Kidney Week

Abstract: FR-PO329

Characterization of the Human Podocin Short Isoform In Vivo Using a New Mouse Model

Session Information

• Genetic Diseases: Models, Mechanisms, Treatments
  November 04, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
  Abstract Time: 10:00 AM - 12:00 PM

Category: Genetic Diseases of the Kidneys

• 1102 Genetic Diseases of the Kidneys: Non-Cystic

Authors

• Butt, Linus, Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany

Linus Butt,

• Unnersjö-Jess, David, Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany

David Unnersjö-Jess,

• Rinschen, Markus M., Aarhus Universitet Institut for Biomedicin, Aarhus, Denmark

Markus M. Rinschen,

• Schermer, Bernhard, Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany

Bernhard Schermer,

• Benzing, Thomas, Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany

Thomas Benzing,

• Höhne, Martin, Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Faculty of Medicine and University Hospital Cologne, Cologne, Germany

Martin Höhne,

Background

To date, many mutations affecting podocyte-specific genes are known to cause steroid-resistant nephrotic syndrome, which usually progresses to end-stage renal disease due to a lack of effective therapeutic strategies. NPHS2, encoding the podocyte-specific protein podocin, is among the most frequently mutated genes in patients with hereditary steroid-resistant nephrotic syndromes. Previously, a short isoform of podocin lacking exon 5 was identified in human kidney tissue and characterized in vitro. However, its role in vivo remained elusive.

Methods

The short isoform of podocin is not found in any other organism except humans. To examine its functional relevance in vivo we generated a mouse line expressing the equivalent podocin isoform (podocin^Δexon5) using CRISPR/Cas9 mediated genome editing. We characterized the phenotype of mice expressing podocin^Δexon5 resembling the human short isoform. Targeted mass spectrometry and qPCR were used to compare protein and RNA levels of podocin^wildtype and podocin^Δexon5. STED microscopy following immunolabeling of slit diaphragm components was applied to visualize alterations of the podocytes’ foot process morphology.

Results

Homozygous podocin^Δexon5 mice born heavily albuminuric, hardly produced any urine and did not survive past the first day after birth. Targeted mass spectrometry revealed decreased protein levels of podocin^Δexon5 whereas RNA abundance was not different to the canonical form (podocin^wildtype). STED microscopy revealed the complete absence of podocin at the podocytes’ slit diaphragm and severe morphological alterations of podocyte foot processes. Mice heterozygous for podocin^Δexon5 were phenotypically unaffected but decreased podocin levels and altered foot process morphology could be detected.

Conclusion

The murine equivalent to the human short isoform of podocin is unable to stabilize the lipid-protein complex at the slit diaphragm. Reduction of podocin levels at the site of the slit diaphragm complex has a detrimental effect on podocyte morphology and decreases protein abundance of nephrin, the core protein of the extracellular slit diaphragm complex. In conclusion, our data support the hypothesis that podocin acts an orchestrator of the slit diaphragm complex, which allows for kidney ultrafiltration. The functional role of the short human isoform remains elusive.

Funding

• Government Support – Non-U.S.