#2450 Alternative splicing in podocytes under mechanical stress: a new perspective for the pathogenic effect of glomerular hypertension
Authors: Mattias Francescapaola , Kliewe Felix , Hammer Elke , Ameling Sabine , Tsoy Olga , Gress Alexander , Simm Stefan , Kalinina Olga V , Endlich Karlhans , Baumbach Jan , Völker Uwe , Endlich Nicole 
Published: May 2024

Abstract

Background and Aims
High blood pressure is considered one of the most important risk factors leading to chronic kidney disease. The pathogenesis of hypertension-induced damage to podocytes remains still elusive. Since recent reports have shown that alternative splicing (AS) has a significant impact on disease progression, we investigated alternative splicing of mechanically stretched podocytes, a well-established model for glomerular hypertension, in the Sys_CARE (Systems Medicine Investigation of AS in Cardiac and Renal Diseases) project. Our results provide new insights into the molecular mechanisms that might drive the progression of hypertensive nephropathy.

Method
Differentiated murine podocytes were exposed to mechanical stretch for 3 days under low and high stretch conditions. Subsequently, mRNA and proteins were analyzed by RNA-Seq and LC-MS/MS, respectively. Enrichment analysis identified transcripts which were classified according to the related biological processes, molecular function, and cellular components. Splicing and transcript expression were evaluated with bioinformatical tools such as MAJIQ, Multivariate Analysis of Transcript Splicing (rMATS), leafcutter, Whippet and IsoformSwitchAnalyzeR.

Results
Transcriptome analyses of mechanically stretched podocytes by RNA sequencing showed that mechanical stress leads to a high number of differentially expressed genes (1323 with log2 fc ± 1) compared to unstretched podocytes. Detailed gene set enrichment analysis for GO-Terms showed an enrichment among up-regulated transcripts related to mRNA processing and RNA splicing. In contrast, most transcripts with decreased expression upon stretch are associated with cytoskeleton function.
To identify AS events, different AS tools were used. We found a wide variety of splice events. The most frequent event was exon skipping (between 63-82% of all AS events), followed by intron retention and alternative 5′ or 3′ splice site. 290 alternatively spliced genes were detected after mechanical stretch by three different splice analysis tools (rMATS, leafcutter, Whippet). Out of these candidates, the IsoformSwitchAnalyzeR identified 17 genes exhibiting significant isoform switches. To prioritize the candidates, we performed a screening that included only those genes that were identified by proteomic analysis and that are expressed in podocytes in vivo or showed an altered expression pattern in glomerular disease. This screening led to the two candidates Shroom3 and Myl6.
Shroom3, an actin-binding protein, is crucial for podocyte morphology and function. Genetic variants of Shroom3 have been linked with chronic kidney disease by GWAS. Here we found that two of four Shroom3 isoforms showed significant expression changes due to mechanical stretch. Isoform 2 which showed the highest expression in cultured podocytes, significantly decreased, but the shorter isoform X1 was significantly up-regulated after mechanical stretch. Both changes were verified by qRT-PCR as well as by in situ hybridization in mouse tissue.
Myosin light chain 6 (Myl6), a component of the myosin motor protein complex, has two isoforms which are detected in cultured podocytes as well as in vivo. We found an expression switch from isoform 1 to 2 due to mechanical stretch. Our analysis showed that this is accompanied by a change of the amino acid sequence AFVRHILS to ELVRMVLN, sequences that are well conserved in mice, rat, and human. The analysis of the protein structure by FoldX revealed that this change in the amino acid sequence could lead to an impairment of the binding of the motor protein complex to actin and might therefore influence the functionality of the actin cytoskeleton, which is highly important for proper podocyte foot process morphology.

Conclusion
Mechanical stretch of cultured mouse podocytes, which is an excellent model to simulate hypertensive nephropathy, leads to alterations in isoform expression due to alternative splicing. These changes in the expression of isoforms, such as Shroom 3 and Myl6, have the potential to play a key role in the pathophysiology of hypertension-induced nephropathy.