The Role And Mechanism Of M6A Methyltransferase METTL3 On Acute Kidney Injury

BackgroundAcute kidney injury(AKI)is a common and severe clinical complication resulting in rapid decline in kidney function and high clinical mortality.Clinically,the causes of AKI mainly include hemorrhagic shock,rhabdomyolysis,direct renal trauma,mass blood transfusion,and various endogenous as well as exogenous nephrotoxins.AKI is associated with 10%-15%in hospitalized patients and the 90-day mortality rate of AKI is as high as 28%.Notably,AKI was strongly associated with prolonged hospital stay,increased mortality,and progression to end-stage kidney disease(CKD).There is a lack of specific and effective treatment except dialysis.Therefore,it is of great significance to explore the molecular mechanism of acute kidney injury and the development of prevention and treatment drugs.N6-adenylate methylation(m6A),as the most abundant chemical modification at the post-transcriptional level,can effectively regulate the processing,translation and degradation rate of pre-mRNA,especially in eukaryotic messenger RNA.More and more growing evidence indicated that m6A methylation is widely involved in kidney development and disease progression,including renal cell carcinoma(RCC),acute kidney injury,and chronic kidney disease,but the specific mechanisms have not been fully understood.Prestudy results show that METTL3 expression is elevated in human biopsies as well as renal tubules in different acute kidney injury(AKI)models and cultured tubular epithelial cells.ObjectiveIn this study,we investigated the function and underlying mechanism of m6A methylation mediated by METTL3 in acute kidney injury,in order to provide theoretical and experimental basis for new clinical intervention strategies for acute kidney injury.1.Investigating the role of METTL3 in regulating kidney injury and inflammatory response in AKI.2.Elucidating the mechanisms by which METTL3 mediates TAB3 methylation modifications and thus regulates inflammatory responses in AKI.3.Investigating whether METTL3 is expected to be a new target for AKI prevention and treatment.Methods1.m6A RNA modifications level and related regulatory enzyme expression was detected in renal tubules in different AKI models as well as in human biopsies and cultured tubular epithelial cells(TECs).(1)AKI clinical sample collection:AKI renal perforation samples were collected to evaluate the methylation level of kidney m6A by immunohistochemistry,m6A enzyme-linked immunosorbent assay and other techniques.(2)In vivo:we established cisplatin-,LPS-and I/R-induced AKI mouse models were established to detect changes in m6A methylation levels,expression and localization of m6A methylation-related enzymes(METTL3,METTL14,WTAP,ALKBH5,FTO),respectively.(3)In vitro:we established TNF-α-,cisplatin-and LPS-stimulated models of inflammation and injury in HK2 cells,respectively,and to detect changes in m6A methylation levels and expression of m6A methylation-related enzymes.(4)The potential mechanism of METTL3 induction in AKI:the transcription factor binding sites of its promoter region were predicted using the JASPAR database.2.The function of METTL3 in renal inflammation(1)We established cisplatin-,I/R-,and LPS-induced AKI mice model in METTL3 cKO mice,renal function,macrophage infiltration,inflammatory cytokines and chemokines levels,and activation of NF-κB signaling pathway were detected.(2)We evaluated the function of METTL3 in HK2 cells in response to inflammatory stimuli.Knockdown and overexpression of METTL3 by transfection of siRNA or overexpression plasmid.We establish TNF-α-,cisplatin-,and LPS-induced inflammatory model,respectively.To detect the m6A RNA modifications level,inflammatory cytokines and chemokines levels,and activation of NF-κB signaling pathway.3.The regulatory mechanism of METTL3 in renal inflammation(1)We conducted MeRIP-sequencing and RNA sequencing analysis in TNF-α-stimulated HK2 cells with or without silencing of METTL3.The downstream target gene TAB3 was found by MeRIP-sequencing.To detected the methylation level and expression level of TAB3 were regulated by METTL3 by using MeRIP-qPCR and Real-time qPCR.Furthermore,we mutate TAB3 m6A methylation modification site,using luciferase reporter assay to detected the involvement of METTL3 in mediating the m6A methylation modification of TAB3.(2)Apply RNA immunoprecipitation assay to search identify TAB3 m6A modified reading protein IGF2BP2,and to detected the effect of silencing IGF2BP2 on the expression level of TAB3.Transcriptional inhibitors were used to detect the effect of IGF2BP2 on the stability of TAB3 mRNA.4.Whether METTL3 can be used as a new target for anti-renal inflammationWe use two strategies to assess the therapeutic potential of METTL3 in AKI mouse models.(1)To detected the protective effect on renal inflammation and kidney injury by the Adeno-associated virus 9(AAV9)-mediated METTL3 silencing.(2)To detected the protective effect of METTL3 small molecule inhibitors on renal inflammation and kidney injury.Results1.METTL3 expression is elevated in renal tubules in different AKI models as well as in human biopsies and cultured tubular epithelial cells(TECs)Using an m6A enzyme-linked immunosorbent assay(ELISA),we found that m6A RNA modifications were substantially elevated in cisplatin-induced AKI.METTL3 was significantly increased in cisplatin-,I/R-,and LPS-induced AKI mice,as well as in human biopsies and TNF-α-,cisplatin-,and LPS-induced tubular epithelial cells.In addition,METTL3 abundance was mainly localized in renal tubules.To explore the potential mechanism of METTL3 induction in AKI,the transcription factor binding sites of its promoter region were predicted using the JASPAR database.Jun potential binding sites,with high scores,were found in the promoter region of METTL3.It was found that the expression of nuclear transcription factor c-Jun protein was up-regulated in a time-dependent manner.Silencing c-Jun inhibited METTL3 protein and mRNA levels,while overexpression of c-Jun promoted METTL3 transcription.In addition,chromatin immunoprecipitation(ChIP)assay showed enhanced binding of c-Jun to the METTL3 promoter region under cisplatin stimulation.2.METTL3 enhances inflammatory response and cell apoptosis in HK2 cells treated with proinflammatory stimuli(1)In vivo:Compared with WT mice,METTL3 deficiency attenuated cisplatin-and I/R-induced renal injury,F4/80+macrophage infiltration,and inhibited the production of pro-inflammatory factors as well as the phosphorylated NF-κBp65 activation.(2)In vitro:METTL3 knockdown reduced the TNF-α-,cisplatin-and LPS-induced inflammation response and cell death,overexpression of METTL3 had the opposite effect.3.TAB3 is a direct target of METTL3(1)KEGG enrichment analysis indicated that inflammation-related signaling pathways(such as the TNF-α pathway)were highly correlated with METTL3-mediated m6A modifications.Through the intersection analysis of "genes with significantly reduced m6A methylation level" and "genes with significantly changed mRNA" after METTL3 silencing,we found that a total of 32 potential genes.Among them,TGF-β-activated kinase 1 binding protein(TAB3)attracted our attention.Silencing METTL3 reduces TAB3 protein and mRNA levels.In addition,visual analysis of gene m6A showed that a m6A peak was detected around the stop codon of TAB3 mRNA in TNF-α-stimulated HK2 cells,which was diminished upon METTL3 knockdown.A luciferase reporter assay showed that the amount of wild-type TAB3 transcript but not the mutant gene decreased in the absence of METTL3.(2)An RNA immunoprecipitation(RIP)analysis that the interaction between IGF2BP2 and TAB3 mRNA.Silencing IGF2BP2 inhibits TAB3 protein and mRNA levels and reduces the stability of its mRNA.4.Inhibition of METTL3 protects against renal damage in AKI murine models(1)AAV9-mediated silencing of METTL3 alleviated cisplatin-and LPS-induced renal injury,F4/80+macrophage infiltration,and inhibited the production of pro-inflammatory factors as well as the phosphorylated NF-κBp65 activation.(2)We identified a METTL3 inhibitor using a hybrid virtual screening strategy,followed by evaluations of its reno-protective and anti-enzyme activity.Among the top 100 compounds screened from ChemDiv(containing 430,000 compounds)and MCE(containing 100,000 compounds),Cpd-564 showed the best reno-protective effect.Molecular docking and cellular thermal shift analysis(CETSA)supports potential interactions between Cpd-564 and METTL3.Cpd-564 treatment alleviates cisplatin induced cell damage,inflammation and apoptosis in vitro and in vivo.ConclusionIn conclusion,our results shown that METTL3 expression is highly induced in TECs in response to various AKI stimuli in a c-Jun-dependent manner,and METTL3 promotes renal inflammation and injury by increasing TAB3 m6A RNA methylation while increasing TAB3 mRNA stability via IGF2BP2-dependent mechanisms.In this study,the molecular mechanism of m6A methylation modification in AKI was investigated,and the abnormal activation of METTL3/TAB3 axis was shown to be one of the important mechanisms driving kidney inflammation and injury in AKI.