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. Here we identifiedAS events that play a role in the development and progression of HN. For this, murine podocytes were exposed to mechanical stretch to model HN, after which proteins and mRNA were measured by proteomics, RNA-Seq, and analyzed by several AS detection tools. 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 and differential gene expression, which together with protein regulation improve the understanding of the pathophysiology of hypertension-induced nephropathy. Overall design: To investigate the role of alternative splicing in glomerular hypertension we performed mechanical stress experiments on conditionally immortalised podocytes (SVI; CLS Cell Line Service GmbH, Eppelheim, Germany) Differentiated mouse podocytes were seeded on flexible silicon membranes of a six well plate (Bioflex, Flexcell® International Corporation, Burlington, NC, USA). Pressure amplitude was chosen to give a maximum up- and downward deflection of the membrane center of 6 mm (low stretch) and a higher amplitude (8 mm). We then performed gene expression profiling using the data from RNA-Seq of unstretched, high-stretched, and low stretched podocytes. Comparative gene expression and alternative splicing analysis of unstretched podocytes versus low-stretch and unstretched podocytes and high-stretch