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Abstract: FR-PO767

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

Session Information

Category: Glomerular Diseases

  • 1401 Glomerular Diseases: Mechanisms, including Podocyte Biology

Authors

  • Endlich, Nicole, Department of Anatomy and Cell Biology, University Medicine Greifswald, Greifswald, Germany
  • Kliewe, Felix, Department of Anatomy and Cell Biology, University Medicine Greifswald, Greifswald, Germany
  • Mattias, Francescapaola, Department of Anatomy and Cell Biology, University Medicine Greifswald, Greifswald, Germany
  • Hammer, Elke, Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, Greifswald, Germany
  • Tsoy, Olga, Institute for Computational Systems Biology, University of Hamburg, Hamburg, Germany
  • Gress, Alexander, Department of Drug Bioinformatics, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Centre for Infection Research (HZI), Saarbrücken, Germany
  • Simm, Stefan, Institute of Bioinformatics, University Medicine Greifswald, Greifswald, Germany
  • Baumbach, Jan, Institute for Computational Systems Biology, University of Hamburg, Hamburg, Germany
  • Völker, Uwe, Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, Greifswald, Germany
  • Endlich, Karlhans, Department of Anatomy and Cell Biology, University Medicine Greifswald, Greifswald, Germany

Group or Team Name

  • Endlich Lab.
Background

Alterations in pre-mRNA splicing play an important role in disease pathophysiology. However, the 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 was to identify AS events that play a role in the development and progression of HN.

Methods

Cultured mouse podocytes were exposed to mechanical stretch for 3 days under low and high stretch conditions using the Stretchy apparatus (NIPOKA GmbH, Greifswald). Subsequently, mRNAs and proteins were analyzed by RNA-Seq and LC-MS/MS. Splicing and transcript expressions were studied with bioinformatical tools.

Results

Transcriptome analyses of mechanically stretched podocytes by RNA-seq showed that mechanical stress leads to a high number of differentially expressed genes compared to unstretched podocytes. GO analysis revealed that a higher number of actin-binding genes were down- and mRNA processing genes were up-regulated. To identify alternative splicing (AS) events, different AS tools were used. We found a wide variety of splice events. The most frequent event was exon skipping, followed by intron retention. We found 290 alternatively spliced genes after mechanical stretch, which were detected with 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, are expressed in podocytes in vivo or showed an altered expression patterns in glomerular disease. This screening led to the two candidates Shroom3, an actin-binding protein, which is crucial for podocyte morphology and function and Myosin light chain 6 (Myl6), a component of the myosin motor protein complex.

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.