Involvement of Histone Lysine Methylation in p21 Gene Expression in Rat Kidney In Vivo and Rat Mesangial Cells In Vitro under Diabetic Conditions

Abstract

Diabetic nephropathy (DN), a common complication associated with type 1 and type 2 diabetes mellitus (DM), characterized by glomerular mesangial expansion, inflammation, accumulation of extracellular matrix (ECM) protein, and hypertrophy, is the major cause of end-stage renal disease (ESRD). Increasing evidence suggested that p21-dependent glomerular and mesangial cell (MC) hypertrophy play key roles in the pathogenesis of DN. Recently, posttranscriptional modifications (PTMs) have uncovered novel molecular mechanisms involved in DN. However, precise regulatory mechanism of histone lysine methylation (HKme) mediating p21 related hypertrophy associated with DN is not clear. We evaluated the roles of HKme and histone methyltransferase (HMT) SET7/9 in p21 gene expression in glomeruli of diabetic rats and in high glucose- (HG-) treated rat mesangial cells (RMCs). p21 gene expression was upregulated in diabetic rats glomeruli; chromatin immunoprecipitation (ChIP) assays showed decreased histone H3-lysine9-dimethylation (H3K9me2) accompanied with enhanced histone H3-lysine4-methylation (H3K4me1/3) and SET7/9 occupancies at the p21 promoter. HG-treated RMCs exhibited increased p21 mRNA, H3K4me level, SET7/9 recruitment, and inverse H3K9me, which were reversed by TGF-β1 antibody. These data uncovered key roles of H3Kme and SET7/9 responsible for p21 gene expression in vivo and in vitro under diabetic conditions and confirmed preventive effect of TGF-β1 antibody on DN.

Figure 1

Results of p21 gene expression in the glomeruli of type 1 diabetic rats. (a) Eight weeks after male Wistar rats were successfully induced to be diabetic models with STZ (55 mg/kg), mRNA levels of p21 gene and housekeeping gene cyclophilin A (CypA) of glomeruli in control and STZ groups were analyzed by RT-qPCR. Gene expression was normalized to internal control β-actin gene; results were expressed as fold over control (mean ± SEM; ^# P < 0.05 compared to control, n = 6/group). (b) Western blot analysis of extracted proteins from control and STZ groups glomeruli using p21 and β-actin antibodies; quantitative analyses were expressed as fold over control (mean ± SEM; ^# P < 0.05 compared to control, n = 6/group).

Figure 2

H3K9me2/3 levels at p21 gene promoter in the glomeruli of type 1 diabetic rats. Bar graphs showing H3K9me2 and H3K9me3 levels at p21 and CypA promoters in glomeruli of control and STZ groups. ChIP assays were performed with H3K9me2 and H3K9me3 antibodies, immunoprecipitated DNA and input DNA were subjected to RT-qPCR with primers for the respective promoter, data were analyzed by the 2^−ΔΔCt method, and results normalized to input DNA were expressed as fold over the control group (mean ± SEM; ^# P < 0.05 compared to control, n = 6/group).

Figure 3

H3K4me1/2/3 levels at p21 gene promoter in the glomeruli of type 1 diabetic rats. H3K4me1, H3K4me2, and H3K4me3 levels at p21 and CypA promoters in glomeruli of control and STZ groups. ChIP assays were performed as described in Figure 2 with respective specific antibodies, and results normalized to input DNA were expressed as fold over the control group (mean ± SEM; ^# P < 0.05 compared to control).

Figure 4

TGF-β1-specific antibody reversed HG-induced p21 gene expression and H3K9me2/3 changes at p21 gene promoter in RMCs. (a) p21 gene mRNA levels in RMCs. Serum-depleted RMCs were treated as described in Materials and Methods. Gene expression was analyzed by RT-qPCR, and results were expressed as fold over NG group (mean ± SEM; ^# P < 0.05 compared to NG; ^& P < 0.05 compared to HG, n = 3). (b) H3K9me2/3 levels at p21 and CypA gene promoters in RMCs. ChIP assays were performed as described in Materials and Methods. Results were expressed as fold over NG (mean ± SEM; ^# P < 0.05 compared to NG; ^& P < 0.05 compared to HG, n = 3).

Figure 5

TGF-β1-specific antibody reversed HG-induced H3K4me1/2/3 levels at p21 gene promoter in RMCs. Bar graphs showing H3K4me1, H3K4me2, and H3K4me3 levels at p21 and CypA gene promoters in RMCs. Serum-depleted RMCs were treated as described in Materials and Methods. ChIP assays were performed as described in Materials and Methods. Results were expressed as fold over NG (mean ± SEM; ^# P < 0.05 compared to NG; ^& P < 0.05 compared to HG, n = 3).

Figure 6

SET7/9 recruitment at p21 gene promoter was enhanced under diabetic conditions, and TGF-β1-specific antibody could reverse HG-induced SET7/9 occupancy in RMCs. (a) SET7/9 recruitment at p21 and CypA gene promoters in glomeruli of control and STZ groups. ChIP assays were performed and results were expressed as described in Materials and Methods (mean ± SEM; ^# P < 0.05 compared to control). (b) Reversion of HG-induced SET7/9 enrichment at p21 promoter by TGF-β1-specific antibody. ChIP assays were performed and results were expressed as described in Materials and Methods (mean ± SEM; ^# P < 0.05 compared to NG; ^& P < 0.05 compared to HG, n = 3).