Determining the Role of Canonical Notch Signalling in the Regulation of the Glucocorticoid Receptor in Steroid Resistant Nephrotic Syndrome
Lead Research Organisation:
University College London
Department Name: Institute of Child Health
Abstract
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.
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.
Technical Summary
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.
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.
Planned Impact
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.
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.
Publications
Asfahani RI
(2018)
Activation of podocyte Notch mediates early Wt1 glomerulopathy.
in Kidney international
Balogh E
(2020)
Pseudouridylation defect due to DKC1 and NOP10 mutations causes nephrotic syndrome with cataracts, hearing impairment, and enterocolitis.
in Proceedings of the National Academy of Sciences of the United States of America
Chanell O Haley
(2018)
Malformations of the Murine Kidney are associated with loss of Cenp-F function
Chesher D
(2018)
Outcome of adult patients with X-linked hypophosphatemia caused by PHEX gene mutations.
in Journal of inherited metabolic disease
Haley CO
(2019)
Malformations in the Murine Kidney Caused by Loss of CENP-F Function.
in Anatomical record (Hoboken, N.J. : 2007)
Kari JA
(2014)
Clinico-pathological correlations of congenital and infantile nephrotic syndrome over twenty years.
in Pediatric nephrology (Berlin, Germany)
Kim JJ
(2015)
The clinical spectrum of hemolytic uremic syndrome secondary to complement factor H autoantibodies.
in Clinical nephrology
Noone D
(2014)
Successful treatment of DEAP-HUS with eculizumab.
in Pediatric nephrology (Berlin, Germany)
Tahoun M
(2020)
Mutations in LAMB2 Are Associated With Albuminuria and Optic Nerve Hypoplasia With Hypopituitarism.
in The Journal of clinical endocrinology and metabolism
Tahoun M
(2020)
Mutations in LAMB2 are associated with albuminuria and optic nerve hypoplasia with hypopituitarism
in Yearbook of Paediatric Endocrinology
Description | ERKNet: European Reference Network for Rare Kidney Diseases |
Geographic Reach | Europe |
Policy Influence Type | Membership of a guideline committee |
Guideline Title | Rituximab in Nephrotic Syndrome |
Description | Support for Commisioning for Rituximab use in Nephrotic Syndrome |
Geographic Reach | National |
Policy Influence Type | Citation in clinical guidelines |
URL | https://www.england.nhs.uk/commissioning/wp-content/uploads/sites/12/2015/10/e03pb-rituxmb-nephrtc-s... |
Description | John Feehally Stoneygate Research Awards |
Amount | £49,658 (GBP) |
Funding ID | JFS_IN_005_20160916 |
Organisation | Kidney Research UK |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 03/2017 |
End | 03/2018 |
Description | Targetting Glomerulosclerosis: Novel Strategies for Steroid Resistant Nephrotic Syndrome |
Amount | £198,000 (GBP) |
Funding ID | Paed_RP_011_20170929 |
Organisation | Kidney Research UK |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 09/2018 |
End | 09/2022 |
Title | Nephrotic Syndrome Database |
Description | Database of nephrotic patients attending Nephrotic Clinic at Great Ormond Street Hospital |
Type Of Material | Database/Collection of data |
Provided To Others? | No |
Impact | Identifies patients that have refractory nephrotic syndrome that requires genetic testing, evaluation of new treatment strategies, in addition to relapsing forms of nephrotic syndrome that are immunologically mediated |
Title | UK STEC-HUS Registry |
Description | This database aims to prospectively collect clinical, biochemical, microbiological data on all UK STEC-HUS cases with the aim of providing a future platform for research in STEC-HUS. |
Type Of Material | Database/Collection of data |
Provided To Others? | No |
Impact | This is in the early stages of development |
Description | Investigating the role of PAR2 in the renal glomerulus |
Organisation | Memorial University of Newfoundland |
Country | Canada |
Sector | Academic/University |
PI Contribution | We have identified a novel pathogenic mutation in the PAR2 gene in a kindred with autoimmune thrombocytopaenia and renal thrombotic microangiopathy. |
Collaborator Contribution | Our collaborators have par2 knockout mice and are sending us tissue for histological analysis. |
Impact | No outputs yet |
Start Year | 2013 |
Description | Regulatory RNA Modification |
Organisation | Yale University |
Country | United States |
Sector | Academic/University |
PI Contribution | Dr Wendy Gilbert is a biologist who is interested in pseudouridylation profiles of regulatory RNAs. We have identified mutations in genes implicated pseudouridylation of telomerase in syndromic nephrotic syndrome. MRC Harwell team have awarded my group a point mutant mouse model in which we will explore pseudouridylation profiles of regulatory RNAs in podocyte injury. Dr Gilbert will be undertaking pseudouridylation sequencing in primary podocytes harvested from these mice. |
Collaborator Contribution | MRC Harwell team have awarded my group a point mutant mouse model in which we will explore pseudouridylation profiles of regulatory RNAs in podocyte injury. Dr Gilbert will be undertaking pseudouridylation sequencing in primary podocytes harvested from these mice. |
Impact | MRC Harwell team have awarded my group a GEMM award that will allow study of pseudouridylation profiles of regulatory RNAs in primary podocytes harvested from the point mutant mouse model that recapitulates the human phenotype. |
Start Year | 2017 |
Description | UK STEC-HUS Rare Disease Working Group |
Organisation | Renal Association |
Country | United Kingdom |
Sector | Charity/Non Profit |
PI Contribution | I am coordinating the development of a national registry for Shiga-Toxin Associated Haemolytic Uraemic Syndrome (STEC-HUS) in collaboration with the Rare Renal Disease Registry (RaDaR). We aim to prospectively collect a national cohort of STEC-HUS for future research studies. |
Collaborator Contribution | My partners are coordinating data collection from their respective centres nation-wide. |
Impact | No outputs yet |
Start Year | 2012 |
Title | ECUSTEC: A Randomised Double Blind Placebo Controlled Trial of Eculizumab on Disease Severity in E Colic Haemolytic Uraemic Syndrome |
Description | A humanized monoclonal anti-C5 antibody, already approved for the management of atypical haemolytic uraemic syndrome and paroxysmal nocturnal haemoglobinuria will be tested in a Phase 2 trial in E Coli-Associated Haemolytic Uraemic Syndrome to determine if intervention will reduce disease severity. |
Type | Therapeutic Intervention - Drug |
Current Stage Of Development | Early clinical assessment |
Development Status | Actively seeking support |
Clinical Trial? | Yes |
Impact | The above outcomes will be tested as trial has not yet begun. |
URL | http://www.isrctn.com/ISRCTN89553116?q=&filters=&sort=&offset=96&totalResults=15354&page=1&pageSize=... |
Description | ECUSTEC trial meeting with HUSH patient representative group |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | Requesting participation and input from patient representative group on how to best engage potential participants for newly funded clinical trial to assess the benefits of Eculizumab in STEC-HUS |
Year(s) Of Engagement Activity | 2016 |
Description | International Paediatric Nephrology Conference |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | 1. Invited Speaker: Pneumococcal Haemolytic Uraemic Syndrome - lecture to international Consultant and Trainee Paediatric Nephrology colleagues. 2. Selected Free Communication: Expression of Ascl1 Precedes Podocyte Notch Activation in Wt1 Glomerulopathy |
Year(s) Of Engagement Activity | 2016 |
URL | http://www.ipna2016.com/content/program-glance |
Description | International eHUSc Consortium Teleconferences |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | Yes |
Type Of Presentation | Workshop Facilitator |
Geographic Reach | International |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | For working group development, PG3 and international colleagues joined our steering group to enhance our strategic agenda of developing an international consortium to facilitate the host biological determinants of VTEC-HUS. PG3 Annual Conference asked for VTEC-HUS Steering Committee member to join at conference |
Year(s) Of Engagement Activity | 2013 |
Description | Lecture on MSc Child Health Module for UCL 2017 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Professional Practitioners |
Results and Impact | Teaching on Nephrotic Syndrome |
Year(s) Of Engagement Activity | 2017 |
Description | Lecture on Steroid Resistant Nephrotic Syndrome, Annual Nephrology Course for General Paediatricians |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | 50 clinicians attended which provoked a question and answer session afterwards. Improved other practitioners understanding |
Year(s) Of Engagement Activity | 2013,2014 |
Description | Nephrogenesis Workshop |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | Free Communication: Expression of Ascl1 Precedes Podocyte Notch Activation in Wt1 Glomerulopathy |
Year(s) Of Engagement Activity | 2016 |
Description | Organisation of A Session on Nephrotic Syndrome: Annual GOSH-ICH Nephro-Urology Week 2017 |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Organisation of Guest Speaker and Session Speakers on Nephrotic Syndrome |
Year(s) Of Engagement Activity | 2016 |
URL | https://www.ucl.ac.uk/ich/short-courses-events/short-courses-events-publication/continuing-education... |
Description | Organised Seminar on Haemolytic Uraemic Syndrome for Annual GOSH Nephro-Urology Course |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Over 50 clinicians attended this seminar and improved their understanding of the latest developments in the field. Attention to the prospective UK STEC-HUS data collection for the Rare Renal Disease Registry was made. |
Year(s) Of Engagement Activity | 2013,2014 |
Description | Overview of Childhood Nephrotic Syndrome |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Postgraduate students |
Results and Impact | Teaching postgraduate students at UCL who are undertaking a medical degree |
Year(s) Of Engagement Activity | 2018 |
Description | Podocyte Notch Activation Mediates Early WT1 Glomeulopathy |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | NOTCH X Meeting, Athens |
Year(s) Of Engagement Activity | 2017 |
Description | UK Kidney Week |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | Free Communication: Expression of Ascl1 Precedes Podocyte Notch Activation in Wt1 Glomerulopathy |
Year(s) Of Engagement Activity | 2016 |
Description | UK STEC-HUS RDWG Teleconferences |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Participants in your research and patient groups |
Results and Impact | National engagement of paediatric nephrologists treating STEC-HUS (etsimated to be >=50) for a proof of concept study investigating a therapeutic strategy for STEC-HUS. No impact yet |
Year(s) Of Engagement Activity | 2014 |