Epigenetic Mechanisms in Sepsis-Associated Acute Kidney Injury.

Sepsis, the dysregulated immune response of the host to infections, leads to numerous complications, including multiple organ dysfunction with sepsis-associated acute kidney injury (SA-AKI) being a frequent complication associated with increased risk of mortality and the progression toward chronic kidney disease (CKD). Several mechanisms have been widely investigated in understanding the complex pathophysiology of SA-AKI, including hemodynamic alterations, inflammation, oxidative stress, and direct cellular injury driven by pathogens or cell-derived products (pathogen-associated molecular patterns and damage-associated molecular patterns). Despite advancements in the management of septic patients, the prognosis of SA-AKI patients remains significantly poor and is associated with high in-hospital mortality and adverse long-term outcomes. Therefore, recent research has focused on the early identification of specific SA-AKI endotypes and subphenotypes through epigenetic analysis and the use of potential biomarkers, either alone or in combination with clinical data, to improve prognosis. Epigenetic regulation, such as DNA methylation, histone modifications, and noncoding RNA modulation, is crucial in modulating gene expression in response to stress and renal injury in SA-AKI. At the same time, these modifications are dynamic and reversible processes that can alter gene expression in several pathways implicated in the context of SA-AKI, including inflammation, immune response, and tolerance status. In addition, specific epigenetic modifications may exacerbate renal damage by causing persistent inflammation or cellular metabolic reprogramming, leading to progression toward CKD. This review aims to provide a comprehensive understanding of the epigenetic characteristics that define SA-AKI, also exploring targeted therapies that can improve patient outcomes and limit the chronic progression of this syndrome.

The p.E152K-STIM1 mutation deregulates Ca2+ signaling contributing to chronic pancreatitis.

Since deregulation of intracellular Ca2+ can lead to intracellular trypsin activation, and stromal interaction molecule-1 (STIM1) protein is the main regulator of Ca2+ homeostasis in pancreatic acinar cells, we explored the Ca2+ signaling in 37 STIM1 variants found in three pancreatitis patient cohorts. Extensive functional analysis of one particular variant, p.E152K, identified in three patients, provided a plausible link between dysregulated Ca2+ signaling within pancreatic acinar cells and chronic pancreatitis susceptibility. Specifically, p.E152K, located within the STIM1 EF-hand and sterile α-motif domain, increased the release of Ca2+ from the endoplasmic reticulum in patient-derived fibroblasts and transfected HEK293T cells. This event was mediated by altered STIM1-sarco/endoplasmic reticulum calcium transport ATPase (SERCA) conformational change and enhanced SERCA pump activity leading to increased store-operated Ca2+ entry (SOCE). In pancreatic AR42J cells expressing the p.E152K variant, Ca2+ signaling perturbations correlated with defects in trypsin activation and secretion, and increased cytotoxicity after cholecystokinin stimulation.This article has an associated First Person interview with the first author of the paper.

GCN5 contributes to intracellular lipid accumulation in human primary cardiac stromal cells from patients affected by Arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is a genetic disease associated with sudden cardiac death and cardiac fibro-fatty replacement. Over the last years, several works have demonstrated that different epigenetic enzymes can affect not only gene expression changes in cardiac diseases but also cellular metabolism. Specifically, the histone acetyltransferase GCN5 is known to facilitate adipogenesis and modulate cardiac metabolism in heart failure. Our group previously demonstrated that human primary cardiac stromal cells (CStCs) contribute to adipogenesis in the ACM pathology. Thus, this study aims to evaluate the role of GCN5 in ACM intracellular lipid accumulation. To do so, CStCs were obtained from right ventricle biopsies of ACM patients and from samples of healthy cadaveric donors (CTR). GCN5 expression was increased both in ex vivo and in vitro ACM samples compared to CTR. When GCN5 expression was silenced or pharmacologically inhibited by the administration of MB-3, we observed a reduction in lipid accumulation and a mitigation of reactive oxygen species (ROS) production in ACM CStCs. In agreement, transcriptome analysis revealed that the presence of MB-3 modified the expression of pathways related to cellular redox balance. Altogether, our findings suggest that GCN5 inhibition reduces fat accumulation in ACM CStCs, partially by modulating intracellular redox balance pathways.

Specialized Pro-Resolving Lipid Mediators in Cystic Fibrosis.

In cystic fibrosis (CF), impaired airway surface hydration (ASL) and mucociliary clearance that promote chronic bacterial colonization, persistent inflammation, and progressive structural damage to the airway wall architecture are typically explained by ion transport abnormalities related to the mutation of the gene coding for the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. However, the progressive and unrelenting inflammation of the CF airway begins early in life, becomes persistent, and is excessive relative to the bacterial burden. Intrinsic abnormalities of the inflammatory response in cystic fibrosis have been suggested but the mechanisms involved remain poorly understood. This review aims to give an overview of the recent advances in the understanding of the defective resolution of inflammation in CF including the abnormal production of specialized pro-resolving lipid mediators (lipoxin and resolvin) and their impact on the pathogenesis of the CF airway disease.

SERCA and PMCA pumps contribute to the deregulation of Ca2+ homeostasis in human CF epithelial cells.

Cystic Fibrosis (CF) disease is caused by mutations in the CFTR gene (CF transmembrane conductance regulator). F508 deletion is the most represented mutation, and F508del-CFTR is absent of plasma membrane and accumulates into the endoplasmic reticulum (ER) compartment. Using specific Ca2+ genetics cameleon probes, we showed in the human bronchial CF epithelial cell line CFBE that ER Ca2+ concentration was strongly increased compared to non-CF (16HBE) cells, and normalized by the F508del-CFTR corrector agent, VX-809. We also showed that ER F508del-CFTR retention increases SERCA (Sarcoplasmic/Reticulum Ca2+ ATPase) pump activity whereas PMCA (Plasma Membrane Ca2+ ATPase) activities were reduced in these CF cells compared to corrected CF cells (VX-809) and non-CF cells. We are showing for the first time CFTR/SERCA and CFTR/PMCA interactions that are modulated in CF cells and could explain part of Ca2+ homeostasis deregulation due to mislocalization of F508del-CFTR. Using ER or mitochondria genetics Ca2+ probes, we are showing that ER Ca2+ content, mitochondrial Ca2+ uptake, SERCA and PMCA pump, activities are strongly affected by the localization of F508del-CFTR protein.

Rio1 downregulates centromeric RNA levels to promote the timely assembly of structurally fit kinetochores.

Kinetochores assemble on centromeres via histone H3 variant CENP-A and low levels of centromere transcripts (cenRNAs). The latter are ensured by the downregulation of RNA polymerase II (RNAPII) activity, and cenRNA turnover by the nuclear exosome. Using S. cerevisiae, we now add protein kinase Rio1 to this scheme. Yeast cenRNAs are produced either as short (median lengths of 231 nt) or long (4458 nt) transcripts, in a 1:1 ratio. Rio1 limits their production by reducing RNAPII accessibility and promotes cenRNA degradation by the 5'-3'exoribonuclease Rat1. Rio1 similarly curtails the concentrations of noncoding pericenRNAs. These exist as short transcripts (225 nt) at levels that are minimally two orders of magnitude higher than the cenRNAs. In yeast depleted of Rio1, cen- and pericenRNAs accumulate, CEN nucleosomes and kinetochores misform, causing chromosome instability. The latter phenotypes are also observed with human cells lacking orthologue RioK1, suggesting that CEN regulation by Rio1/RioK1 is evolutionary conserved.

CF Patients' Airway Epithelium and Sex Contribute to Biosynthesis Defects of Pro-Resolving Lipids.

Specialized pro-resolving lipid mediators (SPMs) as lipoxins (LX), resolvins (Rv), protectins (PD) and maresins (MaR) promote the resolution of inflammation. We and others previously reported reduced levels of LXA4 in bronchoalveolar lavages from cystic fibrosis (CF) patients. Here, we investigated the role of CF airway epithelium in SPMs biosynthesis, and we evaluated its sex specificity. Human nasal epithelial cells (hNEC) were obtained from women and men with or without CF. Lipids were quantified by mass spectrometry in the culture medium of hNEC grown at air-liquid interface and the expression level and localization of the main enzymes of SPMs biosynthesis were assessed. The 5-HETE, LXA4, LXB4, RvD2, RvD5, PD1 and RvE3 levels were significantly lower in samples derived from CF patients compared with non-CF subjects. Within CF samples, the 12-HETE, 15-HETE, RvD3, RvD4, 17-HODHE and PD1 were significantly lower in samples derived from females. While the mean expression levels of 15-LO, 5-LO and 12-LO do not significantly differ either between CF and non-CF or between female and male samples, the SPMs content correlates with the level of expression of several enzymes involved in SPMs metabolism. In addition, the 5-LO localization significantly differed from cytoplasmic in non-CF to nucleic (or nuclear envelope) in CF hNEC. Our studies provided evidence for lower abilities of airway epithelial cells derived from CF patients and more markedly, females to produce SPMs. These data are consistent with a contribution of CF airway epithelium in the abnormal resolution of inflammation and with worse pulmonary outcomes in women.

PAR2, Keratinocytes, and Cathepsin S Mediate the Sensory Effects of Ciguatoxins Responsible for Ciguatera Poisoning.

Ciguatera fish poisoning is caused by the consumption of fish contaminated with ciguatoxins (CTXs). The most distressing symptoms are cutaneous sensory disturbances, including cold dysesthesia and itch. CTXs are neurotoxins known to activate voltage-gated sodium channels, but no specific treatment exists. Peptidergic neurons have been critically involved in ciguatera fish poisoning sensory disturbances. Protease-activated receptor-2 (PAR2) is an itch- and pain-related G protein‒coupled receptor whose activation leads to a calcium-dependent neuropeptide release. In this study, we studied the role of voltage-gated sodium channels, PAR2, and the PAR2 agonist cathepsin S in the cytosolic calcium increase and subsequent release of the neuropeptide substance P elicited by Pacific CTX-2 (P-CTX-2) in rat sensory neurons and human epidermal keratinocytes. In sensory neurons, the P-CTX-2‒evoked calcium response was driven by voltage-gated sodium channels and PAR2-dependent mechanisms. In keratinocytes, P-CTX-2 also induced voltage-gated sodium channels and PAR2-dependent marked calcium response. In the cocultured cells, P-CTX-2 significantly increased cathepsin S activity, and cathepsin S and PAR2 antagonists almost abolished P-CTX-2‒elicited substance P release. Keratinocytes synergistically favored the induced substance P release. Our results demonstrate that the sensory effects of CTXs involve the cathepsin S-PAR2 pathway and are potentiated by their direct action on nonexcitable keratinocytes through the same pathway.

Measuring Calumenin Impact on ER-Calcium Depletion Using Transient Calumenin Overexpression and Silencing.

Calumenin is a secretory pathway protein regulating different endoplasmic reticulum (ER) proteins such as the sarco-endoplasmic reticulum calcium ATPase (SERCA) pumps. Combined with its diverse cellular distribution, its calcium-binding ability, and its interaction with proteins involved in calcium signaling, it is easy to speculate on future description of important roles of calumenin in calcium homeostasis in many cell types, as it was initially observed in muscle cells. In this chapter, we describe basic techniques to modulate calumenin expression and detect its impact on ER calcium content using classic transfection and Western blot techniques, as well as ER calcium measurement using microplate reader.

Major Role for TRPV1 and InsP3R in PAR2-Elicited Inflammatory Mediator Production in Differentiated Human Keratinocytes.

PAR2 activation in basal keratinocytes stimulates inflammation via the Ca2+-dependent production of mediators such as IL-1ß, TNF-α, and TSLP. In this study, we investigated PAR2 calcium signaling and the consequent production of inflammatory mediators in differentiated human primary keratinocytes (DhPKs). Stimulation with the PAR2-activating peptide SLIGKV promoted Ca2+ store depletion in both undifferentiated human primary keratinocytes and DhPKs. SLIGKV-evoked Ca2+ store depletion did not trigger the store-operated Ca2+ entry (i.e., SOCE) through ORAI1 in DhPKs compared with undifferentiated human primary keratinocytes. The inhibition of phospholipase C and the concomitant inhibition of TRPV1 and inositol triphosphate receptor in DhPKs abrogated the SLIGKV-evoked Ca2+ store depletion; NF-κB activity; and the production of inflammatory mediators such as IL-1ß, TNF-α, and TSLP. Taken together, these results indicate a key role for both InsP3R and TRPV1 in Ca2+ internal stores in the PAR2-evoked Ca2+ release and consequent skin inflammation in DhPKs. These findings may provide clues to understanding the pathological role of DhPKs in skin disorders in which PAR2 is known to be involved, such as atopic dermatitis, Netherton syndrome, and psoriasis.

Calumenin contributes to ER-Ca2+ homeostasis in bronchial epithelial cells expressing WT and F508del mutated CFTR and to F508del-CFTR retention.

Cystic Fibrosis (CF) is the most frequent fatal genetic disease in Caucasian populations. Mutations in the chloride channel CF Transmembrane Conductance Regulator (CFTR) gene are responsible for functional defects of the protein and multiple associated dysregulations. The most common mutation in patients with CF, F508del-CFTR, causes defective CFTR protein folding. Thus minimal levels of the receptor are expressed at the cell surface as the mutated CFTR is retained in the endoplasmic reticulum (ER) where it correlates with defective calcium (Ca2+) homeostasis. In this study, we discovered that the Ca2+ binding protein Calumenin (CALU) is a key regulator in the maintenance of ER-Ca2+ calcium homeostasis in both wild type and F508del-CFTR expressing cells. Calumenin modulates SERCA pump activity without drastically affecting ER-Ca2+ concentration. In addition, reducing Calumenin expression in CF cells results in a partial restoration of CFTR activity, highlighting a potential function of Calumenin in CFTR maturation. These findings demonstrate a pivotal role for Calumenin in CF cells, providing insights into how modulation of Calumenin expression or activity may be used as a potential therapeutic tool to correct defects in F508del-CFTR.