The Roles Of Tet2-mediated DNA Demethylation In Kidney Development And Renal Disease

Renal disease is an important disease affecting the health of childrem.Its pathogenesis is complicated and to be elucidated,as it is easy to relapse,and difficult for treatment.Recent studies have found that 5-hydroxymethylcytosine(5-hmC)-mediated epigenetic regulation is involved in various biological processes,but the role of Tet2-mediated DNA demethylation in the kidney development and renal disease is not clear.Purpose:Previous studies have shown that 5-hmC-mediated epigenetic regulation may play an important role in kidney disease.Therefore,we hope to further verify that DNA demethylation is involved in the development of kidney and renal disease through related experiments.Methods:1.To detect the expression of 5-hmC level and Tet2 level in mouse kidney cells and cultured podocytes by immunofluorescence staining.Then to detect the expression of Tetl,Tet2,Tet3 in the different developmental stages,on the first day,the second week and the eighth week of wild type mouse kidney by qRT-PCR.2.After Tet2 knockout,to detect the content of 5-hmC in kidney in both wild type and mutant type(Tetl knockout,Tet2 knockout mice)on the first day,the second week and the eighth week by dot blot.Furthermore,to detect the changes of kidney morphology and renal function after Tet2 knockout by non-target metabolomics analysis and the IPA software.3.To detect the expression of 5-hmC level in patients with renal biopsy tissues by immunofluorescence staining and to detect the change of Tetl,Tet2 mRNA in clinical nephrotic syndrome patients of renal tissues specimens by qRT-PCR.Results:1.5-hmC widely exists in the DNA of mammalian kidney,the expression of 5-hmC in the nuclei of glomerular cells and renal tubule cells was demonstrated by immunofluorescence staining.Further detection showed that the expression level of Tet1,Tet2,and Tet3 in renal tissue was gradually decreased with the increase of age,while the expression level of Tet2 in the three groups remained at a high level.Mcoreover,Tet2 exists in the genomic DNA of mouse glomerular cells and renal tubule cells.2.There is a significant difference in the effect of Tet2 knockout on the expression of 5-hmC,indicating that Tet2 plays an important role in DNA demethylation at different stages of kidney development.At the same time,Tet2 knockout can induce the change of inflammatory factors,so inducing some pathological process which associated with inflammation,through non-targeted metabolomics analysis and the IPA analysis.Thus,we can infer that Tet2 can inhibit the inflammatory response caused by metabolic disorders.3.5-hmC is abundant expressing in the human kidney and the expression of Tet2 in MCNS was increased than that of normal persons by renal biopsy,which indicating that there may be an abnormal DNA methylation pathway in the kidney.These results suggesting that DNA demethylation is upsteam in the pathogenesis of MCNS,and suggesting that Tet2-mediated DNA demethylation may reduce and delay the occurrence and the development of MCNS.5-hmC may be a negative feedback regulation mechanism which can protect the kidney.Conclusions:Tet2-mediated DNA demethylation may be involved in the kidney development and renal disease.