| Part I The relationship between Klotho methylation levels in uremic patients and vascular calcificationBackground:Recent study confirmed that Klotho can directly interact with human vascular smooth muscle cell, and local vascular Klotho expression involved in the occurrence and development of vascular calcification, but the mechanism is not known. DNA methylation, an epigenetic approach, is a regulation of gene expression, via CpG island in the promoter region (300-3000 BP CpG rich region of the dinucleotide) methylation silencing effects on gene expression. The purpose of this study was to explore local hypermethylation of Klotho gene and vascular calcification in patients with ESRD.Methods:We selected 30 ESRD patients undergoing artereovenias fistula plastic operation, and 10 age-matched control group undergoing coronary artery bypass graft. H.E. staining was used to observe the thickness of blood vessels. Alizarin red staining was used to determine the calcium deposition. Osteoblast specific proteins expression such as osteopontin and core binding factor alpha 1 were detected by immunohistochemical method. Vascular smooth muscle cells specific protein alpha-smooth muscle actin. Pyrosequencing assay were used to detect vascular Klotho methylation level. Klotho protein expression and serum indoxyl sulfate level were examed.Results:The average artery pressure, serum phosphorus and iPTH were higher in uremic patients than that of in control group. IMT of radial artery in uremic group was significantly higher than control group (0.68±0.53 vs 0.18±0.13 mm, P<0.05). Radial artery wall/vascular diameter ratio in uremic group was also significantly higher than that in the control group (0.34±0.19 vs 0.23±0.09, P<0.05). The serum IS level in uremic patients was higher than that of control group. The serum IS levels of 21 patients with arterial calcification was higher than that of 9 patients without arterial calcification (34.6±4.13 pg/m,29.8±4.23 pg/ml, P<0.001). Klotho methylation in uremic patients was significantly higher than control group (43.2±1.7% vs.8.7±0.8%, P<0.001); Klotho expression was positive in radial artery of 30 cases; DNMT1 expression in uremic patients was significantly higher than that in the control group. Alizarin red staining showed 21 cases vascular calcification in 30 cases of radial artery specimens, the incidence rate was 70%, the control group had no calcification (P<0.01). Immunohistochemical staining showed 26 cases (87%) with OPN positive deposition in 30 cases of radial artery, while OPN staining was negative in the control group (P<0.001); Cbf al were positive in 28 cases of radial artery in uremic patients while the control group had no positive expression; 28 cases of radial artery were negitive with a-SMA in uremic patients which were significantly lower than the control group. Pearson correlation analysis showed that the serum IS and Klotho methylation was positively (r=0.587, P<0.01); Klotho methylation and degree of arterial calcification was positively correlated (r=0.549, P< 0.01); Pearson correlation analysis showed that the serum IS was positively correlated with vascular calcification (r= 0.763, P<0.01).Conclusion:Obvious calcification exsited in uremic patients. Serum IS levels in uremic patients were significantly higher than the normal controls.Serum IS was positively correlated with the severy of vascular calcification. Klotho hypermethylation exsited in uremic patients.Part II Klotho gene hypermethylation potentiates vascular calcification induced by indoxyl sulfate in HASMCsBackground:IS is related with cardiovascular disease and all-cause mortality in ESRD patients. IS can promote vascular smooth muscle cells differentiation to osteoblast, but its mechanism is still unclear. The first part of our study confirmed that hypermethylation of Klotho gene, serum concentration of IS and vascular Klotho gene methylation is closely related to vascular calcification in ESRD patients. The purpose of this study is to to explore the role hypermethylation of Klotho gene in vascular smooth muscle cells osteoblast transformation induced by IS.Methods:The standard IS were purchersed from Sigma company. IS concentration of 0,200,500,1000 μmol/L were used. Other group were added with culture medium IS 1000 μmol/L and 0,1,10 mol/L of 5-aza-2-deoxycytidine for 6 days. Klotho methylation level were evaluated by pyrosequencing assay; Cbf a 1, osteopontin, a-SMA, Klotho, DNMT1, DNMT3a, DNMT3b were examed by Real-time PCR. Western blot was used to exame the protein expression of osteopontin, a-SMA, Klotho and DNMT1.Results:Alizarin red staining showed that IS promoted HASMCs calcification. IS induced Klotho gene hypermethylation in HASMCs. Klotho mRNA levels were decreased with the increase of IS concentration. Klotho protein also decreased with increasing the concentration of IS. OPN and Cbfal mRNA expression were increased after 6days of IS incubation, OPN expression in control group and IS group were (1.02±0.12 vs.6.11±0.68). Expression of OPN in IS group was higher than that of control group (P<0.01). After 6D incubation, Cbfal in control group and IS group (1000 mol/L) expression was (1.02±0.12 vs 5.21±1.08). a-SMA expression was significantly decreased. IS increased the expression of DNMT1. 5-aza-2-deoxycytidine reduced the degree of Klotho methylation. 5-aza-2-deoxycytidine decreased the expression of DNMT1, while calcification.were significantly reduced in HASMCs.Conclusion:IS increased Klotho methylation level in HASMCs. IS induced vascular calcification via hypermethylation of Klotho gene hypermethylation. 5-aza-2-deoxycytidine inhibited vascular calcification.via decreasing Klotho gene methylation level.Part Ⅲ Klotho gene hypermethylation promotes vascular calcification induced by indoxyl sulfate in CKD rat modelBackground:Vascular calcification is prevalent in uremic patients. Vascular Klotho gene hypermethylation and serum IS are closely related to vascular calcification. Indoxyl sulfate can induce vascular smooth muscle cells deferitiate into osteoblasts. IS can increase Klotho methylation level by inhibit the activity of DNMT1 in vascular smooth muscle cells, and decressed the expression of Klotho protein. The purpose of this study was to to explore the hypermethylation of Klotho gene in vascular calcification in uremic rats induced by IS.Methods:24 SD rats underwent 5/6 nephrectomy were randomly divided into 3 groups. Each group were administered intervention.The control group (8 rats), PBS/48h, intraperitoneal injection,24 weeks; Experimental group (8 rats), IS/100mg/Kg/48h, intraperitoneal injection,24 weeks; Experimental group (8 rats), IS/100mg/Kg/48h+5-aza-2-deoxycytidine/10mg/Kg/48h, intraperitoneal injection,24 weeks. After 24 weeks, blood and urine were collected. To observe the effect of thickness of HE staining, alizarin red staining of vascular calcification, immunohistochemistry staining of Cbf a 1, OPN, a-SMA, Klotho, DNMTl protein expression condition. Aortic specimens to extract DNA, take RNA and protein, and pathological histology paraformaldehyde treatment. Pyrosequencing detection of Klotho methylation level were measured; Real-time PCR for Cbf a 1, OPN, α-SMA, Klotho, DNMT1, DNMT3a, DNMT3b. Western blot method for the determination of Cbf a 1 of each nucleus, OPN, α-SMA, Klotho and DNMT1.Results:Alizarin red staining showed that IS promoted calcium deposition vassels. IS induced Klotho gene hypermethylation. Compared with the control group and the experimental group, IS group had Klotho gene methylation increased significantly. Control group vs IS in experimental group (29± 5% vs.6%±2) (*P<0.001). The mRNA and protein levels of Klotho in IS group were significantly decreased.The expression of DNMT1 in IS group was significantly increased. Osteopontin and Cbfal increased while a-SMA decreased in IS group than the control group. Klotho methylation level decreased significantly in IS+5-aza-2-deoxycytidine group than that in IS group. Klotho protein expression increased in IS+5-aza-2-deoxycytidine group than that in IS group, while the expression of DNMT1 protein decreased. At the same time, Alizarin red staining showed calcification decreased significantly in IS+5-aza-2-deoxycytidine group than that in IS group. Osteopontin and Cbfal protein expression decreased in IS+5-aza-2-deoxycytidine group, while the expression of a-SMA increased,showing the degree of vascular calcification significantly reduced.Conclusions:IS induced vascular calcification by hypermethylation of Klotho gene in vivo experiments.5-aza-2-deoxycytidine inhibited the hypermethylation of Klotho gene and further inhibit vascular calcification. |
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