Transformation of DNA demethylation in the regulatory region of TGFB1 gene in patients with diabetic nephropathy

Abstract

Objective To investigate the role of DNA methylation changes in the regulatory region of TGFB1 gene in patients with diabetic nephropathy (DN). Methods According to the WHO 1999 guideline for diabetes mellitus diagnosis and classification standard, 91 patients who were hospitalized in June 2013 to May 2015 and diagnosed as diabetes mellitus were selected, including 42 patients with diabetes mellitus (DM group) and 49 with diabetic nephropathy (DN group). Thirty cases with health examination were selected as healthy control group (Con group). DNA was extracted from all the subjects' peripheral blood and modified by sodium bisulfite. DNA methylation status of TGFB1 gene regulatory region was screened by methylation specific PCR and the DNA methylation level was detected by bisulfite sequencing PCR. ELISA was used to test serum TGF-β1. Blood urea nitrogen, creatinine, fasting blood sugar, postprandial blood sugar, glycosylated hemoglobin and urinary albumin to creatinine ratio (UACR) were detected by automatic biochemistry analyzer. Pearson correlation analysis and multiple stepwise regression were used to analysis TGF-β1-related factors, the correlation between the level of serum TGF-β1 and the pathological grade was analyzed in DN patients. Results There were 12.2% patients in DN group with DNA methylation of TGFB1 gene regulation region, lower than those in DM group (42.8%) with Con group (73.3%) (all P<0.05). The methylation level of TGFB1 regulatory region was 12.5%±8.1% in DN group, significantly lower than those in DM group (35.6%±6.0%) and Con group (66.7%±9.1%) (all P<0.05). Moreover, compared with that in DM group (1367.22±126.13 ng/L) and Con group [(296.38±74.37) ng/L], TGF-β1 expression was increased significantly in DN group [(2885.73±411.36 ng/L] (all P<0.01). In DN patients serum TGF-β1 was correlated with eGFR (β=-0.690, P<0.01) and the methylation (β=-0.302, P<0.01), and the serum TGF-β1 was negatively correlated with DNA methylation level (r=-0.925, P<0.01), but positively correlated with the pathological scores among glomerulus, tubulus and arterioles (rs=0.847, P<0.01). Meanwhile the methylation level related to the pathological grade (χ2=23.667, P=0.04). Conclusion Demethylation in the regulatory region of TGFB1 may play an important role in the activation of TGFB1 induced by high glucose in mesangial cells, so as to participate in the occurrence and development of DN. Key words: Diabetic nephropathies; Transforming growth factor beta1; DNA methylation