#3172 Regulation of mRNA, miRNAs, and alternative splicing events in an FSGS-like zebrafish injury model

Abstract Background and Aims Focal Segmental Glomerulosclerosis (FSGS) is a renal disease characterized by podocyte cytoskeletal damage, resulting in impaired podocyte foot process morphology and kidney function. Despite significant advancements in our understanding of FSGS, the molecular mechanisms underlying glomerular damage and disease progression remain largely elusive. MiRNAs and alternative splicing (AS) of pre-mRNAs are predicted to play a substantial role in the physiology and pathology of kidney diseases. The present study aims to identify changes in miRNAs and pre-mRNA splicing events that may be responsible for the pathogenesis of FSGS. Method The present study used an established FSGS study model based on transgenic zebrafish larvae expressing bacterial nitroreductase and the fluorophore mCherry at podocyte levels. The addition of the prodrug Nifurpirinol to zebrafish larvae at 4 days post-fertilization (dpf) led to its enzymatic conversion into a toxin, causing glomerular damage that models FSGS pathology. Glomeruli and whole larvae were collected for RNA sequencing at 5 and 6 dpf, followed by gene set enrichment analysis based on biological processes. To identify AS events, RNA-Seq data were analyzed using different bioinformatic tools such as rMATS, leafcutter, Whippet, and IsoformSwitchAnalyzeR. Results Zebrafish larvae exposed to Nifurpirinol developed periorbital and abdominal edema, along with fluorescence accumulation in proximal tubules, indicating podocyte loss. These effects were more pronounced 48 hours post-treatment. Transcriptome analysis revealed a marked increase in differentially expressed genes in whole larvae and isolated glomeruli. Podocyte-specific genes, including nephrin, podocin, podocalyxin, and wt1, were significantly downregulated, confirming the injury model. Gene ontology analysis showed enrichment in glomerular development, rRNA processing, catabolic processes, extracellular matrix, and cytoskeleton organization. MiRNAs dre-mir-21-2 and dre-mir-193, previously associated with CKD and FSGS, were found to be upregulated. AS analysis detected ∼7,000 events in isolated glomeruli, with exon skipping as the most frequent event (∼80%). Differential splicing was notable in the serine/arginine-rich splicing factors (SRSF) family. Across methods (rMATS, Leafcutter, Whippet), 612 and 136 alternatively spliced genes were identified in Nifurpirinol-treated glomeruli at 6 and 5 dpf, respectively. Two candidates, epb41l5 and fgfr1a, showed significant isoform changes and were selected for further studies. Novel splicing variants, including altered epb41l5 isoforms critical for podocyte adhesion and fgfr1a isoforms linked to fibroblast growth signaling, may contribute to podocyte dysfunction. Conclusion The present study identified alterations in pre-mRNAs expression, including epb41l5 and fgfr1a, using a zebrafish model mimicking FSGS. Additionally, up-regulation of specific miRNAs were identified, suggesting a crucial role in the development and progression of FSGS. These findings offer new insights into the pathogenesis of FSGS, supporting the discovery of disease biomarkers and the development of targeted therapeutic strategies.