Normothermic Ex-vivo Kidney Perfusion in a Porcine Auto-Transplantation Model Preserves the Expression of Key Mitochondrial Proteins: An Unbiased Proteomics Analysis

AbstractNormothermic ex-vivo kidney perfusion (NEVKP) results in significantly improved graft function in porcine auto-transplant models of DCD injury compared to static cold storage (SCS); however, the molecular mechanisms underlying these beneficial effects remain unclear. We performed an unbiased proteomics analysis of 28 kidney biopsies obtained at 3 time points from pig kidneys subjected to 30-minutes of warm ischemia, followed by 8 hours of NEVKP or SCS, and auto-transplantation. 70/6593 proteins quantified were differentially expressed between NEVKP and SCS groups (FDR<0.05). Proteins increased in NEVKP mediated key metabolic processes including fatty acid ß-oxidation, the TCA-cycle and oxidative phosphorylation. Comparison of our findings with external datasets of ischemia-reperfusion, and other models of kidney injury confirmed that 47 of our proteins represent a common signature of kidney injury reversed or attenuated by NEVKP. We validated key metabolic proteins (ETFB, CPT2) by immunoblotting. Transcription factor databases identified PPARGC1A, PPARA/G/D and RXRA/B as the upstream regulators of our dataset, and we confirmed their increased expression in NEVKP with RT-PCR. The proteome-level changes observed in NEVKP mediate critical metabolic pathways that may explain the improved graft function observed. These effects may be coordinated by PPAR-family transcription factors, and may represent novel therapeutic targets in ischemia-reperfusion injury.

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Ischemia-Reperfusion Injury in Marginal Liver Grafts and the Role of Hypothermic Machine Perfusion: Molecular Mechanisms and Clinical Implications

Journal of Clinical Medicine ◽

10.3390/jcm9030846 ◽

2020 ◽

Vol 9 (3) ◽

pp. 846 ◽

Cited By ~ 4

Author(s):

Zoltan Czigany ◽

Isabella Lurje ◽

Moritz Schmelzle ◽

Wenzel Schöning ◽

Robert Öllinger ◽

...

Keyword(s):

Reperfusion Injury ◽

Molecular Mechanisms ◽

Ischemia Reperfusion Injury ◽

Ex Vivo ◽

Ischemia Reperfusion ◽

Graft Function ◽

Organ Shortage ◽

Machine Perfusion ◽

Hypothermic Machine Perfusion ◽

Critical Organ

Ischemia-reperfusion injury (IRI) constitutes a significant source of morbidity and mortality after orthotopic liver transplantation (OLT). The allograft is metabolically impaired during warm and cold ischemia and is further damaged by a paradox reperfusion injury after revascularization and reoxygenation. Short-term and long-term complications including post-reperfusion syndrome, delayed graft function, and immune activation have been associated with IRI. Due to the current critical organ shortage, extended criteria grafts are increasingly considered for transplantation, however, with an elevated risk to develop significant features of IRI. In recent years, ex vivo machine perfusion (MP) of the donor liver has witnessed significant advancements. Here, we describe the concept of hypothermic (oxygenated) machine perfusion (HMP/HOPE) approaches and highlight which allografts may benefit from this technology. This review also summarizes clinical applications and the main aspects of ongoing randomized controlled trials on hypothermic perfusion. The mechanistic aspects of IRI and hypothermic MP—which include tissue energy replenishment, optimization of mitochondrial function, and the reduction of oxidative and inflammatory damage following reperfusion—will be comprehensively discussed within the context of current preclinical and clinical evidence. Finally, we highlight novel trends and future perspectives in the field of hypothermic MP in the context of recent findings of basic and translational research.

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Restoring Mitochondrial Function While Avoiding Redox Stress: The Key to Preventing Ischemia/Reperfusion Injury in Machine Perfused Liver Grafts?

International Journal of Molecular Sciences ◽

10.3390/ijms21093132 ◽

2020 ◽

Vol 21 (9) ◽

pp. 3132 ◽

Cited By ~ 3

Author(s):

Julia Hofmann ◽

Giorgi Otarashvili ◽

Andras Meszaros ◽

Susanne Ebner ◽

Annemarie Weissenbacher ◽

...

Keyword(s):

Reperfusion Injury ◽

Ischemia Reperfusion Injury ◽

Ex Vivo ◽

Ischemia Reperfusion ◽

Graft Function ◽

Ros Production ◽

Machine Perfusion ◽

Redox Stress ◽

Molecular Events ◽

Regulate Cell Death

Mitochondria sense changes resulting from the ischemia and subsequent reperfusion of an organ and mitochondrial reactive oxygen species (ROS) production initiates a series of events, which over time result in the development of full-fledged ischemia-reperfusion injury (IRI), severely affecting graft function and survival after transplantation. ROS activate the innate immune system, regulate cell death, impair mitochondrial and cellular performance and hence organ function. Arresting the development of IRI before the onset of ROS production is currently not feasible and clinicians are faced with limiting the consequences. Ex vivo machine perfusion has opened the possibility to ameliorate or antagonize the development of IRI and may be particularly beneficial for extended criteria donor organs. The molecular events occurring during machine perfusion remain incompletely understood. Accumulation of succinate and depletion of adenosine triphosphate (ATP) have been considered key mechanisms in the initiation; however, a plethora of molecular events contribute to the final tissue damage. Here we discuss how understanding mitochondrial dysfunction linked to IRI may help to develop novel strategies for the prevention of ROS-initiated damage in the evolving era of machine perfusion.

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Molecular Mechanisms of Renal Ischemic Conditioning Strategies

European Surgical Research ◽

10.1159/000437352 ◽

2015 ◽

Vol 55 (3) ◽

pp. 151-183 ◽

Cited By ~ 12

Author(s):

Casper Kierulf-Lassen ◽

Gertrude J. Nieuwenhuijs-Moeke ◽

Nicoline V. Krogstrup ◽

Mihai Oltean ◽

Bente Jespersen ◽

...

Keyword(s):

Reperfusion Injury ◽

Molecular Mechanisms ◽

Defense Mechanism ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Kidney Injury ◽

Clinical Settings ◽

Ischemic Conditioning ◽

Protective Signaling ◽

Survival Mechanisms

Ischemia-reperfusion injury is the leading cause of acute kidney injury in a variety of clinical settings such as renal transplantation and hypovolemic and/or septic shock. Strategies to reduce ischemia-reperfusion injury are obviously clinically relevant. Ischemic conditioning is an inherent part of the renal defense mechanism against ischemia and can be triggered by short periods of intermittent ischemia and reperfusion. Understanding the signaling transduction pathways of renal ischemic conditioning can promote further clinical translation and pharmacological advancements in this era. This review summarizes research on the molecular mechanisms underlying both local and remote ischemic pre-, per- and postconditioning of the kidney. The different types of conditioning strategies in the kidney recruit similar powerful pro-survival mechanisms. Likewise, renal ischemic conditioning mobilizes many of the same protective signaling pathways as in other organs, but differences are recognized.

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Molecular Mechanisms of Ferroptosis and Its Roles in Hematologic Malignancies

Frontiers in Oncology ◽

10.3389/fonc.2021.743006 ◽

2021 ◽

Vol 11 ◽

Author(s):

Yan Zhao ◽

Zineng Huang ◽

Hongling Peng

Keyword(s):

Cell Death ◽

Molecular Mechanisms ◽

Hematological Malignancies ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Lipid Peroxide ◽

Kidney Injury ◽

Hematologic Malignancies ◽

Regulated Cell Death ◽

Normal Metabolism

Cell death is essential for the normal metabolism of human organisms. Ferroptosis is a unique regulated cell death (RCD) mode characterized by excess accumulation of iron-dependent lipid peroxide and reactive oxygen species (ROS) compared with other well-known programmed cell death modes. It has been currently recognized that ferroptosis plays a rather important role in the occurrence, development, and treatment of traumatic brain injury, stroke, acute kidney injury, liver damage, ischemia–reperfusion injury, tumor, etc. Of note, ferroptosis may be explained by the expression of various molecules and signaling components, among which iron, lipid, and amino acid metabolism are the key regulatory mechanisms of ferroptosis. Meanwhile, tumor cells of hematological malignancies, such as leukemia, lymphoma, and multiple myeloma (MM), are identified to be sensitive to ferroptosis. Targeting potential regulatory factors in the ferroptosis pathway may promote or inhibit the disease progression of these malignancies. In this review, a systematic summary was conducted on the key molecular mechanisms of ferroptosis and the current potential relationships of ferroptosis with leukemia, lymphoma, and MM. It is expected to provide novel potential therapeutic approaches and targets for hematological malignancies.

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Nicotinamide Mononucleotide Attenuates Renal Interstitial Fibrosis After AKI by Suppressing Tubular DNA Damage and Senescence

Frontiers in Physiology ◽

10.3389/fphys.2021.649547 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yan Jia ◽

Xin Kang ◽

Lishan Tan ◽

Yifei Ren ◽

Lei Qu ◽

...

Keyword(s):

Dna Damage ◽

Ischemia Reperfusion Injury ◽

Interstitial Fibrosis ◽

Ischemia Reperfusion ◽

Kidney Injury ◽

Recovery Phase ◽

Tubular Cell ◽

Dna Damage And Repair ◽

Kidney Fibrosis ◽

Beneficial Effects

Acute kidney injury (AKI) is a worldwide health problem currently lacking therapeutics that directly promote renal repair or prevent the occurrence of chronic fibrosis. DNA damage is a feature of many forms of kidney injury, and targeting DNA damage and repair might be effective strategies for kidney protection in AKI. Boosting nicotinamide adenine dinucleotide (NAD+) levels is thought to have beneficial effects on DNA damage repair and fibrosis in other organs. However, no kidney-related studies of such effects have been performed to date. Here, we have shown that NMN (an NAD+ precursor) administration could significantly reduce tubular cell DNA damage and subsequent cellular senescence induced by hydrogen peroxide and hypoxia in human proximal tubular cells (HK-2 cells). The DNA damage inhibition, antiaging and anti-inflammatory effects of NMN were further confirmed in a unilateral ischemia-reperfusion injury (uIRI) mouse model. Most importantly, the antifibrosis activity of NMN was also shown in ischemic AKI mouse models, regardless of whether NMN was administered in advance or during the recovery phase. Collectively, these results suggest that NMN could significantly inhibit tubular cell DNA damage, senescence and inflammation. NMN administration might be an effective strategy for preventing or treating kidney fibrosis after AKI.

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Huaier Extract Attenuates Acute Kidney Injury to Chronic Kidney Disease Transition by Inhibiting Endoplasmic Reticulum Stress and Apoptosis via miR-1271 Upregulation

BioMed Research International ◽

10.1155/2020/9029868 ◽

2020 ◽

Vol 2020 ◽

pp. 1-8

Author(s):

Jing-Ying Zhao ◽

Yu-Bin Wu

Keyword(s):

Chronic Kidney Disease ◽

Acute Kidney Injury ◽

Endoplasmic Reticulum ◽

Kidney Disease ◽

Endoplasmic Reticulum Stress ◽

Molecular Mechanisms ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Kidney Injury

Endoplasmic reticulum stress (ERS) is strongly associated with acute kidney injury (AKI) to chronic kidney disease (CKD) transition. Huaier extract (HE) protects against kidney injury; albeit, the underlying mechanism is unknown. We hypothesized that HE reduces kidney injury by inhibiting ERS. In this study, using an AKI-CKD mouse model of ischemia-reperfusion injury (IRI), we evaluated the effect of HE on AKI-CKD transition. We also explored the underlying molecular mechanisms in this animal model and in the HK-2 human kidney cell line. The results showed that HE treatment improved the renal function, demonstrated by a significant decrease in serum creatinine levels after IRI. HE appreciably reduced the degree of kidney injury and fibrosis and restored the expression of the microRNA miR-1271 after IRI. Furthermore, HE reduced the expression of ERS markers glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) and inhibited apoptosis in the IRI group. This in vivo effect was supported by in vitro results in which HE inhibited apoptosis and decreased the expression of CHOP and GRP78 induced by ERS. We demonstrated that CHOP is a target of miR-1271. In conclusion, HE reduces kidney injury, probably by inhibiting apoptosis and decreasing the expression of GRP78 and CHOP via miR-1271 upregulation.

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In Vitro/Ex Vivo Models for the Study of Ischemia Reperfusion Injury during Kidney Perfusion

International Journal of Molecular Sciences ◽

10.3390/ijms21218156 ◽

2020 ◽

Vol 21 (21) ◽

pp. 8156

Author(s):

Sebastien Giraud ◽

Raphaël Thuillier ◽

Jérome Cau ◽

Thierry Hauet

Keyword(s):

Reperfusion Injury ◽

Ischemia Reperfusion Injury ◽

Ex Vivo ◽

Ischemia Reperfusion ◽

Kidney Graft ◽

Kidney Preservation ◽

Hypothermic Machine Perfusion ◽

Kidney Perfusion ◽

Marginal Donors

Oxidative stress is a key element of ischemia–reperfusion injury, occurring during kidney preservation and transplantation. Current options for kidney graft preservation prior to transplantation are static cold storage (CS) and hypothermic machine perfusion (HMP), the latter demonstrating clear improvement of preservation quality, particularly for marginal donors, such as extended criteria donors (ECDs) and donation after circulatory death (DCDs). Nevertheless, complications still exist, fostering the need to improve kidney preservation. This review highlights the most promising avenues of in kidney perfusion improvement on two critical aspects: ex vivo and in vitro evaluation.

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p53 target Siva regulates apoptosis in ischemic kidneys

AJP Renal Physiology ◽

10.1152/ajprenal.00591.2010 ◽

2011 ◽

Vol 300 (5) ◽

pp. F1130-F1141 ◽

Cited By ~ 22

Author(s):

Kurinji Singaravelu ◽

Babu J. Padanilam

Keyword(s):

Cytochrome C ◽

Molecular Mechanisms ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Tumor Necrosis Factor Receptor ◽

Kidney Injury ◽

Cytochrome C Release ◽

Apoptosis Inhibition ◽

Apoptosis Inducing Factor

The role of p53 in inducing apoptosis following acute kidney injury is well-established; however, the molecular mechanisms remain largely unknown. We report here that the p53 proapoptotic target Siva and its receptor CD27, a member of the tumor necrosis factor receptor family, are upregulated following renal ischemia-reperfusion injury (IRI). Inhibition of Siva using antisense oligonucleotides conferred functional and morphological protection, and it prevented apoptosis postrenal IRI in mice. Renal IRI in CD27-deficient mice displayed functional protection and partial inhibition of apoptosis, suggesting an incomplete role for CD27 in Siva-mediated apoptosis. To further elucidate mechanisms by which Siva elicits apoptosis, in vitro studies were performed. In Siva-transfected LLC-PK1 cells, Siva is persistently expressed in the nucleus at 3 h onwards and its translocation to mitochondria and the plasma membrane occurred at 6 h. Moreover, Siva overexpression induced mitochondrial permeability, cytochrome c release, caspase-8 and -9 activation, translocation of apoptosis-inducing factor (AIF) to the nucleus, and apoptosis. Inhibition of Siva in ischemic kidneys prevented mitochondrial release of cytochrome c and AIF. These data indicate that Siva function is pivotal in regulating apoptosis in the pathology of renal IRI. Targeting Siva may offer a potential therapeutic strategy for renal IRI.

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Procaine–The Controversial Geroprotector Candidate: New Insights Regarding Its Molecular and Cellular Effects

Oxidative Medicine and Cellular Longevity ◽

10.1155/2021/3617042 ◽

2021 ◽

Vol 2021 ◽

pp. 1-18

Author(s):

Daniela Gradinaru ◽

Anca Ungurianu ◽

Denisa Margina ◽

Maria Moreno-Villanueva ◽

Alexander Bürkle

Keyword(s):

Molecular Mechanisms ◽

Ischemia Reperfusion Injury ◽

Therapeutic Potential ◽

Ischemia Reperfusion ◽

Experimental Models ◽

Beneficial Effects ◽

Cellular Effects ◽

Treatment And Prevention ◽

Age Related ◽

Myocardial Ischemia Reperfusion

Since its discovery in 1905 and its employment in everyday medical practice as a local anesthetic, to its highly controversial endorsement as an “anti-aging” molecule in the sixties and seventies, procaine is part of the history of medicine and gerontoprophylaxis. Procaine can be considered a “veteran” drug due to its long-time use in clinical practice, but is also a molecule which continues to incite interest, revealing new biological and pharmacological effects within novel experimental approaches. Therefore, this review is aimed at exploring and systematizing recent data on the biochemical, cellular, and molecular mechanisms involved in the antioxidant and potential geroprotective effects of procaine, focusing on the following aspects: (1) the research state-of-the-art, through an objective examination of scientific literature within the last 30 years, describing the positive, as well as the negative reports; (2) the experimental data supporting the beneficial effects of procaine in preventing or alleviating age-related pathology; and (3) the multifactorial pathways procaine impacts oxidative stress, inflammation, atherogenesis, cerebral age-related pathology, DNA damage, and methylation. According to reviewed data, procaine displayed antioxidant and cytoprotective actions in experimental models of myocardial ischemia/reperfusion injury, lipoprotein oxidation, endothelial-dependent vasorelaxation, inflammation, sepsis, intoxication, ionizing irradiation, cancer, and neurodegeneration. This analysis painted a complex pharmacological profile of procaine: a molecule that has not yet fully expressed its therapeutic potential in the treatment and prevention of aging-associated diseases. The numerous recent reports found demonstrate the rising interest in researching the multiple actions of procaine regulating key processes involved in cellular senescence. Its beneficial effects on cell/tissue functions and metabolism could designate procaine as a valuable candidate for the well-established Geroprotectors database.

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PPARβ/δ Is Required for Mesenchymal Stem Cell Cardioprotective Effects Independently of Their Anti-inflammatory Properties in Myocardial Ischemia-Reperfusion Injury

Frontiers in Cardiovascular Medicine ◽

10.3389/fcvm.2021.681002 ◽

2021 ◽

Vol 8 ◽

Author(s):

Nitirut Nernpermpisooth ◽

Charlotte Sarre ◽

Christian Barrere ◽

Rafaël Contreras ◽

Patricia Luz-Crawford ◽

...

Keyword(s):

Infarct Size ◽

Ischemia Reperfusion Injury ◽

Ex Vivo ◽

Ischemia Reperfusion ◽

Therapeutic Effects ◽

Beneficial Effects ◽

Adult Mesenchymal Stem Cells ◽

Myocardial Ischemia Reperfusion Injury ◽

Anti Inflammatory ◽

Myocardial Ischemia Reperfusion

Myocardial infarction ranks first for the mortality worldwide. Because the adult heart is unable to regenerate, fibrosis develops to compensate for the loss of contractile tissue after infarction, leading to cardiac remodeling and heart failure. Adult mesenchymal stem cells (MSC) regenerative properties, as well as their safety and efficacy, have been demonstrated in preclinical models. However, in clinical trials, their beneficial effects are controversial. In an experimental model of arthritis, we have previously shown that PPARβ/δ deficiency enhanced the therapeutic effect of MSC. The aim of the present study was to compare the therapeutic effects of wild-type MSC (MSC) and MSC deficient for PPARβ/δ (KO MSC) perfused in an ex vivo mouse model of ischemia-reperfusion (IR) injury. For this purpose, hearts from C57BL/6J mice were subjected ex vivo to 30 min ischemia followed by 1-h reperfusion. MSC and KO MSC were injected into the Langendorff system during reperfusion. After 1 h of reperfusion, the TTC method was used to assess infarct size. Coronary effluents collected in basal condition (before ischemia) and after ischemia at 1 h of reperfusion were analyzed for their cytokine profiles. The dose-response curve for the cardioprotection was established ex vivo using different doses of MSC (3.105, 6.105, and 24.105 cells/heart) and the dose of 6.105 MSC was found to be the optimal concentration. We showed that the cardioprotective effect of MSC was PPARβ/δ-dependent since it was lost using KO MSC. Moreover, cytokine profiling of the coronary effluents collected in the eluates after 60 min of reperfusion revealed that MSC treatment decreases CXCL1 chemokine and interleukin-6 release compared with untreated hearts. This anti-inflammatory effect of MSC was also observed when hearts were treated with PPARβ/δ-deficient MSC. In conclusion, our study revealed that the acute cardioprotective properties of MSC in an ex vivo model of IR injury, assessed by a decreased infarct size at 1 h of reperfusion, are PPARβ/δ-dependent but not related to their anti-inflammatory effects.

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