Proteomic analysis of mitochondria associated membranes in renal ischemic reperfusion injury.

BACKGROUND: The mitochondria and endoplasmic reticulum (ER) communicate via contact sites known as mitochondria associated membranes (MAMs). Many important cellular functions such as bioenergetics, mitophagy, apoptosis, and calcium signaling are regulated by MAMs, which are thought to be closely related to ischemic reperfusion injury (IRI). However, there exists a gap in systematic proteomic research addressing the relationship between these cellular processes. METHODS: A 4D label free mass spectrometry-based proteomic analysis of mitochondria associated membranes (MAMs) from the human renal proximal tubular epithelial cell line (HK-2 cells) was conducted under both normal (N) and hypoxia/reperfusion (HR) conditions. Subsequent differential proteins analysis aimed to characterize disease-relevant signaling molecules. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was applied to total proteins and differentially expressed proteins, encompassing Biological Process (BP), Cell Component (CC), Molecular Function (MF), and KEGG pathways. Further, Protein-Protein Interaction Network (PPI) exploration was carried out, leading to the identification of hub genes from differentially expressed proteins. Notably, Mitofusion 2 (MFN2) and BCL2/Adenovirus E1B 19-kDa interacting protein 3(BNIP3) were identified and subsequently validated both in vitro and in vivo. Finally, the impact of MFN2 on MAMs during hypoxia/reoxygenation was explored through regulation of gene expression. Subsequently, a comparative proteomics analysis was conducted between OE-MFN2 and normal HK-2 cells, providing further insights into the underlying mechanisms. RESULTS: A total of 4489 proteins were identified, with 3531 successfully quantified. GO/KEGG analysis revealed that MAM proteins were primarily associated with mitochondrial function and energy metabolism. Differential analysis between the two groups showed that 688 proteins in HR HK-2 cells exhibited significant changes in expression level with P-value < 0.05 and HR/N > 1.5 or HR/N < 0.66 set as the threshold criteria. Enrichment analysis of differentially expressed proteins unveiled biological processes such as mRNA splicing, apoptosis regulation, and cell division, while molecular functions were predominantly associated with energy metabolic activity. These proteins play key roles in the cellular responses during HR, offering insights into the IRI mechanisms and potential therapeutic targets. The validation of hub genes MFN2 and BNIP3 both in vitro and vivo was consistent with the proteomic findings. MFN2 demonstrated a protective role in maintaining the integrity of mitochondria associated membranes (MAMs) and mitigating mitochondrial damage following hypoxia/reoxygenation injury, this protective effect may be associated with the activation of the PI3K/AKT pathway. CONCLUSIONS: The proteins located in mitochondria associated membranes (MAMs) are implicated in crucial roles during renal ischemic reperfusion injury (IRI), with MFN2 playing a pivotal regulatory role in this context.

Narirutin Attenuates Cerebral Ischemia-Reperfusion Injury by Suppressing the TXNIP/NLRP3 Pathway.

Narirutin (Nar) is a flavonoid that is abundantly present in citrus fruits and has attracted considerable attention because of its diverse pharmacological activities and low toxicity. Here, we evaluated the preventive effects of Nar in middle cerebral artery occlusion/reperfusion (MCAO/R)-injured mice and oxygen-glucose deprivation/reperfusion (OGD/R)-injured bEnd.3 cells. Pretreatment with Nar (150 mg/kg) for 7 days effectively reduced infarct volume, improved neurological deficits, and significantly inhibited neuronal death in the hippocampus and cortex in MCAO/R-injured mice. Moreover, anti-apoptotic effects of Nar (50 µM) were observed in OGD/R-injured bEnd.3 cells. In addition, Nar pre-administration regulated blood-brain barrier function by increasing tight junction-related protein expression after MCAO/R and OGD/R injury. Nar also inhibited NOD-like receptor protein 3 (NLRP3) inflammasome activation by reducing the expression of thioredoxin-interacting protein (TXNIP) in vivo and in vitro. Taken together, these results provide new evidence for the use of Nar in the prevention and treatment of ischemic stroke.

Liver-specific delivery of spherical DNA frameworks for alleviation of hepatic ischemic reperfusion injury.

DNA nanostructures have long been developed for biomedical purposes, but their controlled delivery in vivo proposes a major challenge for disease theranostics. We previously reported that DNA nanostructures on the scales of tens and hundreds nanometers showed preferential renal excretion or kidney retention, allowing for sensitive evaluation and effective protection of kidney function, in response to events such as unilateral ureter obstruction or acute kidney injury. Encouraged by the positive results, we redirected our focus to the liver, specifically targeting organs noticeably lacking DNA materials, to explore the interaction between DNA nanostructures and the liver. Through PET imaging, we identified SDF and M13 as DNA nanostructures exhibiting significant accumulation in the liver among numerous candidates. Initially, we investigated and assessed their biodistribution, toxicity, and immunogenicity in healthy mice, establishing the structure-function relationship of DNA nanostructures in the normal murine. Subsequently, we employed a mouse model of liver ischemia-reperfusion injury (IRI) to validate the nano-bio interactions of SDF and M13 under more challenging pathological conditions. M13 not only exacerbated hepatic oxidative injury but also elevated local apoptosis levels. In contrast, SDF demonstrated remarkable ability to scavenge oxidative responses in the liver, thereby mitigating hepatocyte injury. These compelling results underscore the potential of SDF as a promising therapeutic agent for liver-related conditions. This aimed to elucidate their roles and mechanisms in liver injury, providing a new perspective for the biomedical applications of DNA nanostructures.

Sex-Dependent Differences in the Ischemia/Reperfusion-Induced Expression of AMPA Receptors.

Following ischemia/reperfusion, AMPA receptors (AMPARs) mediate pathologic delayed neuronal death through sustained expression of calcium-permeable AMPARs, leading to excitotoxicity. Preventing the surface removal of GluA2-containing AMPARs may yield new therapeutic targets for the treatment of ischemia/reperfusion. This study utilized acute organotypic hippocampal slices from aged male and female Sprague Dawley rats and subjected them to oxygen-glucose deprivation/reperfusion (OGD/R) to examine the mechanisms underlying the internalization and degradation of GluA2-containing AMPARs. We determined the effect of OGD/R on AMPAR subunits at the protein and mRNA transcript levels utilizing Western blot and RT-qPCR, respectively. Hippocampal slices from male and female rats responded to OGD/R in a paradoxical manner with respect to AMPARs. GluA1 and GluA2 AMPAR subunits were degraded following OGD/R in male rats but were increased in female rats. There was a rapid decrease in GRIA1 (GluA1) and GRIA2 (GluA2) mRNA levels in the male hippocampus following ischemic insult, but this was not observed in females. These data indicate a sex-dependent difference in how AMPARs in the hippocampus respond to ischemic insult, and may help explain, in part, why premenopausal women have a lower incidence/severity of ischemic stroke compared with men of the same age.

Focusing on Ischemic Reperfusion Injury in the New Era of Dynamic Machine Perfusion in Liver Transplantation.

Liver transplantation is the most effective treatment for end-stage liver disease. Transplant indications have been progressively increasing, with a huge discrepancy between the supply and demand of optimal organs. In this context, the use of extended criteria donor grafts has gained importance, even though these grafts are more susceptible to ischemic reperfusion injury (IRI). Hepatic IRI is an inherent and inevitable consequence of all liver transplants; it involves ischemia-mediated cellular damage exacerbated upon reperfusion and its severity directly affects graft function and post-transplant complications. Strategies for organ preservation have been constantly improving since they first emerged. The current gold standard for preservation is perfusion solutions and static cold storage. However, novel approaches that allow extended preservation times, organ evaluation, and their treatment, which could increase the number of viable organs for transplantation, are currently under investigation. This review discusses the mechanisms associated with IRI, describes existing strategies for liver preservation, and emphasizes novel developments and challenges for effective organ preservation and optimization.

Neat1 promotes acute kidney injury to chronic kidney disease by facilitating tubular epithelial cells apoptosis via sequestering miR-129-5p.

Acute kidney injury (AKI) is increasingly identified as a crucial risk factor for progression to CKD. However, the factors governing AKI to CKD progression remain largely unknown. By high-throughput RNA sequencing, we found that Neat1_2, a transcript variant of Neat1, was upregulated in 40-min ischemia/reperfusion injury (IRI), which resulted in the development of renal fibrotic lesions. The upregulation of Neat1_2 in hypoxia-treated TECs was attributed to p53 transcriptional regulation. Gain- and loss-of-function studies, both in vitro and in vivo, demonstrated that Neat1_2 promoted apoptosis of injured TECs induced by IRI and caused tubulointerstitial inflammation and fibrosis. Mechanistically, Neat1_2 shares miRNA response elements with FADD, CASP-8, and CASP-3. Neat1_2 competitively binds to miR-129-5p and prevents miR-129-5p from decreasing the levels of FADD, CASP-8, and CASP-3, and ultimately facilitates TEC apoptosis. Increased expression of Neat1_2 associated with kidney injury and TEC apoptosis was recapitulated in human AKI, highlighting its clinical relevance. These findings suggest that preventing TEC apoptosis by hindering Neat1_2 expression may be a potential therapeutic strategy for AKI to CKD progression.

Immunohistochemical Pattern of Histone H2A Variant Expression in an Experimental Model of Ischemia-Reperfusion-Induced Acute Kidney Injury.

Ischemia-reperfusion injury (IRI) is a frequent cause of AKI, resulting in vasoconstriction, cellular dysfunction, inflammation and the induction of oxidative stress. DNA damage, including physical DNA strand breaks, is also a potential consequence of renal IRI. The histone H2A variants, primary H2AX and H2AZ participate in DNA damage response pathways to promote genome stability. The aim of this study was to evaluate the immunohistochemical pattern of histone H2A variants' (H2AX, γH2AX(S139), H2AXY142ph and H2AZ) expression in an experimental model of ischemia-reperfusion-induced acute kidney injury in spontaneously hypertensive rats. Comparing the immunohistochemical nuclear expression of γH2AX(S139) and H2AXY142ph in AKI, we observed that there is an inverse ratio of these two histone H2AX variants. If we follow different regions from the subcapsular structures to the medulla, there is an increasing extent gradient in the nuclear expression of H2AXY142ph, accompanied by a decreasing nuclear expression of γH2AX. In addition, we observed that different structures dominated when γH2AX and H2AXY142ph expression levels were compared. γH2AX was expressed only in the proximal tubule, with the exception of when they were dilated. In the medulla, H2AXY142ph is predominantly expressed in the loop of Henle and the collecting ducts. Our results show moderate sporadic nuclear H2AZ expression mainly in the cells of the distal tubules and the collecting ducts that were surrounded by dilated tubules with PAS (periodic acid-Schiff stain)-positive casts. These findings may indicate the degree of DNA damage, followed by postischemic AKI, with potential clinical and prognostic implications regarding this condition.

Effect of the Ligation and Reperfusion Timeframe on Maximal Ischemia-Reperfusion Injury in Diverse Rat Models.

Background: Little is known about the effect that different time sequences for coronary ligation and reperfusion have on ischemic-reperfusion (IR) injury. Objective: To investigate the relationship between the extent of IR injury and the timeframe for coronary ligation/reperfusion in three animal models. Methods: Three rat models were used: normal Sprague-Dawley rats, diabetes mellitus (DM) rats, and fat rats. The rats in each model were divided into four groups based on the coronary ligation period (L): 30, 60, 120, and 180 min, and then divided into seven sub-groups based on the reperfusion period (R): 0, 30, 60, 120, 180, 270, and 360 min. R0 was the IR injury baseline for each sub-group. The hearts were harvested and stained with Evans blue and 2,3,5-triphenyl tetrazolium chloride dye to distinguish the different myocardial injury areas: area at risk (AAR) and myocardial necrosis. The difference between each subgroup and baseline (R0) for the necrotic area/AAR was calculated. Results: In the normal rats, the highest IR injury differences compared with the baseline group occurred at L120, with a reperfusion time of > 180 min. The highest IR injury difference compared to the baseline group occurred at L30, with a reperfusion time of > 180 min in the DM rats and at L60R270, L120R180 in the fat rats. Conclusions: IR injury, as induced by different coronary ligation and reperfusion time intervals, had diverse expression profiles in the different animal models. Optimal animal models with optimal coronary ligation/reperfusion protocols to achieve maximal IR injury will affect the results and interpretation of future studies.

Neuroprotective Effect of Piclamilast-Induced Post-Ischemia Pharmacological Treatment in Mice.

Various studies have evidenced the neuroprotective role of PDE4 inhibitors. However, whether PDE4 inhibitor, Piclamilast pharmacological post-treatment is protective during cerebral ischemia reperfusion-induced injury remains unknown. Therefore, this study design included testing the hypothesis that Piclamilast administered at the beginning of a reperfusion phase (Piclamilast pPost-trt) shows protective effects and explores & probes underlying downstream mechanisms. Swiss albino male mice were subjected to global ischemic and reperfusion injury for 17 min. The animals examined cerebral infarct size, biochemical parameters, inflammatory mediators, and motor coordination. For memory, assessment mice were subjected to morris water maze (MWM) and elevated plus maze (EPM) test. Histological changes were assessed using HE staining. Piclamilast pPost-trt significantly reduced I/R injury-induced deleterious effects on biochemical parameters of oxidative stress, inflammatory parameters, infarct size, and histopathological changes, according to the findings. These neuroprotective effects of pPost-trt are significantly abolished by pre-treatment with selective CREB inhibitor, 666-15. Current study concluded that induced neuroprotective benefits of Piclamilast Post-trt, in all probability, maybe mediated through CREB activation. Hence, its neuroprotective effects can be further explored in clinical settings.

Silencing of circCDC14A prevents cerebral ischemia-reperfusion injury via miR-23a-3p/CXCL12 axis.

Ischemic stroke is one of the main causes of death and disability. Circular RNAs (circRNAs) have received extensive attention in the pathogenesis of ischemic stroke. Here, we evaluated the role of circCDC14A in cerebral ischemia-reperfusion (CI/R) injury in vivo and in vitro. The expression of circCDC14A was significantly upregulated in the middle cerebral artery occlusion (MCAO) model and oxygen and glucose deprivation/reoxygenation (OGD/R)-treated HT22 cells. Knockdown of circCDC14A suppressed the cell viability reduction caused by OGD/R, as well as cell damage and apoptosis. Mechanistically, circCDC14A acted as a sponge for miR-23a-3p and promoted the expression of chemokine stromal-derived factor-1 (CXCL12) by negatively regulating miR-23a-3p. Rescue experiments further confirmed that miR-23a-3p inhibitor or circCDC14A-overexpression vectors blocked the beneficial effects of circCDC14A knockdown in OGD/R-induced HT22 cells. Moreover, knockdown of circCDC14A suppressed MCAO-induced cerebral infarction and neurological damage, as well as the brain tissue damage and neuronal apoptosis in vivo. Consistently, miR-23a-3p antagomir treatment abolished the cerebral protective effects of circCDC14A knockdown on MCAO mice. In conclusion, circCDC14A promoted CI/R injury by regulating the miR-23a-3p/CXCL12 axis, which suggested that circCDC14A may become a potential therapeutic target for CI/R injury.

Renal denervation alleviates renal ischemic reperfusion injury-induced acute and chronic kidney injury in rats partly by modulating miRNAs.

BACKGROUND: Renal denervation (RDN) has been used to promote kidney injury repair, whereas miRNAs have been found to be involved in the pathophysiology of renal injury. However, the miRNA alterations that occur after RDN and the related protective mechanisms remain to be determined. METHODS: Renal ischemic reperfusion injury (IRI) rat model was established and RDN was performed. Animals were killed at 24 h and 2 weeks following the operation. Tyrosine hydroxylase (TH) levels, renal function, tubular cell apoptosis and histological sections were examined at 24 h, whereas renal fibrosis and capillary vessels were assessed at 2 weeks. Furthermore, the expression of miRNAs in the injured kidney was determined using micro-array and the target genes were analyzed. RESULTS: We found that TH was eliminated and that renal function was improved in the denervation group at 24 h. RDN reduced tubular cell apoptosis and mitigated the histological lesion. Furthermore, an increase of capillary vessel density and reduction of renal fibrosis were observed after 2 weeks. Moreover, the numbers of miRNAs were up-regulated after RDN treatment, and the miRNAs targeted pro-angiogenic, anti-fibrotic and inflammatory pathways. CONCLUSIONS: RDN is a reliable method for alleviating IRI-induced acute and chronic kidney injury, and modulating the miRNA-related pro-angiogenic, anti-fibrotic or inflammatory pathways involved in this process.

Is there life on the airway tree? A pilot study of bronchial cell vitality and tissue morphology in the ex vivo lung perfusion (EVLP) era of lung transplantation.

BACKGROUND: Ex vivo lung perfusion (EVLP) is a relevant procedure to increase the lung donor pool but could potentially increase the airway tree ischemic injury risk. METHODS: This study aimed to evaluate the direct effect of EVLP on the airway tree by evaluating bronchial cell vitality and tissue signs of injury on a series of 117 bronchial rings collected from 40 conventional and 19 EVLP-treated lung grafts. Bronchial rings and related scraped bronchial epithelial cells were collected before the EVLP procedure and surgical anastomosis. RESULTS: The preimplantation interval was significantly increased in the EVLP graft group (p < 0.01). Conventional grafts presented cell viability percentages of 47.07 ± 23.41 and 49.65 ± 21.25 in the first and second grafts which did not differ significantly from the EVLP group (first graft 50.54 ± 25.83 and second graft 50.22 ± 20.90 cell viability percentage). No significant differences in terms of histopathological features (edema, inflammatory infiltrate, and mucosa ulceration) were observed comparing conventional and EVLP samples. A comparison of bronchial cell viability and histopathology of EVLP samples retrieved at different time intervals revealed no significant differences. Accordingly, major bronchial complications after lung transplant were not observed in both groups. CONCLUSIONS: Based on these data, we observed that EVLP did not significantly impact bronchial cell vitality and airway tissue preservation nor interfere with bronchial anastomosis healing, further supporting it as a safe and useful procedure.

Regulation of the autophagy plays an important role in acute kidney injury induced acute lung injury.

AIM: This study aimed to investigate the regulatory role of autophagy in acute kidney injury (AKI) induced acute lung injury (ALI). METHODS: The male Sprague-Dawley rats were divided into four groups: normal saline-treated sham rats (sham group), normal saline-treated ischemia-reperfusion injury rats (IRI group), 3-methyladenine-treated IRI rats (3-MA group), and rapamycin-treated IRI rats (RA group). The rats in the IRI rat model received the nephrectomy of the right kidney and was subjected to 60 mins of left renal pedicle occlusion, followed by 12, 24, 48, and 72 h of reperfusion. The levels of Scr, BUN, wet-to-dry ratio of lung, inflammatory cytokines, and oxidative stress were determined. The damage to tissues was detected by histological examinations. The western blot and immunohistochemistry methods were conducted to determine the expression of indicated proteins. RESULTS: Renal IRI could induce the pulmonary injury after AKI, which caused significant increases in the function index of pulmonary and renal, the levels of inflammatory cytokines, and biomarkers of oxidative stress. In comparison to the IRI group, the RA group showed significantly decreased P62 and Caspase-3 expression and increased LC-II/LC3-I, Beclin-1, Bcl-2, and unc-51-like autophagy activating kinase 1 expression. Meanwhile, by suppressing the inflammation and oxidative stress, as well as inhibiting the pathological lesions in kidney and lung tissues, the autophagy could effectively ameliorate IRI-induced AKI and ALI. CONCLUSIONS: Autophagy plays an important role in AKI-induced ALI, which could be used as a new target for AKI therapy and reduce the mortality caused by the complication.

A Proof-of-Concept Preclinical Study Using a Novel Thermal Insulation Device in a Porcine Kidney Auto-Transplantation Model.

Ischemia-reperfusion injury remains a fundamental problem during organ transplantation logistics. One key technical factor is the rapid allograft rewarming during the time of vascular reconstruction in the recipient. In this pilot study, a new thermal insulation bag (TIB) for organ transplantation was used. Insulation capacity, tissue compatibility, and usability were tested initially ex vivo on porcine kidneys (n = 24) followed by the first in vivo usage. Fourteen female German landrace pigs underwent kidney auto-transplantation after 24 h cold storage (4 °C). During the implantation process the kidney was either insulated with the new TIB, or it was not thermo-protected at all, which represents the clinical standard. In this proof-of-concept study, the usability (knife-to-skin-time) and the general thermal capacity (30 min warm storage at 38 °C ex vivo p < 0.001) was shown. The clinical outcome showed significant differences in the determination of CRP and pi-GST levels. Syndecan-1 Antibody staining showed clear significant higher counts in the control group (p < 0.01) indicating epithelial damage. However, the effect on renal outcomes in not severely pre-damaged kidneys does not appear to be conclusively significant. A close follow-up study is warranted, especially in the context of marginal organs or in cases where anastomosis-times are prolonged due to surgical complexity (e.g., multiple vessels and complex reconstructions).

Head-to-head comparison of multiple cardiovascular magnetic resonance techniques for the detection and quantification of intramyocardial haemorrhage in patients with ST-elevation myocardial infarction.

OBJECTIVES: T2*-weighted (T2*w) is deemed as a reference standard for post-infarction intramyocardial haemorrhage (IMH). However, high proportion of T2* images is affected by off-resonance artefacts hampering image interpretation. Diagnostic accuracy and precision of alternative techniques for IMH diagnosis and quantification have been seldomly investigated. METHODS AND RESULTS: Between April 2016 and May 2017, 50 ST-segment elevation myocardial infarction patients (66% male, 57 ± 17 years) and 15 healthy controls (60% male, 58 ± 13) were consecutively enrolled. Subjects underwent head-to-head comparison of single mid-infarct slice acquired on black-blood T2-weighted short-TI-inversion recovery (T2w-STIR), bright-blood T2prep-steady-state-free precession (T2prep-SSFP), and T2/T1 maps for IMH diagnosis and quantification against T2*w. All images were graded for quality (grade 1: very poor; grade 4: excellent) and diagnostic confidence (Likert scale, 1: very unsure and 5: highly confident). Reduced relaxation time/hypointense region (hypocore) embedded in infarct-related oedema on T2 map, T1 map, and T2w-STIR had the best overall diagnostic accuracy (per-subject: 91%, 86%, and 86%, respectively; per segment: 95%, 93%, and 93%, respectively). By mixed-effects analysis, image quality, and diagnostic confidence were higher for T2 map and T1 maps than T2*w (p < 0.05 for both scores). For IMH quantification, hypocore on T2 map and T1 map strongly correlated (Spearman's r > 0.7, p < 0.001 for both) with IMH extent on T2*w and presented an overall excellent agreement on Bland-Altman analysis. By linear mixed model analysis, absolute hypocore size did not differ among T1-, T2 map, and T2*w. T2/T1 maps had the best intra- and inter-observer reproducibility among CMR techniques. CONCLUSION: Hypocore on T2/T1 map is the best alternative technique to T2*w for diagnosing and quantifying IMH in post-STEMI patients. KEY POINT: • Mapping techniques are the best alternatives for diagnosing post-infarction intramyocardial haemorrhage. • Mapping techniques are valuable tools for imaging intramyocardial haemorrhage.

Ischemic postconditioning improves the outcome of organs from donors after cardiac death in a pig liver transplantation model and provides synergistic protection with hypothermic machine perfusion.

AIM: This study investigated whether ischemic postconditioning (IPO) improved the outcome of organs from donors after cardiac death and had a synergistic effect with hypothermic machine perfusion (HMP) in a pig liver transplantation model. METHODS: A donor after cardiac death (DCD) model was developed in 48 healthy Bama miniature pigs randomly divided into four groups: simple cold storage group (SCS group), IPO group, HMP group, HMP-IPO group. The levels of serum alanine aminotransferase (ALT), total bilirubin, histopathological findings, apoptotic activity of hepatocytes, international normalized ratio (INR), tumor necrosis factor-α (TNF-α), and Malondialdehyde (MDA) were compared. RESULTS: All recipients in the SCS group died within 6 h after transplantation. The livers of the recipients in the IPO had 50% survival on day 5. HMP allowed 83.3% survival and HMP-IPO allowed 100% survival. After reperfusion, the recipients in the IPO and HMP-IPO group had lower ALT and total bilirubin levels, less Suzuki score, less apoptosis, and less injury to hepatocytes and biliary ducts and attenuated inflammatory response and oxidative load. CONCLUSIONS: IPO improved the outcome of organs from donors after cardiac death and had a synergistic effect with HMP in the pig liver transplantation model.

microRNA-186 alleviates oxygen-glucose deprivation/reoxygenation-induced injury by directly targeting hypoxia-inducible factor-1α.

Previous studies have suggested that microRNA-186 (miR-186) can be induced under hypoxic conditions, and is associated with apoptosis. This study was undertaken to explore the exact role of this microRNA (miRNA) in the apoptotic death of neurons during cerebral ischemic/reperfusion (I/R) injury. To model cerebral ischemia/reperfusion (I/R) injuries, we utilized a transient middle cerebral artery occlusion approach in rats, as well as a model of oxygen-glucose deprivation/reoxygenation (OGD/R) in Neuro2a cells. We found that in both in vitro and in vivo models of cerebral I/R injuries, levels of miR-186 were markedly decreased. When we overexpressed miR-186, this was associated with a reduction in the apoptotic death of neuroblastoma cells in the OGD/R model system, whereas the opposite was true when this miRNA was instead inhibited. We further found miR-186 to directly target hypoxia-inducible factor 1α (HIF-1α) by interacting with the 3'-untranslated region of this mRNA. When we knocked down HIF-1α, this partially overcame the apoptotic death of cells in response to OGD/R injury and associated miR-186 downregulation. Our findings indicate that miR-186 is able to reduce ischemic injury to neurons at least in part through downregulating HIF-1α, suggesting that the miR-186/HIF-1α axis is a potential therapeutic target for the treatment of ischemic stroke.

Assessing Kidney Graft Viability and Its Cells Metabolism during Machine Perfusion.

Kidney transplantation is the golden treatment for end-stage renal disease. Static cold storage is currently considered the standard method of preservation, but dynamic techniques, such as machine perfusion (MP), have been shown to improve graft function, especially in kidneys donated by extended criteria donors and donation after circulatory death. With poor organ quality being a major reason for kidneys not being transplanted, an accurate, objective and reliable quality assessment during preservation could add value and support to clinicians' decisions. MPs are emerging technologies with the potential to assess kidney graft viability and quality, both in the hypothermic and normothermic scenarios. The aim of this review is to summarize current tools for graft viability assessment using MP prior to implantation in relation to the ischemic damage.

Oxidative Stress Underlies the Ischemia/Reperfusion-Induced Internalization and Degradation of AMPA Receptors.

Stroke is the fifth leading cause of death annually in the United States. Ischemic stroke occurs when a blood vessel supplying the brain is occluded. The hippocampus is particularly susceptible to AMPA receptor-mediated delayed neuronal death as a result of ischemic/reperfusion injury. AMPA receptors composed of a GluA2 subunit are impermeable to calcium due to a post-transcriptional modification in the channel pore of the GluA2 subunit. GluA2 undergoes internalization and is subsequently degraded following ischemia/reperfusion. The subsequent increase in the expression of GluA2-lacking, Ca2+-permeable AMPARs results in excitotoxicity and eventually delayed neuronal death. Following ischemia/reperfusion, there is increased production of superoxide radicals. This study describes how the internalization and degradation of GluA1 and GluA2 AMPAR subunits following ischemia/reperfusion is mediated through an oxidative stress signaling cascade. U251-MG cells were transiently transfected with fluorescently tagged GluA1 and GluA2, and different Rab proteins to observe AMPAR endocytic trafficking following oxygen glucose-deprivation/reperfusion (OGD/R), an in vitro model for ischemia/reperfusion. Pretreatment with Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), a superoxide dismutase mimetic, ameliorated the OGD/R-induced, but not agonist-induced, internalization and degradation of GluA1 and GluA2 AMPAR subunits. Specifically, MnTMPyP prevented the increased colocalization of GluA1 and GluA2 with Rab5, an early endosomal marker, and with Rab7, a late endosomal marker, but did not affect the colocalization of GluA1 with Rab11, a marker for recycling endosomes. These data indicate that oxidative stress may play a vital role in AMPAR-mediated cell death following ischemic/reperfusion injury.

Rapamycin Alternatively Modifies Mitochondrial Dynamics in Dendritic Cells to Reduce Kidney Ischemic Reperfusion Injury.

Dendritic cells (DCs) are unique immune cells that can link innate and adaptive immune responses and Immunometabolism greatly impacts their phenotype. Rapamycin is a macrolide compound that has immunosuppressant functions and is used to prevent graft loss in kidney transplantation. The current study evaluated the therapeutic potential of ex-vivo rapamycin treated DCs to protect kidneys in a mouse model of acute kidney injury (AKI). For the rapamycin single (S) treatment (Rapa-S-DC), Veh-DCs were treated with rapamycin (10 ng/mL) for 1 h before LPS. In contrast, rapamycin multiple (M) treatment (Rapa-M-DC) were exposed to 3 treatments over 7 days. Only multiple ex-vivo rapamycin treatments of DCs induced a persistent reprogramming of mitochondrial metabolism. These DCs had 18-fold more mitochondria, had almost 4-fold higher oxygen consumption rates, and produced more ATP compared to Veh-DCs (Veh treated control DCs). Pathway analysis showed IL10 signaling as a major contributing pathway to the altered immunophenotype after Rapamycin treatment compared to vehicle with significantly lower cytokines Tnfa, Il1b, and Il6, while regulators of mitochondrial content Pgc1a, Tfam, and Ho1 remained elevated. Critically, adoptive transfer of rapamycin-treated DCs to WT recipients 24 h before bilateral kidney ischemia significantly protected the kidneys from injury with a significant 3-fold improvement in kidney function. Last, the infusion of DCs containing higher mitochondria numbers (treated ex-vivo with healthy isolated mitochondria (10 µg/mL) one day before) also partially protected the kidneys from IRI. These studies demonstrate that pre-emptive infusion of ex-vivo reprogrammed DCs that have higher mitochondria content has therapeutic capacity to induce an anti-inflammatory regulatory phenotype to protect kidneys from injury.