The Molecular Mechanism Of Macrophages Regulating Diabetic Kidney Disease Through ITGB2

Background: Diabetic kidney disease(DKD)is one of the more common chronic microvascular complications in diabetes and can lead to end-stage renal failure.The causative factors of diabetic kidney disease are complicated,including a variety of factors such as abnormal glucose metabolism,hemodynamic changes,oxidative stress and inflammatory factors involved in the pathogenesis and progression of DKD.At present,there are a large number of genetic chip sequencing data of clinical samples or animal models of diseases in patients with diabetic kidney disease.Data analysis of a single genus can screen some related disease markers in DKD,but there are certain species limitations.A common comprehensive analysis of gene chip data between different species,finding genes with common differences,and in-depth functional network analysis of common differential genes,may be able to prompt the screening of more valuable information.In this study,the kidney gene chip data of diabetic kidney disease patients and diabetic kidney disease mouse animal disease models were comprehensively analyzed to find common differential genes,and then further in-depth bioinformatics analysis was performed to find new targets,and the role of new targets in diabetic kidney disease is investigated.Objective: Through in-depth analysis of gene chip data of kidney specimens from clinical patients with diabetic kidney disease and kidney specimens from mouse animal models of diabetic kidney disease,various bioinformatics analysis methods of gene and protein analysis are comprehensively applied to find new targets in diabetic kidney disease and mechanism analysis of new targets.Methods: 1.Bioinformatics analysis of the molecular interaction network of diabetic kidney disease:(1)Select two sets of diabetic kidney disease gene chip data from GSE30528 and GSE33744 in the NCBI-GEO database.GSE30528 is the kidney genome expression data of DKD patients.GSE33744 is the kidney genome expression data of db / db mouse diabetes model.The common differential genes of the two groups were searched by Morpheus software.(2)Perform gene ontology(GO)enrichment function and KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway analysis on common differential genes to find meaningful gene expression sites and key signaling pathways.(3)Use Ingenuity Pathway Analysis(IPA)analysis software to analyze protein networks and biological functions of common differential genes,and screen for important role networks and gene protein sites with regulatory significance.2.In db/db mouse model of diabetic kidney disease,q PCR was carried out to verify the accuracy of gene chip data for the significantly differentially expressed genes with regulatory significance obtained through bioinformatics analysis.The identified key gene protein sites were verified by immunohistochemistry and western blot.Chromatin immunoprecipitation method and luciferase assay were used to verify the binding relationship between the identified regulatory genes and upstream transcription factors.3.The key gene loci screened out by bioinformatics analysis were analyzed by si RNA,plasmid expression technology and cell co-culture method,and the possible mechanism of their action in diabetic kidney disease was analyzed by western blot and other methods.Results: 1.Through a comprehensive comparative analysis of GSE30528 and GSE33744 data,89 common differentially expressed genes were found,of which 51 were up-regulated and 38down-regulated;analysis of GO enrichment function suggested that the major functional enrichment of common differentially expressed genes was in cell adhesion,extracellular area,extracellular matrix and so on.Analysis of KEGG pathway indicates that co-differentially expressed gene signaling pathways are mainly enriched in cell adhesion molecules,extracellular matrix-receptor interactions,and focal adhesions;IPA bioinformatics analysis suggests that co-differentiated genes are enriched classical signaling pathways include the complement system,primary immunodeficiency signaling,and B cell development,suggesting that the complement system is a significant regulatory pathway in diabetic kidney disease.Disease and biological function enrichment analysis showed that common differentially expressed genes are closely related to diabetes mellitus,glucose metabolism disorders,cell movement of blood cells,activation of blood cells and leukocyte migration.By upstream regulatory factor analysis of IPA,ETS1(ETS Proto Oncogene 1),an important transcriptional regulator,was identified.Combined with the TRUSTA database,we found that ETS1 has an important downstream target,ITGB2(Integrin β2).ETS1 and ITGB2 were selected as the next research focus.2.An animal model of diabetic kidney disease in db/db mice was established,and the establishment of db / db mouse diabetic kidney disease model was confirmed through analysis of body weight,kidney weight,blood glucose,24-hour urine protein quantification,urinary microalbumin creatinine ratio,serum creatinine,urea nitrogen and renal pathological PAS staining.Western blot results suggest that protein expression of ETS1 and ITGB2 in kidney tissue of db/db mice is significantly higher than that of db / m mice;chromatin immunoprecipitation method and luciferase assay confirm that the transcription factor ETS1 can directly regulate downstream ITGB2.3.Established a model of co-culture of macrophages and fibroblasts,western blot results showed that the expression levels of ITGB2,p-SYK,p-ERK,and TGF-β protein in macrophages were significantly increased in a high glucose environment,suggesting that ITGB2 can regular the Syk-ERK pathway and promote the expression of TGF-β in macrophages,further leading to increased expression of α-SMA,type I collagen and fibronectin in fibroblasts,and promotes the process of fibrosis.Conclusions: By studying the use of the kidney gene expression profile database of diabetic kidney disease patients and db/db mice,the important sites of transcription factors ETS1 and ITGB2 were screened.Basic experiments proved that ITGB2 can promote the expression of TGF-β in macrophages through the Syk-ERK signaling pathway,and further act on fibroblasts.It promotes the progression of fibrosis in diabetic kidney disease.