Mechanisms Of RANK And PPBP Participate In The Podocyte Damage In Type 2 Diabetic Kidney Disease

Part1.RANK promotes the podocyte damage in type 2 diabetic kidney disease through endoplasmic reticulum stressBackgrounddiabetic kidney disease(DKD)is a common and troublesome kidney disease.Endoplasmic reticulum stress is the key link in podocyte injury and plays a central role in the progression of diabetic kidney disease.RANK belongs to the tumor necrosis factor receptor superfamily.Previous studies in by our group have demonstrated that RANK may promote podocyte injury in type 1 diabetic kidney disease through promoting oxidative stress.but this effect is incomplete.Whether there are other channels also related to it is unclear.Type 2 diabetic kidney disease is different from type 1 diabetic kidney disease from pathogenesis to pathology.Whether RANK plays the same role in podocyte injury of type 2 diabetes is not clear.The expression of RANK in type 2 diabetic kidney disease patients was higher than that in normal subjects.To establish a podocyte specific overexpression and silencing RANK mouse model of type 2 diabetic kidney disease,establish a podocyte model of diabetic kidney disease in vitro,and explore whether RANK can mediate podocyte injury through type 2 diabetic kidney disease through endoplasmic reticulum stress.Establish a podocyte specific overexpression and silencing RANK mouse model of type 2 diabetic kidney disease,establish a podocyte model of diabetic kidney disease in vitro,explore whether RANK can mediate podocyte injury through type 2 diabetic kidney disease through endoplasmic reticulum stress.ObjectivesTo explore the role of RANK in podocyte injury in in type 2 diabetes and its relationship with endoplasmic reticulum stress.Methods1.The expression of RANK was detected by immunohistochemistry in patients with type 2 diabetic kidney disease and normal paracancerous tissues.2.Establishment of podocyte-specific overexpression and knocking out of RANK db/db model mice.3.PCR detection of gene expression in model mice and grouping.4.RNAscope and immunofluorescence were used to detect the expression of RANK in mouse podocytes.5.The body weight,blood glucose,protein/Creatinine and blood Creatinine of mice in each group were measured.6.The pathology of each group was observed by PAS staining and electron microscope.7.The expression of GRP78 and P-PERK was detected by immunohistochemistry.8.Mouse podocytes were cultured in vitro,to establish RANK overexpression and RANK silencing podocytes in vitro,and to verify the transfection efficiency.9.The expressions of GRP78,P-PERK,ATF4,P-eIF2a and Podocin in podocytes cultured in vitro were detected by WB.10.The effect of RANK on the migration of podocytes was verified by scratch experiments,and endoplasmic reticulum stress promoted the migration of RANK on podocytes.ResultsThe expression of RANK in renal biopsy specimens of type 2 diabetic kidney disease patients was increased by immunohistochemistry.Mouse tail cutting PCR was used to detect the gene of model mice.RNAscope and immunofluorescence were used to detect the expression of RANK mRNA and protein in mouse podocytes of each group.After overexpression of RANK,the protein/Creatinine ratio was higher,the blood Creatinine was higher,the glomerular mesangial matrix was significantly increased by PAS,the glomerular basement membrane was significantly thickened by electron microscope,and the expression of GRP78 and P-PERK protein was significantly higher by immunofluorescence.These changes were significantly improved after silencing RANK in mice.WB results showed that the expression of RANK increased after high glucose stimulated podocytes in vitro.The efficiency of podocyte overexpression and knockout of RANK were verified respectively.WB results showed that the expression of RANK in podocytes cultured in vitro was positively correlated with the expression of endoplasmic reticulum stress-related protein and negatively correlated with the expression of podocin.Scratch assay showed that podocyte migration increased when RANK was overexpressed,and decreased when RANK was knocked out.Endoplasmic reticulum stress inhibitor can inhibit the inCrease of RANK on podocyte migration.ConclusionThe expression of RANK increased in type 2 diabetic kidney disease patients.Kidney injury aggravated in podocyte specific overexpression of RANK in type 2 diabetic kidney disease mourse.RANK silencing reduced renal injury in rats.GRP78 and P-PERK positively correlated with RANK expression.In vitro cultured podocytes confirmed that RANK can stimulate podocyte migration through endoplasmic reticulum stress,and finally aggravate type 2 diabetic kidney disease injury.Part2.PPBP participates in podocyte damage in type 2 diabetic kidney disease via Bioinformatics Analysisdiabetic kidney disease(DKD)is a type of kidney injuries associated with diabetes mellitus and the prevalence of DKD has inCreased dramatically.However,DKD still pose problems in therapy,and prognosis.Finding new type 2 diabetes nephropathy podocyte injury factors will help reduce the incidence of diabetic kidney disease and develop new prevention methods.ObjectivesThe bioinformatics method was used to identify the factors affecting podocyte injury in type 2 diabetes nephropathy.Methods1.from GSE36336 dataset with DKD glomeruli samples,we sCreened for 238 differentially expressed genes.2.Enrichment analysis were performed to find out biological function and diseases of DEGs.3.Next,depended on protein-protein interaction network,We identified top 10 hub genes.4.In Nephroseq V5,external data sets were used to examine the correlation between central gene expression and clinical characteristics of diabetic kidney disease.5.Transcriptome sequencing was carried out in vitro on podocyte diabetic kidney disease model.6.In order to verify the effectiveness of the selected genes as biomarkers,we carried out real-time quantitative PCR,renal histological analysis,immunofluorescence and other experiments.ResultsThe data set GSE36336 was downloaded from GEO.After differential expression analysis,238 differentially expressed genes were sCreened,of which 153 were upregulated and 85 were down regulated.In the form of heat map,the differentially expressed genes and their expression quantities in the top 30 of the degree of difference are presented.The differential expression analysis was enriched by go,KEGG and mesh.According to the protein protein interaction network,we sCreened the first 10 candidate genes(Serpine1,Cxcl10,Cfd,PPBP,Retn,Socs2,Ccr5,Mmp8,Pf4,Cxcl9)in diabetic kidney disease,and initially identified PPBP as a central gene.In the Hodgin Diabetes Mouse Glom dataset of Nephroseq V5,Ccr5,Cfd,Cxcl10,Pf4,PPBP and Pf4 showed significant differences in the expression levels between diabetic kidney disease and normal samples.The transcriptome of podocytes was sequenced to verify the reliability of PPBP as a central gene again.Compared with control mice,db/db mice had significantly increased body weight,blood glucose level,serum Creatinine and urinary protein/Creatinine.For histopathological examination,PAS staining showed obvious proliferation of glomerular mesangial cells in db/db rats.The expression of PPBP mRNA increased in podocytes stimulated by high glucose.In the immunohistochemical experiment,compared with the normal control group,the expression of PPBP in the glomerulus of db/db mice was significantly inCreased.In immunofluorescence analy sis,compared with the normal control group,the expression of PPBP in renal podocytes of db/db mice inCreased.ConclusionPPBP is involved in podocyte injury in type 2 diabetes mellitus.