Alternative Splicing in Mechanically Stretched Podocytes as a Model of Glomerular Hypertension

Key Points Mechanical stretch induced over 3000 alternative splicing events in podocytes, affecting gene expression and protein abundance. Seventeen genes showed consistent splicing events across multiple analysis tools, with key isoform changes. Shroom3 and Myl6 underwent isoform switches under mechanical stretch, altering the C-terminal sequence and interaction properties of Myl6. Background Alterations in pre-mRNA splicing are crucial to the pathophysiology of various diseases. However, the effects of alternative splicing of mRNA on podocytes in hypertensive nephropathy are still unknown. The Sys_CARE project aimed to identify alternative splicing events involved in the development and progression of glomerular hypertension. Methods Murine podocytes were exposed to mechanical stretch, after which proteins and mRNA were analyzed by proteomics, RNA sequencing, and several bioinformatic alternative splicing tools. Results Using transcriptomic and proteomic analysis, we identified significant changes in gene expression and protein abundance because of mechanical stretch. RNA-Seq identified over 3000 alternative spliced genes after mechanical stretch, including all types of alternative splicing events. Among these, 17 genes exhibited an alternative splicing event across four different splicing analysis tools. From this group, we focused on Myl6 , a component of the myosin protein complex, and Shroom3 , an actin-binding protein essential for podocyte function. We identified two Shroom3 isoforms with significant expression changes under mechanical stretch, which was validated by quantitative RT-PCR and in situ hybridization. In addition, we observed an expression switch of two Myl6 isoforms after mechanical stretch, accompanied by an alteration in the C-terminal amino acid sequence. Conclusions A comprehensive RNA-Seq analysis of mechanically stretched podocytes identified novel potential podocyte-specific biomarkers and highlighted significant alternative splicing events, notably in the mRNA of Shroom3 and Myl6 .