Identification Of Diagnostic Markers Associated With M6A Modification In Diabetic Kidney Disease And Study On Mechanisms Of Renal Proper Cell Pyroptosis And Inflammatory Injury Mediated By FTO Demethylation

Objective(s):Diabetic kidney disease is one of the most common complications of diabetes mellitus and one of the main causes of End Stage Renal Disease(ESRD).Patients were often accompanied by clinical manifestations such as albuminuria,decreased estimated Glomerular Filtration Rate(e GFR),increased Serum Creatinine(SCR),and glucose and lipid metabolism disorder,which seriously threatened human health.The pathogenesis of DKD is complex,and the effectiveness of existing therapeutic methods is limited.Therefore,further exploration of the molecular mechanism of DKD will help to find new potential therapeutic targets and provide a new scheme for the early diagnosis and treatment of DKD.In recent years,studies have confirmed that m6 A methylation modification and pyroptosis play an important role in the pathogenesis of diabetes complications.Therefore,based on bioinformatics analysis,this study identified m6 A related genes in DKD,explored the signaling pathways and biological processes involved in them,and analyzed their regulatory mechanisms.Secondly,the results of bioinformatics analysis were verified by in vitro experiments,providing new clues for the development of DKD.Methods:1.The microarray data sets(GSE99339,GSE104948,GSE96804,GSE30122)were downloaded from GEO database,and the differentially expressed genes(DEGs)between normal and DKD samples were screened,after removing the duplicates,the gene sets of m6A-related regulators were obtained.2.Speraman correlation was used to calculate the correlation between DEGs and m6 A in DKD samples,go and KEGG enrichment analyses were performed on DEGs with an absolute value greater than 0.5 of correlation with m6 A regulators to reveal the biological function of m6A-DEGs.3.The experimentally validated set of m6 A methylation-modifying genes involved in DM was downloaded from the RMVAR(m6AVar)database and intersected with m6A-degs to obtain key m6A-degs target genes.4.Verify the expression of key m6A-DEGs target genes in DKD by Nephroseq database,and the correlation with the clinical symptoms of DKD patients.5.Gene set enrichment analysis(GSEA)looked at the enrichment of key m6A-DEGS target gene bioinformation and assessed the abundance of immune cell infiltration between control samples and DKD samples using the ss GSEA algorithm,the relationship between key m6A-DEGs target genes and immune cell infiltration was calculated.6.Molecular docking was used to verify the interaction between key m6A-DEGs target genes and m6 A regulators,and mi RNA-m RNA visual regulatory network was constructed by mi RNA prediction database,to reveal the regulatory mechanism of key m6A-DEGS target genes and m6 A regulators.7.The m6 A content of total RNA in human glomerular podiatocytes(HPC)was determined by colorimetric method using m6 A methylation quantitative kit.8.Immunoco-precipitation(Co-i P)was used to verify the interaction between immune-associated m6A-DEGs target genes and m6 A regulatory factors in HPC cells cultured with high glucose and lipopolysaccharide.9.Western blotting verified the expressions of immune-related m6A-DEGs target genes,markers of podocyte injury,proteins associated with classical pyroptosis pathway,and inflammatory injury factors in HPC and human renal tubular epithelial cells(HK-2)induced by high glucose and lipopolysaccharide.10.IPA analysis verified the regulatory relationship between m6 A demethylation enzyme FTO and pyroptosis-related genes.Results:1.A total of 161 DEGs were identified between normal glomeruli and DKD glomeruli by difference analysis,and 121 m6A-DEGs were screened by overlap analysis.2.121 m6A-DEGs were analyzed by GO and Kegg.The results showed that m6A-DEGs mainly concentrated in the process of immune regulation,collagen fibers,extracellular matrix tissue,positive regulation of immune effect,PI3K-AKT signal pathway,AGE-RAGE signal pathway and complement system,the results suggest that 121 m6A-DEGs are closely related to the immune and inflammatory regulation of DKD.3.The m6 A target gene sets in DM or DKD were downloaded from the m6 AVar database.A total of 251 m6 A target genes were obtained,and 251 m6 A target genes were intersected with 121 m6A-degs,the key m6 A target genes CD36 and COL6A3 were obtained,and the RMVAR database showed the presence of m6 A modification sites on CD36 and COL6A3 in DM or DKD.4.Correlation Analysis of CD36 and COL6A3 with m6 A regulators in the training set found that CD36 and COL6A3 were negatively correlated with FTO in the DKD samples.CD36 and COL6A3 were also negatively associated with FTO in the independent validation set GSE96804.CD36 and COL6A3 were also negatively correlated with FTO in another independent validation set,GSE30122,suggesting that CD36 and COL6A3 expression might be significantly correlated with FTO.5.In the combined training set and two independent verification sets,CD36 and COL6A3 gene expressions were significantly increased in DKD samples,while FTO was significantly decreased in DKD samples.The receiver ROC curve was drawn based on the expressions of CD36 and COL6A3 genes in DKD samples and control samples.The results showed that CD36 and COL6A3 genes had high diagnostic value for DKD,with AUC greater than 0.85.Two independent verification sets also showed that the area under the AUC curve was greater than 0.7.It is suggested that the key m6A-DEGs target genes CD36 and COL6A3 can better distinguish DKD from normal control samples.6.The expressions of CD36,COL6A3 and FTO genes were correlated with e GFR and SCR in glomerular and tubule interstitial tissue by Nephroseq chip database.Compared with normal kidney donors,the expressions of CD36 and COL6A3 genes in glomeruli and tubule interstitial tissues of DKD patients were significantly up-regulated,while the expressions of FTO were significantly decreased,which was consistent with the current trend of this study.Meanwhile,correlation analysis also found that the expression of CD36 in glomeruli and tubule interstitium was negatively correlated with e GFR in DKD patients,and positively correlated with SCR.COL6A3 expression in glomeruli and tubule interstitium was also negatively correlated with e GFR in DKD patients and positively correlated with SCR.The expression of FTO in glomeruli and tubule interstitium was positively correlated with e GFR and negatively correlated with SCR in DKD patients.7.GSEA enrichment analysis showed that CD36 and COL6A3 were mainly enriched in chemokine signaling pathways,ECM receptors,leukocyte migration across endothelial cells,natural killer cell-mediated cytotoxicity,Nod-like receptor signaling pathways,complement systems,primary immunodeficiency and toll-like receptors.8.The abundance of immune cell infiltration in normal control samples and DKD samples was evaluated by ss GSEA algorithm.The results showed that the percentages of activated B cells,activated CD4 T cells,activated CD8 T cells,activated dendritic cells,natural killer cells,myeloid inhibitory cells,macrophages,regulatory T cells,type 1 helper T cells and type 2 helper T cells in DKD samples were significantly higher than those in normal control samples.The proportion of eosinophils,neutrophils,plasmacytoid dendritic cells and T helper cells type 17 was lower than that of normal control samples.Correlation analysis showed that CD36 and COL6A3 were positively correlated with the abundance of immune cell infiltration,suggesting that key m6A-DEGs target genes CD36 and COL6A3 were involved in the regulation of immune cell infiltration in DKD.9.The mi RNA prediction database predicted the upstream mirnas targeting CD36 and COL6A3 and FTO,and the intersection was taken to obtain the relationship pairs of hsa-mi R-150-5P-CD36 and hsa-mi R-150-5P-FTO,which further explained the regulatory relationship between CD36 and FTO in DKD.10.The results of molecular docking showed that lysine 398 and lysine 403 of CD36(chain A)and aspartic acid ASP 676 of FTO(chain B)formed hydrogen bond interaction.In addition,glutamic acid No.152 GLN,aspartic acid ASN 151,TYR Tyrosine No.149 and Proline No.193 PRO of chain A have hydrogen bond interaction with ASN No.583 aspartic acid,GLN No.648 glutamic acid and LYS no.653 of chain B.The 164 th lysine LYS of the a-chain forms a salt bridge with the glutamic acid GLN of 672 nd of the b-chain,and 21 hydrophobic forces are also formed between the a-chain and the b-chain.These results indicate that CD36 and FTO geometry,hydrogen bond,salt bridge,hydrophobic interaction may interact with each other and complement each other.11.m6 A RNA methylation Quantitative analysis showed that the overall m6 A level in HPC cells was increased by HG combined with LPS,and further increased after FTO knockdown,however,the overall m6 A level decreased after overexpression of FTO.12.Co-immunoprecipitation assay confirmed the interaction between FTO and CD36,and western blotting analysis showed that the interaction between FTO and CD36 in HPC and HK-2 cells induced by HG combined with LPS,the markers of podocyte injury(Nephrin and Podocin)and FTO were significantly decreased,while CD36 and NLRP3,GSDMD,Pro-Caspase-1,il-1β,IL-18 were significantly increased.However,knockdown of FTO further enhanced the expression of coke-death-related proteins,further reduced podocyte injury markers,and simultaneously enhanced abnormal migration rate of HK-2 cells.In contrast,overexpression of FTO reversed the reduction of HG-and LPS-induced podocyte damage marker proteins and the upregulation of pyroptosis and inflammation-related proteins,and decreased abnormal migration rate of HK-2 cells.13.IPA analysis showed that activation of FTO could inhibit the activation of NLRP3,thereby inhibiting the expression of Caspase-1,GSDMD,IL-18 and il-1β,while inhibition of FTO could activate NLRP3,it leads to the activation of Caspase-1,GSDMD,IL-18 and il-1β Proinflammatory cytokine.Conclusion(s):1.CD36 and COL6A3 can be used as potential diagnostic markers for DKD.The expression level of CD36 and COL6A3 is significantly correlated with m6 A demethylase FTO.2.CD36 and FTO were negatively regulated in HPC cells,and CD36 was significantly up-regulated in the combination of high glucose and LPS.Knockdown of FTO could enhance the expression of CD36,while overexpression of FTO could reduce the expression of CD36.3.NLRP3 inflammatory body activation was found in the renal proper cell induced by high glucose and lipopolysaccharide.Overexpression of FTO could reverse the occurrence of pyroptosis and inflammation in the renal proper cell.The mechanism may be related to the regulation of NLRP3/Caspase-1/GSDMD pyroptosis signaling pathway.The inhibitory effect of FTO on NLRP3 inflammatory body may be mediated by CD36.The mechanism of m6 A demethylase FTO involved in the development and protection of DKD was elucidated.