Steroid Resistance in Nephrotic Syndrome

说明： 激素抵抗型特发性肾病综合征(SRINS)是导致儿童肾功能衰竭和终末期肾病的重要原因。类固醇耐药的原因尚不清楚，但有证据表明T细胞功能障碍是原因之一。基于SRINS中T细胞核因子-kB亚型组成异常的初步数据，建议的项目将确定导致SRINS患者糖皮质激素抵抗的T细胞信号机制缺陷。这些实验将比较新诊断的SRINS患者、类固醇敏感患者和健康对照组儿童的T细胞。将评估下列特定信号转导机制中的潜在缺陷，可能与核因子-kB功能异常有关：1)体外地塞米松作用下糖皮质激素受体(GCR)的核移位；2)STAT5的产生增加，并通过与STAT5的异源二聚体干扰GCR的核转移；3)地塞米松刺激IL-2的产生；4)地塞米松诱导细胞凋亡。这些方法将包括标准T细胞分离，实时RT-PCR检测基因表达，电泳迁移率改变分析检测核质组分中的因子，免疫沉淀分析检测复杂的GCRs，以及细胞凋亡

DESCRIPTION: Steroid-resistant idiopathic nephrotic syndrome (SRINS) is an important cause of renal failure and end-stage renal disease in children. The cause of steroid resistance is unknown but evidence suggests that T cell dysfunction is responsible. Based on preliminary data on abnormal T cell NF-kB isoform composition in SRINS, the proposed project will identify the defects in T cell signaling mechanisms that lead to glucocorticoid resistance in patients with SRINS. The experiments will compare T cells from newly diagnosed SRINS patients with those from steroid-sensitive patients and healthy control children. Potential defects in the following specific signal transduction mechanisms, which may be associated with abnormal NF-kB function, will be evaluated: 1) Nuclear translocation of the glucocorticoid receptor (GCR) in response to dexamethasone exposure in vitro; 2) Increased production of STAT5 and interference of nuclear transfer of GCR by heterodimerizing with STAT5; 3) Dexamethasone-stimulated IL-2 production; 4) Induction of apoptosis in response to dexamethasone exposure. The methods will include standard T cell isolation, real time RT-PCR for gene expression, electrophoretic mobility shift assay for quantifying factors in nuclear vs cytoplasmic fractions, immunoprecipitation assay to detect complexed GCRs, as well as apoptosis