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

Abstract Background and Aims It has been shown that mRNA splicing plays a relevant role in disease pathophysiology. However, the possible role of alternative splicing (AS) in hypertensive nephropathy (HN) has not been investigated. The purpose of the Sys_CARE (Systems Medicine Investigation of AS in Cardiac and Renal Diseases) project is to investigate the possible implication of AS events in HN development and progression. Method Differentiated murine podocytes were seeded on a flexible silicone membrane, and mechanically stretched using the Stretchy apparatus (NIPOKA GmbH, Greifswald). Cells were stretched for 3 days under low and high stretch conditions with a frequency of 0.5 Hz. The mRNA and proteins were isolated and analyzed using RNA_Seq and LC-MS/MS techniques. Enrichment analysis identified proteins which were classified according to the related biological processes, molecular function and cellular components. Splicing and transcript expression were evaluated with bioinformatical tools (rMATS, leafcutter, Whippet, MAJIQ, IsoformSwitchAnalyzeR). Results Proteomic analysis of cultured podocytes revealed significant different expression levels for 135 proteins (54 increased and 81 decreased) and 424 proteins (195 increased and 229 decreased) under low and high stretch conditions, respectively, compared to unstretched conditions. Interestingly, most of the proteins with decreased intensity upon stretch are cytoskeleton and actin-binding proteins. In contrast, the up-regulated proteins are more associated in clusters affecting mRNA processing and splicing. By RNA_Seq we identified over 1000 different splicing events including all types of alternative splicing events. We screened for candidates that showed an alternative splicing event in multiple tools, were found in the proteomics, were podocyte-specific, or showed altered expression in glomerulopathies such as diabetic nephropathy. In this regard, we found an isoform switch of Myl6 and Shroom3 after mechanical stretch. Conclusion Using mechanically stretched podocytes as a model of HN, we found significant up- and down-regulated proteins. In addition, by RNA_Seq we identified an isoform switch of Myl6 and Shroom3 that might be essential for the development of HN.