The effect of serum from children with nephrotic syndrome on transcriptome of human immortalized podocytes cultured in vitro

Nephrotic syndrome (NS) is an unfavorable disease with heterogeneous causes and variable prognosis. One of the clinically crucial prognostic feature is the response to initial glucocorticoid treatment. Whereas in some children with steroid-sensitive nephrotic syndrome (SSNS) the treatment may induce long-term remission of the disease, steroid-resistant cases (SRNS) are at risk of renal failure requiring renal replacement therapy. The aim of this project was to explore whether sera from children with the two main clinically defined types of NS induce changes in transcriptome of in-vitro cultures of immortalized human podocytes. After 3 days of culture, total RNA was isolated and then library from polyadenylated RNA was prepared and RNA sequencing was performed. Log2 (fold-changes) were calculated to describe differential gene expression. Within the Reactome online tool, Camera methodology was implemented to identify functionally linked gene groups (pathways) that could distinguish the two patient groups. Significant transcriptome differences were found between samples from children with SSNS and SRNS. This may help to reveal the molecular mechanisms of the disease and open up for an effective individualized treatment. SSNS serum samples (n = 9) were obtained at disease manifestation, before the administration of glucocorticoids. SRNS serum samples (n = 4) were obained from patients, who had long-term remission and did not take any immunosuppressive drugs for at least 6 months before blood sampling. Fully differentiated immortalized human podocytes were incubated with 10 % serum samples at 37 °C in 5 % CO2 for 72 hours, in triplicates. The cells were lysed and the RNA was isolated using spin-column method. The RNA libraries were prepared from polyadenylated RNA and sequenced by NextSeq instrument. Sequencing data were processed by STAR program and DESeq2. Pathway analysis was performed by Reactome online software.

肾病综合征（Nephrotic Syndrome, NS）是一类病因异质性强、预后差异显著的不良疾病。临床中最为关键的预后特征之一，为患者对初始糖皮质激素治疗的应答反应。部分激素敏感型肾病综合征（steroid-sensitive nephrotic syndrome, SSNS）患儿可通过该治疗获得疾病长期缓解，而激素耐药型肾病综合征（steroid-resistant nephrotic syndrome, SRNS）患儿则面临肾衰竭风险，需接受肾脏替代治疗。本研究旨在探究两类临床分型的肾病综合征患儿血清，是否会诱导永生化人足细胞体外培养模型的转录组发生改变。体外培养3天后，提取总RNA，随后制备聚腺苷酸化RNA（polyadenylated RNA）文库，并开展RNA测序。通过计算log₂(倍数变化值)以描述差异基因表达情况。借助Reactome在线工具，采用Camera分析方法，识别可区分两类患者群体的功能关联基因集（通路）。研究发现激素敏感型与激素耐药型肾病综合征患儿的样本间存在显著转录组差异，该结果或有助于揭示疾病的分子机制，并为开发高效个体化治疗方案提供思路。激素敏感型肾病综合征血清样本（n=9）采集于疾病确诊时、糖皮质激素治疗前；激素耐药型肾病综合征血清样本（n=4）取自达到长期缓解且在采血前至少6个月未使用任何免疫抑制药物的患者。将完全分化的永生化人足细胞与10%血清样本共孵育，孵育条件为37℃、5%二氧化碳环境，时长72小时，设置3次平行重复。收集细胞并裂解，采用离心柱法提取RNA。以聚腺苷酸化RNA为模板构建RNA文库，并通过NextSeq测序仪完成测序。测序数据通过STAR程序与DESeq2工具进行处理，通路分析则借助Reactome在线软件完成。