Transcriptome Analysis On Metabolic Memory In Epithelial-Mesenchymal Transition Of Podocytes Induced By High Glucose

Background and Objective:Diabetic kidney disease(DKD),a common diabetic microvascular complication,is one of the major contributing factor to end-stage renal failure.Although the occurrence and development of DKD have been reduced by the comprehensive intervention of multiple factors,the residual risk of DKD is still remain high.To our surprise,kidney impairment caused by previous high glucose continues to progress even though adequate control of blood glucose is achieved in the later period,this phenomenon is called "hyperglycemia memory",but its mechanism has not been fully clarified.Recently,podocyte,as an important part of the glomerular filtration barrier,can be impaired and transformed into mesenchymal cells in the high glucose medium.Thus,the pathophysiology of podocyte impairment attracted more and more attention.But,it must be noted that previous studies often focus on the known signal pathways,which is difficult to avoid subjectivity.Therefore,in this paper,the metabolicmemory in podocyte EMT induced by high glucose was simulated in vitro,and high-throughput transcriptome technology was used to explore the new target molecules for the podocyte EMT.Methods:1.Podocytes were cultured in four groups: normal glucose group(NG,5mmol/L D-glucose×48h);high glucose group(HG,30mmol/L D-glucose×48h);metabolic memory group(MG,30mmol/L D-glucose×48h+5.5mmol/L D-glouse×48h);osmotic pressure group(OSM,5.5mmol/L D-glucose +24.5mmol/L mannitol×48h).Western blot and immunofluorescence were used to detect the EMT indicators of each group.2.A high throughput RNA sequencing(RNA-seq)using the Illumina Hiseq 2000 was applied to NG,HG and MG,GO enrichment analysis and KEGG pathway enrichment analysis of DEGs were performed.3.The selected differentially expressed genes were verified by quantitative Real-time PCR.Results:1.Compared with NG,both Westren blot and immunofluorescence showed that high glucose could induce the nephrin down and ?-SMA up,and these abnormalities persisted existence even in subsequent normal glucose culture.2.RNA-Seq showed that 194 genes were differentially expressed between the HG and NG,among which 119 were up-regulated and 75 weredown-regulated.However,532 genes were differentially expressed between the MG and NG,including 316 up-regulation genes and 216 downregulation genes.Further,it showed 108 genes(35 up-regulation and 73down-regulation)had similar expression patterns in the HG and MG by comparing the above genes.Then,data mining analysis of the common differentially expressed genes were performed: the GO analysis showed that 10 GO terms were significantly enriched,and the KEGG pathway analysis showed that 10 pathways were significantly enriched.Finally,the analysis found that Smad9,Id2,Snai1,Mapkapk3,Igf1,Fgf22,Lama1 and Zfhx3 might have significant changes in podocyte EMT.3.Quantitative Real-time PCR showed that the expression of Smad9,Id2,Snail1,Mapkapk3,Igf1,Fgf22,Lama1 and Zfhx3 involved in the significant enriched GO terms and KEGG pathways were similar to the RNA-seq except for Id2.Conclusion:1.High glucose can induce podocyte EMT in vitro,and remain existence even after normal glucose culture.2.RNA sequencing analysis have revealed some new molecules,among them,Smad9,Snai1 and Mapkapk3 were up-regulated,while Igf1,Fgf22,Lama1 and Zfhx3 were down-regulated,and those genes were potentially related to TGF-? signaling pathway,focal adhesion signaling pathway,Rap1 signaling pathway and pluripotent regulation of stem cellssignaling pathway.