Forsøksdyr: Establishment of a novel zebrafish model of Fabry nephropathy

Godkjenningsdato 02.07.2018

Chronic kidney disease is a serious, life-threatening condition with highly increasing incidence. Although the pathways leading to the development of chronic kidney diseases are diverse, the final picture of a progressed disease shows a uniform picture of renal fibrosis, represented by profound glomerular and tubulointerstitial injury. The purpose of this experiment is to develop a novel model to study kidney diseases which could be used for development of novel therapies of kidney diseases. Zebrafish has shown to be a useful vertebrate model to study genetic human disease. High genetic conservation with humans is reflected in the highly conserved microstructure of the kidney. Especially, the renal filtration unit, the glomerulus, which is highly conserved between the species. This has been used in the past to study glomerular diseases, such as chemically induced podocyte attrition. Our laboratory in Bergen has a long lasting interest and experience with studying the renal consequences of a monogenic lysosomal storage disease - Fabry disease, referred to as Fabry nephropathy. The condition develops as a result of a mutation in the gene coding for alpha-galactosidase A (GLA). The lack of the enzyme results in accumulation of globotriaosylceramide (Gb3) and other glycosphingolipids inside cells. The kidney is one of the organs seriously affected by such deposits.
The CRISPR/Cas9 technique has revolutionized and simplified the process of development of novel zebrafish models of human genetic diseases. We aim to establish a zebrafish model of Fabry nephropathy using CRISPR/Cas9 to knock-out the GLA gene. In addition, we plan to investigate an alternative pathway involved in the pathomechanisms behind the development of the Fabry nephropathy. Acid ceramidase (Asah1) is an alternative enzyme involved in the removal of Gb3. Hence, in total, we plan to establish three different zebrafish lines, with systemic knock out of GLA, Asah-1 and a combination of both.
This new model will be the basis for better understanding of the disease and testing of novel anti-fibrotic therapies with possible implications for patients with other types of kidney diseases. The level of distress will be minimal (tail clipping) to moderate (edema induced by kidney disease).
The total number of embryos will be 600. The total number of fish in F1 generation will be 300. A maximum of 90 fish from the F1 generation will be used to keep the line. The lowest possible amount of embryos/fish to maintain sufficient microinjection and propagation efficiency will be used.
Considering replacement, reduction and refinement, the number of the animals was calculated based on previous work by Gagnon et al. 2014, in order to use the minimal number of animals to yield the desired results. Moreover, for high throughput genotyping of zebrafish, the High Resolution Melting Analysis (HRMA) will be used to distinguish wildtype, heterozygous mutants and homozygous mutants. This technique is known for its effectiveness in reducing the number of zebrafish needed for each transgenic line (Parant et al., 2009).