Akt Inhibition as Preconditioning Treatment to Protect Kidney Cells against Anoxia

Lesions issued from the ischemia/reperfusion (I/R) stress are a major challenge in human pathophysiology. Of human organs, the kidney is highly sensitive to I/R because of its high oxygen demand and poor regenerative capacity. Previous studies have shown that targeting the hypusination pathway of eIF5A through GC7 greatly improves ischemic tolerance and can be applied successfully to kidney transplants. The protection process correlates with a metabolic shift from oxidative phosphorylation to glycolysis. Because the protein kinase B Akt is involved in ischemic protective mechanisms and glucose metabolism, we looked for a link between the effects of GC7 and Akt in proximal kidney cells exposed to anoxia or the mitotoxic myxothiazol. We found that GC7 treatment resulted in impaired Akt phosphorylation at the Ser473 and Thr308 sites, so the effects of direct Akt inhibition as a preconditioning protocol on ischemic tolerance were investigated. We evidenced that Akt inhibitors provide huge protection for kidney cells against ischemia and myxothiazol. The pro-survival effect of Akt inhibitors, which is reversible, implied a decrease in mitochondrial ROS production but was not related to metabolic changes or an antioxidant defense increase. Therefore, the inhibition of Akt can be considered as a preconditioning treatment against ischemia.

Subnormothermic isolated organ perfusion with Nicorandil increased cold ischemic tolerance of liver in experimental model

BACKGROUND: The cold ischemia –reperfusion injury may lead to microcirculatory disturbances, hepatocellular swelling, inflammation, and organ dysfunction. Nicorandil is an anti-ischemic, ATP-sensitive potassium (KATP) channel opener drug and has proved its effectiveness against hepatic Ischemia/Reperfusion (I/R) injury. OBJECTIVE: This study aimed to investigate the effect of Nicorandil on mitochondrial apoptosis, oxidative stress, inflammation, histopathological changes, and cold ischemic tolerance of the liver in an ex vivo experimental isolated-organ-perfusion model. METHODS: We used an ex vivo isolated rat liver perfusion system for this study. The grafts were retrieved from male Wistar rats (n = 5 in each), preserved in cold storage (CS) for 2 or 4 hours (group 1, 2), or perfused for 2 or 4 hours (group 3, 4) immediately after removal with Krebs Henseleit Buffer (KHB) solution or Nicorandil containing KHB solution under subnormothermic (22–25°C) conditions (group 5, 6). After 15 minutes incubation at room temperature, the livers were reperfused with acellular, oxygenated solution under normothermic condition for 60 minutes. RESULTS: In the Nicorandil perfused groups, significantly decreased liver enzymes, GLDH, TNF-alpha, and IL-1ß were measured from the perfusate. Antioxidant enzymactivity was higher in the perfused groups. Histopathological examination showed ameliorated tissue deterioration, preserved parenchymal structure, decreased apoptosis, and increased Bcl-2 activity in the Nicorandil perfused groups. CONCLUSIONS: Perfusion with Nicorandil containing KHB solution may increase cold ischemic tolerance of the liver via mitochondrial protection which can be a potential therapeutic target to improve graft survival during transplantation.

Loss of ischemic tolerance with age: can we protect an old kidney

The most abundant and vulnerable cohort of patients with acute kidney injury (AKI) is represented by the older people. It is well-known, the kidney tissue undergoes some changes with age, both at the morphological and molecular level. Therefore, when treating AKI in older patients, it is necessary to take into account the morphofunctional features of aging kidney tissue and metabolic alterations. We have shown that the kidney of old rats does not perceive signals from the most well-known protective approaches such as ischemic preconditioning (IPC) and caloric restriction (CR). Although the old kidney did not develop more severe AKI after ischemia, we found no pronounced effect on attempts to increase its resistance by IPC and CR. Analysis of the mechanisms underlying this loss of tolerance has shown that the most affected pathways are the mechanism of mitochondrial quality control, the effectiveness of autophagy, and the proliferative potential of kidney cells. However, several protective pathways activated in the young kidney were also active in the old one in response to the CR. In particular, an increase in SIRT1 deacetylase, antiapoptotic Bcl-xL, and a decrease in oxidative stress were observed. Our results show that some defense systems demonstrating their effectiveness in young organisms lose their beneficial effect in old organisms, while others still can be activated by protective approaches. Thus, it is necessary to carefully analyze the possibilities of increasing ischemic tolerance for old organisms. This work was supported by the Russian science foundation (grant #21-75-30009).

Tissue-type plasminogen activator induces TNF-α-mediated preconditioning of the blood-brain barrier

Ischemic tolerance is a phenomenon whereby transient exposure to a non-injurious preconditioning stimulus triggers resistance to a subsequent lethal ischemic insult. Despite the fact that not only neurons but also astrocytes and endothelial cells have a unique response to preconditioning stimuli, current research has been focused mostly on the effect of preconditioning on neuronal death. Thus, it is unclear if the blood-brain barrier (BBB) can be preconditioned independently of an effect on neuronal survival. The release of tissue-type plasminogen activator (tPA) from perivascular astrocytes in response to an ischemic insult increases the permeability of the BBB. In line with these observations, treatment with recombinant tPA increases the permeability of the BBB and genetic deficiency of tPA attenuates the development of post-ischemic edema. Here we show that tPA induces ischemic tolerance in the BBB independently of an effect on neuronal survival. We found that tPA renders the BBB resistant to an ischemic injury by inducing TNF-α-mediated astrocytic activation and increasing the abundance of aquaporin-4-immunoreactive astrocytic end-feet processes in the neurovascular unit. This is a new role for tPA, that does not require plasmin generation, and with potential therapeutic implications for patients with cerebrovascular disease.

Ischemic Tolerance – Blessing or Curse

Application of knowledge about ischemic tolerance to clinic requires the solid understanding of mechanism of creation of this phenomenon. This review summarizes research that has been carried out in many laboratories over a long period of time, but the main focus will be on own experimental research. The main emphasis is devoted to the possibility of preparing full tolerance in the donor's body and its transfer to the patient in the form of activated blood plasma. Such plasma could be administered as soon as the patient is transported to the hospital and would take effect immediately after administration to the patient's bloodstream. One chapter is also devoted to anticonditioning, i.e. the possibility of preventing the activation of tolerance. Anticonditioning could be used to treat oncologic patients. We expect that this method could increase effectiveness of cancer treatment. Cross-tolerance with a wide range of diverse stressors gives us the courage to assume that activated plasma can significantly help with a wide range of pathological events.

Genome-Wide Transcription Analysis of Electroacupuncture Precondition-Induced Ischemic Tolerance on SD Rat With Ischemia–Reperfusion Injury

Acupuncture promotes the recovery of neurological function by the overall improvement of ischemic brain injury. It is not only regarded as a rehabilitative treatment but also a pretreatment method for stroke. However, its mechanism has not been fully elucidated. In this study, rats were treated with electroacupuncture (EA) at Baihui (GV20) for 30 min/day for 6 days, ahead of conducting cerebral ischemia–reperfusion (I/R) injury. Infarction volume, Evans blue leakage, and neurological deficits were evaluated at 24 h after I/R injury. Then, the ipsilateral ischemic brain was isolated for RNA sequencing (RNA-Seq) to identify molecular consequences. The results showed that EA pretreatment decreased blood–brain barrier (BBB) permeability, reduced brain infarction volume, and improved neurological outcomes. EA pretreatment could upregulate expression of antivirus and immunity activity-associated genes (such as Ifit1, Ifit3, Irf7, and Oasla) and downregulate expression of matrix disruption-associated genes (Col24a1, Col11a1, Col27a1, etc.) in healthy rats. In addition, it could partially reverse or ameliorate genome-wide transcription changes of the ipsilateral ischemic brain. For the first time, this study provides insight into genomic network modulation of a healthy rat with EA treatment and a EA-preconditioned rat under subsequent I/R injury, which is helpful in explaining acupuncture precondition-induced ischemic tolerance of stroke. It also provides new strategies and targets for the prevention of ischemic stroke.

Inhibition of Arginyl-tRNA Synthetase Promotes the Protection of Neuronal Ischemic Tolerance in Vitro Author Imformation

BackgroundsStudies have shown that the metabolic rate of mammals decreases during hibernation, suggesting that effective reduction of the energy consumption of ischemic cells may be the basis of the protective effect of ischemic tolerance. Anderson et al. reported after inhibition of the gene encoding arginyl-tRNA synthetase, the protein translation and mortality in C. elegans under anoxic conditions decreased significantly.PurposesWhether inhibition of arginyl-tRNA synthetase (RARS), in addition to combating hypoxic injury in C. elegans, protects rat neurons from ischemic damage remains unknown. The aim of this study is to determine whether knockdown of arginyl-tRNA synthetase improves the tolerance of primary cultured rat neurons to ischemic anoxia. Methods For the primary neuronal cutlure, cerebral cortex tissues were collected from newborn 24 hours Sprague-Dawley rats. Different viral vectors were transfected into cultured primary neurons, and the optimal viral vector and time points for gene silencing were determined by detecting the expression of RARS gene and protein. The adenovirus vector expressing shRNA-RARS with the highest silencing efficiency was transfected into rat primary cultured cortical neurons. The time point at which the viral vector exhibits optimal gene silencing efficiency was selected as the detection time point for subsequent experiments. The neurons after OGD treatment were divided into 2 groups: the experimental group and the control group. Each group was divided into 3 subgroups: the normal group, the control shRNA group and the shRNA-RARS group. After 3 hours of OGD treatment, cell survival, ATP levels and RARS protein expression were evaluated , and the data was analyzed. ResultsPrimary cultured neurons were identified and purified. The adenoviral vector expressing RARS-RNAi (27394-1) exhibited the highest silencing efficiency of the three adenoviral vectors, and 3 days after transfection of the adenoviral vector was selected as the optimal time point for gene silencing. Transfection of adenoviral vectors expressing shRNA-RARS reduced RARS protein expression, ATP consumption and neuronal death, and increased CCK8 activity in neurons after OGD insult.Conclusions Our work revealed neurons transfected with adenoviral vectors expressing shRNA-RARS exhibited stronger tolerance to ischemia and hypoxia, which was due to the inhibition of RARS activity andreduced cell energy metabolism rate. These results suggested that RARS inhibition reduced protein translation and energy consumption, and played a protective role in ischemic tolerance.

Virtual test occlusion for assessing ischemic tolerance using computational fluid dynamics

Background: Ischemic tolerance has been evaluated by the balloon test occlusion (BTO) for cerebral aneurysms and tumors that might require parent artery occlusion during surgery. However, because of its invasiveness, a non-invasive evaluation method is needed. In this study, we assessed the possibility of virtual test occlusion using computational fluid dynamics (CFD) as a non-invasive alternative to BTO for evaluating ischemic tolerance. Methods: Twenty-one patients who underwent BTO were included in the study. Virtual test occlusion was performed using CFD analysis, and the flow rate (FR) and wall shear stress (WSS) of the middle cerebral artery on the occlusion side were calculated. The correlations between these parameters and examination data including the parameters of computed tomography perfusion during BTO were assessed and the cutoff value of CFD parameters for detecting the good collateral group was calculated. Results: The FR was strongly correlated with mean transit time (MTT) during BTO and moderately correlated with collateral flow grade based on angiographic appearance. The WSS was moderately correlated with collateral flow grade, mean stump pressure (MSP), and MTT. Furthermore, the FR and WSS were strongly correlated with the total FR and the diameters of the inlet vessels. The cutoff value of FR for detecting the good collateral group was 126.2 mL/min, while that of the WSS was 4.54 Pa. Conclusion: The parameters obtained through CFD analysis were correlated with collateral flow grade and MSP in addition to MTT. CFD analysis may be useful to evaluate ischemic tolerance as a non-invasive alternative to BTO.