Developing a Clinically Relevant Hemorrhagic Shock Model in Rats.

Over the recent decades, the development of animal models allowed us to better understand various pathologies and identify new treatments. Hemorrhagic shock, i.e., organ failure due to rapid loss of a large volume of blood, is associated with a highly complex pathophysiology involving several pathways. Numerous existing animal models of hemorrhagic shock strive to replicate what happens in humans, but these models have limits in terms of clinical relevance, reproducibility, or standardization. The aim of this study was to refine these models to develop a new model of hemorrhagic shock. Briefly, hemorrhagic shock was induced in male Wistar Han rats (11-13 weeks old) by a controlled exsanguination responsible for a drop in the mean arterial pressure. The next phase of 75 min was to maintain a low mean arterial blood pressure, between 32 mmHg and 38 mmHg, to trigger the pathophysiological pathways of hemorrhagic shock. The final phase of the protocol mimicked patient care with an administration of intravenous fluids, Ringer Lactate solution, to elevate the blood pressure. Lactate and behavioral scores were assessed 16 h after the protocol started, while hemodynamics parameters and plasmatic markers were evaluated 24 h after injury. Twenty-four hours post-hemorrhagic shock induction, the mean arterial and diastolic blood pressure were decreased in the hemorrhagic shock group (p < 0.05). Heart rate and systolic blood pressure remained unchanged. All organ damage markers were increased with the hemorrhagic shock (p < 0.05). The lactatemia and behavioral scores were increased compared to the sham group (p < 0.05). In conclusion, we demonstrated that the protocol described here is a relevant model of hemorrhagic shock that can be used in subsequent studies, particularly to evaluate the therapeutic potential of new molecules.

EMT and primary ciliogenesis: For better or worse in sickness and in health.

Epithelial-mesenchymal transition (EMT) and primary ciliogenesis are two cell-biological programs that are essential for development of multicellular organisms and whose abnormal regulation results in many diseases (i.e., developmental anomalies and cancers). Emerging studies suggest an intricate interplay between these two processes. Here, we discuss physiological and pathological contexts in which their interconnections promote normal development or disease progression. We describe underlying molecular mechanisms of the interplay and EMT/ciliary signaling axes that influence EMT-related processes (i.e., stemness, motility and invasion). Understanding the molecular and cellular mechanisms of the relationship between EMT and primary ciliogenesis may provide new insights in the etiology of diseases related to EMT and cilia dysfunction.

A Rat Model of Clinically Relevant Extracorporeal Circulation Develops Early Organ Dysfunctions.

In clinical practice, extracorporeal circulation (ECC) is associated with coagulopathy and inflammation, eventually leading to organ injuries without preventive systemic pharmacological treatment. Relevant models are needed to reproduce the pathophysiology observed in humans and preclinical tests. Rodent models are less expensive than large models but require adaptations and validated comparisons to clinics. This study aimed to develop a rat ECC model and to establish its clinical relevance. One hour of veno-arterial ECC or a sham procedure were achieved on mechanically ventilated rats after cannulations with a mean arterial pressure objective > 60 mmHg. Five hours post-surgery, the rats' behavior, plasmatic/blood biomarkers, and hemodynamics were measured. Blood biomarkers and transcriptomic changes were compared in 41 patients undergoing on-pump cardiac surgery. Five hours post-ECC, the rats presented hypotension, hyperlactatemia, and behavioral alterations. The same patterns of marker measurements (Lactate dehydrogenase, Creatinine kinase, ASAT, ALAT, and Troponin T) were observed in both rats and human patients. Transcriptome analyses showed similarity in both humans and rats in the biological processes involved in the ECC response. This new ECC rat model seems to resemble both ECC clinical procedures and the associated pathophysiology, but with early organ injury corresponding to a severe phenotype. Although the mechanisms at stake in the post-ECC pathophysiology of rats or humans need to be described, this new rat model appears to be a relevant and costless preclinical model of human ECC.

An overview of tools to decipher O-GlcNAcylation from historical approaches to new insights.

O-GlcNAcylation is a post-translational modification which affects approximately 5000 human proteins. Its involvement has been shown in many if not all biological processes. Variations in O-GlcNAcylation levels can be associated with the development of diseases. Deciphering the role of O-GlcNAcylation is an important issue to (i) understand its involvement in pathophysiological development and (ii) develop new therapeutic strategies to modulate O-GlcNAc levels. Over the past 30 years, despite the development of several approaches, knowledge of its role and regulation have remained limited. This review proposes an overview of the currently available tools to study O-GlcNAcylation and identify O-GlcNAcylated proteins. Briefly, we discuss pharmacological modulators, methods to study O-GlcNAcylation levels and approaches for O-GlcNAcylomic profiling. This review aims to contribute to a better understanding of the methods used to study O-GlcNAcylation and to promote efforts in the development of new strategies to explore this promising modification.

The Endothelial Dysfunction Could Be a Cause of Heart Failure with Preserved Ejection Fraction Development in a Rat Model.

50% of patients with heart failure have a preserved ejection fraction (HFpEF). Numerous studies have investigated the pathophysiological mechanisms of HFpEF and have shown that endothelial dysfunction plays an important role in HFpEF. Yet no studies answered whether endothelial dysfunction could be the cause or is the consequence of HFpEF. Recently, we have shown that the endothelial overexpression of human ß 3-adrenoreceptor (Tgß 3) in rats leads to the slow development of diastolic dysfunction over ageing. The aim of the study is to decipher the involvement of endothelial dysfunction in the HFpEF development. For that, we investigated endothelial and cardiac function in 15-, 30-, and 45-week-old wild-type (WT) and Tgß 3 rats. The aortic expression of • NO synthase (NOS) isoforms was evaluated by Western blot. Finally, electron paramagnetic resonance measurements were performed on aortas to evaluate • NO and O2 •- production. Vascular reactivity was altered as early as 15 weeks of age in response to isoproterenol in Tgß 3 aortas and mesenteric arteries. NOS1 (neuronal NOS) expression was higher in the Tgß 3 aorta at 30 and 45 weeks of age (30 weeks: WT: 1.00 ± 0.21; Tgß 3: 6.08 ± 2.30; 45 weeks: WT: 1.00 ± 0.12; Tgß 3: 1.55 ± 0.17; p < 0.05). Interestingly, the endothelial NOS (NOS3) monomer form is increased in Tgß 3 rats at 45 weeks of age (ratio NOS3 dimer/NOS3 monomer; WT: 1.00 ± 0.37; Tgß 3: 0.13 ± 0.05; p < 0.05). Aortic •NO production was increased by NOS2 (inducible NOS) at 15 weeks of age in Tgß 3 rats (+52% vs. WT). Aortic O2 •- production was increased in Tgß 3 rats at 30 and 45 weeks of age (+75% and+76%, respectively, vs. WT, p < 0.05). We have shown that endothelial dysfunction and oxidative stress are present as early as 15 weeks of age and therefore conclude that endothelial dysfunction could be a cause of HFpEF development.

Study of the Role of the Tyrosine Kinase Receptor MerTK in the Development of Kidney Ischemia-Reperfusion Injury in RCS Rats.

Renal ischaemia reperfusion (I/R) triggers a cascade of events including oxidative stress, apoptotic body and microparticle (MP) formation as well as an acute inflammatory process that may contribute to organ failure. Macrophages are recruited to phagocytose cell debris and MPs. The tyrosine kinase receptor MerTK is a major player in the phagocytosis process. Experimental models of renal I/R events are of major importance for identifying I/R key players and for elaborating novel therapeutical approaches. A major aim of our study was to investigate possible involvement of MerTK in renal I/R. We performed our study on both natural mutant rats for MerTK (referred to as RCS) and on wild type rats referred to as WT. I/R was established by of bilateral clamping of the renal pedicles for 30' followed by three days of reperfusion. Plasma samples were analysed for creatinine, aspartate aminotransferase (ASAT), lactate dehydrogenase (LDH), kidney injury molecule -1 (KIM-1), and neutrophil gelatinase-associated lipocalin (NGAL) levels and for MPs. Kidney tissue damage and CD68-positive cell requirement were analysed by histochemistry. monocyte chemoattractant protein-1 (MCP-1), myeloperoxidase (MPO), inducible nitric oxide synthase (iNOS), and histone 3A (H3A) levels in kidney tissue lysates were analysed by western blotting. The phagocytic activity of blood-isolated monocytes collected from RCS or WT towards annexin-V positive bodies derived from cultured renal cell was assessed by fluorescence-activated single cell sorting (FACS) and confocal microscopy analyses. The renal I/R model for RCS rat described for the first time here paves the way for further investigations of MerTK-dependent events in renal tissue injury and repair mechanisms.

An O-GlcNAcylomic Approach Reveals ACLY as a Potential Target in Sepsis in the Young Rat.

Sepsis in the young population, which is particularly at risk, is rarely studied. O-GlcNAcylation is a post-translational modification involved in cell survival, stress response and metabolic regulation. O-GlcNAc stimulation is beneficial in adult septic rats. This modification is physiologically higher in the young rat, potentially limiting the therapeutic potential of O-GlcNAc stimulation in young septic rats. The aim is to evaluate whether O-GlcNAc stimulation can improve sepsis outcome in young rats. Endotoxemic challenge was induced in 28-day-old rats by lipopolysaccharide injection (E. Coli O111:B4, 20 mg·kg-1) and compared to control rats (NaCl 0.9%). One hour after lipopolysaccharide injection, rats were randomly assigned to no therapy, fluidotherapy (NaCl 0.9%, 10 mL·kg-1) ± NButGT (10 mg·kg-1) to increase O-GlcNAcylation levels. Physiological parameters and plasmatic markers were evaluated 2h later. Finally, untargeted mass spectrometry was performed to map cardiac O-GlcNAcylated proteins. Lipopolysaccharide injection induced shock with a decrease in mean arterial pressure and alteration of biological parameters (p < 0.05). NButGT, contrary to fluidotherapy, was associated with an improvement of arterial pressure (p < 0.05). ATP citrate lyase was identified among the O-GlcNAcylated proteins. In conclusion, O-GlcNAc stimulation improves outcomes in young septic rats. Interestingly, identified O-GlcNAcylated proteins are mainly involved in cellular metabolism.

Emerging therapeutic strategies for transplantation-induced acute kidney injury: protecting the organelles and the vascular bed.

INTRODUCTION: Renal ischemia-reperfusion injury (IRI) is a significant clinical challenge faced by clinicians in a broad variety of clinical settings such as perioperative and intensive care. Renal IRI induced acute kidney injury (AKI) is a global public health concern associated with high morbidity, mortality, and health-care costs. Areas covered: This paper focuses on the pathophysiology of transplantation-related AKI and recent findings on cellular stress responses at the intersection of 1. The Unfolded protein response; 2. Mitochondrial dysfunction; 3. The benefits of mineralocorticoid receptor antagonists. Lastly, perspectives are offered to the readers. Expert opinion: Renal IRI is caused by a sudden and temporary impairment of blood flow to the organ. Defining the underlying cellular cascades involved in IRI will assist us in the identification of novel interventional targets to attenuate IRI with the potential to improve transplantation outcomes. Targeting mitochondrial function and cellular bioenergetics upstream of cellular damage may offer several advantages compared to targeting downstream inflammatory and fibrosis processes. An improved understanding of the cellular pathophysiological mechanisms leading to kidney injury will hopefully offer improved targeted therapies to prevent and treat the injury in the future.

Stretch-activated Piezo1 Channel in Endothelial Cells Relaxes Mouse Intrapulmonary Arteries.

In intrapulmonary arteries (IPA), endothelial cells (EC) respond to mechanical stimuli by releasing vasoactive factors to set the vascular tone. Piezo1, a stretch-activated, calcium-permeable channel, is a sensor of mechanical stress in EC. The present study was undertaken to investigate the implication of Piezo1 in the endothelium-dependent regulation of IPA tone and potential involvement of Piezo1 in pulmonary hypertension, the main disease of this circulation. IPA tone was quantified by means of a myograph in control Piezo1+/+ mice and in mice lacking endothelial Piezo1 (EC-Piezo1-/-). Endothelial intracellular calcium concentration ([Ca2+]i) and nitric oxide (NO) production were measured, in mouse or human EC, with Fluo-4 or DAF-FM probe, respectively. Immunofluorescent labeling and patch-clamp experiments revealed the presence of Piezo1 channels in EC. Yoda1, a Piezo1 agonist, induced an endothelium-dependent relaxation that was significantly reduced in pulmonary arteries in EC-Piezo1-/- compared with Piezo1+/+ mice. Yoda1 as well as mechanical stimulation (by osmotic stress) increased [Ca2+]i in mouse or human EC. Consequently, both stimuli increased the production of NO. NO and [Ca2+]i increases were reduced in EC from Piezo1-/- mice or in the presence of Piezo1 inhibitors. Furthermore, deletion of Piezo1 increased α-adrenergic agonist-mediated contraction. Finally, in chronically hypoxic mice, a model of pulmonary hypertension, Piezo1 still mediated arterial relaxation, and deletion of this channel did not impair the development of the disease. The present study thus demonstrates that endothelial Piezo1 contributes to intrapulmonary vascular relaxation by controlling endothelial [Ca2+]i and NO production and that this effect is still present in pulmonary hypertension.