Figure 5 - available from: Scientific Reports
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Functional annotations for the 3 groups. Representative gene symbols for each category are shown in the middle panel. Fold enrichment scores for each category from DAVID are shown in the bar graphs in the right panel.
Source publication
Tubulointerstitial fibrosis has been recently reported to be caused by the collapse of the epigenetic regulation of kidney diseases. We examined whether pharmacological inhibition of histone modification is effective against renal fibrosis. DZNep (3-deazaneplanocin A) was originally developed as an anti-cancer drug to inhibit the repressive histone...
Citations
... Previous reports utilizing pharmacological agents to reduce H3K27Me3 abundance in vivo suggest its involvement in the regulation of fibrogenesis (41)(42)(43). Specifically, systemic administration of EZH2 inhibitors (lowering H3K27Me3 abundance) associates with a decreased expression of extracellular matrix genes, including collagens and fibronectin (41)(42)(43). In Collagen expression is reduced in H3K27Me3-deficient HUVECs. ...
... Previous reports utilizing pharmacological agents to reduce H3K27Me3 abundance in vivo suggest its involvement in the regulation of fibrogenesis (41)(42)(43). Specifically, systemic administration of EZH2 inhibitors (lowering H3K27Me3 abundance) associates with a decreased expression of extracellular matrix genes, including collagens and fibronectin (41)(42)(43). In Collagen expression is reduced in H3K27Me3-deficient HUVECs. ...
Objective
Endothelial-to-mesenchymal transition (EndMT) is a transdifferentiation process in which endothelial cells (ECs) adopt a mesenchymal-like phenotype. Over the past few years, it became clear that EndMT can contribute to several cardiovascular pathologies. However, the molecular pathways underlying the development of EndMT remain incompletely understood. Since the epigenetic enzyme Enhancer of Zeste Homolog 2 (EZH2) and its concomitant mark H3K27Me3 have been shown to be elevated in many cardiovascular diseases that associate with EndMT, we hypothesized that H3K27Me3 is a determinant for the susceptibility of EndMT.
Methods
To study the association between H3K27Me3 and EndMT, a knockdown model of EZH2 in human endothelial cells (HUVEC) was utilized to reduce H3K27Me3 abundance, followed by induction of EndMT using TGFβ1. The expression of molecular markers of EndMT and fibrogenesis were analysed.
Results
In cultured HUVECs, a reduction of H3K27Me3 abundance facilitates EndMT but mitigates fibrogenesis as shown by a decreased expression of collagen I and III. In HUVEC, H3K27Me3 abundance directly affects the expression of miR29c, a collagen-targeting miRNA. Additionally, knockdown of miR-29c in HUVEC with low H3K27Me3 abundance partly restored the expression of collagen I and III. Expectedly, in rats with perivascular fibrosis an increased abundance of H3K27Me3 associated with a decreased expression of miR-29c.
Conclusion
our data shows that endothelial fibrogenesis underlies an epigenetic regulatory pathway and we demonstrate that a decreased abundance of H3K27Me3 in ECs blunts fibrogenesis in part in a miR-29c dependent manner. Therefore, a reduction of H3K27Me3 could serve as a novel therapeutical strategy to mitigate fibrogenesis and may prove to be beneficial in fibrogenic diseases including atherosclerosis, cardiac fibrosis, and PAH.
... Moreover, there was elevated expression of tissue inhibitor of metalloproteinase 2 (TIMP2), a pro-fibrotic factor, in I/R injury. However, this mechanism was suppressed by Dznep (Mimura et al., 2018). The drug inhibits not only Ezh2 but also S-adenosylhomocysteine hydrolase. ...
Epidemiological studies have shown that patients who recovered from acute kidney injury (AKI) may subsequently develop chronic kidney disease (CKD). AKI is primarily caused by renal hypoxia, and it causes epigenetic alterations, known as hypoxic memory. 3‐Deazaneplanocin A (Dznep), an inhibitor of histone modification, suppresses renal fibrosis and the expression of tissue inhibitor of metalloproteinases‐2 (TIMP2), a profibrotic factor, in mouse ischemia–reperfusion models. The current study investigated the epigenetic regulation of TIMP2 in human kidney 2 (HK‐2) cells. The expression of TIMP2 was upregulated in HK‐2 cells under hypoxic conditions and was suppressed by Dznep. ChIP‐qPCR showed that Dznep reduced the amount of H3K4me3 at the promoter region of the TIMP2 gene under hypoxic condition. Formaldehyde‐assisted isolation of regulatory elements‐qPCR of the TIMP2 gene showed that Dznep reduced open chromatin area. In addition, based on ChIP‐qPCR of hypoxia‐inducible factor 1 alpha (HIF1α), Dznep inhibited the binding of HIF1α to the TIMP2 gene under hypoxic conditions. The reporter assays for the binding region of HIF1α showed enhanced transcriptional activity by hypoxia. Dznep suppresses the expression of TIMP2 under hypoxic conditions by inhibiting the binding of HIF1α to the TIMP2 gene.
... Moreover, there was elevated expression of tissue inhibitor of metalloproteinase 2 (TIMP2), a pro-brotic factor, in I/R injury. However, this mechanism was suppressed by Dznep [15]. The drug inhibits not only the expression of Ezh2 but also the expression of Sadenosylhomocysteine hydrolase. ...
... Reporter assay for the HIF1α-binding site of the TIMP2 gene The HIF1α-binding site of the TIMP2 gene was extracted from our previous HIF-1α Chip-seq data [15]. The gene area of this region was ampli ed via PCR with the primers shown in Table 1. ...
IntroductionEpidemiological studies have shown that patients who recovered from acute kidney injury (AKI) may subsequently develop chronic kidney disease (CKD). AKI is primarily caused by renal hypoxia, and it causes epigenetic alterations, known as hypoxic memory. 3-Deazaneplanocin A (Dznep), an inhibitor of histone modification, suppresses renal fibrosis and the expression of tissue inhibitor of metalloproteinases-2 (TIMP2), a profibrotic factor, in mouse ischemia–reperfusion models. The current study investigated the epigenetic regulation of TIMP2 in tubular cells.Methods and ResultsThe expression of TIMP2 was upregulated in human kidney 2 cells under hypoxic conditions and was suppressed by Dznep. ChIP-qPCR showed that Dznep reduced the expression of H3K4me3 at the promoter region of the TIMP2 gene under hypoxic condition. Formaldehyde-assisted isolation of regulatory elements-qPCR of the TIMP2 gene showed that Dznep reduced open chromatin area. In addition, based on ChIP-qPCR of hypoxia-inducible factor 1 alpha (HIF1α), Dznep inhibited the binding of HIF1α to the TIMP2 gene under hypoxic conditions.Conclusion
Dznep suppresses the expression of TIMP2 under hypoxic conditions by altering the histone methylations of the TIMP2 gene, decreasing open chromatin area, and inhibiting the binding of HIF1α to the TIMP2 gene.
... DZNep was studied as an antitumor drug, and in rodents, it exhibits favorable pharmacokinetics for treating acute pulmonary infections (Bray et al, 2000;Peer et al, 2013;Sun et al, 2015). It has also been shown to support tissue regeneration (Xiao et al, 2016;Zeybel et al, 2017;Mimura et al, 2018), which is essential to mitigate virus-associated long-term complications. In order to test whether DZNep treatment is antiviral against SARS-CoV-2 in vivo, we infected C57BL/6 mice with SARS-CoV-2 beta variant (B.1.351, ...
... Furthermore, we show that DZNep treatment alone or in context of SARS-CoV or SARS-CoV-2 infections reduces abundance of pulmonary fibrosis markers (e.g., SERPINE1, MMP14, and COL4A1) and increases levels of factors with antifibrotic activity (e.g., HOPX, PI3/ELAFIN, and SLPI; Fig 4D). These observations are in line with previous reports describing antifibrotic activity of DZNep in lungs (Xiao et al, 2016), liver (Zeybel et al, 2017), and kidneys (Mimura et al, 2018), which was linked to drug-induced inhibition of EZH2. Similar modulation of fibrosis-related proteins may be induced by other SCIs beyond DZNep, which may also perturb EZH2 activity in a metabolitemediated manner. ...
The SARS-CoV-2 infection cycle is a multi-stage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2'-O-ribose cap needed for viral immune escape. We find that the host cap 2'-O-ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS-CoV-2 replication. Using in silico target-based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti-SARS-CoV-2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co-substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in vitro, ex vivo and in a mouse infection model, and synergizes with existing COVID-19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection-induced hyperinflammation, and reduces lung fibrosis markers ex vivo. Thus, multi-specific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID-19.
... The EZH2 inhibitor, 3-deazaneplanocin A (DZNeP), decreased fibrosis in the UUO model, decreasing signaling from several receptors including TGF-β1, EGFR, and platelet-derived growth factor b receptor (PDGFbR), and PTEN, which may exert a therapeutic effect [94]. Our group performed a genome-wide analysis of TECs using RNA-seq in vivo and in vitro and discovered that DZNeP decreased the expression of profibrotic genes and inhibited tubulointerstitial fibrosis in a murine IRI model of AKI-to-CKD progression [100]. The SET7/9 inhibitor, sinefungin, and the G9a inhibitor, BIX01294I, also decreased fibrosis and reduced the levels of H3K4me1 or H3K9me1, respectively, in kidneys from UUO mice [101,102]. ...
Acute kidney injury (AKI) was previously thought to be a merely transient event; however, recent epidemiological evidence supports the existence of a causal relationship between AKI episodes and subsequent progression to chronic kidney disease (CKD). Although the pathophysiology of this AKI-to-CKD transition is not fully understood, it is mediated by the interplay among multiple components of the kidney including tubular epithelial cells, endothelial cells, pericytes, inflammatory cells, and myofibroblasts. Epigenetic alterations including histone modification, DNA methylation, non-coding RNAs, and chromatin conformational changes, are also expected to be largely involved in the pathophysiology as a “memory” of the initial injury that can persist and predispose to chronic progression of fibrosis. Each epigenetic modification has a great potential as a therapeutic target of AKI-to-CKD transition; timely and target-specific epigenetic interventions to the various temporal stages of AKI-to-CKD transition will be the key to future therapeutic applications in clinical practice. This review elaborates on the latest knowledge of each mechanism and the currently available therapeutic agents that target epigenetic modification in the context of AKI-to-CKD transition. Further studies will elucidate more detailed mechanisms and novel therapeutic targets of AKI-to-CKD transition.
... Histone modification inhibitors are being studied as a potential treatment for these epigenetic changes. The histone deacetylase inhibitors, such as vorinostat, valproate, sodium butyrate, and trichostatin, have been reported to reduce proteinuria and improve oxidative stress, fibrosis, glomerular damage, and inflammation in the DKD rat model [121][122][123][124][125]. In addition, Dznep, an inhibitor of Ezh2, H3K27 methyltransferase, was reported to inhibit renal fibrosis in mice with UUO and AKI-to-CKD transition [126,127]. Our group showed that AKI damage caused an elevated level of tissue inhibitor of matrix metalloproteinases 2 (TIMP2), which is considered a profibrotic gene, and that TIMP2 was downregulated by Dznep, which meant that TIMP2 was regulated epigenetically and that Dznep recovered this change. ...
Diabetic kidney disease (DKD) is the major cause of end-stage kidney disease. However, only renin-angiotensin system inhibitor with multidisciplinary treatments is effective for DKD. In 2019, sodium-glucose cotransporter 2 (SGLT2) inhibitor showed efficacy against DKD in Canagliflozin and Renal Events in Diabetes with Established Nephropathy Clinical Evaluation (CREDENCE) trial, adding a new treatment option. However, the progression of DKD has not been completely controlled. The patients with transient exposure to hyperglycemia develop diabetic complications, including DKD, even after normalization of their blood glucose. Temporary hyperglycemia causes advanced glycation end product (AGE) accumulations and epigenetic changes as metabolic memory. The drugs that improve metabolic memory are awaited, and AGE inhibitors and histone modification inhibitors are the focus of clinical and basic research. In addition, incretin-related drugs showed a renoprotective ability in many clinical trials, and these trials with renal outcome as their primary endpoint are currently ongoing. Hypoxia-inducible factor prolyl hydroxylase inhibitors recently approved for renal anemia may be renoprotective since they improve tubulointerstitial hypoxia. Furthermore, NF-E2-related factor 2 activators improved the glomerular filtration rate of DKD patients in Bardoxolone Methyl Treatment: Renal Function in chronic kidney disease/Type 2 Diabetes (BEAM) trial and Phase II Study of Bardoxolone Methyl in Patients with Chronic Kidney Disease and Type 2 Diabetes (TSUBAKI) trial. Thus, following SGLT2 inhibitor, numerous novel drugs could be utilized in treating DKD. Future studies are expected to provide new insights.
... Moreover, most studies have been carried out to date on animal models, explanted organs, or tumor explants without limitations on the amount or restriction in the sample processing (Kohda et al., 2000;Mimura et al., 2018). In addition, the diagnostic methods, routinely used for preparing tissue samples, frequently make the sample not suitable for molecular analysis. ...
Renal biopsy (RBx) is an essential tool in the diagnostic and therapeutic process of most native kidney diseases and in the renal transplanted graft. Laser capture microdissection (LCM), combined with molecular biology, might improve the diagnostic power of RBx. However, the limited amount of available renal tissue is often an obstacle for achieving a satisfactory qualitative and quantitative analysis. In our work we present a method which allows us to obtain good quality and quantity of RNA from formalin-fixed and paraffin-embedded (FFPE) renal tissue derived from RBx performed in transplanted patients.
Histology, immunohistochemistry, LCM, pre-amplify system and qRT-PCR of biomarkers related to tubular damage, inflammation and fibrosis on FFPE RBx were performed. Glomeruli, tubules and interstitium of three RBx (RB-A: no alteration; RB-B and -C: the progressive rise of creatinine) were compared.
The method proposed, could well be useful in future clinical practice. It is quick, easy to perform and allows the analyses of many biomarkers. In addition, it could be extended to all types of RBx without any limitation on the sample amount. Nevertheless, the need for a higher number of well-trained technicians might represent some limitation, counterbalanced by the opportunity to elaborate more accurate diagnosis and, consequently, more targeted therapies.
... EZH2 was associated with cytokine gene expression in T cells (13,23). As an inhibitor of histone EZH2, DZNep was reported to have protective effects in ischemic brain injury (18) and tubulointerstitial fibrosis (24). In our study, DZNep ameliorated the renal IRI and injury of the renal tubular cells in vivo and in vitro and such protective effects persisted and improved the survival rate in our 3-days follow-up. ...
Renal ischemia-reperfusion injury (IRI) after renal transplantation often leads to the loss of kidney graft function. However, there is still a lack of efficient regimens to prevent or alleviate renal IRI. Our study focused on the renoprotective effect of 3-Deazaneplanocin A (DZNep), which is a histone methylation inhibitor. We found that DZNep significantly alleviated renal IRI by suppressing nuclear factor kappa-B (NF-κB), thus inhibiting the expression of inflammatory factors in renal tubular epithelial cells in vivo or in vitro. After treatment with DZNep, T cell activation was impaired in the spleen and kidney, which correlated with the downregulated expression of T-cell immunoglobulin mucin (TIM)-1 on T cells and TIM-4 in macrophages. In addition, pretreatment with DZNep was not sufficient to protect the kidney, while administration of DZNep from before to after surgery significantly ameliorated IRI. Our findings suggest that DZNep can be a novel strategy for preventing renal IRI following kidney transplantation.
... The apoptosis of renal tubular cells plays an important role in the pathogenesis of kidney injury, including acute kidney injury (AKI) [1,2]. Recent studies have found that epigenetic factors are involved in signal transduction and information transmission in the initiation and progression of renal tubular cell apoptosis [3,4]. ...
Background:
The enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase and induces the trimethylation of histone H3 lysine 27 (H3K27me3) in the promoter of many key genes; EZH2 acts as a transcriptional repressor and is an epigenetic regulator for several cancers. However, the role of EZH2 in nonneoplastic diseases, such as kidney diseases, is unknown and has been investigated.
Materials and method:
NRK-52E cells were treated with DZNep, a potent inhibitor of EZH2, with different concentrations and for different times to evaluate the apoptosis level of NRK-52E cells by Western blot and Flow cytometry analysis. The binding of EZH2 to the Deptor promoter was determined by ChIP assay.
Results:
The inhibition of EZH2 with 3-deazaneplanocin A (DZNep), a specific inhibitor of EZH2, led to the apoptosis of NRK-52E cells and the inhibition of mTORC1 and mTORC2 activity. A ChIP assay demonstrated that EZH2 bound the promoter region of Deptor, an endogenous inhibitor of mTORC1 and mTORC2, and regulated the transcription of Deptor by modulating H3K27me3 in its promoter region. Further experiments were performed to examine the effects of EZH2 inhibition on cisplatin-induced injured cells. Cisplatin induced the activation of mTORC1 and mTORC2 and apoptosis in NRK-52E cells, and DZNep inhibited mTORC1 and mTORC2 activity and aggravated cell apoptosis.
Conclusions:
These data suggested that EZH2 inhibition increased the transcription of Deptor by modifying H3K27me3 in its promoter region, subsequently inhibited mTORC1 and mTORC2 activities, downregulated the expression of apoptosis suppressor genes, and finally led to apoptosis in renal tubular cells. The inhibition of EZH2 aggravated the cisplatin-induced injury in renal tubular cells by inactivating the mTOR complexes. The present study provides new insight into renal protection and suggests that EZH2 might be a target.
... Upregulation of enhancer of zeste homologue 2 (EZH2) -a histone methyltransferase that specifically mediates trimethylation of lysine 27 on histone H3 (H3K27m3) -has been documented in fibrotic kidneys from mice with UUO and in patients with CKD, suggesting that this methyltransferase has profibrotic functions 72,73 . Pharmacological inhibition of EZH2 decreased H3K27m3 and attenuated renal fibrosis in mice with IRI by suppressing the expression of genes encoding profibrotic proteins, such as collagen type 3a1 (COL3A1) and tissue inhibitor of metalloproteinase 2 (TIMP2) 74 . ...
Acute kidney injury (AKI) is a major public health concern associated with high morbidity and mortality. Despite decades of research, the pathogenesis of AKI remains incompletely understood and effective therapies are lacking. An increasing body of evidence suggests a role for epigenetic regulation in the process of AKI and kidney repair, involving remarkable changes in histone modifications, DNA methylation and the expression of various non-coding RNAs. For instance, increases in levels of histone acetylation seem to protect kidneys from AKI and promote kidney repair. AKI is also associated with changes in genome-wide and gene-specific DNA methylation; however, the role and regulation of DNA methylation in kidney injury and repair remains largely elusive. MicroRNAs have been studied quite extensively in AKI, and a plethora of specific microRNAs have been implicated in the pathogenesis of AKI. Emerging research suggests potential for microRNAs as novel diagnostic biomarkers of AKI. Further investigation into these epigenetic mechanisms will not only generate novel insights into the mechanisms of AKI and kidney repair but also might lead to new strategies for the diagnosis and therapy of this disease.
