Epigenetic Mechanism Of MEN1 Deficiency Leads To Renal Fibrosis By Suppressing The Transcriptional Activation Of HGF/Adamts5 Genes

Objective:To investigate the biological role of MEN1 in the kidney and elucidate the epigenetic mechanism of MEN1 regulating epithelial-to-mesenchymal transition during renal fibrogenesis.The present study will provide a theoretical basis for developing epigenetic-therapy drugs in renal fibrosis treatment targeting MEN1 signal axis.Methods:Diabetic nephropathy（db/db）and Unilateral Ureteral Obstruction（UUO）-induced renal interstitial fibrosis mouse models were established,and renal damage and fibrosis were evaluated by hematoxylin-eosin（HE）,Masson trichrome and Sirius red staining.The expression of MEN1 in fibrotic mouse kidneys and human diabetic nephropathy（DN）was detected RT-q PCR,Western blot and immunohistochemistry（IHC）.Immunofluorescence（IF）or Western blot was used to detect the expression of menin and fibrosis-related proteins（α-SMA,Collagen 1）in m RTECs,NRK-52E and HK-2 cells,which were treated with TGF-βor aristolochic acid（AA）.Men1 wild-type(Men1^f/f)and Men1 knockout(Men1^Δ/Δ)mice were established,and the degree of renal damage and fibrosis was evaluated by HE,Masson trichrome,Sirius red and IHC staining.RT-q PCR was used to detects the expression of Kim-1（an acute kidney injury marker）and fibrosis-related genes,including ACTA2,Fibronectin1,Col1α1and Osteopontin.RNA sequencing（RNA-Seq）was performed on the kidney tissues of Men1^f/f and Men1^Δ/Δmice and the differentially expressed genes were analyzed by GO analysis,KEGG and Reactome signaling pathway enrichment analysis.RT-q PCR was used to verified m RNA levels of differentially expressed genes in kidney tissue.CRISPR/Cas9 gene editing technology was used to establish MEN1 gene knockout HK-2 cells,named HK-2-MEN1-KO,and the wild type was named HK-2-MEN1-WT,F-actin antibody was used to observe cell morphology and F-actin expression,as determined by IF staining.Overexpressed MEN1,（MEN1）,MEN1-KO and MEN1knockdown（sh MEN1）renal tubular epithelial cells were treated with TGF-βor IL-β,and the expressions of EMT-related proteins E-cadherin,Desmin,N-cadherin and Vimentin were detected by Western blot.IHC and Western blot were used to detect the expression of EMT-related proteins,H3K4me1/2/3 and H3K9me3 in the kidney tissues of Men1^f/f,Men1^Δ/Δand UUO mice.IF staining was used to detect the level of H3K4me3 in HK-2 cells treated with TGF-β.Histone was isolated from MEN1-WT,MEN1-KO and sh MEN1 HK-2 cells,and the abundance of chromatin histone（H3K4me1/2/3 and H3K9me3）were detected by Western blot.F-actin antibody was used for IF staining to observe the cell morphology in HK-2 cells treated with MI-3（a menin/MLL1 interaction inhibitor）.Western blot was used to detect the expression of EMT-related proteins and histone modification levels in m RTECs treated with TGF-βand MI-3 alone or in combination,and HK-2-MEN1 cells treated with MI-3.Mouse kidney tissues were subjected to chromatin immunoprecipitation and sequencing（Ch IP-seq）with menin and H3K4me3 antibodies for GO enrichment analysis.Integrated analysis of RNA-seq and Ch IP-seq data identified 105 target genes regulated by Men1,focusing on Hgf and Adamts5.The m RNA and protein expressions of Hgf and Adamts5 were detected by RT-q PCR,Western blot or IHC in Men1^Δ/Δmice kidney tissues,TGF-β-treated m RTECs or HK-2 cells.Western blot was used to detect the expression of menin,HGF and Adamts5 in HK-2 cells treated with knockdown（KD）of HGF（si HGF）or exogenous recombinant human HGF（r HGF）.Ch IP-q PCR was used to detect the binding of menin and H3K4me3 in the promoter regions of Hgf and Adamts5 genes.The expressions of menin and renal fibrosis-related proteins were detected in renal tubular epithelial cells treated with r HGF.The UUO mouse models of Men1^f/f and Men1^Δ/Δwere established.HE,Masson trichrome and Sirius red staining were used to detect renal injury and fibrosis in UUO mouse models of Men1^f/f and Men1^Δ/Δwhich were treated by r HGF.The expression of renal fibrosis-related proteins was detected by IHC and Western blot to evaluate the therapeutic effect of r HGF on renal fibrosis.Flow cytometry was used to analyzed Cell cycle.The expressions of cell cyclin-related proteins,JNK and ERK signaling pathway-related proteins were detected by IHC,IF or Western blot.Results:MEN1,MLL1 and H3K4me3 were down-regulated in the kidney tissues of UUO mice,db/db diabetic nephropathy mice and human diabetic nephropathy.The expression of MEN1 was decreased in renal tubular epithelial cells induced by TGF-βor aristolochic acid.Comparing with Men1^f/f mice,morphological observation revealed severe kidney swelling and a significant kidney weight increase in Men1^Δ/Δmice.The normal renal architecture was exhibited in the Men1^Δ/Δmice at 1 month of age.By 4 months,mild kidney oedema started to appear accompanied by local inflammatory cell infiltration in some of the Men1^Δ/Δmice.By 8 months,fibrotic changes began to develop,and most animals presented moderately dilated renal tubules and reduced internal clearance between glomeruli and occasionally cystic appearing.By 12 months,the kidney became severely swollened and the kidney weight obviously enhanced in the Men1^Δ/Δmice when compared with the Men1^f/fanimals.The m RNA expression levels of Kim-1（an acute kidney injury marker）,collagen fibers and renal fibrosis-related genes were increased in Men1^Δ/Δmice.With the extension of Men1 deficiency time,the pathological changes gradually aggravated,and the expression of renal injury markers,collagen fibers and renal fibrosis-related genes gradually increased.Masson trichrome and Sirius red staining showed that the level of fibrosis in the kidney tissue of Men1^Δ/Δmice was significantly higher than that of Men1^f/f mice.RNA-seq data showed that renal fibrosis-related molecular events such as collagen trimer,extracellular matrix and contractile fibers were significantly enriched in the kidney tissues of Men1^Δ/Δmice.KEGG and Reactome pathway enrichment analysis showed that EMT-related pathways such as JAK-STAT,PI3k-Akt and G2/M checkpoint were significantly enriched.MEN1-KO or sh MEN1 inhibited the protein expression of E-cadherin and promoted the protein expression of N-cadherin,Vimentin andα-SMA in renal tubular epithelial cells.MEN1 deletion reduced the level of chromatin H3K4me3 and H3K9me3,but not H3K4me2 and H3K4me1.Renal tubular epithelial cells treated with MI-3 reduced the level of H3K4me3 modification,inhibited the protein expression of E-cadherin,and increased the protein expression of N-cadherin,Vimentin andα-SMA.Integrated analysis of RNA-seq and Ch IP-seq revealed that renal fibrosis-related molecular events such as Menin-dependent H3K4me3-mediated EMT,Wnt pathway,fibroblast proliferation and epithelial cell differentiation were significantly enriched in the kidney tissues of Men1^Δ/Δmice.RT-q PCR results showed that Men1 deficiency down-regulated the expression of Hgf,Adamts5,Gdf11,Pdgfra,ECM deposition,and renal fibrogenesis genes.MEN1 deficiency inhibited Hgf and Adamts5 expression,while menin expression was not affected by si HGF or r HGF in the renal fibrosis model.Ch IP-q PCR results showed that menin and H3K4me3 co-bound to the Hgf and Adamts5 promoter regions.Deletion of MEN1 reduced H3K4me3 modification in Hgf/Adamts5 promoter region,then inhibited the transcriptional activation of Hgf/Adamts5 genes.Compared with Men1^f/fmice,Men1^Δ/Δmice induced kidney fibrosis Collagen accumulation,relatively severe kidney injury,and increased protein expression of Collagen 1 andα-SMA in UUO kidneys.Men1^Δ/Δmice treated with r HGF reduced the protein expression of Collagen 1 andα-SMA and collagen accumulation.r HGF can increase the expression of Adamts5,but had no effect on the expression of menin.Flow cytometry analysis showed that MEN1 deficiency caused G2/M phase arrest and activated JNK and ERK signaling pathways to induce the development of renal fibrosis.Conclusions:Conclusions of this study are as follows:1.The low expression of MEN1 in renal fibrotic tissues and renal tubular epithelial cells induced by fibrotic factors suggests that MEN1 is involved in the occurrence and development of renal fibrotic diseases.2.MEN1 deficiency causes chronic renal injury and fibrosis,as well as activates the EMT pathway,indicating that MEN1 is an important protective factor of renal function.3.Menin/MLL complex mediated H3K4me3 modification regulates Hgf/Adamts5 gene expression inhibits EMT pathway,and reduces ECM accumulation to prevent the pathogenesis of renal fibrosis through epigenetic mechanism.4.Targeted activation of MEN1-Hgf-Adamts5 signaling axis partially improves renal dysfunction caused by fibrotic factors and UUO induced renal interstitial fibrosis.5.MEN1 plays a protective role in renal function by regulating multiple signaling pathways.Chronic renal fibrosis caused by MEN1 dysfunction is related to the activation of EMT,JNK or ERK signaling pathways and G2/M phase arrest of the cell cycle.