Supplementary Material for: Plasma transcriptome profile associated with CKD progression

Introduction: Understanding the molecular signals associated with the progression of kidney disease is vital for risk stratification and targeted treatment. Recent advances in RNA-sequencing technique has enabled us to characterize extracellular transcriptome profiles for precision diagnostics.
Method: We evaluated the plasma mRNA profile of participants exhibiting slow (n=119) and fast (n=119) decline in estimated glomerular filtration rate (eGFR) among the Chronic Renal Insufficiency Cohort (CRIC) in a nested case control study. The two groups were matched for age, sex, race, baseline eGFR, proteinuria and diabetes status. The next generation sequencing data was analyzed using edgeR to identify differentially expressed genes (DEGs) and Ingenuity Pathway Analysis (IPA) was done to identify the associated pathways. We also compared the top plasma DEGs with gene expression in microdissected human CKD kidney.
Results: We identified fragments from ~28,000 annotated genes, of which 783 transcripts exhibited differential expression between slow and fast CKD progressors. Among 629 protein coding genes, 469 were overexpressed in slow progressors, while 157 showed increased expression in fast progressors. Expression of GLI2, CUX1, NOTCH1 and LRP1 transcripts were amplified in slow progressors. Pathway analysis linked these differentially expressed genes to WNT/β-catenin signaling, IL-12 signaling and production in macrophages, Netrin-1 Signaling and Epithelial-Mesenchymal Transition pathways. Many of the plasma differentially expressed genes were also upregulated in microdissected human CKD kidney.
Conclusion: Warranting further validation, circulating levels of aberrantly expressed transcripts hold potential to be used as biomarkers for fast CKD progression.

引言：解析与肾脏病进展相关的分子信号，对于风险分层与靶向治疗至关重要。近年来RNA测序（RNA-sequencing）技术的进步，使我们能够表征细胞外转录组谱，从而实现精准诊断。
方法：本研究为巢式病例对照研究，基于慢性肾功能不全队列（CRIC），纳入估算肾小球滤过率（eGFR）分别呈缓慢下降（n=119）与快速下降（n=119）的参与者，对其血浆mRNA谱进行评估。两组在年龄、性别、种族、基线eGFR、蛋白尿情况及糖尿病状态上进行了匹配。采用edgeR分析下一代测序数据以筛选差异表达基因（DEGs），并通过Ingenuity通路分析（IPA）鉴定相关通路。此外，我们还将筛选得到的Top血浆差异表达基因与显微切割的人类慢性肾脏病（CKD）肾脏组织中的基因表达进行了对比。
结果：本研究共鉴定到约28000个注释基因的转录本，其中783个转录本在慢性肾脏病缓慢进展者与快速进展者中存在差异表达。在629个蛋白编码基因中，469个在缓慢进展者中呈高表达，157个在快速进展者中呈高表达。GLI2、CUX1、NOTCH1及LRP1转录本在缓慢进展者中表达上调。通路分析将这些差异表达基因关联至WNT/β-连环蛋白信号通路、巨噬细胞中白细胞介素12（IL-12）的信号通路与产生通路、Netrin-1信号通路以及上皮-间质转化通路。诸多血浆差异表达基因在显微切割的人类慢性肾脏病肾脏组织中同样呈上调表达。
结论：尽管尚需进一步验证，但异常表达的循环转录本水平有望作为慢性肾脏病快速进展的生物标志物。