›› 2012, Vol. 32 ›› Issue (11): 1455-.doi: 10.3969/j.issn.1674-8115.2012.11.013

• Original article (Basic research) • Previous Articles     Next Articles

DNA methylation of promoter of apoptosis-related FADD and APAF1 genes in rats with adriamycin nephropathy

LIU Jian, ZHONG Fang, XU Li-li, ZHOU Qiao, LU Ying, HAO Xu, WANG Wei-ming, CHEN Nan   

  1. Department of Nephrology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China
  • Online:2012-11-28 Published:2012-11-30
  • Supported by:

    National Natural Science Foundation of China, 81270782；National Basic Research Program of China, “973” Program, 2012CB517700；Shanghai Science and Technology Committee Foundation, 08dz1900502

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Abstract:

Objective To investigate the relationship between DNA methylation of apoptosis-related genes in renal tissues and disease progression in rats with adriamycin nephropathy. Methods Twelve male SD rats were randomly divided into adriamycin nephropathy group and normal control group, with 6 rats in each group. Adriamycin nephropathy model was established in adriamycin nephropathy group by injection of 5 mg/kg adriamycin on the first day and 2.5 mg/kg adriamycin one week later through penis vein. Blood samples were collected via abdominal aorta 8 weeks after model establishment, then rats were sacrificed, and renal tissues were harvested. The biochemical parameters such as serum creatinine (Crea), blood urea nitrogen (BUN), serum albumin (ALB) and urinary albumin to creatinine ratio (UACR) were measured. The pathological changes of renal tissues were observed with Masson staining. The apoptosis of renal tubular cells and glomerular cells was detected by TUNEL staining. The DNA methylation in the promoter of Fas-association protein with death (FADD) and apoptotic protease activating factor-1 (APAF1) genes was determined by high-resolution melting (HRM) analysis, and the expression of FADD mRNA was detected by Real-Time PCR. Results Crea, BUN and UACR in adriamycin nephropathy group were significantly higher than those in normal control group (P<0.05 or P<0.01), while ALB in adriamycin nephropathy group was significantly lower than that in normal control group (P<0.01). Pathological examination revealed that compared with normal control group, there were increased glomerular mesangial cell proliferation, swelling of renal tubular epithelial cells and inflammatory cell infiltration in adriamycin nephropathy group. TUNEL assay indicated that the number of apoptotic tubular cells and glomerular cells in adriamycin nephropathy group was significantly higher than that in normal control group (P<0.01). HRM analysis and Real-Time PCR demonstrated that compared with normal control group, the promoter of FADD was relatively hypermethylated, and the expression of FADD mRNA was significantly up-regulated in adriamycin nephropathy group (P<0.05). Conclusion In rats with adriamycin nephropathy, the DNA in the promoter of FADD is hypermethylated, but it may not be the major factor affecting the expression of FADD mRNA.

Key words: apoptosis, DNA methylation, high-resolution melting curve, kidney