Using zebrafish to investigate new genes implicated in kidney disease

Supervisors: Dr David Long, Professor Steve Wilson, Professor Adrian Woolf

Background:
The main job of the kidney is to remove waste products from the blood. To do this the blood is filtered through a unit called the glomerulus, a structure which is conserved across diverse species including humans, mice and zebrafish. In normal physiology, macromolecules such as albumin are almost completely excluded from entering the filtrate by the glomerular filtration barrier consisting of endothelia, podocytes and glomerular basement membrane (GBM)(1). Major disruption of the glomerular filtration barrier occurs in children and adults with nephrotic syndrome and other kidney diseases leading to massive protein leakage into the urine(1). Therefore, devising new treatments that maintain the structure and integrity of the glomerular filtration barrier and prevent protein leakage are of critical importance to improve the quality of life of patients with kidney disease. Recently, we have identified several unsuspected new molecules whose expression levels in the glomerulus are associated with the development of proteinuria and may represent novel biomarkers and therapeutic targets for early kidney and vascular disease(2). In this proposal, we will use zebrafish to answer the question whether these changes in gene expression are simply reactions to albuminuria, or if they may actually drive glomerular pathobiology.

Methods:
The student will:

1) Establish a model of protein leakage in zebrafish(3) by abrogating the activity of a key glomerular gene, nephrin. We will utilise several techniques to assess the effect on the biology of the glomerulus including histology, examination of low molecular weight dextran and using vitamin-D binding protein (VDBP) as a tracer of proteinuria(4-5).
2) Examine expression of candidate genes in the zebrafish protein leakage model.
3) Use transgenic approaches to overexpress or knock-down candidate genes in the zebrafish glomerulus and examine the effect on protein leakage.

The student will gain a broad experience in the following fields and techniques working not only at ICH, but also in the laboratory of Professor Steve Wilson at UCL: understanding mechanisms of kidney developmental and cell biology; working with zebrafish; manipulation of gene expression in zebrafish using morpholinos and transgenic approaches, histology; expression analyses at the protein and mRNA levels (in-situ hybridisation, immunohistochemistry, western blotting and quantitative RT-PCR.

References:
(1) Patrakka J, Tryggvason K. Biochem Biophys Res Commun 2010; 396: 164-16
(2) Long DA et al. Kidney Int 2013, (Feb 27, e-pub ahead of print)
(3) Drummond IA. J Am Soc Nephrol 2005, 16: 299-304
(4) Ferrante MI et al. Hum Mol Genet 2009, 18: 289-303
(5) Zhou W, Hildebrandt F. J Am Soc Nephrol 2012 23: 1039-1047