Palmitic Acid-Induced Long Noncoding RNA PARAIL Regulates Inflammation via Interaction With RNA-Binding Protein ELAVL1 in Monocytes and Macrophages.

BACKGROUND: Obesity and diabetes are associated with elevated free fatty acids like palmitic acid (PA), which promote chronic inflammation and impaired inflammation resolution associated with cardiometabolic disorders. Long noncoding RNAs (lncRNAs) are implicated in inflammatory processes; however, their roles in PA-regulated inflammation and resolution are unclear. METHODS: We performed RNA-sequencing analysis to identify PA-regulated coding genes and novel lncRNAs in CD14+ monocytes from healthy volunteers. We investigated the regulation and function of an uncharacterized PA-induced lncRNA PARAIL (PA-regulated anti-inflammatory lncRNA). We examined its role in inflammation resolution by employing knockdown and overexpression strategies in human and mouse macrophages. We also used RNA pulldown coupled with mass spectrometry to identify PARAIL interacting nuclear proteins and their mechanistic involvement in PARAIL functions in human macrophages. RESULTS: Treatment of human CD14+ monocytes with PA-induced several lncRNAs and genes associated with inflammatory phenotype. PA strongly induced lncRNA PARAIL expressed near RIPK2. PARAIL was also induced by cytokines and infectious agents in human monocytes/macrophages and was regulated by NF-κB (nuclear factor-kappa B). Time course studies showed PARAIL was induced during inflammation resolution phase in PA-treated macrophages. PARAIL knockdown with antisense oligonucleotides upregulated key inflammatory genes and vice versa with PARAIL overexpression. We found that PARAIL interacts with ELAVL1 (ELAV-like RNA-binding protein 1) protein via adenylate/uridylate-rich elements (AU-rich elements; AREs). ELAVL1 knockdown inhibited the anti-inflammatory functions of PARAIL. Moreover, PARAIL knockdown increased cytosolic localization of ELAVL1 and increased the stability of ARE-containing inflammatory genes. Mouse orthologous Parail was downregulated in macrophages from mice with diabetes and atherosclerosis. Parail overexpression attenuated proinflammatory genes in mouse macrophages. CONCLUSIONS: Upregulation of PARAIL under acute inflammatory conditions contributes to proresolution mechanisms via PARAIL-ELAVL1 interactions. Conversely, PARAIL downregulation in cardiometabolic diseases enhances ELAVL1 function and impairs inflammation resolution to further augment inflammation. Thus, inflammation-resolving lncRNAs like PARAIL represent novel targets to combat inflammatory cardiometabolic diseases.

Angiotensin II-Induced Long Non-Coding RNA Alivec Regulates Chondrogenesis in Vascular Smooth Muscle Cells.

Long non-coding RNAs (lncRNAs) play key roles in Angiotensin II (AngII) signaling but their role in chondrogenic transformation of vascular smooth muscle cells (VSMCs) is unknown. We describe a novel AngII-induced lncRNA Alivec (Angiotensin II-induced lncRNA in VSMCs eliciting chondrogenic phenotype) implicated in VSMC chondrogenesis. In rat VSMCs, Alivec and the nearby gene Acan, a chondrogenic marker, were induced by growth factors AngII and PDGF and the inflammatory cytokine TNF-α. AngII co-regulated Alivec and Acan through the activation of AngII type1 receptor signaling and Sox9, a master transcriptional regulator of chondrogenesis. Alivec knockdown with GapmeR antisense-oligonucleotides attenuated the expression of AngII-induced chondrogenic marker genes, including Acan, and inhibited the chondrogenic phenotype of VSMCs. Conversely, Alivec overexpression upregulated these genes and promoted chondrogenic transformation. RNA-pulldown coupled to mass-spectrometry identified Tropomyosin-3-alpha and hnRNPA2B1 proteins as Alivec-binding proteins in VSMCs. Furthermore, male rats with AngII-driven hypertension showed increased aortic expression of Alivec and Acan. A putative human ortholog ALIVEC, was induced by AngII in human VSMCs, and this locus was found to harbor the quantitative trait loci affecting blood pressure. Together, these findings suggest that AngII-regulated lncRNA Alivec functions, at least in part, to mediate the AngII-induced chondrogenic transformation of VSMCs implicated in vascular dysfunction and hypertension.

Emerging Role of Long Non-Coding RNAs in Diabetic Vascular Complications.

Chronic metabolic disorders such as obesity and diabetes are associated with accelerated rates of macrovascular and microvascular complications, which are leading causes of morbidity and mortality worldwide. Further understanding of the underlying molecular mechanisms can aid in the development of novel drug targets and therapies to manage these disorders more effectively. Long non-coding RNAs (lncRNAs) that do not have protein-coding potential are expressed in a tissue- and species-specific manner and regulate diverse biological processes. LncRNAs regulate gene expression in cis or in trans through various mechanisms, including interaction with chromatin-modifying proteins and other regulatory proteins and via posttranscriptional mechanisms, including acting as microRNA sponges or as host genes of microRNAs. Emerging evidence suggests that major pathological factors associated with diabetes such as high glucose, free fatty acids, proinflammatory cytokines, and growth factors can dysregulate lncRNAs in inflammatory, cardiac, vascular, and renal cells leading to altered expression of key inflammatory genes and fibrotic genes associated with diabetic vascular complications. Here we review recent reports on lncRNA characterization, functions, and mechanisms of action in diabetic vascular complications and translational approaches to target them. These advances can provide new insights into the lncRNA-dependent actions and mechanisms underlying diabetic vascular complications and uncover novel lncRNA-based biomarkers and therapies to reduce disease burden and mortality.

lncRNA DRAIR is downregulated in diabetic monocytes and modulates the inflammatory phenotype via epigenetic mechanisms.

Long noncoding RNAs (lncRNAs) are increasingly implicated in the pathology of diabetic complications. Here, we examined the role of lncRNAs in monocyte dysfunction and inflammation associated with human type 2 diabetes mellitus (T2D). RNA sequencing analysis of CD14+ monocytes from patients with T2D versus healthy controls revealed downregulation of antiinflammatory and antiproliferative genes, along with several lncRNAs, including a potentially novel divergent lncRNA diabetes regulated antiinflammatory RNA (DRAIR) and its nearby gene CPEB2. High glucose and palmitic acid downregulated DRAIR in cultured CD14+ monocytes, whereas antiinflammatory cytokines and monocyte-to-macrophage differentiation upregulated DRAIR via KLF4 transcription factor. DRAIR overexpression increased antiinflammatory and macrophage differentiation genes but inhibited proinflammatory genes. Conversely, DRAIR knockdown attenuated antiinflammatory genes, promoted inflammatory responses, and inhibited phagocytosis. DRAIR regulated target gene expression through interaction with chromatin, as well as inhibition of the repressive epigenetic mark H3K9me2 and its corresponding methyltransferase G9a. Mouse orthologous Drair and Cpeb2 were also downregulated in peritoneal macrophages from T2D db/db mice, and Drair knockdown in nondiabetic mice enhanced proinflammatory genes in macrophages. Thus, DRAIR modulates the inflammatory phenotype of monocytes/macrophages via epigenetic mechanisms, and its downregulation in T2D may promote chronic inflammation. Augmentation of endogenous lncRNAs like DRAIR could serve as novel antiinflammatory therapies for diabetic complications.

Role of epigenetic mechanisms regulated by enhancers and long noncoding RNAs in cardiovascular disease.

PURPOSE OF REVIEW: Hyperlipidemia, hypertension, diabetes and related metabolic disorders increase the risk for cardiovascular disease (CVD). Despite significant progress in the identification of key mechanisms and genetic polymorphisms linked to various CVDs, the rates of CVDs continue to escalate, underscoring the need to evaluate additional mechanisms for more effective therapies. Environment and lifestyle changes can alter epigenetic mechanisms mediated by histone modifications and long noncoding RNAs (lncRNAs) which play important roles in gene regulation. The review summarizes recent findings on the role of epigenetic mechanisms in CVD. RECENT FINDINGS: Recent studies identified dysregulated histone modifications and chromatin modifying proteins at cis-regulatory elements, including enhancers/super-enhancers, mediating the expression of genes associated with CVD in vascular and immune cells in response to growth factors and inflammatory mediators. Several lncRNAs have also been reported to contribute to pathological gene expression via cis and trans mechanisms involving interactions with nuclear proteins, co-operation with enhancers/super enhancers and acting as microRNA sponges. SUMMARY: Epigenomic approaches in cells affected in CVDs can be exploited to understand the function of genetic polymorphisms at cis-regulatory elements and crosstalk between enhancers and lncRNAs associated with disease susceptibility and progression. The reversible nature of epigenetics provides opportunities for the development of novel therapeutic strategies for CVD.

Novel Long Noncoding RNA, Macrophage Inflammation-Suppressing Transcript (MIST), Regulates Macrophage Activation During Obesity.

OBJECTIVE: Systemic low-grade inflammation associated with obesity and metabolic syndrome is a strong risk factor for the development of diabetes mellitus and associated cardiovascular complications. This inflammatory state is caused by release of proinflammatory cytokines by macrophages, especially in adipose tissue. Long noncoding RNAs regulate macrophage activation and inflammatory gene networks, but their role in macrophage dysfunction during diet-induced obesity has been largely unexplored. Approach and Results: We sequenced total RNA from peritoneal macrophages isolated from mice fed either high-fat diet or standard diet and performed de novo transcriptome assembly to identify novel differentially expressed mRNAs and long noncoding RNAs. A top candidate long noncoding RNA, macrophage inflammation-suppressing transcript (Mist), was downregulated in both peritoneal macrophages and adipose tissue macrophages from high-fat diet-fed mice. GapmeR-mediated Mist knockdown in vitro and in vivo upregulated expression of genes associated with immune response and inflammation and increased modified LDL (low-density lipoprotein) uptake in macrophages. Conversely, Mist overexpression decreased basal and LPS (lipopolysaccharide)-induced expression of inflammatory response genes and decreased modified LDL uptake. RNA-pull down coupled with mass spectrometry showed that Mist interacts with PARP1 (poly [ADP]-ribose polymerase-1). Disruption of this RNA-protein interaction increased PARP1 recruitment and chromatin PARylation at promoters of inflammatory genes, resulting in increased gene expression. Furthermore, human orthologous MIST was also downregulated by proinflammatory stimuli, and its expression in human adipose tissue macrophages inversely correlated with obesity and insulin resistance. CONCLUSIONS: Mist is a novel protective long noncoding RNA, and its loss during obesity contributes to metabolic dysfunction and proinflammatory phenotype of macrophages via epigenetic mechanisms.

Dysregulation of histone H3 lysine 27 trimethylation in transforming growth factor-ß1-induced gene expression in mesangial cells and diabetic kidney.

Transforming growth factor-ß1 (TGF-ß)-induced fibrotic and inflammatory genes in renal mesangial cells (MCs) play important roles in glomerular dysfunction associated with diabetic nephropathy (DN). TGF-ß regulates gene expression in MCs by altering key chromatin histone modifications at target gene promoters. However, the role of the repressive histone H3 lysine 27 trimethylation (H3K27me3) modification is unclear. Here we show that TGF-ß reduces H3K27me3 at the Ctgf, Serpine1, and Ccl2 gene promoters in rat MCs (RMCs) and reciprocally up-regulates the expression of these pro-fibrotic and inflammatory genes. In parallel, TGF-ß down-regulates Enhancer of Zeste homolog 2 (Ezh2), an H3K27me3 methyltransferase, and decreases its recruitment at Ctgf and Ccl2 but not Serpine1 promoters. Ezh2 knockdown with siRNAs enhances TGF-ß-induced expression of these genes, supporting its repressive function. Mechanistically, Ezh2 down-regulation is mediated by TGF-ß-induced microRNA, miR-101b, which targets Ezh2 3'-UTR. TGF-ß also up-regulates Jmjd3 and Utx in RMCs, suggesting a key role for these H3K27me3 demethylases in H3K27me3 inhibition. In RMCs, Utx knockdown inhibits hypertrophy, a key event in glomerular dysfunction. The H3K27me3 regulators are similarly altered in human and mouse MCs. High glucose inhibits Ezh2 and increases miR-101b in a TGF-ß-dependent manner. Furthermore, in kidneys from rodent models of DN, fibrotic genes, miR-101b, and H3K27me3 demethylases are up-regulated, whereas Ezh2 protein levels as well as enrichment of Ezh2 and H3K27me3 at target genes are decreased, demonstrating in vivo relevance. These results suggest that H3K27me3 inhibition by TGF-ß via dysregulation of related histone-modifying enzymes and miRNAs augments pathological genes mediating glomerular mesangial dysfunction and DN.

A Novel Angiotensin II-Induced Long Noncoding RNA Giver Regulates Oxidative Stress, Inflammation, and Proliferation in Vascular Smooth Muscle Cells.

RATIONALE: AngII (angiotensin II)-mediated vascular smooth muscle cell (VSMC) dysfunction plays a major role in hypertension. Long noncoding RNAs have elicited much interest, but their molecular roles in AngII actions and hypertension are unclear. OBJECTIVE: To investigate the regulation and functions of a novel long noncoding RNA growth factor- and proinflammatory cytokine-induced vascular cell-expressed RNA ( Giver), in AngII-mediated VSMC dysfunction. METHODS AND RESULTS: RNA-sequencing and real-time quantitative polymerase chain reactions revealed that treatment of rat VSMC with AngII increased the expression of Giver and Nr4a3, an adjacent gene encoding a nuclear receptor. Similar changes were observed in rat and mouse aortas treated ex vivo with AngII. RNA-FISH (fluorescence in situ hybridization) and subcellular fractionation showed predominantly nuclear localization of Giver. AngII increased Giver expression via recruitment of Nr4a3 to Giver promoter. Microarray profiling and real-time quantitative polymerase chain reaction validation in VSMC showed that Giver knockdown attenuated the expression of genes involved in oxidative stress ( Nox1) and inflammation ( Il6, Ccl2, Tnf) but increased Nr4a3. Conversely, endogenous Giver overexpression showed opposite effects supporting its role in oxidative stress and inflammation. Chromatin immunoprecipitation assays showed Giver overexpression also increased Pol II (RNA polymerase II) enrichment and decreased repressive histone modification histone H3 trimethylation on lysine 27 at Nox1 and inflammatory gene promoters. Accordingly, Giver knockdown inhibited AngII-induced oxidative stress and proliferation in rat VSMC. RNA-pulldown combined with mass spectrometry showed Giver interacts with nuclear and chromatin remodeling proteins and corepressors, including NONO (non-pou domain-containing octamer-binding protein). Moreover, NONO knockdown elicited similar effects as Giver knockdown on the expression of key Giver-regulated genes. Notably, GIVER and NR4A3 were increased in AngII-treated human VSMC and in arteries from hypertensive patients but attenuated in hypertensive patients treated with ACE (angiotensin-converting enzyme) inhibitors or angiotensin receptor blockers. Furthermore, human GIVER also exhibits partial functional conservation with rat Giver. CONCLUSIONS: Giver and its regulator Nr4a3 are important players in AngII-mediated VSMC dysfunction and could be novel targets for antihypertensive therapy.

Diabetes Mellitus-Induced Long Noncoding RNA Dnm3os Regulates Macrophage Functions and Inflammation via Nuclear Mechanisms.

Objective- Macrophages play key roles in inflammation and diabetic vascular complications. Emerging evidence implicates long noncoding RNAs in inflammation, but their role in macrophage dysfunction associated with inflammatory diabetic complications is unclear and was therefore investigated in this study. Approach and Results- RNA-sequencing and real-time quantitative PCR demonstrated that a long noncoding RNA Dnm3os (dynamin 3 opposite strand) is upregulated in bone marrow-derived macrophages from type 2 diabetic db/db mice, diet-induced insulin-resistant mice, and diabetic ApoE-/- mice, as well as in monocytes from type 2 diabetic patients relative to controls. Diabetic conditions (high glucose and palmitic acid) induced Dnm3os in mouse and human macrophages. Promoter reporter analysis and chromatin immunoprecipitation assays demonstrated that diabetic conditions induce Dnm3os via NF-κB activation. RNA fluorescence in situ hybridization and real-time quantitative PCRs of subcellular fractions demonstrated nuclear localization and chromatin enrichment of Dnm3os in macrophages. Stable overexpression of Dnm3os in macrophages altered global histone modifications and upregulated inflammation and immune response genes and phagocytosis. Conversely, RNAi-mediated knockdown of Dnm3os attenuated these responses. RNA pull-down assays with macrophage nuclear lysates identified nucleolin and ILF-2 (interleukin enhancer-binding factor 2) as protein binding partners of Dnm3os, which was further confirmed by RNA fluorescence in situ hybridization immunofluorescence. Furthermore, nucleolin levels were decreased in diabetic conditions, and its knockdown enhanced Dnm3os-induced inflammatory gene expression and histone H3K9-acetylation at their promoters. Conclusions- These results demonstrate novel mechanisms involving upregulation of long noncoding RNA Dnm3os, disruption of its interaction with nucleolin, and epigenetic modifications at target genes that promote macrophage inflammatory phenotype in diabetes mellitus. The data could lead to long noncoding RNA-based therapies for inflammatory diabetes mellitus complications.

Regulation of angiotensin II actions by enhancers and super-enhancers in vascular smooth muscle cells.

Angiotensin II (AngII) promotes hypertension and atherosclerosis by activating growth-promoting and pro-inflammatory gene expression in vascular smooth muscle cells (VSMCs). Enhancers and super-enhancers (SEs) play critical roles in driving disease-associated gene expression. However, enhancers/SEs mediating VSMC dysfunction remain uncharacterized. Here, we show that AngII alters vascular enhancer and SE repertoires in cultured VSMCs in vitro, ex vivo, and in AngII-infused mice aortas in vivo. AngII-induced enhancers/SEs are enriched in binding sites for signal-dependent transcription factors and dependent on key signaling kinases. Moreover, CRISPR-Cas9-mediated deletion of candidate enhancers/SEs, targeting SEs with the bromodomain and extra-terminal domain inhibitor JQ1, or knockdown of overlapping long noncoding RNAs (lncRNAs) blocks AngII-induced genes associated with growth-factor signaling and atherosclerosis. Furthermore, JQ1 ameliorates AngII-induced hypertension, medial hypertrophy and inflammation in vivo in mice. These results demonstrate AngII-induced signals integrate enhancers/SEs and lncRNAs to increase expression of genes involved in VSMC dysfunction, and could uncover novel therapies.

Regulation of Vascular Smooth Muscle Cell Dysfunction Under Diabetic Conditions by miR-504.

OBJECTIVE: Diabetes mellitus accelerates proatherogenic and proinflammatory phenotype of vascular smooth muscle cell (VSMC) associated with vascular complications. Evidence shows that microRNAs (miRNAs) play key roles in VSMC functions, but their role under diabetic conditions is unclear. We profiled miRNAs in VSMC from diabetic mice and examined their role in VSMC dysfunction. APPROACH AND RESULTS: High throughput small RNA-sequencing identified 135 differentially expressed miRNAs in VSMC from type 2 diabetic db/db mice (db/dbVSMC) versus nondiabetic db/+ mice. Several of these miRNAs were known to regulate VSMC functions. We further focused on miR-504, because it was highly upregulated in db/dbVSMC, and its function in VSMC is unknown. miR-504 and its host gene Fgf13 were significantly increased in db/dbVSMC and in aortas from db/db mice. Bioinformatics analysis predicted that miR-504 targets including signaling adaptor Grb10 and transcription factor Egr2 could regulate growth factor signaling. We experimentally validated Grb10 and Egr2 as novel targets of miR-504. Overexpression of miR-504 in VSMC inhibited contractile genes and enhanced extracellular signal-regulated kinase 1/2 activation, proliferation, and migration. These effects were blocked by miR-504 inhibitors. Grb10 knockdown mimicked miR-504 functions and increased inflammatory genes. Egr2 knockdown-inhibited anti-inflammatory Socs1 and increased proinflammatory genes. Furthermore, high glucose and palmitic acid upregulated miR-504 and inflammatory genes, but downregulated Grb10. CONCLUSIONS: Diabetes mellitus misregulates several miRNAs including miR-504 that can promote VSMC dysfunction. Because changes in many of these miRNAs are sustained in diabetic VSMC even after in vitro culture, they may be involved in metabolic memory of vascular complications. Targeting such mechanisms could offer novel therapeutic strategies for diabetic complications.

Epigenetic Histone Modifications Involved in Profibrotic Gene Regulation by 12/15-Lipoxygenase and Its Oxidized Lipid Products in Diabetic Nephropathy.

AIMS: Epigenetic mechanisms, including histone post-translational modifications and DNA methylation, are implicated in the pathogenesis of diabetic nephropathy (DN), but the mediators are not well known. Moreover, although dyslipidemia contributes to DN, epigenetic changes triggered by lipids are unclear. In diabetes, increased expression of 12/15-lipoxygenase (12/15-LO) enhances oxidized lipids such as 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], which promote oxidant stress, glomerular and mesangial cell (MC) dysfunction, and fibrosis, and mediate the actions of profibrotic growth factors. We hypothesized that 12/15-LO and its oxidized lipid products can regulate epigenetic mechanisms mediating profibrotic gene expression related to DN. RESULTS: 12(S)-HETE increased profibrotic gene expression and enrichment of permissive histone lysine modifications at their promoters in MCs. 12(S)-HETE also increased protein levels of SET7, a histone H3 lysine 4 methyltransferase, and promoted its nuclear translocation and enrichment at profibrotic gene promoters. Furthermore, SET7 (Setd7) gene silencing inhibited 12(S)-HETE-induced profibrotic gene expression. 12/15-LO (Alox15) gene silencing or genetic knockout inhibited transforming growth factor-ß1 (TGF-ß1)-induced expression of Setd7 and profibrotic genes and histone modifications in MCs. Furthermore, 12/15-LO knockout in mice ameliorated key features of DN and abrogated increases in renal SET7 and profibrotic genes. Additionally, 12/15-LO siRNAs in vivo blocked increases in renal SET7 and profibrotic genes in diabetic mice. INNOVATION AND CONCLUSION: These novel results demonstrate for the first time that 12/15-LO-derived oxidized lipids regulate histone modifications associated with profibrotic gene expression in MCs, and 12/15-LO can mediate similar actions of TGF-ß1 and diabetes. Targeting 12/15-LO might be a useful strategy to inhibit key epigenetic mechanisms involved in DN.

Anti-Inflammatory Role of MicroRNA-146a in the Pathogenesis of Diabetic Nephropathy.

Inflammation has a critical role in the pathogenesis of diabetic complications, including diabetic nephropathy (DN). MicroRNAs have recently emerged as important regulators of DN. However, the role of microRNAs in the regulation of inflammation during DN is poorly understood. Here, we examined the in vivo role of microRNA-146a (miR-146a), a known anti-inflammatory microRNA, in the pathogenesis of DN. In a model of streptozotocin-induced diabetes, miR-146a(-/-) mice showed significantly exacerbated proteinuria, renal macrophage infiltration, glomerular hypertrophy, and fibrosis relative to the respective levels in control wild-type mice. Diabetes-induced upregulation of proinflammatory and profibrotic genes was significantly greater in the kidneys of miR-146a(-/-) than in the kidneys of wild-type mice. Notably, miR-146a expression increased in both peritoneal and intrarenal macrophages in diabetic wild-type mice. Mechanistically, miR-146a deficiency during diabetes led to increased expression of M1 activation markers and suppression of M2 markers in macrophages. Concomitant with increased expression of proinflammatory cytokines, such as IL-1ß and IL-18, markers of inflammasome activation also increased in the macrophages of diabetic miR-146a(-/-) mice. These studies suggest that in early DN, miR-146a upregulation exerts a protective effect by downregulating target inflammation-related genes, resulting in suppression of proinflammatory and inflammasome gene activation. Loss of this protective mechanism in miR-146a(-/-) mice leads to accelerated DN. Taken together, these results identify miR-146a as a novel anti-inflammatory noncoding RNA modulator of DN.

Recent developments in epigenetics of acute and chronic kidney diseases.

The growing epidemic of obesity and diabetes, the aging population as well as prevalence of drug abuse has led to significant increases in the rates of the closely associated acute and chronic kidney diseases, including diabetic nephropathy. Furthermore, evidence shows that parental behavior and diet can affect the phenotype of subsequent generations via epigenetic transmission mechanisms. These data suggest a strong influence of the environment on disease susceptibility and that, apart from genetic susceptibility, epigenetic mechanisms need to be evaluated to gain critical new information about kidney diseases. Epigenetics is the study of processes that control gene expression and phenotype without alterations in the underlying DNA sequence. Epigenetic modifications, including cytosine DNA methylation and covalent post-translational modifications of histones in chromatin, are part of the epigenome, the interface between the stable genome and the variable environment. This dynamic epigenetic layer responds to external environmental cues to influence the expression of genes associated with disease states. The field of epigenetics has seen remarkable growth in the past few years with significant advances in basic biology, contributions to human disease, as well as epigenomics technologies. Further understanding of how the renal cell epigenome is altered by metabolic and other stimuli can yield novel new insights into the pathogenesis of kidney diseases. In this review, we have discussed the current knowledge on the role of epigenetic mechanisms (primarily DNAme and histone modifications) in acute and chronic kidney diseases, and their translational potential to identify much needed new therapies.

Epigenetic mechanisms in diabetic complications and metabolic memory.

The incidence of diabetes and its associated micro- and macrovascular complications is greatly increasing worldwide. The most prevalent vascular complications of both type 1 and type 2 diabetes include nephropathy, retinopathy, neuropathy and cardiovascular diseases. Evidence suggests that both genetic and environmental factors are involved in these pathologies. Clinical trials have underscored the beneficial effects of intensive glycaemic control for preventing the progression of complications. Accumulating evidence suggests a key role for epigenetic mechanisms such as DNA methylation, histone post-translational modifications in chromatin, and non-coding RNAs in the complex interplay between genes and the environment. Factors associated with the pathology of diabetic complications, including hyperglycaemia, growth factors, oxidant stress and inflammatory factors can lead to dysregulation of these epigenetic mechanisms to alter the expression of pathological genes in target cells such as endothelial, vascular smooth muscle, retinal and cardiac cells, without changes in the underlying DNA sequence. Furthermore, long-term persistence of these alterations to the epigenome may be a key mechanism underlying the phenomenon of 'metabolic memory' and sustained vascular dysfunction despite attainment of glycaemic control. Current therapies for most diabetic complications have not been fully efficacious, and hence a study of epigenetic mechanisms that may be involved is clearly warranted as they can not only shed novel new insights into the pathology of diabetic complications, but also lead to the identification of much needed new drug targets. In this review, we highlight the emerging role of epigenetics and epigenomics in the vascular complications of diabetes and metabolic memory.

Regulation of inflammatory phenotype in macrophages by a diabetes-induced long noncoding RNA.

The mechanisms by which macrophages mediate the enhanced inflammation associated with diabetes complications are not completely understood. We used RNA sequencing to profile the transcriptome of bone marrow macrophages isolated from diabetic db/db mice and identified 1,648 differentially expressed genes compared with control db/+ mice. Data analyses revealed that diabetes promoted a proinflammatory, profibrotic, and dysfunctional alternatively activated macrophage phenotype possibly via transcription factors involved in macrophage function. Notably, diabetes altered levels of several long noncoding RNAs (lncRNAs). Because the role of lncRNAs in diabetes complications is unknown, we further characterized the function of lncRNA E330013P06, which was upregulated in macrophages from db/db and diet-induced insulin-resistant type 2 diabetic (T2D) mice, but not from type 1 diabetic mice. It was also upregulated in monocytes from T2D patients. E330013P06 was also increased along with inflammatory genes in mouse macrophages treated with high glucose and palmitic acid. E330013P06 overexpression in macrophages induced inflammatory genes, enhanced responses to inflammatory signals, and increased foam cell formation. In contrast, small interfering RNA-mediated E330013P06 gene silencing inhibited inflammatory genes induced by the diabetic stimuli. These results define the diabetic macrophage transcriptome and novel functional roles for lncRNAs in macrophages that could lead to lncRNA-based therapies for inflammatory diabetes complications.

Losartan reverses permissive epigenetic changes in renal glomeruli of diabetic db/db mice.

Epigenetic mechanisms such as chromatin histone H3 lysine methylation and acetylation have been implicated in diabetic vascular complications. However, histone modification profiles at pathologic genes associated with diabetic nephropathy in vivo and their regulation by the angiotensin II type 1 receptor (AT1R) are not clear. Here we tested whether treatment of type 2 diabetic db/db mice with the AT1R blocker losartan not only ameliorates diabetic nephropathy, but also reverses epigenetic changes. As expected, the db/db mice had increased blood pressure, mesangial hypertrophy, proteinuria, and glomerular expression of RAGE and PAI-1 vs. control db/+ mice. This was associated with increased RNA polymerase II recruitment and permissive histone marks as well as decreased repressive histone marks at these genes, and altered expression of relevant histone modification enzymes. Increased MCP-1 mRNA levels were not associated with such epigenetic changes, suggesting post-transcriptional regulation. Losartan attenuated key parameters of diabetic nephropathy and gene expression, and reversed some but not all the epigenetic changes in db/db mice. Losartan also attenuated increased H3K9/14Ac at RAGE, PAI-1, and MCP-1 promoters in mesangial cells cultured under diabetic conditions. Our results provide novel information about the chromatin state at key pathologic genes in vivo in diabetic nephropathy mediated in part by AT1R. Thus, combination therapies targeting epigenetic regulators and AT1R could be evaluated for more effective treatment of diabetic nephropathy.

Epigenetic modifications in the pathogenesis of diabetic nephropathy.

Diabetic nephropathy (DN) is a leading cause of end-stage renal disease. Diabetic vascular complications such as DN can progress despite subsequent glycemic control, suggesting a metabolic memory of previous exposure to hyperglycemia. Diabetes profoundly impacts transcription programs in target cells through activation of multiple signaling pathways and key transcription factors leading to aberrant expression of pathologic genes. Emerging evidence suggests that these factors associated with the pathophysiology of diabetic complications and metabolic memory also might be influenced by epigenetic mechanisms in chromatin such as DNA methylation, histone lysine acetylation, and methylation. Key histone modifications and the related histone methyltransferases and acetyltransferases have been implicated in the regulation of inflammatory and profibrotic genes in renal and vascular cells under diabetic conditions. Advances in epigenome profiling approaches have provided novel insights into the chromatin states and functional outcomes in target cells affected by diabetes. Because epigenetic changes are potentially reversible, they can provide a window of opportunity for the development of much-needed new therapies for DN in the future. In this review, we discuss recent developments in the field of epigenetics and their relevance to diabetic vascular complications and DN pathogenesis.