Abstract: SA-PO595
Modelling Alport Syndrome in Zebrafish
Session Information
- Noncystic Mendelian Diseases
November 04, 2017 | Location: Hall H, Morial Convention Center
Abstract Time: 10:00 AM - 10:00 AM
Category: Genetic Diseases of the Kidney
- 802 Non-Cystic Mendelian Diseases
Authors
- Naylor, Richard William, University of Manchester, Manchester, United Kingdom
- Gasiunas, Saule N, University of Manchester, Manchester, United Kingdom
- Lennon, Rachel, University of Manchester, Manchester, United Kingdom
Background
Alport syndrome is a hereditary renal disorder that manifests in early childhood with haematuria followed by proteinuria and ultimate progression to end stage renal disease. Genetic analysis has shown that patients with Alport syndrome carry mutations in genes encoding three isomers of collagen type IV: COL4A3, COL4A4 and COL4A5. Type IV collagens are the most abundant collagens in basement membranes and exist as three different trimeric protomers, α1α1α2(IV), α3α4α5(IV) and α5α5α6(IV). In the glomerulus, podocytes deposit the α3α4α5(IV) trimer and the endothelium deposits the α1α1α2(IV) trimer. The fusion of these two extracellular matrices forms the glomerular basement membrane (GBM). In Alport syndrome, mutations in either COL4A3, COL4A4 and COL4A5 lead to depletion of the α3α4α5(IV) trimer in the GBM. This loss of α3α4α5(IV) initially creates a thinner GBM that at later stages of the disease develops a ‘basket-weave’ appearance. Treatment of Alport syndrome is limited to angiotensin converting enzyme inhibitors but the mechanism of their action on the glomerulus has not been fully elucidated.
Methods
To improve our understanding of disease and treatment mechanisms we aimed to generate a zebrafish model of Alport syndrome. The zebrafish is a highly tractable system that has become a premier organism for disease modelling. Many of the cellular components of the glomerular filter are conserved between zebrafish and humans. We have used in situ hybridisation with RNA probes and immunofluorescence with collagen type IV antibodies to observe if the molecular components of the glomerular filter are also conserved. In addition, we have used the CRISPR/Cas9 system to generate col4a3, col4a4 and col4a5 knockout mutant lines.
Results
We find zebrafish podocytes express col4a3, col4a4 and col4a5 and have also identified collagen type IV isoforms in the zebrafish GBM. We have also found phenotypes in the glomerulus of our knockout lines and have performed functional and ultrastructural analyses.
Conclusion
We have demonstrated expression of the α3α4α5(IV) network in the zebrafish GBM and have created a new in vivo model for Alport syndrome. This model will allow the use of new approaches to investigate disease mechanisms in Alport syndrome and will facilitate high throughput compound screening for drug discovery.
Funding
- Government Support - Non-U.S.