The Role Of LSD1 In Tubulointerstitial Inflammation In Hepatitis B Virus-Associated Glomerulonephritis

Objective:The pathogenesis of hepatitis B virus-associated glomerulonephritis(HBV-GN)is unclear,and there is still a lack of effective means to delay its progress.China is a country with high incidence of chronic hepatitis B,HBV-GN has become one of the main secondary kidney diseases in China.Recent studies have confirmed that HBV infection can directly cause renal tubular epithelial cell injury,tubulointerstitial inflammatory cell infiltration,and eventually progress to renal tubulointerstitial fibrosis,but the specific mechanism is still unclear.Lysine-specific demethylase 1(LSD1)is the first histone demethylase found to specifically remove both the mono-or di-methylation of histone 3 lysine 4(H3K4me1/2)and histone 3 lysine 9(H3K9 me1/2),thereby regulating the transcriptional activity of target genes.It has been found that LSD1 regulates the expression of immune-and inflammatory-related genes by mediating histone demethylation,and participates in immune and inflammatory responses.In the present study,we observed the role of LSD1 in the formation of renal tubular interstitial inflammation induced by HBV in vivo and in vitro,and investigated the possible mechanisms.Methods:(1)Fifty three patients diagnosed as HBV-GN by medical history,clinical examination and renal biopsy from January 2015 to December 2017 at the Department of Nephrology of Shanghai Jiaotong University Affiliated First People’s Hospital were included in this study.Other kidney diseases were excluded.According to the pathological type and serum HBV antigens,we divided samples into four groups:group I,the HBV-GN group,53 samples;group II,the HBV-positive primary glomerulonephritis(PGN)group(PGN and serum HBsAg or HBV DNA positive)including 35 samples;group III,the HBV-negative PGN group(PGN and serum HBsAg or HBV DNA negative),including 50 samples;and group IV,normal control group(15 samples from the adjacent normal renal tissues of tumor resection specimens).The co-expression of HBsAg and LSD1 in renal tissue was detected by immunofluorescence double labeling,and the expression of inflammatory markers and LSD1 was detected by immunohistochemistry,and the relationship between them was analyzed.(2)We transfected the plasmid pCMV-HBV-1.3 containing 1.3-fold full-length HBV genome into human proximal tubular epithelial cells(HK-2)to establish a cell model of HBV infection.The expression and secretion of proinflammatory mediators,IL-1β,IL-6,TNF-αand MCP-1,were examined by Realtime RT-PCR and ELISA,and the expression of LSD1 was examined by Realtime RT-PCR and Western blot.We constructed LSD1-overexpression plasmids(pcDNA3.1-myc-LSD1)and LSD1 shRNA plasmids(shLSD1)and transfected them into HBV infected HK-2 cells.The expression and secretion of above mentioned proinflammatory mediators were detected by Realtime RT-PCR and ELISA.(3)The differentially expressed genes of the cells before and after silencing LSD1 were detected by RNA-seq,and the expression of H3K4me1/2,H3K9me1/2 and TLR4 was examined by Western blot.The levels of LSD1,H3K4me1/2 and H3K9me1/2 at the promoter of TLR4 in HK-2 cells were examined by chromatin immunoprecipitation(ChIP)-qPCR.TLR4-overexpression plasmids(pcDNA3.1-myc-TLR4)and TLR4 shRNA plasmids(shTLR4)were constructed and co-transfected with LSD1 shRNA plasmids into HBV infected HK-2cells.The secretion of proinflammatory mediators was detected by ELISA.TLR4downstream NF-κΒand MAPK signal pathways in HK-2 cells were detected by Western blot.After intervention with NF-κB or MAPK inhibitors,the secretion of proinflammatory mediators was determined.Results:(1)Compared with the control group,the HBV-GN patients were characterized by tubulointerstitial infiltration of CD4~+T cells and macrophages and increased IL-1β,IL-6,MCP-1 and LSD1 expression in renal tubular epithelial cells.LSD1 was seen distributed in the renal tubular area,where the distribution of HBsAg had similar intensity.Further study found that the expression of LSD1 in HBV-GN patients was closely related to the degree of tubular and interstitial lesions,and was most closely related to interstitial inflammatory cell infiltration,tubular atrophy and interstitial fibrosis.(2)After transfection of HBV genome,the expression of HBV DNA,HBsAg and HBeAg in culture medium of HK-2 cells was upregulated,suggesting that the cell model of HBV infection was successfully constructed.Moreover,the expression and secretion of proinflammatory mediators in HK-2 cells were increased,and the expression of LSD1 was upregulated.After LSD1 overexpression or silencing intervention,the expression and secretion of proinflammatory mediators in HK-2 cells were increased or decreased accordingly.(3)RNA-seq analysis found that there were altogether 983 mRNAs revealed≥1.5-fold increased abundance;conversely,557 genes exhibited a decline in abundance(≤1.5-fold)owing to the silencing of LSD1.And many of these genes are involved in inflammatory response processes.Furthermore,among the most highly expressed genes,many renowned genes,such as TLR4,IL1B,TNFAIP3and SOCS2,are associated with inflammation.Further analysis found that LSD1 could not directly bind to the promoter regions of IL1B and IL-6 to regulate their expression,while LSD1 could directly bind to the promoter regions of TLR4 to mediate H3K9me1/2 rather than H3K4me1/2 demethylation,and then regulate its expression.Moreover,silencing of TLR4 could inhibit the secretion of inflammatory cytokines induced by HBV in HK-2 cells,and overexpression of TLR4 could restore the decrease of inflammatory cytokines secretion induced by silencing of LSD1.In addition,silencing of LSD1 could inhibit the phosphorylation of IKKα/β,IκBα,JNK1/2,ERK1/2and p38MAPK induced by HBV,while overexpression of TLR4 could restore the phosphorylation of these proteins.NF-κB or JNK inhibitors could inhibit the secretion of inflammatory cytokines induced by HBV in HK-2 cells.Conclusion:(1)LSD1 was significantly upregulated in renal tissue(mainly renal tubules)of HBV-GN patients,and its expression was positively correlated with tubular and interstitial inflammation injury,suggesting that LSD1 might be related to tubulointerstitial inflammation of HBV-GN.(2)The cell model of HBV infection was successfully constructed.HBV could induce the secretion of inflammatory cytokines and upregulation of LSD1 expression in HK-2 cells.Furthermore,overexpression or silencing of LSD1 could promote or inhibit the secretion of inflammatory cytokines induced by HBV in HK-2 cells,which might depend on histone demethylation of LSD1.(3)RNA-seq analysis revealed that TLR4 was the downstream target gene of LSD1.Moreover,LSD1 could directly bind to TLR4 promoter to mediate H3K9me1/2demethylation,and then regulate its expression.In addition,LSD1 could activate NF-κB and JNK signaling pathways through direct regulation of TLR4 expression,thereby promoting HBV-induced secretion of proinflammatory mediators in HK-2 cells.