Abstract: TH-PO068

In Vivo Characterization of Podocin Variants Based on a CRISPR/Cas9 Based Genome Editing Approach

Session Information

Category: Glomerular

  • 1002 Glomerular: Basic/Experimental Pathology

Authors

  • Butt, Linus, Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany
  • Ebert, Lena K., Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany
  • Rinschen, Markus M., Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany
  • Höhne, Martin, Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany
  • Zevnik, Branko, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany
  • Brinkkoetter, Paul T., Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany
  • Schermer, Bernhard, Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany
  • Benzing, Thomas, Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany
Background

NPHS2 encodes for Podocin, a membrane protein at the inner leaflet of the plasma membrane of podocytes, and is the most frequently mutated gene in patients with Steroid-Resistant Nephrotic Syndrome (SRNS). Analyzing the role of posttranscriptional and -translational regulations of Podocin in the maintenance of the architecture and functional integrity of the slit diaphragm will contribute to the understanding of the underlying pathomechanisms. In previous studies, we confirmed the expression of a short isoform of Podocin in the human kidney, characterized its biochemical properties in vitro and showed an altered localization of the protein. In addition, we identified phosphorylation sites within Podocin by analyzing the glomerular phosphoproteome and found evidence for their biological significance, e.g. for Podocin multimerization (p.T234).

Methods

We used different CRISPR/Cas9 based genome editing strategies that enabled us to alter the murine Nphs2 gene in a targeted manner. The endonuclease Cas9 forms a complex with a guide RNA which selectively binds to DNA and induces double strand breaks. Subsequently, repair mechanisms can either lead to random indel mutations via Non-Homologous End-Joining (NHEJ) or precise mutations via Homology-Directed Repair (HDR). By pronuclear injection we generated a mouse line that, in analogy to the human short isoform, lacks the entire exon 5. In an additional approach we eliminated a phosphorylation site within the PHB domain (p.T234I) by integrating a point mutation.

Results

Strikingly, in vivo data of compound-heterozygous animals show a rapid development of proteinuria and elevated retention parameters indicating a loss of kidney function. Histological examinations reveal Fokal-Segmental Glomerulosclerosis (FSGS), a hallmark of glomerular disease. Mice homozygous for the T234I allele do not display an overt phenotype and up to now we did not observe living offspring homozygous for the short isoform allele.

Conclusion

CRISPR/Cas9 technology allows us to create a fast and efficient pipeline to generate mutant alleles mimicking human genetic diseases and our pioneer project already provided novel insights into the pathogenesis of SRNS.