Ischemia-reperfusion Injury Damage Research Articles

CTGF regulated by ATF6 inhibits vascular endothelial inflammation and reduces hepatic ischemia–reperfusion injury

Vascular endothelial inflammation is crucial in hepatic ischemia–reperfusion injury (IRI). Our previous research has shown that connective tissue growth factor (CTGF), secreted by endothelial cells, protects against acute liver injury, but its upstream mechanism is unclear. We aimed to clarify the protective role of CTGF in endothelial cell inflammation during IRI and reveal the regulation between endoplasmic reticulum stress-induced activating transcription factor 6 (ATF6) and CTGF. Hypoxia/reoxygenation in endothelial cells, hepatic IRI in mice and clinical specimens were used to examine the relationships between CTGF and inflammatory factors and determine how ATF6 regulates CTGF and reduces damage. We found that activating ATF6 promoted CTGF expression and reduced liver damage in hepatic IRI. In vitro, activated ATF6 upregulated CTGF and downregulated inflammation, while ATF6 inhibition had the opposite effect. Dual-luciferase assays and chromatin immunoprecipitation confirmed that activated ATF6 binds to the CTGF promoter, enhancing its expression. Activated ATF6 increases CTGF and reduces extracellular regulated protein kinase 1/2 (ERK1/2) phosphorylation, decreasing inflammatory factors. Conversely, inhibiting ATF6 decreases CTGF and increases the phosphorylation of ERK1/2, increasing inflammatory factor levels. ERK1/2 inhibition reverses this effect. Clinical samples have shown that CTGF increases after IRI, inversely correlating with inflammatory cytokines. Therefore, ATF6 activation during liver IRI enhances CTGF expression and reduces endothelial inflammation via ERK1/2 inhibition, providing a novel target for diagnosing and treating liver IRI.

Ozone Administration Reduces Myocardial Ischemia Reperfusion Injury in Streptozotocin Induced Diabetes Mellitus Rat Model.

This study aimed to demonstrate whether ozone has cardioprotective effects on the myocardial ischemia-reperfusion injury (IRI) in rats with streptozotocin(STZ)-induced diabetes. A total of 38 male Wistar Albino rats were divided into five groups as follows: control group (group C,n=6), diabetic group (group D,n=6), diabetic ozone group (group DO,n=6), diabetic-ischemia/reperfusion (group DIR,n=6), diabetic-ischemia/reperfusion-ozone (group DIRO,n=6). Six rats died during this period and two died because of surgical complications. A myocardial ischemia-reperfusion model was created using a thoracotomy incision from 4th intercostal space. The LAD was ligated using an 8-0 prolene suture for 30min. Ozone was administered intraperitoneally(1mg/kg) 5min before reperfusion. The reperfusion time was 120min. At the end of the reperfusion procedure, myocardial tissue histopathological examinations, and serum biochemical analyses were performed. The percentage of TUNEL(+) cardiomyocytes/HPF was significantly higher in the DIR group than in the C, D, and DO groups. Conversely, TUNEL positivity was significantly lower in the DIRO group than in the DIR group. The IRI score was significantly higher in the DIR and DIRO groups than that in the C, D, and DO groups. In contrast, the IRI damage score in the DIRO group was significantly lower than that in the DIR group. Serum MDA levels were significantly higher in the DIR group than in the C, D, and DO groups. Similarly, MDA levels were significantly higher in the DIRO group than in the C and D groups. CAT activity was significantly higher in the DIR group than in the C and D groups. SOD activity was significantly higher in the DIR group than in the C and DO groups. Our study showed that ozone exerts cardioprotective effects in STZ-induced diabetic rats through its antioxidant role against oxidative stress. Both biochemical and histological analyses clearly revealed that ozone has beneficial effects against IRI in the diabetic rat myocardium.

The Effect of Cerium Oxide (CeO2) on Ischemia-Reperfusion Injury in Skeletal Muscle in Mice with Streptozocin-Induced Diabetes.

Objective: Lower extremity ischemia-reperfusion injury (IRI) may occur with trauma-related vascular injury and various vascular diseases, during the use of a tourniquet, in temporary clamping of the aorta in aortic surgery, or following acute or bilateral acute femoral artery occlusion. Mitochondrial dysfunction and increased basal oxidative stress in diabetes may cause an increase in the effects of increased reactive oxygen species (ROS) and mitochondrial dysfunction due to IRI. It is of great importance to examine therapeutic approaches that can minimize the effects of IRI, especially for patient groups under chronic oxidative stress such as DM. Cerium oxide (CeO2) nanoparticles mimic antioxidant enzymes and act as a catalyst that scavenges ROS. In this study, it was aimed to investigate whether CeO2 has protective effects on skeletal muscles in lower extremity IRI in mice with streptozocin-induced diabetes. Methods: A total of 38 Swiss albino mice were divided into six groups as follows: control group (group C, n = 6), diabetes group (group D, n = 8), diabetes-CeO2 (group DCO, n = 8), diabetes-ischemia/reperfusion (group DIR, n = 8), and diabetes-ischemia/reperfusion-CeO2 (group DIRCO, n = 8). The DCO and DIRCO groups were given doses of CeO2 of 0.5 mg/kg intraperitoneally 30 min before the IR procedure. A 120 min ischemia-120 min reperfusion period with 100% O2 was performed. At the end of the reperfusion period, muscle tissues were removed for histopathological and biochemical examinations. Results: Total antioxidant status (TAS) levels were found to be significantly lower in group DIR compared with group D (p = 0.047 and p = 0.022, respectively). In group DIRCO, total oxidant status (TOS) levels were found to be significantly higher than in group DIR (p < 0.001). The oxidative stress index (OSI) was found to be significantly lower in group DIR compared with group DCO (p < 0.001). Paraoxanase (PON) enzyme activity was found to be significantly increased in group DIR compared with group DCO (p < 0.001). The disorganization and degeneration score for muscle cells, inflammatory cell infiltration score, and total injury score in group DIRCO were found to be significantly lower than in group DIR (p = 0.002, p = 0.034, and p = 0.001, respectively). Conclusions: Our results confirm that CeO2, with its antioxidative properties, reduces skeletal muscle damage in lower extremity IRI in diabetic mice.

CHOP-mediated Gasdermin E expression promotes pyroptosis, inflammation, and mitochondrial damage in renal ischemia-reperfusion injury

In clinical practice, renal ischemia-reperfusion injury (IRI) is a common cause of acute kidney injury (AKI), often leading to acute renal failure or end-stage renal disease (ESRD). The current understanding of renal IRI mechanisms remains unclear, and effective therapeutic strategies and clear targets are lacking. Therefore, the need to find explicit and effective ways to reduce renal IRI remains a scientific challenge. The current study explored pyroptosis, a type of inflammation-regulated programmed cell death, and the role of Gasdermins E (GSDME)-mediated pyroptosis, mitochondrial damage, and inflammation in renal IRI. The analysis of human samples showed that the expression levels of GSDME in normal human renal tissues were higher than those of GSDMD. Moreover, our study demonstrated that GSDME played an important role in mediating pyroptosis, inflammation, and mitochondrial damage in renal IRI. Subsequently, GSDME-N accumulated in the mitochondrial membrane, leading to mitochondrial damage and activation of caspase3, which generated a feed-forward loop of self-amplification injury. However, GSDME knockout resulted in the amelioration of renal IRI. Moreover, the current study found that the transcription factor CHOP was activated much earlier in renal IRI. Inhibition of BCL-2 by CHOP leaded to casapse3 activation, resulting in mitochondrial damage and apoptosis; not only that, but CHOP positively regulated GSDME thereby causing pyroptosis. Therefore, this study explored the transcriptional mechanisms of GSDME during IRI development and the important role of CHOP/Caspase3/GSDME mechanistic axis in regulating pyroptosis in renal IRI. This axis might serve as a potential therapeutic target.

Inhibition of Sirt3 activates the cGAS-STING pathway to aggravate hepatocyte damage in hepatic ischemia–reperfusion injury mice

Hepatic ischemia–reperfusion injury (IRI) typically manifests during subtotal hepatectomy and inflicts substantial damage to liver function in the perioperative period. Although the central role of cGAS-STING-mediated immune inflammation in hepatocyte damage during hepatic IRI is acknowledged, the precise regulatory mechanisms remain elusive. The current study aims to elucidate how Sirt3 inhibition activates the cGAS-STING pathway and exacerbates hepatocyte damage in hepatic IRI. We established both in vivo and in vitro models by creating hepatic IRI mice model and subjecting AML-12 hepatocyte cell lines to oxygen-glucose deprivation/reperfusion (OGD/R). Hepatic IRI compromised liver and mitochondrial function while elevating cytosolic mitochondrial DNA (mtDNA) levels in hepatocytes. Additionally, both in vivo hepatic IRI and in vitro OGD/R induced increased phosphorylation and activation of cGAS, STING, and IRF3, accompanied by heightened levels of pro-inflammatory factors, including TNF-α, IL-1β, and type I interferon (IFN-β). Importantly, knockdown of cGAS or STING through siRNA effectively attenuated hepatic IRI-induced inflammation and ameliorated liver function in both experimental settings, underscoring the dynamic involvement of the cGAS-STING pathway in hepatic IRI-induced inflammation. Furthermore, we observed a significant reduction in Sirt3 expression following hepatic IRI, both in vivo and in vitro. Then we generated Sirt3-deficient mice and applied Sirt3 knockdown in AML-12 hepatocytes. Notably, Sirt3 deficiency led to increased phosphorylation and activation of cGAS, STING, and IRF3, coupled with elevated TNF-α, IL-1β, and IFN-β levels in both in vivo and in vitro conditions. Moreover, upon silencing various downstream targets of Sirt3, such as transcription factors Sp1, Pu1, and p65, we observed that specifically knocking down p65 in AML-12 hepatocytes reduced cGAS mRNA levels. Co-immunoprecipitation assays confirmed a direct interaction between Sirt3 and p65. The absence of Sirt3 significantly increased nuclear translocation of p65 in mice, whereas Sirt3 knockdown in AML-12 hepatocytes heightened nuclear translocation of p65. ChIP-PCR assays demonstrated that Sirt3 deficiency notably enhanced the binding of p65 to two cGAS promoters, ultimately promoting cGAS transcription. Collectively, our results underscored that inhibition of Sirt3 activates the cGAS-STING pathway to aggravate hepatocyte damage by increasing cytosolic mtDNA and promoting nuclear translocation of p65 to promote cGAS transcription in hepatic IRI. These findings hold promise for potential therapeutic interventions in hepatic IRI by targeting the Sirt3-cGAS-STING axis, offering new avenues for the development of clinical strategies to mitigate liver damage during the perioperative period.

Banaba Restricts Brain Damage: Neuroprotective Role in Cerebral Ischemiareperfusion Injury

Background:: Glucose regulation and energy homeostasis mitigate energy crises milieu in reperfusion injury. We investigated Banaba for its outcomes on cerebral ischemia reperfusion (IR) injury using artery occlusion in rats. The pleiotropic activity of Banaba on various debilitating mechanisms inducing reperfusion injury was evaluated. Aim:: This study aimedto evaluate the pharmacological activity of Banaba (Lagerstroemia speciosa) extract on reperfusion injury and investigate the effect of Banaba on vascular permeability, oxidative stress and cellular damage in ischemia reperfusion injury in rats. Methods:: Transient ischemia and reperfusion through occlusion of the middle cerebral artery (MCAO) lead to Cerebral IR injury in Wistar rats; it was treated with oral administration of Banaba extract (100mg/kg and 200mg/kg). The injury outcomes were evaluated after 22 hours of reperfusion by determining cellular injury, its impact on musculoskeletal coordination, multiple free radical scavenging measures (SOD, GSH, LPO) and vascular permeability of the blood-brain barrier. Result:: Banaba treatment led to a marked improvement in neurological outcomes by enhanced coordination and reduced cerebral infarct in comparison to vehicle control ischemic group. Free radical scavenging activity (SOD and GSH) was significantly better, and lipid peroxidation was reduced by Banaba treatment; it also reduced the vascular permeability of the blood-brain barrier. We observed that a lower dose of Banaba (100 mg/kg) was more effective than the higher (200 mg/kg) in ischemic rats. The anti-inflammatory and anti-oxidant activity could drive the neuroprotective outcomes of Banaba in cerebral IR injury. The critical factor of the beneficial effect of Banaba in cerebral injury is the optimization of dose in this experimental setup of reperfusion injury using rats. Conclusion:: The recovery of injury could be attributed to Banaba’s multi-factorial effect targeting free radicals, inflammation, and necrosis during ischemiareperfusion injury.

Mitochondrial succinate transport as a novel therapeutic target for cardiac ischemia/reperfusion injury

Abstract Background The accumulation of the mitochondrial metabolite succinate during ischemia and its rapid oxidation by succinate dehydrogenase (SDH) upon reperfusion drives ischemia/reperfusion injury (IRI) in myocardial infarction. Succinate oxidation leads to the production of reactive oxygen species (ROS) by reverse electron transport (RET) at mitochondrial complex I, orchestrating a cascade of events leading to cardiomyocyte death. Modulating succinate metabolism, such as SDH inhibition, is a promising therapeutic strategy. During ischemia, the mitochondria produced succinate is transported into the cytosol, enabling the accumulation of a large succinate pool. On reperfusion, the succinate is transported back into mitochondria and oxidised by SDH, thus we hypothesised that targeting succinate mitochondrial transport could be a novel therapeutic target to prevent cardiac IRI. Purpose To assess the role of succinate mitochondrial transport as a novel therapeutic target to prevent cardiac IRI. Methods Isolated heart mitochondria were used to assess mitochondrial respiration by Oroboros, RET-ROS production by Amplex Red and butylmalonate delivery. Cultured H9C2 myoblasts and isolated primary adult cardiomyocytes from C57BL/6J mice were used to assess butylmalonate uptake and delivery from esters, as well as their impact on succinate accumulation and oxidation in vitro. LAD ligation and reperfusion in C57BL/6J mice was used as an in vivo MI model. Metabolite levels were measured by LC-MS/MS. Results Butylmalonate, an inhibitor of the mitochondrial dicarboxylate carrier (DIC), blocks succinate-dependent respiration and RET-ROS production in isolated mitochondria. However, butylmalonate entry into cells was inefficient and does not impact infarct size after IRI in vivo. We developed novel butylmalonate esters to improve butylmalonate delivery to cardiomyocytes in vivo. We found that diacetoxymethyl butylmalonate (DAB) effectively delivered butylmalonate and could prevent succinate accumulation and oxidation in vitro. DAB could also prevent succinate-mediated respiration and RET-ROS at lower concentrations than butylmalonate alone. Finally, DAB reduced acute infarct size in vivo when administered at reperfusion. Conclusions Blocking succinate transport back into mitochondria at reperfusion prevents the damage in IRI by preventing the oxidation of succinate by SDH and downstream RET-ROS production. Therefore, targeting the mitochondrial transport of succinate may be a promising novel strategy in preventing cardiac IRI.

Protective effect of IAXO-102 on renal ischemia-reperfusion injury in rats.

Ischemia/reperfusion injury (IRI) is a common cause of kidney damage, characterized by oxidative stress and inflammation. In this study, we investigated the potential protective effects of IAXO-102, a chemical compound, on experimentally induced IRI in male rats. The bilateral renal IRI model was used, with 24 adult male rats randomly divided into four groups (N=6): sham group (laparotomy without IRI induction), control group (laparotomy plus bilateral IRI for 30 minutes followed by 2 hours of reperfusion), vehicle group (same as control but pre-injected with the vehicle), and treatment group (similar to control but pre-injected with IAXO-102). We measured several biomarkers involved in IRI pathophysiology using enzyme-linked immunosorbent assay (ELISA), including High mobility group box1 (HMGB1), nuclear factor kappa b-p65 (NF-κB p65), interleukin beta-1 (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), 8-isoprostane, Bcl-2 associated X protein (BAX), heat shock protein 27 (HSP27), and Bcl-2. Statistical analysis was performed using one-way ANOVA and Tukey post hoc tests. Our results showed that IAXO-102 significantly improved kidney function, reduced histological alterations, and decreased the inflammatory response (IL-1, IL-6, and TNF) caused by IRI. IAXO-102 also decreased apoptosis by reducing pro-apoptotic Bax and increasing anti-apoptotic Bcl-2 without impacting HSP27. In conclusion, our findings suggest that IAXO-102 had a significant protective effect against IRI damage in the kidneys.

Role of Mesenchymal Stem/Stromal Cells in Modulating Ischemia/Reperfusion Injury: Current State of the Art and Future Perspectives

Ischemia/reperfusion injury (IRI) is a multistep damage that occurs in several tissues when a blood flow interruption is inevitable, such as during organ surgery or transplantation. It is responsible for cell death and tissue dysfunction, thus leading, in the case of transplantation, to organ rejection. IRI takes place during reperfusion, i.e., when blood flow is restored, by activating inflammation and reactive oxygen species (ROS) production, causing mitochondrial damage and apoptosis of parenchymal cells. Unfortunately, none of the therapies currently in use are definitive, prompting the need for new therapeutic approaches. Scientific evidence has proven that mesenchymal stem/stromal cells (MSCs) can reduce inflammation and ROS, prompting this cellular therapy to also be investigated for treatment of IRI. Moreover, it has been shown that MSC therapeutic effects were mediated in part by their secretome, which appears to be involved in immune regulation and tissue repair. For these reasons, mediated MSC paracrine function might be key for injury amelioration upon IRI damage. In this review, we highlight the scientific literature on the potential beneficial use of MSCs and their products for improving IRI outcomes in different tissues/organs, focusing in particular on the paracrine effects mediated by MSCs, and on the molecular mechanisms behind these effects.

2-Mercaptoethanol protects against DNA double-strand breaks after kidney ischemia and reperfusion injury through GPX4 upregulation.

Kidney ischemia reperfusion injury (IRI) is characterized by tubular cell death. DNA double-strand breaks is one of the major sources of tubular cell death induced by IRI. 2-Mercaptoethanol (2-ME) is protective against DNA double-strand breaks derived from calf thymus and bovine embryo. Here, we sought to determine whether treatment with 2-ME attenuated DNA double-strand breaks, resulting in reduced kidney dysfunction and structural damage in IRI. Kidney IRI or sham-operation in mice was carried out. The mice were treated with 2-ME, Ras-selective lethal 3, or vehicle. Kidney function, tubular injury, DNA damage, antioxidant enzyme expression, and DNA damage response (DDR) kinases activation were assessed. Treatment with 2-ME significantly attenuated kidney dysfunction, tubular injury, and DNA double-strand breaks after IRI. Among DDR kinases, IRI induced phosphorylation of ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR), but IRI reduced phosphorylation of other DDR kinases including ataxia telangiectasia and Rad3 related, checkpoint kinase 1 (Chk1), Chk2, and Chinese hamster cells 1 (XRCC1). Treatment with 2-ME enhanced phosphorylation of ATM and ATM-mediated effector kinases in IRI-subjected kidneys, suggesting that 2-ME activates ATM-mediated DDR signaling pathway. Furthermore, 2-ME dramatically upregulated glutathione peroxidase 4 (GPX4) in IRI-subjected kidneys. Inhibition of GPX4 augmented adverse IRI consequences including kidney dysfunction, tubular injury, DNA double-strand breaks, and inactivation of ATM-mediated DDR signaling pathway after IRI in 2-ME-treated kidneys. We have demonstrated that exogenous 2-ME protects against DNA double-strand breaks after kidney IRI through GPX4 upregulation and ATM activation.

Phosphoinositide 3 Kinase γ Plays a Critical Role in Acute Kidney Injury.

Inflammatory cells contribute to the pathogenesis of renal ischemia-reperfusion injury (IRI). However, the signaling mechanisms underlying the infiltration of inflammatory cells into the kidney are not well understood. In this study, we examined the effects of phosphoinositide 3 kinase γ (PI3Kγ) on inflammatory cells infiltration into the kidney in response to ischemia-reperfusion injury. Compared with wild-type mice, PI3Kγ knockout mice displayed less IRI in the kidney with fewer tubular apoptotic cell. Furthermore, PI3Kγ deficiency decreased the number of infiltrated neutrophils, macrophages, and T cells in the kidney, which was accompanied by a decrease in the expression of pro-inflammatory cytokines in the kidney. Moreover, wild-type mice treated with AS-605240, a selective PI3Kγ inhibitor, displayed less tubular damage, accumulated fewer inflammatory cells, and expressed less proinflammatory molecules in the kidney following IRI. These results demonstrate that PI3Kγ has a critical role in the pathogenesis of kidney damage in IRI, indicating that PI3Kγ inhibition may serve as a potential therapeutic strategy for the prevention of ischemia-reperfusion-induced kidney injury.

Human Amnion-Derived Mesenchymal Stromal/Stem Cells Pre-Conditioning Inhibits Inflammation and Apoptosis of Immune and Parenchymal Cells in an In Vitro Model of Liver Ischemia/Reperfusion.

Ischemia/reperfusion injury (IRI) represents one of the leading causes of primary non-function acute liver transplantation failure. IRI, generated by an interruption of organ blood flow and the subsequent restoration upon transplant, i.e., reperfusion, generates the activation of an inflammatory cascade from the resident Kupffer cells, leading first to neutrophils recruitment and second to apoptosis of the parenchyma. Recently, human mesenchymal stromal/stem cells (hMSCs) and derivatives have been implemented for reducing the damage induced by IRI. Interestingly, sparse data in the literature have described the use of human amnion-derived MSCs (hAMSCs) and, more importantly, no evidence regarding hMSCs priming on liver IRI have been described yet. Thus, our study focused on the definition of an in vitro model of liver IRI to test the effect of primed hAMSCs to reduce IRI damage on immune and hepatic cells. We found that the IFNγ pre-treatment and 3D culture of hAMSCs strongly reduced inflammation induced by M1-differentiated macrophages. Furthermore, primed hAMSCs significantly inhibited parenchymal apoptosis at early timepoints of reperfusion by blocking the activation of caspase 3/7. All together, these data demonstrate that hAMSCs priming significantly overcomes IRI effects in vitro by engaging the possibility of defining the molecular pathways involved in this process.

Ischemic-Free Liver Transplantation Reduces the Recurrence of Hepatocellular Carcinoma After Liver Transplantation.

Ischemia reperfusion injury (IRI) is an adverse factor for hepatocellular carcinoma (HCC) recurrence after liver transplantation. Ischemic-free liver transplantation (IFLT) is a novel transplant procedure that can largely reduce or even prevent IRI, but the clinical relevance of IFLT and the recurrence of HCC after liver transplantation are still unknown. This retrospective study compared survival outcomes, HCC recurrence, perioperative data and IRI severity following liver transplantation (LT). 30 patients received IFLT and 196 patients received conventional liver transplantation (CLT) were chosen for the entire cohort between June 2017 and August 2020. A 1:3 propensity score matching was performed, 30 IFLT recipients and 85 matched CLT patients were enrolled in propensity-matched cohorts. An univariate and multivariate Cox regression analysis was performed, and showed surgical procedure (CLT vs IFLT) was an independent prognostic factor (HR 3.728, 95% CI 1.172-11.861, P=0.026) for recurrence free survival (RFS) in HCC patients following liver transplantation. In the Kaplan–Meier analysis, the RFS rates at 1 and 3 years after LT in recipients with HCC in the IFLT group were significantly higher than those in the CLT group both in the entire cohort and propensity-matched cohort (P=0.006 and P=0.048, respectively). In addition, patients in the IFLT group had a lower serum lactate level, lower serum ALT level and serum AST level on postoperative Day 1. LT recipients with HCC in the IFLT group had a lower incidence of early allograft dysfunction than LT recipients with HCC in the CLT group. Histological analysis showed no obvious hepatocyte necrosis or apoptosis in IFLT group. In conclusion, IFLT can significantly reduce IRI damage and has the potential to be a useful strategy to reduce HCC recurrence after liver transplantation.

Interferon-γ-Induced Myeloid-Derived Suppressor Cells Aggravate Kidney Ischemia-Reperfusion Injury by Regulating Innate Immune Cells

Objective: Myeloid-derived suppressor cells (MDSCs) are heterogeneous cells which can suppress T-cell functionality. Herein, we evaluated the functional importance of MDSCs in the context of kidney ischemia-reperfusion injury (IRI) and explored their ability to regulate innate and adaptive immune cell function in this context. Methods: The differentiation of MDSCs was induced in vitro by treating cells with GM-CSF and interferon (IFN)-γ. In a murine model of renal IRI, serum creatinine and blood urea nitrogen values were measured to monitor kidney function, while histopathological and immunohistochemical approaches were used to assess kidney injury severity. In addition, flow cytometry was employed to assess the phenotypes and apoptosis of kidney cells in these mice. Results: MDSCs induced by treatment with GM-CSF + IFN-γ could suppress T-cell functionality in vitro. The adoptive transfer of these MDSCs into an IRI mouse model system enhanced kidney damage and impaired renal function following the recruitment of these cells to renal tissues in these mice. Following such adoptive transfer, the relative frequency of MDSCs with a CD11b⁺Ly6G⁻Ly6C^high monocytic-MDSC phenotype decreased, whereas cells with a CD11b⁺Ly6G⁺Ly6C^low polymorphonuclear-MDSC phenotype become more prevalent within kidney tissues following IRI. Adoptive transfer also coincided with increased frequencies of macrophages and dendritic cells (DCs) in the kidney tissues. This suggested that M-MDSCs contributed to early-stage renal IRI damage by differentiating into these deleterious cell types. However, MDSC-induced suppression of CD4⁺ and CD8⁺ T-cell infiltration was not sufficient to prevent the deterioration of renal function in these mice. Conclusions: Herein, we successfully developed a protocol wherein MDSCs were differentiated in vitro through combination GM-CSF/IFN-γ treatment. When these MDSCs were subsequently adoptively transferred into a murine model of renal IRI, they aggravated kidney damage, likely owing to the differentiation of M-MDSCs into deleterious macrophages and DCs.

PPARγ Plays an Important Role in Acute Hepatic Ischemia-Reperfusion Injury via AMPK/mTOR Pathway.

Background Hepatic ischemia-reperfusion (IR) injury is one of the severe complications associated with liver surgery and leads to liver dysfunction. PPARγ is always linked with various physiologic pathways, and it can alleviate liver damage in IR injury. Aim In this study, we explored the potential mechanism of PPARγ in the pathogenesis of hepatic IR injury by mice model. Methods After treated with si-PPARγ or rosiglitazone, mice were subjected to hepatic ischemia-reperfusion. Liver tissue and blood samples were collected to evaluate liver injury and detected relative mRNA and protein expressions. Results The expression of PPARγ was increased after reperfusion. And the alleviation of PPARγ aggravated the liver damage in IR; at the same time, upregulation of the expression of PPARγ released the liver damage. And these effects of PPARγ in IR were related to the AMPK/mTOR/autophagy signaling pathway. Conclusion PPARγ plays an important role in hepatic IR injury at least partly via the AMPK/mTOR/autophagy pathway.

Noscapine alleviates cerebral damage in ischemia-reperfusion injury in rats.

With high unmet medical needs, stroke remains an intensely focused research area. Although noscapine is a neuroprotective agent, its mechanism of action in ischemic-reperfusion (I-R) injury is yet to be ascertained. We investigated the effect of noscapine on the molecular mechanisms of cell damage using yeast, and its neuroprotection on cerebral I-R injury in rats. Yeast, both wild-type and Δtrx2 strains, was evaluated for cell growth and viability, and oxidative stress to assess the noscapine effect at 8, 10, 12, and 20μg/ml concentrations. The neuroprotective activity of noscapine (5 and 10mg/kg; po for 8days) was investigated in rats using middle cerebral artery occlusion-induced I-R injury. Infarct volume, neurological deficit, oxidative stress, myeloperoxidase activity, and histological alterations were determined in I-R rats. In vitro yeast assays exhibited significant antioxidant activity and enhanced cell tolerance against oxidative stress after noscapine treatment. Similarly, noscapine pretreatment significantly reduced infarct volume and edema in the brain. The neurological deficit was decreased and oxidative stress biomarkers, superoxide dismutase activity and glutathione levels, were significantly increased while lipid peroxidation showed significant decrease in comparison to vehicle-treated I-R rats. Myeloperoxidase activity, an indicator of inflammation, was also reduced significantly in treated rats; histological changes were attenuated with noscapine. The study demonstrates the protective effect of noscapine in yeast and I-R rats by improving cell viability and attenuating neuronal damage, respectively. This protective activity of noscapine could be attributed to potent free radical scavenging and inhibition of inflammation in cerebral ischemia-reperfusion injury.

Evaluation of Glutaredoxin 1 as a Novel Marker of Renal Damage after Ischemia‐Reperfusion Injury in Mice

Renal ischemia‐reperfusion injury (IRI) is a significant cause of acute renal failure and delayed graft function in clinical patients. Tissue damage in renal IRI is caused in part by the activation of the immune response and the generation of reactive oxygen species (ROS), both taking place mainly during the reperfusion phase, once the blood supply to the tissue is restored. Glutaredoxin 1 (Grx1), a cytosolic protein that reduces protein disulfides as well as glutathionylated proteins, plays a protective role in ROS‐mediated damage to the cell. In a previous work, we detected increased levels of Grx1 in the urine of mice 24h after renal IRI 1. Recently, Grx1 was shown to be elevated in the serum of patients with chronic kidney disease 2. To determine the usefulness of Grx1 as a possible biomarker of ischemic renal damage we evaluated serum, urinary, as well as intra‐renal levels of Grx1 in a mouse model of renal IRI. Different reperfusion times (4h, 8h, 16h, 24h, 48h, 96h, and 168h) were compared in order to follow Grx1 fluctuation levels in the kidney, serum, and urine. Serum Grx1 levels, analyzed by ELISA, were correlated with several renal function parameters such as serum creatinine, proteinuria, glomerular diameter, and tubular thickness. After IRI, Grx1 serum levels increased much earlier than creatinine (4h versus 24h). Western blot analyses showed a decrease of Grx1 in the kidney of mice with surgically induced IRI, compared to the sham group. Additionally, immunohistochemistry revealed an increased apical localization of Grx1 in cells of the distal convoluted tubules of the mice exhibiting IRI. Urinary levels of Grx1, even if increased in the first hours after renal IRI, were not significantly altered. These findings provide initial supporting evidence for the use of a redox protein such as Grx1 as a novel biomarker for assessing renal damage under experimental conditions. Further studies are necessary to validate these results.Support or Funding InformationThis work was funded by the Center of Integrative Mammalian Research of Ross University School of Veterinary Medicine.

Renal tubular arginase-2 participates in the formation of the corticomedullary urea gradient and attenuates kidney damage in ischemia-reperfusion injury in mice.

Arginase 2 (ARG2) is a mitochondrial enzyme that catalyses hydrolysis of l-arginine into urea and l-ornithine. In the kidney, ARG2 is localized to the S3 segment of the proximal tubule. It has been shown that expression and activity of this enzyme are upregulated in a variety of renal pathologies, including ischemia-reperfusion (IR) injury. However, the (patho)physiological role of ARG2 in the renal tubule remains largely unknown. We addressed this question in mice with conditional knockout of Arg2 in renal tubular cells (Arg2lox/lox /Pax8-rtTA/LC1 or, cKO mice). We demonstrate that cKO mice exhibit impaired urea concentration and osmolality gradients along the corticomedullary axis. In a model of unilateral ischemia-reperfusion injury (UIRI) with an intact contralateral kidney, ischemia followed by 24hours of reperfusion resulted in significantly more pronounced histological damage in ischemic kidneys from cKO mice compared to control and sham-operated mice. In parallel, UIRI-subjected cKO mice exhibited a broad range of renal functional abnormalities, including albuminuria and aminoaciduria. Fourteen days after UIRI, the cKO mice exhibited complex phenotype characterized by significantly lower body weight, increased plasma levels of early predictive markers of kidney disease progression (asymmetric dimethylarginine and symmetric dimethylarginine), impaired mitochondrial function in the ischemic kidney but no difference in kidney fibrosis as compared to control mice. Collectively, these results establish the role of ARG2 in the formation of corticomedullary urea and osmolality gradients and suggest that this enzyme attenuates kidney damage in ischemia-reperfusion injury.

Renal tubular Arginase-2 participates in the formation of the corticomedullary urea gradient and attenuates kidney damage in ischemia-reperfusion injury in mice

Renal tubular Arginase-2 participates in the formation of the corticomedullary urea gradient and attenuates kidney damage in ischemia-reperfusion injury in mice

Labile Heme Aggravates Renal Inflammation and Complement Activation After Ischemia Reperfusion Injury.

Background: Ischemia reperfusion injury (IRI) plays a major role in solid organ transplantation. The length of warm ischemia time is critical for the extent of tissue damage in renal IRI. In this experimental study we hypothesized that local release of labile heme in renal tissue is triggered by the duration of warm ischemia (15 vs. 45 min IRI) and mediates complement activation, cytokine release, and inflammation.Methods: To induce IRI, renal pedicle clamping was performed in male C57BL/6 mice for short (15 min) or prolonged (45 min) time periods. Two and 24 h after experimental ischemia tissue injury labile heme levels in the kidney were determined with an apo-horseradish peroxidase assay. Moreover, renal injury, cytokines, and C5a and C3a receptor (C5aR, C3aR) expression were determined by histology, immunohistochemistry and qPCR, respectively. In addition, in vitro studies stimulating bone marrow-derived macrophages with LPS and the combination of LPS and heme were performed and cytokine expression was measured.Results: Inflammation and local tissue injury correlated with the duration of warm ischemia time. Labile heme concentrations in renal tissue were significantly higher after prolonged (45 min) as compared to short (15 min) IRI. Notably, expression of the inducible heme-degrading enzyme heme oxygenase-1 (HO-1) was up-regulated in kidneys after prolonged, but not after short IRI. C5aR, the pro-inflammatory cytokines IL-6 and TNF-α as well as pERK were up-regulated after prolonged, but not after short ischemia times. Consecutively, neutrophil infiltration and up-regulation of pro-fibrotic cytokines such as CTGF and PAI were more pronounced in prolonged IRI in comparison to short IRI. In vitro stimulation of macrophages with LPS revealed that IL-6 expression was enhanced in the presence of heme. Finally, administration of the heme scavenger human serum albumin (HSA) reduced the expression of pro-inflammatory cytokines, C3a receptor and improved tubular function indicated by enhanced alpha 1 microglobulin (A1M) absorption after IRI.Conclusions: Our data show that prolonged duration of warm ischemia time increased labile heme levels in the kidney, which correlates with IRI-dependent inflammation and up-regulation of anaphylatoxin receptor expression.