#2407 Multiomics analysis of isolated zebrafish glomeruli to unravel the impact of alternative splicing in glomerular diseases

Abstract

Abstract Background and Aims Alternative splicing (AS) is a known key factor in gene regulation. There is evidence that changes in the alternative mRNA splicing can lead to the onset of various diseases. However, the potential role of AS in the pathogenesis of glomerular diseases has only been investigated to a very limited extent. In the past, the zebrafish larva (Danio rerio) has been established as an animal model for the in vivo investigation of kidney diseases, since the glomeruli of zebrafish larvae are genetically, molecularly, and functionally similar to those of mammals. As part of the Sys_CARE (Systems Medicine Investigation of AS in Cardiac and Renal Diseases) project, we wanted to identify specific AS events that might be involved in the development of focal segmental glomerulosclerosis (FSGS). Method For all experiments, the double transgenic strain [Tg (nphs2: GAL4-VP16); (UAS:Eco.nfsB-mCherry)] was used. At 4 dpf (days post fertilization), the zebrafish larvae were divided into two groups. The control group was treated with 0.1% dimethyl sulfoxide (DMSO) in E3 medium. To induce FSGS, zebrafish larvae were treated with 50 nM nifurpirinol (NFP) in 0.1% DMSO in E3 medium for 24 hours. Glomeruli were manually isolated at 5 and 6 dpf and analyzed together with lysates of whole larvae by LC-MS/MS and RNA-Seq. To identify AS events, RNA-Seq data were analyzed by MAJIQ, Multivariate Analysis of Transcript Splicing (rMATS), leafcutter, Whippet as well as IsoformSwitchAnalyzeR. Gene set enrichment analysis (GSEA) identified proteins and genes which were classified according to their related biological processes. Results Proteomics and transcriptomics showed significant differences in the glomerular protein and transcript levels between NFP-treated larvae and the control group. The abundance of the podocyte-specific protein nephrin as well as the podocin promotor-driven expression of the fluorescent dye mCherry was significantly reduced after NFP treatment confirming the onset of the injury. Subsequent analyses identified more than 1000 transcripts that were significantly differentially expressed, as well as several splicing events in the larvae with a FSGS-like damage. There was also a marked shift in isoform patterns in larvae with FSGS. Gene set enrichment analysis indicated an enrichment in the up-regulated transcripts and proteins involved in metabolic processes, especially those related to rRNA and ncRNA processing. Conversely, transcripts and proteins that were down-regulated showed significant clusters in catabolic processes, extracellular matrix, and cytoskeleton organization as well as in nephron and glomerulus development. Conclusion Using our zebrafish model, in-depth transcriptomics and proteomics analysis provided novel insights into the pathogenesis of podocyte-associated glomerular diseases. Data suggest, that pathological changes are accompanied by alternative splice events which might therefore be a new therapeutic approach in glomerular nephropathy.