Figure 7

Figure 7

Multi-omics integration identifies TNFRSF1A as a causal mediator of DKD and validates findings in a zebrafish model: (A) Circular heatmap displaying p-values of MR-identified plasma proteins associated with DKD risk. Each ring represents a method. Color scale denotes statistical significance. (B) Gene Ontology (GO) enrichment analysis of significant genes identified from the MR. The bar plot shows enriched GO terms across three categories: Biological Process (BP), Cellular Component (CC), and Molecular Function (MF). The x-axis represents gene ratio, and color indicates adjusted p-value. (C) KEGG pathway enrichment analysis of hub genes. The bubble plot displays significantly enriched KEGG pathways, with bubble size indicating the number of genes involved and color representing adjusted p-value (D) Hub protein network centered on TNFRSF1A. TNFRSF1A emerges as a central protein with extensive connections to inflammatory mediators including IL1RL1, TNFRSF1B, and ANGPT1. (E) Representative brightfield and fluorescence microscopy images comparing control morpholino oligonucleotide (CoMo)-injected and pdx1 knockdown (pdx1 KD) zebrafish larvae at 2 days post-fertilization (dpf). Fluorescent transgenic markers (cdh17:GFP, wt1b:GFP) label proximal tubules and podocytes, respectively. pdx1 KD larvae exhibit yolk retention (blue arrow), shortened neck segments (yellow arrows), and dilated glomeruli (red arrow), recapitulating previously described phenotypes [18]. Representative images were selected from three independent biological replicates (n = 3), with 50–60 larvae examined per group in each replicate. The described phenotypes were consistently observed across all replicates. Scale bar = 200 μm. (F,G) Quantification of TNFRSF1A mRNA expression in CoMo and pdx1 KD larvae by RT-PCR. For each biological replicate, 45–50 embryos were pooled. Three biological replicates (n = 3) were performed. Relative expression levels normalized to ef1a show significant upregulation of TNFRSF1A in pdx1 KD larvae. (H) Quantification of TNFRSF1A mRNA expression in CoMo and pdx1 KD larvae by qPCR (2^−ΔΔCt method). Each group included three biological replicates (n = 3), and each biological replicate was measured in duplicate (two technical replicates), resulting in six data points per group.