Aberrant NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3) inflammasome activation in innate immune cells, triggered by diverse cellular danger signals, leads to the production of inflammatory cytokines (IL-1β and IL-18) and cell death by pyroptosis. These processes are involved in the pathogenesis of a wide range of diseases such as autoimmune, neurodegenerative, renal, metabolic, vascular diseases and cancer, and during physiological processes such as aging. Epigenetic dynamics mediated by changes in DNA methylation patterns, chromatin assembly and non-coding RNA expression are key regulators of the expression of inflammasome components and its further activation. Here, we review the role of the epigenome in the expression, assembly, and activation of the NLRP3 inflammasome, providing a critical overview of its involvement in the disease and discussing how targeting these mechanisms by epigenetic treatments could be a useful strategy for controlling NLRP3-related inflammatory diseases.

Loss of protein homeostasis (proteostasis) in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR), restoring correct protein folding. Sustained ER stress exacerbates activation of the major UPR branches (IRE1α/XBP1, PERK/ATF4, ATF6), inducing expression of numerous genes involved in inflammation, cell death, autophagy, and oxidative stress. We investigated whether epigenetic dynamics mediated by histone H3K9 and H3K27 methylation might help to reduce or inhibit the exacerbated and maladaptive UPR triggered in tubular epithelial cells. Epigenetic treatments, specific silencing, and chromatin immunoprecipitation assays were performed in human proximal tubular cells subjected to ER stress. Pharmacological blockage of KDM4C and JMJD3 histone demethylases with SD-70 and GSKJ4, respectively, enhanced trimethylation of H3K9 and H3K27 in the ATF4 and XBP1 genes, inhibiting their expression and that of downstream genes. Conversely, specific G9a and EZH2 knockdown revealed increases in ATF4 and XBP1 expression. This is a consequence of the reduced recruitment of G9a and EZH2 histone methylases, diminished H3K9me3 and H3K27me3 levels, and enhanced histone acetylation at the ATF4 and XBP1 promoter region. G9a and EZH2 cooperate to maintain the repressive chromatin structure in both UPR-induced genes, ATF4 and XBP1. Therefore, preserving histone H3K9 and H3K27 methylation could ameliorate the ER stress, and consequently the oxidative stress and the triggered pathological processes that aggravate renal damage.

Background and Aims The vitamin D receptor (VDR) is a nuclear receptor that acts as a ligand-induced transcription factor regulating the renal expression of numerous genes with anti-inflammatory and anti-fibrotic effects, among others. For that, VDR requires the binding of its ligand, 1,25 (OH)2D3, to further heterodimerize with its co-activator, RXR, and translocate into the nucleus. Vitamin D deficiency in patients with renal disease leads to a decrease in VDR-mediated signaling and, consequently, of its beneficial functions. Additionally, these patients have intestinal dysbiosis, which leads to alterations in the production of microbial metabolites. Short-chain fatty acids (SCFAs) are metabolites with a clear anti-inflammatory and immunoregulatory role. Until now, some studies have studied their role as regulators of the inflammation and oxidative stress during the renal damage.1,2 However, their role in the VDR-mediated signaling in the kidney and the potential consequences to prevent the progression of the disease have not yet been explored in detail. Method The tubular epithelial cell line, HK2, was used to analyze the effect of the SCFAs, Propionate (Prop,1-15mM) and Butyrate (But, 0.5-3 mM), in the VDR expression and their target-genes. In vitro treatment with histone deacetylases (HDACs) inhibitors and specific HDAC1 and HDAC3 siRNAs were used to determine the role of both SCFAs as epigenetic remodelers. The binding of VDR to HDAC1/3 and RXR was determined by ChIP and co-immunoprecipitation assays. The in vivo effect of these SCFAs was evaluated in an acute kidney injury mice model induced by folic acid (250 mg/kg) and administration of Prop (200mg/kg) or But (500mg/kg) at different time points. The expression of VDR and its target genes, inflammatory cell infiltration, renal damage markers and renal function parameters were evaluated at shorter (24h) and longer times (40 days). Results Treatment of tubular cells with Prop and But induces, in a dose-dependent manner, the VDR gene transcription. This effect is similar to the one obtained by using specific inhibitors and siRNA treatments against HDAC1/3. We determine using ChIP assay, that HDAC1/3 are recruited to the promoter regions of the VDR gene, blocking its expression. In presence of Prop and But, these HDACs are displaced, increasing the acetylation levels and VDR transcription. Moreover, Prop and But prevent the degradation of VDR by the proteasome, increasing its stability and enhancing VDR protein levels. In the presence of both SCFAs, VDR dimerizes with RXR initiating its translocation to the nucleus and allowing the transcription of its dependent genes, such as Cyp24a1 and E-cadherin. Of note, VDR activation by Prop and But is additive to the effect achieved by the vitamin D alone. In vivo studies reported that administration of Prop or But prevents the loss of VDR expression 24h after induction of the damage, leading to its activation and to the expression of its target genes. Additionally, a decrease in the recruitment of neutrophils to the kidney was observed associated to a reduced expression of IL-6. These changes are accompanied by a decrease of the kidney damage markers (KIM-1 and NGAL) and a significant reduction of the creatinine and blood urea nitrogen serum levels. In a second model of AKI-to CKD transition, administration of both SCFAs shows a decrease in the inflammatory (Ccl20, Ccl2, Lif, Ltb, Csf2SF2, Il18, Ccl5, Tnf-α) and pro-fibrotic markers (Fsp1, α-Sma, Col1a1, Fn1) and a partial recovery of the glomerular filtration rate at long term. Conclusion Propionate and Butyrate, not only induce the VDR gene transcription in renal tubular cells but are also able to stabilize and activate the VDR protein. Accordingly, both metabolites are able to restore the loosed expression and activation of VDR due to induced renal damage, reduce the infiltration of immune cells, and partially recover the renal function. Thus, strategies aimed to increase the propionate and butyrate levels with postbiotics could be useful to ameliorate the AKI renal damage and CKD transition.

BACKGROUND AND AIMS Alterations in protein homeostasis in tubular cells lead to endoplasmic reticulum (ER) stress activating the unfolded protein response (UPR) pathway, which contributes to repair or aggravate the renal damage [1]. This pathway is initiated by three major protein sensors (IRE1α, PERK and ATF6) that activate their corresponding transcription factors (TF), XBP1, ATF4 and ATF6, respectively, to ultimately regulate the transcription of numerous genes essential for cell survival. However, an exacerbated activation of the UPR pathway can also lead to the expression of genes related to inflammation and fibrosis, cell death or autophagy contributing to perpetuate the renal damage [2]. The balance between these two processes (adaptive/maladaptive response) is mediated not only by the length and strength of the initial stimulus, but also by changes in the chromatin structure that may induce or repress gene transcription. Epigenetic changes are mainly mediated by the expression and recruitment of epigenetic enzymes, such as histone methyltransferases (HMTs) and demethylases (HDMs) to target genes in order to modify their expression. Thus, the aim of this study pursues to identify the epigenetic changes mediated by the histone methylation (H3K9 and H3K27) in the UPR pathway and to explore the role of the epigenetic drugs as potential treatments for kidney disease. METHOD The tubular epithelial cell line, HK2, was used to analyse the epigenetic changes in vitro before and after induction of ER stress mediated by Thapsigargin (Tg), an ER Ca2+ 2'-ATPase inhibitor and strongUPR inductor. Specific pharmacological inhibitors of the G9a and EZH2 HMTs, BIX-01 294 and GSK126, respectively, and of the JMJD3 and KDM4C HDMs, GSKJ4 and SD-70, respectively, or small interfering RNAs were used. The recruitment of these epigenetic enzymes and the presence of the H3K9me3 and H3K27me3 repressive histone marks were analysed by chromatin immunoprecipitation (ChIP) and coimmunoprecipitation assays. RESULTS HK-2 treatment with the BIX-01 294 or GSK126 pharmacological inhibitors or specific gene silencing of the G9a and EZH2 enzymes reveals an increase of the expression of ATF4 and XBP1 TFs, without inducing changes in ATF6 transcription. Moreover, this effect is additive to the one observed with Tg, indicating that changes in the chromatin structure are required for a full transcription and UPR activation. These results correlated with a lower recruitment of G9a and EZH2, and decreased H3K9me3 and H3K27me3 levels at the promoter region of ATF4 and XBP1 genes, corresponding with the increased transcription of these TFs. G9a and EZH2 HMTs act in coordination, so inhibition of G9a significantly reduces the recruitment of EZH2 and H3K27me3 levels to the regulatory region of ATF4 and XBP1 genes, and vice versa with EZH2 inhibition. In addition, enrichment in the global acetylation levels at histone H3 and H4 was observed, cooperatively facilitating the opening of the chromatin and the accessibility to transcriptional regulators. In accordance with these results, we demonstrate that blockage of the JMJD3 and KDM4C enzymes, responsible for demethylation of H3K9me3 and H3K27me3 marks, respectively, using the SD-70 and GSKJ4 epigenetic drugs, inhibits ATF4 and XBP1 expression under ER stress conditions and, consequently, the triggering of a maladaptative response. CONCLUSION Changes in the chromatin dynamics mediated by the H3K9me3 and H3K27me3 histone marks are key to regulate the expression of the UPR transcription factors ATF4 and XBP1 after ER stress activation. Pharmacological treatment with the epigenetic drugs SD-70 and GSKJ4 blocks the expression of these TFs and thus, the activation of the pathophysiological processes that contribute to aggravate renal damage triggered by the UPR pathway activation. [1] Yan M., et al. Endoplasmic reticulum stress in ischemic and nephrotoxic acute kidney injury. Ann Med. 2018, 50: 381–390. [2] Hetz, C.; et al. Mechanisms, regulation and functions of the unfolded protein response. Nat Rev Mol Cell Biol. 2020, 21: 421–438.