Splicing Changes Drive Kidney Disease in Zebrafish Model

Researchers at the University of Greifswald and collaborating institutions have identified widespread post-transcriptional regulatory alterations in the kidney cells of zebrafish larvae subjected to a model of focal segmental glomerulosclerosis, or FSGS. Published in EMBO Molecular Medicine, the study demonstrates that podocyte injury triggers extensive changes in alternative splicing and microRNA expression, processes that were previously underappreciated in the molecular pathogenesis of this common cause of nephrotic syndrome. The findings suggest that RNA processing mechanisms, rather than just transcriptional changes, play a central role in the progression of glomerular disease.

FSGS is a histopathological pattern characterized by segmental scarring of the glomeruli, the filtering units of the kidney. It is a major driver of progression to end-stage renal disease in both children and adults. While genetic mutations and transcriptional dysregulation in podocytes have been extensively studied, the contribution of post-transcriptional regulatory mechanisms has remained poorly understood. The study aimed to dissect these regulatory layers by combining a zebrafish podocyte-specific injury model with glomerulus-resolved transcriptomic profiling.

The team utilized a genetically modified zebrafish larval model in which the enzyme nitroreductase and the fluorescent protein mCherry are expressed exclusively in podocytes. Treatment with the chemical Nifurpirinol induced selective podocyte ablation, leading to progressive edema and loss of fluorescence, consistent with previously established characterizations of this model. Whole larvae and manually isolated glomeruli were collected at 5 and 6 days post-fertilization, corresponding to 24 and 48 hours post-treatment, for parallel RNA sequencing, small RNA profiling, and alternative splicing analysis.

Bulk RNA sequencing of isolated glomeruli revealed a progressive transcriptional response to podocyte ablation. At 5 days post-fertilization, 1,007 differentially expressed genes were detected, a number that increased substantially to 2,299 at 6 days post-fertilization. This amplification of the molecular injury response over time indicated a dynamic remodeling of the glomerular transcriptome. Among the most highly regulated genes were canonical podocyte markers such as nphs1, nphs2, and ptpro, which were significantly downregulated in the injured glomeruli.

Gene ontology enrichment analysis indicated distinct functional signatures at early versus later disease stages. At 5 days post-fertilization, downregulated genes were enriched for processes involved in the regulation of gene expression and chromatin dynamics, whereas upregulated genes were associated with cytokine and hormone-related signaling pathways. By 6 days post-fertilization, downregulated genes were frequently associated with immune homeostasis, while upregulated genes were linked to metabolic pathways. This temporal shift suggests a transition from a differentiated podocyte state toward an injury-associated phenotype.

Small RNA sequencing identified 42 significantly regulated microRNAs in isolated glomeruli at 6 days post-fertilization. Notably, miR-21 and miR-193, two microRNAs strongly linked to FSGS and chronic kidney disease, were among the dysregulated species. Bioinformatic target analysis revealed that miR-21 targets included epb41l5, npr3, and ptpro, while miR-193 targets included wt1, col4a3, and foxc1. These key podocyte genes were significantly downregulated in the injured zebrafish glomeruli, demonstrating coordinated repression of podocyte-enriched targets in vivo.

Alternative splicing analysis using multiple complementary tools revealed that exon skipping was the predominant splicing event, accounting for approximately 80% of detected changes. The number of genes exhibiting alternative splicing increased over time, from 136 in isolated glomeruli at 5 days post-fertilization to 612 at 6 days post-fertilization. Many of these alternatively spliced genes were podocyte-enriched and linked to the glomerular filtration barrier, including slit diaphragm proteins, cytoskeletal regulators, and glomerular basement membrane proteins.

A central finding of the study was the identification of disease-associated isoform switches in the splicing factor SRSF3 and the polarity regulator EPB41L5. In zebrafish, a shift toward an SRSF3 isoform lacking a conserved serine-rich C-terminal domain was observed. This domain contains twelve phosphorylation sites required for SRSF3 autoregulation. Immunofluorescence staining of human kidney biopsies showed increased nuclear SRSF3 expression in FSGS patients compared to healthy controls, supporting the conservation of this regulatory change across species.

EPB41L5, a regulator of epithelial polarity and cell-cell contacts, was downregulated in podocytes and switched toward a truncated isoform in the zebrafish model. This isoform switch was accompanied by the downregulation of its interacting partners CRB2 and MPP5, key proteins required for podocyte polarity and integrity. Quantitative immunofluorescence of human biopsies confirmed a significant reduction of EPB41L5 expression exclusively in glomeruli from FSGS biopsies compared to healthy tissue and membranous glomerulopathy controls.

To test the functional relevance of the observed inflammatory signaling, the researchers examined the IL6/JAK/STAT3 axis. They found significant upregulation of stat3 and its downstream target socs3 in injured zebrafish glomeruli and human FSGS glomeruli. However, pharmacological inhibition of the JAK/STAT3 pathway using inhibitors such as Ruxolitinib and Stattic failed to improve the injury. Instead, treatment accelerated disease progression, evidenced by enhanced edema formation and increased mortality rates. This suggests that downstream post-transcriptional pathways may represent a point of limited reversibility in the context of rapid podocyte depletion.

The study also demonstrated strong cross-species concordance between zebrafish, mouse, and human FSGS glomerular transcriptomes. Integration with published single-cell RNA sequencing data from mouse glomeruli in nephrotoxic serum nephritis and doxorubicin models corroborated the findings, showing similar increases in Stat3 and Socs3 expression in podocytes. This conservation validates the zebrafish model as a platform for mechanistic and therapeutic investigation of post-transcriptional regulation in glomerular disease.

The findings establish alternative splicing as a central regulatory mechanism in FSGS and identify SRSF3 and EPB41L5 isoform switches as promising candidates for disease biomarkers and therapeutic targeting. The study provides a comprehensive, glomerulus-resolved map of post-transcriptional regulation in podocyte injury, offering a conceptual framework linking podocyte injury to RNA regulation. While the zebrafish model recapitulates key molecular features of human disease, the lack of rescue from STAT3 inhibition highlights the complexity of signaling networks and the need for further investigation into splicing-corrective strategies.

Beyond isolated glomerular responses, transcriptomic evaluations of whole zebrafish larvae at five and six days post-fertilization captured a broadening injury response, expanding from 230 differentially expressed genes to 707 genes over time. Within isolated glomeruli, downregulated transcripts were linked to developmental programs governing the nephron and glomerulus, whereas upregulated transcripts showed enrichment for RNA processing and ribosome biogenesis. Glomerular comparisons across species revealed that injury-associated transcripts such as fn1, serpine1, thbs2, actn1, and kank1 were elevated in both larval glomeruli and human disease tissue. Conversely, transcription factors including foxc1 and mafb were downregulated across species, alongside phospholipase C epsilon 1 (PLCE1). Microarray profiling confirmed that PLCE1 is enriched in healthy glomeruli relative to the tubulointerstitium, while immunofluorescence localized the protein to podocyte cell bodies, with human biopsies mirroring the expression declines observed in the larval model.

The molecular shift in SRSF3 involves structural modification rather than simple transcriptional upregulation. While total srsf3a expression rose in injured glomeruli, the shift favored isoform 206 over full-length isoform 201. Both variants preserve the N-terminal RNA recognition motif and nuclear localization signal, but isoform 206 lacks a conserved 38-amino-acid serine-rich C-terminal segment harboring twelve phosphorylation sites necessary for normal autoregulation. This truncation corresponds to alternative inclusion of poison exon 4, which introduces an in-frame stop codon. Concurrently, other splicing machinery components underwent alterations, including downregulation of srsf2a, srsf2b, srsf4, and heterogeneous nuclear ribonucleoproteins hnrnpa3 and hnrnpul1. Automated nuclear intensity quantification using the StarDist algorithm in human tissue verified elevated SRSF3 protein signals in 36 focal segmental glomerulosclerosis glomeruli compared with 24 healthy control glomeruli.

The modulation of the receptor tyrosine kinase fgfr1a provided direct evidence that alternative splicing can alter signaling capacity without altering overall transcript abundance. While total fgfr1a mRNA levels remained unchanged after injury induction, isoform analysis revealed a substantial reallocation of transcript variants, marked by a 37 percent decline in isoform 204 and a 176 percent increase in isoform 201. Computational structural modeling using FoldX and StructMAn demonstrated that alternative splicing altered the position of coding exon 7 at the pre-mRNA level. This genomic rearrangement generated isoform-specific amino acid substitutions within the extracellular fibroblast growth factor binding domain, which are predicted to diminish receptor interaction affinity with fibroblast growth factor 2.

Splicing alterations also dismantled the structural polarity complex anchored by EPB41L5. The disease-associated switch generated epb41l5-204, a truncated splice variant that loses its final nine exons. This change coincided with the coordinated downregulation of its physical binding partners, the Crumbs polarity complex components crb2 and mpp5. Cross-species single-cell and transcriptomic datasets confirmed concordant suppression of EPB41L5, CRB2, and MPP5 in mouse models of nephrotoxic serum nephritis and doxorubicin nephropathy, as well as in human patient cohorts. Confocal fluorescence quantification demonstrated that EPB41L5 protein loss was specific to focal segmental glomerulosclerosis across 39 evaluated human glomeruli, showing significant divergence from 38 membranous glomerulopathy glomeruli and 31 healthy control tissues.

The study also delineated specific experimental parameters and procedural boundaries across its functional assays. In pharmacological testing, cohorts of 50 larvae received either the positive control belinostat or one of four pathway inhibitors, ruxolitinib, baricitinib, WP1066, or Stattic, at graded concentrations up to 100 micromolar, with baricitinib capped at 50 micromolar. None yielded clinical protection, instead accelerating mortality and increasing edema scores evaluated on a four-point scale at six, seven, and eight days post-fertilization. Additionally, manual isolation protocols required microdissecting pools of 10 glomeruli using fine tweezers, all larvae were sexually undifferentiated at testing, and experimental designs were unblinded without sample exclusions. Furthermore, researchers cautioned that all reported microRNA target interactions were bioinformatically predicted rather than directly validated through functional binding experiments.

Subject of Research: Post-transcriptional regulation and alternative splicing in podocytes during focal segmental glomerulosclerosis progression

Article Title: Disease-driven post-transcriptional alterations and alternative splicing in podocytes in focal segmental glomerulosclerosis

Article References: Mattias, F., Tsoy, O., Iervolino, A., Simm, S., Hammer, E., Gress, A., Siegerist, F., Schindler, M., Lange, T., Ameling, S., Kacprowski, T., List, M., Kalinina, O., Franzenburg, S., Capasso, G., Endlich, K., Baumbach, J., Völker, U., Endlich, N., & Kliewe, F. (2026). Disease-driven post-transcriptional alterations and alternative splicing in podocytes in focal segmental glomerulosclerosis. EMBO Molecular Medicine, 18(9), 3824-3849. https://doi.org/10.1038/s44321-026-00495-5

Image Credits: AI Generated

DOI: 10.1038/s44321-026-00495-5

Keywords: focal segmental glomerulosclerosis, alternative splicing, podocyte injury, zebrafish model, microRNA, SRSF3, EPB41L5, glomerular disease, RNA regulation, nephrotic syndrome, Disease-driven, post-transcriptional