Regulation of the transcriptome, miRNAs, and alternative splicing in a FSGS zebrafish injury model

Background

Focal Segmental Glomerulosclerosis (FSGS) is a severe kidney disorder with complex and not yet fully understood pathogenesis. Alternative splicing (AS) – the generation of distinct protein isoforms from the same gene – might play a critical role by the regulation of gene functions and disease development.

Methods

To investigate the role of AS in FSGS, we used a zebrafish model, which mimics key human FSGS features, including foot process effacement, matrix accumulation, podocyte detachment and parietal epithelial cell activation.

We performed total RNA sequencing of isolated zebrafish glomeruli and whole larvae, followed by integrative bioinformatic analysis to identify AS events and regulatory miRNAs.

Results

Our data revealed a downregulation of essential podocyte genes ( nphs1, nphs2, podxl, wt1 ) and an inhibition of pathways associated with nephron development and cytoskeletal organization. We also observed increased expression of the transcription factor stat3 and disease-associated miRNAs such as miR-21 and miR-193. AS analysis identified approximately ∼7,000 splicing events, primarily exon skipping (∼80%), affecting genes such as nphs1 , magi2 , and ptpro . A total of 136 and 612 alternatively spliced genes were found at 5 and 6 days post-fertilization (dpf), respectively. Isoform switch analysis uncovered 70 genes affected by AS in FSGS, including epb41l5 (linked to podocyte adhesion), fgfr1a (fibroblast growth signaling), and members of the SRSF splicing factor family (e.g., srsf3a ).

Conclusions

These findings emphasize the importance of transcriptional and post-transcriptional regulation, including AS, in FSGS pathogenesis. Furthermore, they support the zebrafish model as a valuable system for identifying novel mechanisms and potential therapeutic targets for kidney diseases.