Abstract: SA-PO322
Elucidating the Pathogenesis of Focal Segmental Glomerulosclerosis Using CRISPR/Cas9 Mediated Genome Engineering
Session Information
- Cellular Crosstalk in Glomerular Diseases - II
October 27, 2018 | Location: Exhibit Hall, San Diego Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1201 Glomerular Diseases: Fibrosis and Extracellular Matrix
Authors
- Butt, Linus, University Hospital Cologne, Cologne, Germany
- Unnersjö-Jess, David, Royal Institute of Technology, Solna, Sweden
- Rinschen, Markus M., University Hospital Cologne, Cologne, Germany
- Reilly, Dervla, University Hospital Cologne, Cologne, Germany
- Ester, Lioba, University Hospital Cologne, Cologne, Germany
- Brismar, Hjalmar, Royal Institute of Technology, Solna, Sweden
- Brinkkoetter, Paul T., University Hospital Cologne, Cologne, Germany
- Schermer, Bernhard, University Hospital Cologne, Cologne, Germany
- Benzing, Thomas, University of Cologne, Köln, Germany
Background
NPHS2 encodes for the PHB-domain protein podocin and is one of the most frequently mutated genes in patients with Focal Segmental Glomerulosclerosis (FSGS). Currently, loss of podocytes is regarded as the hallmark of the pathogenesis of FSGS. Investigating the initial molecular events causing podocyte injury and depletion is of utmost importance for future preventive and therapeutic strategies.
Methods
Here, we present a mouse model for a late-onset FSGS phenotype. Using CRISPR/Cas9 mediated genome engineering, we separately integrated two point mutations in the Nphs2 gene which, in compound-heterozygosity, represent a commonly found genotype in patients with a late-onset.
Results
In vivo data of compound-heterozygous animals reveal an early onset of mild proteinuria long before the manifestation of FSGS lesions. The proteinuria increases gradually and reaches nephrotic range in young adult mice (> 8 weeks). Histologically, FSGS lesions are found between 8 and 20 weeks. By using STED microscopy at different time points (P0, 2, 4, 8, 20 weeks) we show a shortening and widening of the foot processes and a decreasing containment of the capillary surface by the secondary processes in compound-heterozygous in comparison to control mice. Quantification of podocyte numbers in kidney sections revealed a progressive decline in mutant mice. The glomerular proteome at 3 weeks shows a general decrease in podocyte specific proteins, in particular of polarity related proteins.
Conclusion
In this study, we have generated a late-onset FSGS disease model resembling human genetic disease by using CRISPR/Cas9 mediated genome engineering. Mutant mice develop progressive proteinuria accompanied by a full blown morphological manifestation of FSGS. Delineation of the foot process microarchitecture by STED microscopy shows a loss of the distinct microarchitecture of the podocyte and a decreased containment of the capillary surface as an early pathological sign that precedes the onset of massive proteinuria. Interestingly, the containment is relatively steady throughout the first phase of podocyte depletion, possibly by expansion of individual foot processes. The shift in the glomerular proteome is consistent with a podocyte dedifferentitation and/or loss of polarity.