Alternative splicing in mechanically stretched podocytes as a model of glomerular hypertension

Alterations in pre-mRNA splicing play an important role in disease pathophysiology. However, the role of alternative splicing (AS) for podocytes in hypertensive nephropathy (HN) has not been investigated. The purpose of the Sys_CARE project was to identify AS events that play a role in the development and progression of HN. For this, murine podocytes were exposed to mechanical stretch, after which proteins and mRNA were analyzed by proteomics, RNA-Seq and several bioinformatic AS tools.

Based on transcriptomics and proteomics analysis we could observe significant changes in gene expression and abundance of proteins under mechanical stretch compared to unstretched conditions. By RNA-Seq, we identified over 1000 different splicing events including all types of AS events. We identified 17 genes that showed an AS event in four different splicing analysis tools. We focused on Myl6 , a component of the myosin protein complex, and Shroom3 , an actin-binding protein crucial for podocyte function. We found two Shroom3 isoforms that showed significant changes in expression upon mechanical stretch, which was verified by qRT-PCR and in situ hybridization. Furthermore, we observed an expression switch of two Myl6 isoforms after mechanical stretch. This switch is accompanied by a change in a C-terminally located amino acid sequence.

In summary, mechanical stretch of cultured podocytes is an excellent model to simulate hypertensive nephropathy. In depth RNA-Seq analysis disclosed alternative splicing events, such as in Shroom3 and Myl6 , which may play a crucial role in the pathophysiology of hypertension-induced nephropathy.