Identification of mechanistic chronic kidney disease biomarkers by comprehensive bulk RNA-seq transcriptomics and SWATH proteomics of the rat sub-total nephrectomy model of kidney fibrosis [RNA-Seq_2]

The rat sub-total nephrectomy (SNx) is a functional model of chronic kidney disease (CKD), where the main pathological driver is glomerular hypertension. Comprehensive transcriptomics and proteomics analyses on the rat SNx model were performed to identify biomarkers in plasma or urine that correlate with kidney disease and functional kidney loss. Kidneys were subjected to collagen I and III staining for fibrosis scoring, SWATH proteomics and bulk RNA-sequencing transcriptomics (RNA-seq), with SWATH also performed on plasma and urine. Differential expression analysis demonstrated significant dysregulation of genes and proteins involved in fibrosis, metabolism, and immune response in the SNx rats compared to controls. Gene ontology analysis of the intersecting genes and proteins from both studies demonstrated common biology between animal cohorts that reached the predefined kidney disease thresholds (serum creatinine >2-fold or proteinuria >3-fold increase over sham-operated). Thirteen significantly differential molecules were detected with consistent directional changes in both transcriptomics and proteomics datasets. These molecules were detected independently in kidney (both RNA and protein) and urine (protein only), but not in plasma. The bioinformatics analysis enabled the identification of mechanistic CKD biomarkers including lumican and collagen alpha-1(III) chain, whose co-expression has previously been both implicated in fibrosis and detected in urine in CKD patients.大鼠肾大部切除术（subtotal nephrectomy, SNx）是慢性肾脏病（chronic kidney disease, CKD）的功能性模型，其核心病理驱动因素为肾小球高压。本研究针对大鼠SNx模型开展了全面的转录组学（transcriptomics）与蛋白质组学（proteomics）分析，以筛选与肾脏疾病及肾功能丧失相关的血浆或尿液生物标志物。 研究对肾脏组织进行Ⅰ型和Ⅲ型胶原染色以完成纤维化评分，同时采用SWATH蛋白质组学及批量RNA测序（bulk RNA-sequencing, RNA-seq）转录组学技术对肾脏样本进行检测；此外还对血浆和尿液样本开展了SWATH蛋白质组学分析。 差异表达分析结果显示，与假手术对照组相比，SNx模型大鼠体内涉及纤维化、代谢及免疫应答的基因与蛋白质均存在显著表达失调。对两项组学研究的交集基因和蛋白质进行基因本体论（gene ontology, GO）分析后发现，达到预设肾脏病阈值（血清肌酐较假手术组升高2倍以上，或蛋白尿较假手术组升高3倍以上）的动物队列存在共通的生物学特征。 本研究在转录组学与蛋白质组学数据中共检测到13个差异表达显著的分子，且其表达变化方向完全一致；这些分子可在肾脏组织（RNA和蛋白质层面）及尿液（仅蛋白质层面）中独立检出，但未在血浆样本中检测到。通过生物信息学分析，本研究鉴定出了潜在的CKD机制性生物标志物，包括饰胶蛋白聚糖（lumican）及胶原蛋白α1(Ⅲ)链（collagen alpha-1(III) chain）；既往研究已证实二者的共表达与纤维化过程密切相关，且在CKD患者的尿液中可被检出。