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

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.

The histological appearance of GS in nephrotic syndrome (NS) can be associated with the development of temporal resistance to glucocorticoid therapy over time. Whether Notch activation is associated with the development of glucocorticoid resistance in GS is currently unexplored.
My preliminary data has shown that the glucocorticoid receptor, NR3C1, is expressed in wild-type human podocytes in vitro and in vivo. Furthermore, in primary podocyte cell lines derived from patients with steroid-resistant nephrotic syndrome (SRNS), NR3C1 expression is downregulated compared to that in wild-type control podocytes. As activation of the canonical Notch effector, HES-1, has been previously shown to downregulate NR3C1 and mediate the development of GR in T-ALL, I have determined by immunohistochemistry that HES-1 is temporally upregulated in podocytes of sclerosed glomeruli in kidney biopsy tissue in patients with NS who have been initially steroid sensitive and subsequently developed steroid resistance. These preliminary data support a requirement to investigate further the role of canonical Notch signalling in the development of GR in human NS. I will use a combination of transgenic murine models of GS in addition to cell lines derived from patients with SRNS to examine for Notch pathway activation (gene and protein expression) and determine whether down regulation of the GR and its target genes are coincident with onset of GS and Notch activation. Thereafter I will utilise an inducible murine model of podocyte-specific Notch activation to directly test the role that Notch plays in regulating GR in podocytes. Pharmacological inhibition of Notch will be employed to determine if GR can be rescued in murine models. In parallel, I will utilise next generation sequencing strategies to search for new nephrotic syndrome disease genes.

Nephrotic syndrome (NS) is a rare childhood disease with an incidence of 1/100, 000 children. If new therapeutic strategies are deemed highly effective, then it is anticipated that there will be a large number of research beneficiaries that will include affected patients and their families, local physicians, nurses and community care staff, tertiary centre admissions offices, local and tertiary centre financial annual budgets, government policy makers on the funding of new therapies for rare diseases.

As glomerulosclerosis is the most common cause of acquired childhood kidney disease necessitating renal transplantation, a need for the development of alternate therapeutic strategies is clearly warranted. My aim is to target those patients with nephrotic syndrome who I see in clinic for whom no further treatment options are available other than dialysis and renal transplantation. If progression to end-stage kidney disease can be delayed through the appropriate introduction of effective treatments, the duration spent in hospital would be greatly reduced, time spent in education increased and less financial burden on families and the NHS achieved.


There is an annual Nephrotic Syndrome patient-away day held by the UK Nephrotic Syndrome Trust whereby research updates are provided by researchers in the field. Participation of UK-wide patients affected with nephrotic syndrome are encouraged to attend. If any significant advances are made such as the identification of new disease genes, patients and their families will be contacted through the Nephrotic syndrome Trust (UK), the NephCure Foundation (US) and PodoNet (Europe) to inform them of new findings should they wish to avail of genetic testing for any new genes identified.

As glucocorticoid resistance can be a significant problem of several other diseases that rely on glucocorticoid-based treatment strategies and given the extent of pre-clinical and early phase clinical trials on the efficacy of Notch inhibitors in the field of cancer biology and diseases such as Alzheimer's Disease, I anticipate that there may be interest from the pharmaceutical companies that are already developing these drugs and seeking new indications for their use. Should this be the case, then this may have wider beneficial consequences on cost of in-patient care and overall disease burden in addition to improving quality of life for affected patients and their families.