Supplementary Material for: Weighted Gene Correlation Network Analysis (WGCNA) Detected Loss of MAGI2 Promotes Chronic Kidney Disease (CKD) by Podocyte Damage

Background/Aims: Podocyte damage is associated with proteinuria, glomerulosclerosis and decline of renal function. This study aimed to screen critical genes associated with podocyte injury in chronic kidney disease (CKD) by weighted gene correlation network analysis (WGCNA), and explore related functions. Methods: GSE66107, GSE93798, GSE30528, GSE32591 gene expression data including podocyte injury models or glomeruli in CKD patients were downloaded from the GEO database. R was used for data analysis. Differentially expressed genes (DEGs) (FDR< 0.05 or |Fold Change|≥1.5) in GSE993395 were assessed by WGCNA. According to Gene Ontology (GO) and known podocyte standard genes (PSGs), podocyte injury-associated modules were defined, with hub genes selected based on average intramodular connectivity. The Cytoscape software was used for network visualization. Nephroseq was used to assess the clinical significance of hub genes. Small interfering RNA (siRNA) was used to evaluate the roles of hub genes in podocyte injury Results: Totally 7957 DEGs were screened, with 15 (co.DEGs) altered in all 4 datasets; 4031 DEGs were used for WGCNA, encompassing 12 modules. Green modules (most PSGs and co.DEGs) were significantly enriched in glomerular development, and considered podocyte injury-associated modules. Furthermore, MAGI2 (a hub gene) was also a co.DEG and PSG. Glomerular MAGI2 levels were reduced in various kidney diseases, and positively and negatively associated with glomerular filtration rate and urinary protein levels in CKD patients. Moreover, MAIG2 knockdown reduced NPHS2, CD2AP and SYNPO levels, and induced podocyte rearrangement and apoptosis. Conclusion: MAGI2 identified by WGCNA regulates cytoskeletal rearrangement in podocytes, with its loss predisposing to proteinuria and CKD.

背景与目的：足细胞损伤与蛋白尿、肾小球硬化及肾功能下降密切相关。本研究旨在通过加权基因共表达网络分析（weighted gene correlation network analysis, WGCNA）筛选慢性肾脏病（chronic kidney disease, CKD）中与足细胞损伤相关的关键基因，并探究其相关功能。
方法：从GEO数据库下载包含慢性肾脏病患者足细胞损伤模型或肾小球样本的GSE66107、GSE93798、GSE30528、GSE32591基因表达数据集。采用R语言进行数据分析。针对GSE993395数据集，通过WGCNA分析其差异表达基因（differentially expressed genes, DEGs），筛选标准为错误发现率（false discovery rate, FDR）<0.05或|折叠变化（fold change, FC）|≥1.5。结合基因本体（Gene Ontology, GO）注释与已知足细胞标准基因（podocyte standard genes, PSGs），定义与足细胞损伤相关的共表达模块，并基于模块内平均连通性筛选枢纽基因（hub genes）。采用Cytoscape软件进行网络可视化。利用Nephroseq数据库评估枢纽基因的临床意义。采用小干扰RNA（small interfering RNA, siRNA）验证枢纽基因在足细胞损伤中的作用。
结果：本研究共筛选得到7957个DEGs，其中15个共差异表达基因（co-DEGs）在全部4个数据集均存在表达变化；最终纳入4031个DEGs用于WGCNA分析，共得到12个共表达模块。绿色模块（包含绝大多数PSGs与co-DEGs）显著富集于肾小球发育通路，因此被认定为足细胞损伤相关模块。此外，MAGI2作为枢纽基因之一，同时属于co-DEGs与PSGs。各类肾脏疾病患者的肾小球MAGI2表达水平均出现下调，且其表达量与慢性肾脏病患者的肾小球滤过率呈正相关，与尿蛋白水平呈负相关。进一步实验显示，MAGI2敲低可降低NPHS2、CD2AP及SYNPO的表达水平，并诱导足细胞骨架重排与细胞凋亡。
结论：本研究通过WGCNA筛选得到的枢纽基因MAGI2可调控足细胞骨架重排，其表达缺失会增加蛋白尿与慢性肾脏病的发病风险。