肾病综合征患儿外周血单个核细胞内GR α和GR β表达的意义

Significance of α-and β-isoforms of glucocorticoid receptors expression in children with idiopathic nephrotic syndrome

目的 探讨原发性肾病综合征 (INS)患儿外周血单个核细胞 (PBMC)内两种糖皮质激素受体 (GR)亚型———GRα和GRβ表达水平与糖皮质激素治疗效应之间的关系。方法 应用免疫细胞化学方法、WesternBlot和凝胶电泳迁移率变化方法 (EMSA) ,分别检测不同激素效应INS患儿和正常对照组儿童GRα和GRβ免疫阳性的PBMC数目、PBMC细胞内GRα和GRβ蛋白质表达和GR DNA连接活性。结果 (1)INS患儿PBMC内同时有GRα和GRβ的表达 ,并以GRα表达为主。 (2 )激素敏感型INS患儿GRα阳性的PBMC细胞数为 (5 0 0± 4 5 ) ,激素抵抗型INS患儿为 (4 7 0± 5 5 ) ,两组相比差异无显著意义 (P >0 0 5 ) ;而激素抵抗型INS患儿GRβ阳性PBMC细胞数 (2 2 0± 3 5 )明显高于激素敏感型INS患儿 (7 5± 2 0 ) (P <0 0 1)。 (3)WesternBlot实验结果显示 ,激素抵抗型INS患儿PBMC核内GRα蛋白量明显低于激素敏感型INS患儿 ,而GRβ蛋白量却明显高于激素敏感型患儿。(4 )激素抵抗型INS患儿的GR DNA连接能力明显低于激素敏感型患儿。结论 INS患儿PBMC内GRα和GRβ表达比例失调。

Objective Glucocorticoid (GC) is the principal therapeutic drug in the treatment of idiopathic nephrotic syndrome (INS), and the response to GC treatment is an important indicator for the outcome of INS children. Children with GC-resistant INS are usually incompletely or non responseive to GC, and may herald the progression to end-stage renal failure. However, the detailed mechanism for why some INS children respond to GC and others do not, have still not been clearly elucidated. It is well known that GC action is mediated by the GC receptor (GR), a ligand-dependent transcription factor that belongs to the super-family of nuclear receptors. Two human GR isoforms termed GR α and GR β have been described. Recent studies demonstrated that the β-isoform of GR (GR β) is a potential endogenous inhibitor of the GC action in humans. So far, no research papers have involved in the relationship between the levels of two GR isoforms, GR α and GR β, expression in INS children and the response to GC in INS children. The aim of the present study was to determine the levels of GR α and GR β in INS children with the variable response to the GC treatment. Methods Twenty patients aged 4～13 years with INS were classified as GC-sensitive INS (8 patients) or GC-resistant INS (12 patients), according to their response to an eight-week course of oral prednisone. The heparinized venous blood was collected, and peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque gradient centrifugation. The immunocytochemical assay was used to investigate the expression of two isoforms of GR in PBMC. The cytoplasm protein and nuclear protein extracts from PBMC were obtained, and the quantity of GR α and GR β proteins were analyzed by Western blotting assay. Then the GR-DNA-binding activity was detected by electrophoretic mobility shift assay (EMSA). Results The two isoforms of GR were expressed in PBMC in INS and healthy children. The percentages of GR αpositive staining cells in GC-resistance INS and GC-sensitive INS were significantly lower than that in healthy children (P<0.01), but no significant difference was found between two INS groups. However, the number of GR β positive staining cells in GC-resistant INS was significantly higher than that in GC-sensitive INS (P<0.01). Results of Western blots assay identified the presence of only one specific protein band at the expected molecular weight 94 000 or 90 000, which was consistent with those of GR α and GR β, respectively. In GC-resistant INS children, the quantity of GR α protein in GC-resistant INS group was significantly higher in the cytoplasm, and significantly lower in the nuclei, while the quantity of GR β protein in the nuclei was significantly higher than that of GC-sensitive INS, respectively. The results of EMSA showed that GR-DNA-binding activity in GR-resistant INS group was decreased compared to GR-sensitive INS group. Conclusions The patients with GC-resistant INS have an increased expression of GR β. The imbalanced expression between GR α and GR β, especially increased expression of GR β in PBMC might account for GC-resistant INS. GR β might antagonize GC-mediated GR α function by prohibiting the GR α translocation into the nuclei and depressing GR-DNA binding activity. This finding suggests that GR β might be a critical factor regulating the responsiveness of the target cell to GC. The ability of GR β to repress the action of GR α also illustrates a new insight to the satisfactory explanation of why some INS children respond to GC and others do not.