Study On The Role Of Epigenetic Regulation In Renal Tubular Damage In Diabetic Nephropathy

PurposeDiabetic nephropathy（DN）is the leading cause of end-stage kidney disease（ESKD）.Although the impaired filtration barrier of the glomerulus is its main feature,the abnormalities of the renal tubules in DN also plays an important role in the occurrence and development of DN.However,in the state of diabetes,the mechanism that determines the rapid progression of DN and the key molecule that drives the pathological changes of renal tubules are currently unclear.With the rapid development of epigenetics,it has been revealed that the epigenetic regulations are underlying the mechanisms of cell phenotypic changes caused by the environment at the chromatin level.Therefore,epigenetic regulations are expected to play important roles in the injury of renal tubular in diabetic kidney disease.This study explore the epigenetic regulations during renal tubular injuries both in vitro and in vivo.Through the chromatin accessibility profile and histone modification profile combined with the analysis of the transcriptome,we reveal the important epigenetic changes that mediate renal tubular damage in the diabetic state,discuss the relationship between the epigenetic regulation of renal tubules and the progression of DN,identify the key molecules and explore the possible mechanisms.MethodsPart 1:First,we use hydrogen peroxide（H₂O₂）,transforming growth factorβ1（TGF-β1）and Roxadustat（FG4592）to simulate oxidative stress and fibrosis and hypoxic state respectively,which are damage factors of DN.Through Assay for Transposase Accessible Chromatin using sequencing（ATAC-seq）and RNA Sequencing（RNA-seq）,we captured chromatin accessibility profile and transcriptome data of the proximal tubules epithelial cell line HK2 cells.In order to identify the transcriptional regulation process that has changed in each group of cells,we compared each treatment group with the normal group,looked for different chromatin accessibility sites and gene expression,and explained the potential regulatory relationship between the two.In order to further explore the similarities and differences in the mechanisms of renal tubular injury under different stimuli,we integrated the differential chromatin accessibility sites under all conditions,and carried out cluster analysis,biological function analysis and transcription factor motif analysisPart 2:We use laser capture microdissection/sieving method to separate the glomerulus and tubulointerstitium from renal biopsy samples from patients with DN and adjacent normal tissue from patients with renal carcinoma.H3K27ac Ch IP-seq（4 samples in control group,4 samples in DN group）and RNA-seq（30 samples in control group,100 samples in DN group）were performed on the tubulointerstitial compartment.Through the genome comparison and difference analysis of H3K27ac Ch IP-seq data and transcriptional data,we identified the differential activated chromatin regulatory elements and differentially expressed genes under diabetic conditions.Through the integrated analysis of the two,the gene sets involved in the progression of DN were identified.On the basis of confirming that epigenetic regulation plays an important role in kidney injury,we integrated H3K27ac Ch IP-seq with kidney single cell RNA-seq data and revealed the biological processes that occur in different components/cell types in the state of diabetes.In addition,we further predicted the important transcription factors that mediate renal tubular damage and discussed the biological processes and mechanisms it participates in.ResultsPart1:We first identified the dynamic change of chromatin accessibility under different simulated diabetic nephropathy stimulations,and determined the functional regions that coordinate transcriptional regulation in HK2 cells.The dynamic changes of these regulatory elements present stimulus-specific regulatory characteristics.H₂O₂ induced the inflammation and apoptosis of HK2cells mediated by FRA1 and RUNX1,TGF-β1 induced cell fibrosis mediated by FRA1 and SMAD2,and FG4592 induced the high reactivity of cells to a variety of stimuli mediated by FRA1 and ETS1.We also discovered a set of chromatin regions whose dynamic changes are shared by different stimuli.The integrated analysis of ATAC-seq and RNA-seq shows that the transcription factor HNF1B mediates the shared module of transcription regulation under the three different stimuli.The down-regulation of HNF1B at protein level may be an important mechanism.In addition,our study further confirmed in vivo data that the gene expression network regulated by the transcription factor HNF1B is significantly disrupted in the tubulointerstitium of DN.Part 2:Our study captured the histone H3K27ac modification profile of DN samples that were clearly diagnosed by renal biopsy for the first time.In the joint analysis with RNA-seq,it was found that H3K27ac modification can regulate gene expression and that the genes whose changes are regulated by H3K27ac modification are closely related to kidney function.In detail,the up-regulated genes are significantly negatively correlated with e GFR,while the down-regulated genes are significantly positively correlated with e GFR.Through the integrated analysis of H3K27ac Ch IP-seq and renal single-cell RNA-seq data,we found that in a high-glucose environment,abnormal modification of H3K27ac leads to proximal tubular metabolism and transport dysfunction,collecting duct fibrosis-like changes and immune cell-mediated inflammation in the kidney.In addition,our research further confirmed that the genes related to fatty acid metabolic process are down-regulated caused by the weakening of H3K27ac modification in renal proximal tubule epithelial cells,and the down-regulated genes are significantly positively correlated with kidney function.The occurrence of this process is related to the participation of the transcription factor HNF4A.Conclusion（1）HNF1B-mediated transcriptional regulatory network was destroyed under various simulated stimuli of diabetic kidney disease,which changed the phenotype of HK2 cells;（2）In diabetic environment,the genes whose changes of expression were caused by epigenetic regulation play an important role in the development of DN.Among them,the significant down-regulated genes related to fatty acid metabolic process in proximal tubules is positively correlated with kidney function.This damage process is determined by the reprogramming of H3K27ac modification,which is related to the participation of HNF4A.