Mechanisms of COVID-19-induced kidney injury and current pharmacotherapies.

The COVID-19 pandemic created a worldwide debilitating health crisis with the entire humanity suffering from the deleterious effects associated with the high infectivity and mortality rates. While significant evidence is currently available online and targets various aspects of the disease, both inflammatory and noninflammatory kidney manifestations secondary to COVID-19 infection are still largely underrepresented. In this review, we summarized current knowledge about COVID-19-related kidney manifestations, their pathologic mechanisms as well as various pharmacotherapies used to treat patients with COVID-19. We also shed light on the effect of these medications on kidney functions that can further enhance renal damage secondary to the illness.

Gasoline fume inhalation induces apoptosis, inflammation, and favors Th2 polarization in C57BL/6 mice.

Gasoline exposure has been widely reported in the literature as being toxic to human health. However, the exact underlying molecular mechanisms triggered by its inhalation have not been thoroughly investigated. We herein present a model of sub-chronic, static gasoline vapor inhalation in adult female C57BL/6 mice. Animals were exposed daily to either gasoline vapors (0.86 g/animal/90 min) or ambient air for 5 days/week over 7 consecutive weeks. At the end of the study period, toxic and molecular mechanisms underlying the inflammatory, oxidative, and apoptotic effects triggered by gasoline vapors, were examined in the lungs and liver of gasoline-exposed (GE) mice. Static gasoline exposure induced a significant increase (+21%) in lungs/body weight (BW) ratio in GE versus control (CON) mice along with a pulmonary inflammation attested by histological staining. The latter was consistent with increases in the transcript levels of proinflammatory cytokines [Interleukins (ILs) 4 and 6], respectively by ~ 6- and 4-fold in the lungs of GE mice compared to CON. Interestingly, IL-10 expression was also increased by ~ 10-fold in the lungs of GE mice suggesting an attempt to counterbalance the established inflammation. Moreover, the pulmonary expression of IL-12 and TNF-α was downregulated by 2- and 4-fold, respectively, suggesting the skewing toward Th2 phenotype. Additionally, GE mice showed a significant upregulation in Bax/Bcl-2 ratio, caspases 3, 8, and 9 with no change in JNK expression in the lungs, suggesting the activation of both intrinsic and extrinsic apoptotic pathways. Static gasoline exposure over seven consecutive weeks had a minor hepatic portal inflammation attested by H&E staining along with an increase in the hepatic expression of the mitochondrial complexes in GE mice. Therefore, tissue damage biomarkers highlight the health risks associated with vapor exposure and may present potential therapeutic targets for recovery from gasoline intoxication.

VIP modulates human macrophages phenotype via FPRL1 via activation of RhoA-GTPase and PLC pathways.

OBJECTIVE AND DESIGN: This study is aimed at uncovering the signaling pathways activated by vasoactive intestinal peptide in human macrophages MATERIALS: Human peripheral blood mononuclear cell-derived macrophages were used for the in vitro investigation of the VIP-activated signaling pathways. METHODS AND TREATMENT: Time-course and dose-response experiments and siRNA were used in human macrophages co-challenged with various concentrations of VIP and different MAPK pharmacologic inhibitors to investigate signaling pathways activated by VIP. Flow analysis was performed to assess the levels of CD11b, CD35 and CD66. Luminescence spectrometry was used to measure the levels of the released hydrogen peroxide and the intracellular calcium levels in the media. RESULTS: Macrophages incubated with VIP showed increased phospho-AKT and phospho-ERK1/2 levels in a GTP-RhoA-GTPase-dependent manner. Similarly, VIP increased intracellular release of H2O2 and calcium via PLC and GTP-RhoA-GTPase, in addition to inducing the expression of CD11b, CD35, CD66 and MMP9. Furthermore, VIP activated P38 MAPK through the cAMP/PKA pathway but was independent of both PLC and RhoA signaling. The above-mentioned VIP effects were mediated via activation of the FPRL1 receptor. CONCLUSION: VIP/FPRL1/VPAC/GTP-RhoA-GTPase signaling modulated macrophages phenotype through activation of multiple signaling pathways including ERK1/2, AKT, P38, ROS, cAMP and calcium.

An innovative sequence of hypoxia-reoxygenation on adult mouse cardiomyocytes in suspension to perform multilabeling analysis by flow cytometry.

Cardiovascular diseases remain the leading cause of death worldwide. Although major therapeutic progress has been made during the past decades, a better understanding of the underlying mechanisms will certainly help to improve patient's prognosis. In vitro models, particularly adult mouse cardiomyocytes, have been largely used; however, their fragility and large size are major obstacles to the use of flow cytometry. Conventional techniques, such as cell imaging, require the use of large numbers of animals and are time consuming. Here, we described a new, simple, and rapid one-day protocol using living adult mouse cardiomyocytes in suspension exposed to hypoxia-reoxygenation that allows a multilabeling analysis by flow cytometry. Several parameters can be measured by fluorescent probes labeling to assess cell viability (propidium iodide, calcein-AM, and Sytox Green), mitochondrial membrane potential [DilC1(5) and TMRM], reactive oxygen species production (MitoSOX Red), and mitochondrial mass (MitoTracker Deep Red). We address the robustness and sensitivity of our model using a cardioprotective agent, cyclosporine A. Overall, our new experimental set-up offers a high-speed quantitative multilabeling analysis of adult mouse cardiomyocytes exposed to hypoxia-reoxygenation. Our model might be interesting to investigate other cellular stresses (oxidative and inflammation) or to perform pharmacological screening.

Ghrelin-induced multi-organ damage in mice fed obesogenic diet.

OBJECTIVE AND DESIGN: Ghrelin has a key role in modulating energy metabolism and weight gain. The present study aimed at studying the potential role of ghrelin in the development and/or exacerbation of organ damage in a mouse model of diet-induced obesity. OBJECTIVE AND DESIGN: Adult mice were fed one of two diets for 20 weeks: standard high carbohydrate (HC) or high-fat high-sugar (HFHS). Starting week 17, the animals were given regular intraperitoneal ghrelin (160 µg/kg) or saline injections Abdominal fat, serum creatinine, and glucose levels, as well as kidney, liver and heart weight and pathology were assessed. RESULTS: Ghrelin-injected mice showed significant organ damage, which was more exacerbated in HFHS-fed animals. While the HFHS diet was associated with significant liver damage, ghrelin administration did not reverse it. Interestingly, ghrelin administration induced moderate kidney damage and significantly affected the heart by increasing perivascular and myocardium fibrosis, steatosis as well as inflammation. Moreover, serum creatinine levels were higher in the animal group injected with ghrelin. CONCLUSION: Ghrelin administration was associated with increased functional and structural organ damage, regardless of diet. The present study provides novel evidence of multi-organ physiologic alterations secondary to ghrelin administration.

Critical appraisal of STAT3 pattern in adult cardiomyocytes.

The signal transducer and activator of transcription 3, STAT3, transfers cellular signals from the plasma membrane to the nucleus, acting as a signaling molecule and a transcription factor. Reports proposed an additional non-canonical role of STAT3 that could regulate the activity of complexes I and II of the electron transport chain and the opening of the mitochondrial permeability transition pore (PTP) after ischemia-reperfusion in various cell types. The native expression of STAT3 in heart mitochondria, together with a direct versus an indirect transcriptional role in mitochondrial functions, have been recently questioned. The objective of the present study was to investigate the cellular distribution of STAT3 in mouse adult cardiomyocytes under basal and stress conditions, along with assessing its presence and activity in cardiac mitochondria using structural and functional approaches. The analysis of the spatial distribution of STAT3 signal in the cardiomyocytes interestingly showed that it is transversely distributed along the T-tubules and in the nucleus. This distribution was neither affected by hypoxia nor by hypoxia/re­oxygenation conditions. Focusing on the mitochondrial STAT3 localization, our results suggest that serine-phosphorylated STAT3 (PS727-STAT3) and total STAT3 are detected in crude but not in pure mitochondria of mouse adult cardiomyocytes, under basal and ischemia-reperfusion conditions. The inhibition of STAT3, with a pre-validated non-toxic Stattic dose, had no significant effects on mitochondrial respiration, but a weak effect on the calcium retention capacity. Overall, our results exclusively reveal a unique cellular distribution of STAT3 in mouse adult cardiomyocytes, along the T-tubules and in nucleus, under different conditions. They also challenge the expression and activity of STAT3 in mitochondria of these cells under basal conditions and following ischemia-reperfusion. In addition, our results underline technical methods, complemental to cell fractionation, to evaluate STAT3 roles during hypoxia-reoxygenation and at the interface between nucleus and endoplasmic reticulum.

Deleterious Anti-Inflammatory Macrophage Recruitment in Early Post-Infarction Phase: Unraveling the IL-6/MCP-1/STAT3 Axis.

Using a translational approach with an ST-segment myocardial infarction (STEMI) cohort and mouse model of myocardial infarction, we highlighted the role of the secreted IL-6 and MCP-1 cytokines and the STAT3 pathway in heart macrophage recruitment and activation. Cardiac myocytes secrete IL-6 and MCP-1 in response to hypoxic stress, leading to a recruitment and/or polarization of anti-inflammatory macrophages via the STAT3 pathway. In our preclinical model of myocardial infarction, neutralization of IL-6 and MCP-1 or STAT3 pathway reduced infarct size. Together, our data demonstrate that anti-inflammatory macrophages can be deleterious in the acute phase of STEMI.

Impaired aerobic capacity and premature fatigue preceding muscle weakness in the skeletal muscle Tfam-knockout mouse model.

Mitochondrial diseases are genetic disorders that lead to impaired mitochondrial function, resulting in exercise intolerance and muscle weakness. In patients, muscle fatigue due to defects in mitochondrial oxidative capacities commonly precedes muscle weakness. In mice, deletion of the fast-twitch skeletal muscle-specific Tfam gene (Tfam KO) leads to a deficit in respiratory chain activity, severe muscle weakness and early death. Here, we performed a time-course study of mitochondrial and muscular dysfunctions in 11- and 14-week-old Tfam KO mice, i.e. before and when mice are about to enter the terminal stage, respectively. Although force in the unfatigued state was reduced in Tfam KO mice compared to control littermates (wild type) only at 14 weeks, during repeated submaximal contractions fatigue was faster at both ages. During fatiguing stimulation, total phosphocreatine breakdown was larger in Tfam KO muscle than in wild-type muscle at both ages, whereas phosphocreatine consumption was faster only at 14 weeks. In conclusion, the Tfam KO mouse model represents a reliable model of lethal mitochondrial myopathy in which impaired mitochondrial energy production and premature fatigue occur before muscle weakness and early death.

ANT2-Mediated ATP Import into Mitochondria Protects against Hypoxia Lethal Injury.

Following a prolonged exposure to hypoxia-reoxygenation, a partial disruption of the ER-mitochondria tethering by mitofusin 2 (MFN2) knock-down decreases the Ca2+ transfer between the two organelles limits mitochondrial Ca2+ overload and prevents the Ca2+-dependent opening of the mitochondrial permeability transition pore, i.e., limits cardiomyocyte cell death. The impact of the metabolic changes resulting from the alteration of this Ca2+crosstalk on the tolerance to hypoxia-reoxygenation injury remains partial and fragmented between different field of expertise. >In this study, we report that MFN2 loss of function results in a metabolic switch driven by major modifications in energy production by mitochondria. During hypoxia, mitochondria maintain their ATP concentration and, concomitantly, the inner membrane potential by importing cytosolic ATP into mitochondria through an overexpressed ANT2 protein and by decreasing the expression and activity of the ATP hydrolase via IF1. This adaptation further blunts the detrimental hyperpolarisation of the inner mitochondrial membrane (IMM) upon re-oxygenation. These metabolic changes play an important role to attenuate cell death during a prolonged hypoxia-reoxygenation challenge.

Acute Induction of Translocon-Mediated Ca2+ Leak Protects Cardiomyocytes Against Ischemia/Reperfusion Injury.

During myocardial infarction, dysregulation of Ca2+ homeostasis between the reticulum, mitochondria, and cytosol occurs in cardiomyocytes and leads to cell death. Ca2+ leak channels are thought to be key regulators of the reticular Ca2+ homeostasis and cell survival. The present study aimed to determine whether a particular reticular Ca2+ leak channel, the translocon, also known as translocation channel, could be a relevant target against ischemia/reperfusion-mediated heart injury. To achieve this objective, we first used an intramyocardial adenoviral strategy to express biosensors in order to assess Ca2+ variations in freshly isolated adult mouse cardiomyocytes to show that translocon is a functional reticular Ca2+ leak channel. Interestingly, translocon activation by puromycin mobilized a ryanodine receptor (RyR)-independent reticular Ca2+ pool and did not affect the excitation-concentration coupling. Second, puromycin pretreatment decreased mitochondrial Ca2+ content and slowed down the mitochondrial permeability transition pore (mPTP) opening and the rate of cytosolic Ca2+ increase during hypoxia. Finally, this translocon pre-activation also protected cardiomyocytes after in vitro hypoxia reoxygenation and reduced infarct size in mice submitted to in vivo ischemia-reperfusion. Altogether, our report emphasizes the role of translocon in cardioprotection and highlights a new paradigm in cardioprotection by functionally uncoupling the RyR-dependent Ca2+ stores and translocon-dependent Ca2+ stores.

An Update on the Multifaceted Roles of STAT3 in the Heart.

Signal transducer and activator of transcription 3 (STAT3) is a signaling molecule and transcription factor that plays important protective roles in the heart. The protection mediated by STAT3 is attributed to its genomic actions as a transcription factor and other non-genomic roles targeting mitochondrial function and autophagy. As a transcription factor, STAT3 upregulates genes that are anti-oxidative, anti-apoptotic, and pro-angiogenic, but suppresses anti-inflammatory and anti-fibrotic genes. Its suppressive effects on gene expression are achieved through competing with other transcription factors or cofactors. STAT3 is also linked to the modification of mRNA expression profiles in cardiac cells by inhibiting or inducing miRNA. In addition to these genomic roles, STAT3 is suggested to function protectively in mitochondria, where it regulates ROS production, in part by regulating the activities of the electron transport chain complexes, although our recent evidence calls this role into question. Nonetheless, STAT3 is a key player known to be activated in the cardioprotective ischemic conditioning protocols. Through these varied roles, STAT3 participates in various mechanisms that contribute to cardioprotection against different heart pathologies, including myocardial infarction, hypertrophy, diabetic cardiomyopathy, and peripartum cardiomyopathy. Understanding how STAT3 is involved in the protective mechanisms against these different cardiac pathologies could lead to novel therapeutic strategies to treat them.

A Dynamic Transcriptional Analysis Reveals IL-6 Axis as a Prominent Mediator of Surgical Acute Response in Non-ischemic Mouse Heart.

BACKGROUND: Ischemic heart diseases are a major cause of death worldwide. Different animal models, including cardiac surgery, have been developed over time. Unfortunately, the surgery models have been reported to trigger an important inflammatory response that might be an effect modifier, where involved molecular processes have not been fully elucidated yet. OBJECTIVE: We sought to perform a thorough characterization of the sham effect in the myocardium and identify the interfering inflammatory reaction in order to avoid misinterpretation of the data via systems biology approaches. METHODS AND RESULTS: We combined a comprehensive analytical pipeline of mRNAseq dataset and systems biology analysis to characterize the acute phase response of mouse myocardium at 0 min, 45 min, and 24 h after surgery to better characterize the molecular processes inadvertently induced in sham animals. Our analysis showed that the surgical intervention induced 1209 differentially expressed transcripts (DETs). The clustering of positively co-regulated transcript modules at 45 min fingerprinted the activation of signalization pathways, while positively co-regulated genes at 24 h identified the recruitment of neutrophils and the differentiation of macrophages. In addition, we combined the prediction of transcription factors (TF) regulating DETs with protein-protein interaction networks built from these TFs to predict the molecular network which have induced the DETs. By mean of this retro-analysis of processes upstream gene transcription, we revealed a major role of the Il-6 pathway and further confirmed a significant increase in circulating IL-6 at 45 min after surgery. CONCLUSION: This study suggests that a strong induction of the IL-6 axis occurs in sham animals over the first 24 h and leads to the induction of inflammation and tissues' homeostasis processes.