Determining the Role of Canonical Notch Signalling in the Regulation of the Glucocorticoid Receptor in Steroid Resistant Nephrotic Syndrome

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Scarring of the glomerulus can cause terminal kidney disease. Glucocorticoid treatment has a beneficial effect but resistance (GR) develops over time. Alternative treatments are available with significant side effects and only 30-60% of patients respond. Consequently, new treatments are needed. Recently, we have shown that activation of a critical signalling pathway (Notch) is involved in initiating glomerular scarring. Using knockout mouse models and drugs which block the pathway, we and others, have shown that glomerular scarring can be prevented. Studies in leukaemic patients have shown that Notch activity can inhibit the glucocorticoid response, an effect which can be reversed by inhibiting Notch. My preliminary data shows that the glucocorticoid receptor, NR3C1, is expressed in control human glomerular cells in vitro and in vivo and is downregulated in human glomerular cells derived from patients with steroid-resistance. As activation of the Notch effector gene, HES-1, has been shown to downregulate NR3C1 and mediate the development of GR in leukaemia, I have observed that HES-1 is temporally upregulated in podocytes of sclerosed glomeruli in kidney biopsy tissue in patients with nephrotic syndrome who have been initially glucocorticoid sensitive and subsequently developed GR. These data support a role for Notch activation in steroid-resistant nephrotic syndrome.My research objectives will involve testing a few basic principles.Firstly, I would like to demonstrate that glucocorticoid-responsive gene expression occurs in glomerular epithelial cells when treated with glucocorticoid therapy and then determine, absence of expression of the same genes in glomerular cells derived from kidney biopsies of steroid resistant nephrotic syndrome (SRNS) patients. Next, I propose to determine whether expression of components of the Notch signalling pathway are upregulated in podocyte cell lines of SRNS patients. If this is the case, I will then deplete Notch effector genes to determine if I can rescue expression of glucocorticoid-responsive genes.Secondly, I will use mouse models of genetic forms of glomerulosclerosis (scarring of the glomerulus), to determine whether loss of glucocorticoid-responsive gene expression in glomerular epithelial cells is associated with the development of glomerulosclerosis and whether this is also associated with an up regulation of Notch pathway components.I will next specifically explore whether secondary Notch activation in mouse models of genetic forms of glomerulosclerosis is associated with loss of glucocorticoid-responsive gene expression in glomerular epithelial cells - this will be facilitated by generating transgenic mice of Notch activation and examining whether onset of glomerular scarring as a result of Notch activation is associated with loss of glucocorticoid-responsive gene expression in glomerular epithelial cells. If this proves true, chemicals which inhibit the Notch pathway will be used in mouse models of genetic forms of glomerulosclerosis to see whether it is possible to rescue glucocorticoid-responsiveness concommitant with resolution of glomerular scarring.In parallel, genetic analysis of a large cohort of patients with nephrotic syndrome will be undertaken with collaborators in an attempt to further define molecular mechanisms of glomerulosclerosis which may open up new avenues of investigation for therapeutic strategies in nephrotic syndrome.