慢性肾脏病患者肾组织Klotho基因启动子的超甲基化与临床病理的相关性

目的 评价慢性肾脏病(CKD)患者肾组织和外周血单个核细胞(PBMC)Klotho基因启动子的甲基化水平,探讨Klotho基因启动子超甲基化与CKD患者临床病理特征的关系.方法 以47例接受肾脏活组织病理检查的CKD患者为研究对象,47例肾癌根治术患者肾脏切除标本中的正常肾脏组织和48例健康志愿者的PBMC分别作为肾组织和PBMC的对照组.采用焦磷酸测序(PS)法检测肾组织和PBMC Klotho基因启动子的甲基化水平.结果 CKD患者肾组织Klotho基因启动子甲基化率显著高于对照组,差异有统计学意义(17.04±6.42)％比(9.34±2.43)％,P＜ 0.01]；PBMC Klotho基因启动子甲基化率亦显著高于对照组,差异有统计学意义(14.19±5.86)％比(6.90±2.39)％,P＜ 0.01].CKD患者PBMC与肾组织Klotho基因启动子甲基化率呈正相关(r=0.811,P＜0.01),PBMC Klotho基因启动子甲基化率预测肾组织Klotho基因启动子超甲基化的受试者工作曲线下面积为0.958(P＜0.01).肾组织和PBMC Klotho基因启动子甲基化率与eGFR均呈负相关(分别r=-0.827,P＜0.01;r=-0.626,P＜0.01),与肾小管间质纤维化面积呈正相关(r=0.865,P＜0.01；r=0.748,P＜0.01),与24 h尿蛋白量无相关.结论 CKD患者肾组织和PBMC Klotho基因启动子甲基化率升高,PBMC Klotho基因启动子甲基化率可作为肾组织Klotho基因启动子超甲基化的无创评价指标.Klotho基因启动子超甲基化与肾脏纤维化的关系值得进一步研究.

Klotho hypermethylation in kidney biopsy tissue and peripheral blood mononuclear cells in chronic kidney disease patients by pyrosequencing

ObjectiveTo assess the relationship between levels of Klotho hypermethylation of intrarenal and peripheral blood mononuclear cells(PBMC) and clinical and histological severity of the disease. Methods Intrarenal and PBMC levels of Klotho promoter methylation were quantified in 47 CKD patients by pyroseqencing. The results were compared with 47 nephrectomy specimens and PBMC of 48 healthy volunteers. Results Higher levels of Klotho promoter methylation were observed in both renal biopsy and PBMC at specific CpG sites in CKD patients compared to control(17.04±6.42) % vs (9.34±2.43) %, P＜0.01; (14.19±5.86) % vs (6.90±2.39)%, P＜0.01]. The level of Klotho promoter methylation on PBMC was positively correlated with intrarenal level (r＝0.811，P＜0.01). Using receiver operator characteristic curve analysis, PBMC level of Klotho promoter methylation had an area under the curve (AUC) of 0.958 (P＜0.01) in predicting intralrenal Klotho promoter hypermethylation.Estimated glomerular filtration rate inversely correlated with intrarenal and PBMC levels of Klotho hypermethylation (r=－ 0.827, P＜0.01; r=－ 0.626, P＜0.01). Tubulointerstitial scarring positively correlated with intrarenal level of Klotho hypermethylation, as well as PBMC level of Klotho hypermethylation (r＝0.865, P＜0.01; r＝0.748, P＜0.01). There was no correlation between proteinuria and the level of Klotho promoter methylation of renal tissue or PBMC. Conclusions Klotho promoter methylation levels of intrarenal and PBMC are significantly elevated in CKD patients and correlated with clinical and histological severity. Klotho promoter hypermethylation may play animportant role in renal fibrosis. The level of Klotho promoter methylation of PBMC may be a potential biomarker of intrarenal Klotho promoter hypermethylation.