Modelling Alport syndrome in zebrafish

Alport syndrome (AS) is a hereditary disorder characterised by defects in the kidneys, eyes and ears. ¬AS patients present with microhaematuria in the early years of life and subsequently develop proteinuria as the blood filter degenerates. Current treatment for AS relies on angiotensin-converting-enzyme (ACE) inhibitors, which act to lower blood pressure and reduce damage to the renal filters caused by mechanical loading. Despite ACE inhibitors being effective in delaying the onset of chronic kidney disease (CKD) in AS patients, they do not completely prevent the decline of kidney function and patients develop end stage renal disease, for which the only treatments are dialysis and renal transplantation.

AS is caused by mutations in COL4A3, COL4A4 and COL4A5, which encode collagen Type IV proteins that are important components of basement membranes. In the kidney, the basement membrane found in the blood filter (glomerulus) is enriched with collagen Type IV a3, a4 and a5. These collagens form a heterotrimeric helix in the extracellular matrix that polymerises into a complex tertiary structure. This network acts as a scaffold to provide structural support to the filter and facilitates a number of signalling networks that maintains the health of the glomerulus.

In patients with CKD, proteinuria is a common feature and occurs as a result of damage to the blood filter. Such damage can be observed by changes in the blood filter ultrastructure. The specialised epithelial cells (podocytes) that form the slit diaphragm undergo dedifferentiation, effacement and detachment during CKD.

In addition, an abnormally split or laminated glomerular basement membrane is common in CKD. These pathologies are observed in AS, and therefore AS is considered a robust model of CKD with proteinuria and studies in the AS model are likely to have wider relevance across the spectrum of CKD.

AS research in the laboratory setting has relied primarily on the study of Col4a3-/- mice, which present with the glomerular phenotypes described above. This model has enabled novel paradigms to be discovered about the molecular mechanisms associated with AS. However, the ability to test novel therapies in the Col4a3-/- mouse is limited by low fecundity and high maintenance costs. To overcome these drawbacks, the Lennon laboratory has used gene-editing tools to generate col4a3, col4a4 and col4a5 knockout lines in the zebrafish. Analysis of the blood filters in these fish show they have defective blood filter phenotypes consistent with those observed in human AS patients. All three mutant lines also present with proteinuria at embryonic stages (between 3-5 days post fertilisation).

An important advantage of the zebrafish system over mammalian models is its high fecundity (200-300 eggs laid per female per week). This offers the researcher the ability to perform high throughput chemical screens of compounds that may preclude or delay the onset of disease. The aim of this project is to characterise the zebrafish model of Alport syndrome. This project has the potential to identify novel therapies that could be used to impact the lives of patients with AS and CKD. This project also has the potential to determine novel molecular mechanisms by which collagen IV networks function in the basement membrane, which could lead to future research programmes and discoveries.

The post will be hosted in the Lennon Lab within the Wellcome Trust Centre for Cell-Matrix Research at the University of Manchester. The focus of the research group is glomerular cell and matrix biology, with a particular interest in the regulation of glomerular cell adhesion in health and disease. The Cell-Matrix Centre has an international reputation for excellence in the field of adhesion and matrix biology and offers a fantastic setting for doctoral training and research. The Centre is extremely well supported by Core Facilities offering access to and training in the use of cutting edge technologies including bioimaging, electron microscopy, mass spectrometry, genomic and transgenic technologies. For more information about the Cell-Matrix Centre, please visit our website: http://www.wellcome-matrix.org

Training/techniques to be provided -
The student will be trained in cutting edge molecular biology techniques (CRISPR/Cas9, Gibson Assembly, Gateway cloning, use of snapgene software) and zebrafish manipulation and husbandry. It is expected that the student will become fluent in the use of these techniques, which fit with the MRC aim to advance skills for interdisciplinary techniques (molecular biology) and whole organ/ organism physiology (zebrafish in vivo study). The student will also be trained in the use of confocal microscopy imaging, FRET imaging, histology and RNA-seq (which fulfils the MRCs request for training in omics approaches).