondrial DNA copy number, and decreased cytoplasmic cytochrome C), and alleviated DNA damage after IRI. In conclusion, pretreatment with FG‐4592 may effectively prevent kidney from IRI possibly by via diminishing tubular cells injuries and protection of mitochondrial damage pathway. These results further validate that FG‐4592 may be an effective drug in the clinical treatment of IRI‐AKI. [ABSTRACT FROM AUTHOR]

Aims Systemic inflammation and increased activity of atrial NOX2-containing NADPH oxidases have been associated with the new onset of atrial fibrillation (AF) after cardiac surgery. In addition to lowering LDL-cholesterol, statins exert rapid anti-inflammatory and antioxidant effects, the clinical significance of which remains controversial. Methods and results We first assessed the impact of cardiac surgery and cardiopulmonary bypass (CPB) on atrial nitroso-redox balance by measuring NO synthase (NOS) and GTP cyclohydrolase-1 (GCH-1) activity, biopterin content, and superoxide production in paired samples of the right atrial appendage obtained before (PRE) and after CPB and reperfusion (POST) in 116 patients. The effect of perioperative treatment with atorvastatin (80 mg once daily) on these parameters, blood biomarkers, and the post-operative atrial effective refractory period (AERP) was then evaluated in a randomized, double-blind, placebo-controlled study in 80 patients undergoing cardiac surgery on CPB. CPB and reperfusion led to a significant increase in atrial superoxide production (74% CI 71–76%, n  = 46 paired samples, P  < 0.0001) and a reduction in atrial tetrahydrobiopterin (BH4) (34% CI 33–35%, n  = 36 paired samples, P  < 0.01), and in GCH-1 (56% CI 55–58%, n  = 26 paired samples, P  < 0.001) and NOS activity (58% CI 52–67%, n  = 20 paired samples, P  < 0.001). Perioperative atorvastatin treatment prevented the effect of CPB and reperfusion on all parameters but had no significant effect on the postoperative right AERP, troponin release, or NT-proBNP after cardiac surgery. Conclusion Perioperative statin therapy prevents post-reperfusion atrial nitroso-redox imbalance in patients undergoing on-pump cardiac surgery but has no significant impact on postoperative atrial refractoriness, perioperative myocardial injury, or markers of postoperative LV function. Clinical Trial Registration https://clinicaltrials.gov/ct2/show/NCT01780740 [ABSTRACT FROM AUTHOR]

Extracellular cold‐inducible RNA‐binding protein (CIRP) is a proinflammatory mediator that aggravates ischaemia‐reperfusion injury (IRI). Normothermic machine perfusion (NMP) could effectively alleviate the IRI of the liver, but the underlying mechanism remains to be explored. We show that human DCD livers secreted a large amount of CIRP during static cold storage (CS), which is released into the circulation after reperfusion. The expression of CIRP was related to postoperative IL‐6 levels and liver function. In a rat model, the CIRP expression was upregulated during warm ischaemia and cold storage. Then, rat DCD livers were preserved using CS, hypothermic oxygenated machine perfusion (HOPE) and NMP. C23, a CIRP inhibitor, was administrated in the HOPE group. Compared with CS, NMP significantly inhibited CIRP expression and decreased oxidative stress by downregulating NADPH oxidase and upregulating UCP2. NMP markedly inhibited the mitochondrial fission‐related proteins Drp‐1 and Fis‐1. Further, NMP increased the mitochondrial biogenesis‐related protein, TFAM. NMP significantly reduced inflammatory reactions and apoptosis after reperfusion, and NMP‐preserved liver tissue had higher bile secretion and ICG metabolism compared to the CS group. Moreover, C23 administration attenuated IRI in the HOPE group. Additionally, HL‐7702 cells were stimulated with rhCIRP and C23. High rhCIRP levels increased oxidative stress and apoptosis. In summary, NMP attenuates the IRI of DCD liver by inhibiting CIRP‐mediated oxidative stress and mitochondrial fission. [ABSTRACT FROM AUTHOR]

Background Down-regulation of the enzymes involved in tryptophan-derived nicotinamide (NAM) adenine dinucleotide (NAD+) production was identified after acute kidney injury (AKI), leading to the hypothesis that supplementation with NAM may increase the kidney NAD+ content, rescuing tryptophan pathways and subsequently improving kidney outcomes. Methods Urinary measurement of tryptophan and kynurenin using liquid chromatography–mass spectrometry metabolomics was used in a cohort of 167 cardiac bypass surgery patients along with tests for correlation to the development of postoperative AKI. A mouse model of ischaemic AKI using ischaemia–reperfusion injury (bilateral clamping of renal arteries for 25 min) was also used. Results We identified a significant decrease in urinary tryptophan and kynurenin in patients developing AKI, irrespective of the Kidney Disease: Improving Global Outcomes (KDIGO) stage. Although a significant difference was observed, tryptophan and kynurenin moderately discriminated for the development of all AKI KDIGO stages {area under the curve [AUC] 0.82 [95% confidence interval (CI) 0.75–0.88] and 0.75 [0.68–0.83], respectively} and severe KDIGO Stages 2–3 AKI [AUC 0.71 (95% CI 0.6–0.81) and 0.66 (0.55–0.77), respectively]. Sparked by this confirmation in humans, we aimed to confirm the potential preventive effect of NAM supplementation in wild-type male and female C57BL/6 mice subjected to ischaemic AKI. NAM supplementation had no effect on renal function (blood urea nitrogen at Day 1, sinistrin–fluorescein isothiocyanate glomerular filtration rate), architecture (periodic acid–Schiff staining) and injury or inflammation (kidney injury molecule 1 and IL18 messenger RNA expression). In addition, NAM supplementation did not increase post-AKI NAD+ kidney content. Conclusion Notwithstanding the potential role of NAM supplementation in the setting of basal NAD+ deficiency, our findings in mice and the reanalysis of published data do not confirm that NAM supplementation can actually improve renal outcomes after ischaemic AKI in unselected animals and probably patients. [ABSTRACT FROM AUTHOR]