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INTRODUCTION: Acute kidney injury (AKI) is a common clinical syndrome associated with high morbidity and mortality. Inhibition of the methyltransferase enhancer of zeste homolog 2 (EZH2) by its inhibitor 3-deazaneplanocin A (3-DZNeP) exerts renal benefits in acute renal ischemia-reperfusion injury (IRI). However, the underlying mechanisms are not completely known. This study aimed to elucidate the pathological mechanism of EZH2 in renal IRI by combination of multi-omics analysis and expression profiling in a public clinical cohort. METHODS: In this study, C57BL/6 J mice were used to establish the AKI model, which were treated with 3-DZNeP for 24 h. Kidney samples were collected for RNA-seq analysis, which was combined with publicly available EZH2 chromatin immunoprecipitation sequencing (ChIP-seq) data of mouse embryonic stem cell for a joint analysis to identify differentially expressed genes. Several selected differentially expressed genes were verified by quantitative PCR. Finally, single-nucleus sequencing data and expression profiling in public clinical datasets were used to confirm the negative correlation of the selected genes with EZH2 expression. RESULTS: 3-DZNeP treatment significantly improved renal pathology and function in IRI mice. Through RNA-seq analysis combined with EZH2 ChIP-seq database, 162 differentially expressed genes were found, which might be involved in EZH2-mediated pathology in IRI kidneys. Four differential expressed genes (Scd1, Cidea, Ghr, and Kl) related to lipid metabolism or cell growth were selected based on Gene Ontology and Kyoto Encyclopedia of Genes and Genome enrichment analysis, which were validated by quantitative PCR. Data from single-nucleus RNA sequencing revealed the negative correlation of these four genes with Ezh2 expression in different subpopulations of proximal tubular cells in IRI mice in a different pattern. Finally, the negative correlation of these four genes with EZH2 expression was confirmed in patients with AKI in two clinical datasets. CONCLUSIONS: Our study indicates that Scd1, Cidea, Ghr, and Kl are downstream genes regulated by EZH2 in AKI. Upregulation of EZH2 in AKI inhibits the expression of these four genes in a different population of proximal tubular cells to minimize normal physiological function and promote acute or chronic cell injuries following AKI.
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Injúria Renal Aguda , Adenosina , Adenosina/análogos & derivados , Proteína Potenciadora do Homólogo 2 de Zeste , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão , Animais , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Camundongos , Adenosina/farmacologia , Injúria Renal Aguda/tratamento farmacológico , Injúria Renal Aguda/prevenção & controle , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/etiologia , Masculino , Rim/efeitos dos fármacos , Rim/patologia , Rim/metabolismo , MultiômicaRESUMO
The role of RNA methylation on the sixth N atom of adenylate (m6A) in acute kidney injury (AKI) is unknown. FTO (fat mass and obesity-associated protein) reverses the m6A modification in cisplatin-induced AKI. Here, we aimed to determine FTO's role in AKI. We induced AKI in c57BL/6 mice by intraperitoneal cisplatin injection and treated the animal with vehicle or an FTO inhibitor meclofenamic acid (MA) for 3 days. Moreover, as an in vitro model, human kidney proximal tubular cells (HK2 cells) were treated with cisplatin. We found that the cisplatin treatment reduces FTO expression and increases m6A levels in vivo and in vitro MA aggravated renal damage and increased apoptosis in cisplatin-treated kidneys, phenotypes that were correlated with reduced FTO expression and increased m6A levels. Moreover, MA promoted apoptosis in cisplatin-treated HK2 cells, which was correlated with the reduced FTO expression and increased m6A in HK2 cells. FTO protein overexpression reduced m6A levels and inhibited apoptosis in cisplatin-treated HK2 cells and also blocked the MA-induced increase in m6A levels and apoptosis rates. In agreement, overexpression of the m6A-generating methyltransferase-like 3 and 14 (METTL3 and METTL14) or siRNA-mediated FTO knockdown promoted apoptosis and enhanced m6A levels in cisplatin-treated HK2 cells. MA increased p53 mRNA and protein levels in AKI both in vitro and in vivo, and FTO overexpression reduced p53 expression and reversed the MA-induced p53 increase in AKI. In conclusion, reduced renal FTO expression in cisplatin-induced AKI increases RNA m6A levels and aggravates renal damages.
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Injúria Renal Aguda/patologia , Tecido Adiposo/efeitos dos fármacos , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Cisplatino/farmacologia , Ácido Meclofenâmico/farmacologia , RNA/química , RNA/metabolismo , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/genética , Injúria Renal Aguda/metabolismo , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Dioxigenase FTO Dependente de alfa-Cetoglutarato/deficiência , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Sinergismo Farmacológico , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: Acute kidney injury (AKI) is a clinical syndrome characterized by significant morbidity and a high death rate. Interleukin (IL)-27 is a newly described member of the IL-6/IL-12 heterodimeric cytokine family and displays anti-inflammatory and antiapoptotic properties. OBJECTIVES: To determine the effect and mechanism of IL-27 in AKI. METHOD: We used a mouse model of renal ischemia/reperfusion (I/R) injury to investigate whether IL-27 has a therapeutic potential for the treatment of AKI. For the IL-27 administration group, IL-27 protein was injected 1 h before ischemia. Human proximal tubular epithelial cells were exposed to ischemia for 2 h and followed by 2 h of reperfusion (I2h+R2h treatment) used as an in vitro model to investigate the effect of IL-27. RESULTS: Two IL-27 subunits, Epstein-Barr virus gene 3 and p28, were upregulated in kidneys 24 h after I/R. Renal expression of IL-27 receptor subunits (gp130 and WSX-1) was also increased. Treatment with IL-27 reduced structural/functional damages, ameliorated renal inflammation, inhibited the cleaved caspase-3 expression, upregulated antiapoptotic protein Bcl-2 and downregulated proapoptotic protein Bax in the kidneys of mice subjected to I/R. Meanwhile, the level of IL-27 receptor on renal tubular epithelial cells was increased after I2h+R2h treatment, and IL-27 administration suppressed I2h+R2h-induced epithelial cell apoptosis. Furthermore, IL-27 treatment led to activation of signal transducer and activator of transcription 3 (STAT3) both in vivo and in vitro, and IL-27-mediated protection against I2h+R2h injury was abolished by STAT3 inhibition. CONCLUSIONS: IL-27 protects against renal I/R injury by activating STAT3, suggesting that IL-27 may represent a novel strategy for the treatment of AKI.
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Injúria Renal Aguda/tratamento farmacológico , Interleucina-27/uso terapêutico , Animais , Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Células Cultivadas , Células Epiteliais , Humanos , Interleucina-27/farmacologia , Túbulos Renais Proximais/citologia , Camundongos , Traumatismo por Reperfusão , Fator de Transcrição STAT3RESUMO
BACKGROUND: To investigate the expression of long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in renal ischemia-reperfusion injury and explore its role in acute kidney injury. METHODS: 18 mice were randomly divided into a sham operation group (Sham) and an ischemia-reperfusion group (IR) in which animals were sacrificed at 6 h or 12 h after surgery. The kidneys were harvested to measure the expression of MALAT1 mRNA. HK2 cells were treated with cobalt chloride (CoCl2) to mimic hypoxia or transfected with siRNA to knockdown MALAT1 before CoCl2 treatment. After that, MALAT1 was analyzed by RT-PCR (reverse transcription-polymerase chain reaction). HIF-1É (hypoxia-inducible factor-1 alpha) and NF-κB (nuclear factor-kappa B) was measured by Western blot. The concentrations of IL-6 (interleukin-6) and TNF-É (tumor necrosis factor-alpha) in the media were detected by ELISA (enzyme-linked immunosorbent assay). RESULTS: The expression of MALAT1 in the IR (6 h/12 h) group was significantly higher than that in the sham group. In HK2 cells, MALAT1 was significantly increased at 1 h, 3 h, and 6 h after CoCl2 treatment but had reduced to the basal level at 12 h and 24 h. Knockdown of MALAT1 by siRNA significantly up-regulated the expression of HIF-1É and NF-κB proteins in CoCl2-treated HK2 cells. In addition, the concentrations of IL-6 and TNF-É were increased by MALAT1 siRNA transfection in CoCl2-treated HK2 cells. CONCLUSION: The expression of MALAT1 is increased in renal ischemia-reperfusion injury. It is likely that MALAT1 inhibits the hypoxia-induced inflammatory response through the NF-κB pathway.
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Inflamação/patologia , Rim/patologia , RNA Longo não Codificante/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Linhagem Celular , Cobalto/farmacologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , RNA Longo não Codificante/genética , RNA Interferente Pequeno/análise , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Obstructive sleep apnea syndrome (OSAS), characterized by chronic intermittent hypoxia (CIH), is an independent risk factor for aggravating non-alcoholic steatohepatitis (NASH). The prevailing mouse model employed in CIH research is inadequate for the comprehensive exploration of the impact of CIH on NASH development due to reduced food intake observed in CIH-exposed mice, which deviates from human responses. To address this issue, a pair-feeding investigation with CIH-exposed and normoxia-exposed mice is conducted. It is revealed that CIH exposure aggravates DNA damage, leading to hepatic fibrosis and inflammation. The analysis of genome-wide association study (GWAS) data also discloses the association between Eepd1, a DNA repair enzyme, and OSAS. Furthermore, it is revealed that CIH triggered selective autophagy, leading to the autophagic degradation of Eepd1, thereby exacerbating DNA damage in hepatocytes. Notably, Eepd1 liver-specific knockout mice exhibit aggravated hepatic DNA damage and further progression of NASH. To identify a therapeutic approach for CIH-induced NASH, a drug screening is conducted and it is found that Retigabine dihydrochloride suppresses CIH-mediated Eepd1 degradation, leading to alleviated DNA damage in hepatocytes. These findings imply that targeting CIH-mediated Eepd1 degradation can be an adjunctive approach in the treatment of NASH exacerbated by OSAS.
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Nonalcoholic fatty liver disease (NAFLD) is considered a risk factor for cardiovascular and cerebrovascular disease owing to its close association with coagulant disturbances. However, the precise biological functions and mechanisms that connect coagulation factors to NAFLD pathology remain inadequately understood. Herein, with unbiased bioinformatics analyses followed by functional testing, we demonstrate that hepatic expression of coagulation factor VII (FVII) decreases in patients and mice with NAFLD/nonalcoholic steatohepatitis (NASH). By using adenovirus-mediated F7-knockdown and hepatocyte-specific F7-knockout mouse models, our mechanistic investigations unveil a noncoagulant function of hepatic FVII in mitigating lipid accumulation and lipotoxicity. This protective effect is achieved through the suppression of fatty acid uptake, orchestrated via the AKT-CD36 pathway. Interestingly, intracellular FVII directly interacts with AKT and PP2A, thereby promoting their association and triggering the dephosphorylation of AKT. Therapeutic intervention through adenovirus-mediated liver-specific overexpression of F7 results in noteworthy improvements in liver steatosis, inflammation, injury, and fibrosis in severely afflicted NAFLD mice. In conclusion, our findings highlight coagulation factor FVII as a critical regulator of hepatic steatosis and a potential target for the treatment of NAFLD and NASH.
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Fator VII , Hepatopatia Gordurosa não Alcoólica , Animais , Humanos , Camundongos , Fator VII/genética , Fator VII/metabolismo , Ácidos Graxos/metabolismo , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
OBJECTIVE: Betaine-homocysteine methyltransferase (Bhmt) belongs to the family of methyltransferases and is involved in the one-carbon metabolic cycle, which is associated with the risk of diabetes and adiposity. This study aimed to explore whether Bhmt participated in the development of obesity or its associated diabetes, as well as the mechanism involved. METHODS: The expression levels of Bhmt were examined in stromal vascular fraction cells and mature adipocytes in obesity and nonobesity. Knockdown and overexpression of Bhmt in C3H10T1/2 cells were used to investigate Bhmt's function in adipogenesis. Bhmt's role in vivo was analyzed using an adenovirus-expressing system and a high-fat diet-induced obesity mouse model. RESULTS: Bhmt was highly expressed in stromal vascular fraction cells rather than mature adipocytes of adipose tissue and was upregulated in adipose tissue in obesity and C3H10T1/2-commited preadipocytes. Overexpression of Bhmt promoted adipocyte commitment and differentiation in vitro and exacerbated adipose tissue expansion in vivo, with a concomitant increase in insulin resistance, whereas Bhmt silencing exhibited opposite effects. Mechanistically, Bhmt-induced adipose expansion was mediated by stimulating the p38 MAPK/Smad pathway. CONCLUSIONS: The findings of this study highlight the obesogenic and diabetogenic role of adipocytic Bhmt and propose Bhmt as a promising therapeutic target for obesity and obesity-related diabetes.
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Betaína-Homocisteína S-Metiltransferase , Resistência à Insulina , Animais , Camundongos , Adipócitos/metabolismo , Betaína-Homocisteína S-Metiltransferase/metabolismo , Obesidade/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Acute kidney injury (AKI) is a common clinical complication associated with high mortality in patients. Immune cells and cytokines have recently been described to play essential roles in AKI pathogenesis. Plasmacytoid dendritic cells (pDCs) are a unique DC subset that specializes in type I interferon (IFN) production. Here, we showed that pDCs rapidly infiltrated the kidney in response to AKI and contributed to kidney damage by producing IFN-α. Deletion of pDCs using DTRBDCA2 transgenic (Tg) mice suppressed cisplatin-induced AKI, accompanied by marked reductions in proinflammatory cytokine production, immune cell infiltration and apoptosis in the kidney. In contrast, adoptive transfer of pDCs during AKI exacerbated kidney damage. We further identified IFN-α as the key factor that mediated the functions of pDCs during AKI, as IFN-α neutralization significantly attenuated kidney injury. Furthermore, IFN-α produced by pDCs directly induced the apoptosis of renal tubular epithelial cells (TECs) in vitro. In addition, our data demonstrated that apoptotic TECs induced the activation of pDCs, which was inhibited in the presence of an apoptosis inhibitor. Furthermore, similar deleterious effects of pDCs were observed in an ischemia reperfusion (IR)-induced AKI model. Clinically, increased expression of IFN-α in kidney biopsies was observed in kidney transplants with AKI. Taken together, the results of our study reveal that pDCs play a detrimental role in AKI via IFN-α.