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Genetic or siRNA inhibition of MBD2 attenuates the UUO- and I/R-induced renal fibrosis via downregulation of EGR1.

ABSTRACT: DNA methylation plays a pivotal role in the progression of renal fibrosis. Methyl-CpG-binding domain protein 2 (MBD2), a protein reader of methylation, is involved in the development of acute kidney injury (AKI) caused by vancomycin. However, the role and mechanism of action of MBD2 in renal remain unclear. In this study, MBD2 mediated extracellular matrix (ECM) production induced by TGF-β1 in Boston University mouse proximal tubule (BUMPT) cells，and upregulated the expression EGR1 to promote ECM production in murine embryonic NIH 3T3 fibroblasts. ChIP analysis demonstrated that MBD2 physically interacted with the promoter region of the CpG islands of EGR1 genes and then activated their expression by inducing hypomethylation of the promoter region. In vivo, PT-MBD2-KO attenuated unilateral ureteral obstruction (UUO)-induced renal tubulointerstitial fibrosis via downregulation of EGR1, which was demonstrated by the downregulation of fibronectin (FN), collagen I and IV, α-SMA, and EGR1. Injection of MBD2-siRNA attenuated the UUO- and I/R-induced renal fibrosis. Those molecular changes were verified by biopsies from patients with obstructive nephropathy (OB). These data collectively demonstrated that inhibition of MBD2 reduces renal fibrosis via downregulating EGR1, which could be a target for treatment of fibrotic kidney disease.

SUBMITTER: Ai K

PROVIDER: S-EPMC8933641 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Genetic or siRNA inhibition of MBD2 attenuates the UUO- and I/R-induced renal fibrosis via downregulation of EGR1.

Ai Kai K Li Xiaozhou X Zhang Pan P Pan Jian J Li Huiling H He Zhibiao Z Zhang Hongliang H Yi Lei L Kang Ye Y Wang Yinhuai Y Chen Junxiang J Li Yijian Y Xiang Xudong X Chai Xiangping X Zhang Dongshan D

Molecular therapy. Nucleic acids 20220228

DNA methylation plays a pivotal role in the progression of renal fibrosis. Methyl-CpG-binding domain protein 2 (MBD2), a protein reader of methylation, is involved in the development of acute kidney injury (AKI) caused by vancomycin. However, the role and mechanism of action of MBD2 in renal remain unclear. In this study, MBD2 mediated extracellular matrix (ECM) production induced by TGF-β1 in Boston University mouse proximal tubule (BUMPT) cells，and upregulated the expression EGR1 to promote EC ...[more]

PMID: 35356685

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Project description:Acute kidney injury and chronic renal fibrosis are intractable pathological processes to resolve, yet limited strategies are able to effectively address them. Cardamonin (CAD) is a flavonoid with talented antioxidant, anti-inflammatory capacity, and satisfactory biosafety. In our study, animal and cellular models of renal ischemia/reperfusion (I/R) and unilateral ureteral obstruction (UUO) were successfully constructed to confirm whether CAD confers protective effects and underlying mechanisms. Animal experiments demonstrated that CAD application (100 mg/kg) distinctly ameliorated tissue damage and improved renal function. Meanwhile, the continuous oral administration of CAD after UUO surgery efficiently inhibited renal fibrosis as confirmed by hematoxylin-eosin (H&E), Sirius red, and Masson staining as well as the downregulated mRNA and protein expression of collagen I, α-smooth muscle actin (α-SMA), collagen III, and fibronectin. Interestingly, in transforming growth factor β1 (TGF-β1)-stimulated and hypoxia/reoxygenation (H/R)-exposed human kidney-2 (HK-2) cells, protective effects of CAD were again authenticated. Meanwhile, we performed bioinformatics analysis and constructed the "ingredient-target-pathway-disease" network to conclude that the potential mechanisms of CAD protection may be through the regulation of oxidative stress, inflammation, apoptosis, and mitogen-activated protein kinase (MAPK) pathway. Furthermore, experimental data validated that CAD evidently decreased the reactive oxygen species (ROS) production and malondialdehyde (MDA) content while depressing the mRNA and protein expression of inflammatory markers (tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and Il-1β) and inhibiting apoptosis as evidenced by decreased levels of P53, BAX, cleaved caspase-3, and apoptotic rate in renal I/R and UUO models. In addition, the impact of CAD on restraining oxidative stress and inflammation was attributed to its ability to elevate antioxidant enzyme activities including catalase, superoxide dismutase 1 (SOD1), and superoxide dismutase 2 (SOD2) and to inhibit the inflammation-associated MARK/nuclear factor-κB (MAPK/NF-κB) signaling pathway. In conclusion, cardamonin restored the antioxidative capacity to block oxidative stress and suppressed the MAPK/NF-κB signaling pathway to alleviate inflammatory response, thus mitigating I/R-generated acute kidney injury/UUO-induced renal fibrosis in vivo and in vitro, which indicated the potential therapeutic advantage of cardamonin in attenuating acute and chronic kidney injuries.

Project description:BackgroundRenal tubulointerstitial fibrosis is the hallmark of various chronic kidney diseases. Symmetric dimethylarginine (SDMA) is an independent cardiovascular risk factor in patients with chronic kidney diseases, which is mostly excreted through renal tubules. However, the effect of SDMA on kidneys in a pathological condition is currently unknown. In this study, we investigated the role of SDMA in renal tubulointerstitial fibrosis and explored its underlying mechanisms.MethodsMouse unilateral ureteral obstruction (UUO) and unilateral ischemia-reperfusion injury (UIRI) models were established to study renal tubulointerstitial fibrosis. SDMA was injected into kidneys through ureter retrogradely. TGF-β stimulated human renal epithelial (HK2) cells were used as an in vitro model and treated with SDMA. Signal transducer and activator of transcription-4 (STAT4) was inhibited by berbamine dihydrochloride or siRNA or overexpressed by plasmids in vitro. Masson staining and Western blotting were performed to evaluate renal fibrosis. Quantitative PCR was performed to validate findings derived from RNA sequencing analysis.ResultsWe observed that SDMA (from 0.01 to 10 µM) dose-dependently inhibited the expression of pro-fibrotic markers in TGF-β stimulated HK2 cells. Intrarenal administration of SDMA (2.5 µmol/kg or 25 µmol/kg) dose-dependently attenuated renal fibrosis in UUO kidneys. A significant increase in SDMA concentration (from 19.5 to 117.7 nmol/g, p < 0.001) in mouse kidneys was observed after renal injection which was assessed by LC-MS/MS. We further showed that intrarenal administration of SDMA attenuated renal fibrosis in UIRI induced mouse fibrotic kidneys. Through RNA sequencing analysis, we found that the expression of STAT4 was reduced by SDMA in UUO kidneys, which was further confirmed by quantitative PCR and Western blotting analysis in mouse fibrotic kidneys and renal cells. Inhibition of STAT4 by berbamine dihydrochloride (0.3 mg/ml or 3.3 mg/ml) or siRNA reduced the expression of pro-fibrotic markers in TGF-β stimulated HK2 cells. Furthermore, blockage of STAT4 attenuated the anti-fibrotic effect of SDMA in TGF-β stimulated HK2 cells. Conversely, overexpression of STAT4 reversed the anti-fibrotic effect of SDMA in TGF-β stimulated HK2 cells.ConclusionTaken together, our study indicates that renal SDMA ameliorates renal tubulointerstitial fibrosis through inhibition of STAT4.

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Genetic or siRNA inhibition of MBD2 attenuates the UUO- and I/R-induced renal fibrosis via downregulation of EGR1.

Publications

Genetic or siRNA inhibition of MBD2 attenuates the UUO- and I/R-induced renal fibrosis via downregulation of EGR1.

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