MICA: Signalling pathways to proteinuria - part II. Establishment of b3 integrin and TRPC6 as tractable renal disease targets

Lead Research Organisation: University of Bristol
Department Name: Clinical Science at North Bristol

Abstract

A major factor in morbidity and mortality worldwide is end stage renal disease (ESRD). The UK currently has around 40,000 patients on renal replacement therapy, over 650 per million population, at a cost of over £700 million per year resulting in 2% of the NHS budget being spent on less that 0.1 % of the population. At least 10% of ESRD is caused by steroid resistant nephrotic syndrome (SRNS). This devastating disease is typically associated with oedema, proteinuria, hypertension, microscopic haematuria, and renal insufficiency and usually leads to end stage renal failure despite the use of prolonged and toxic immunosuppression. A difficult and intriguing aspect of SRNS is that in many cases, it will recur following kidney transplantation. The incidence of SRNS, which is particularly common in children, has increased markedly recently with the latest epidemiological study showing a dramatic increase in SRNS as a proportion of primary glomerulopathy from 17 to 59% between 1992 and 2002. Although the cause of SRNS is still unknown, the fact that up to 60% of patients who receive a first kidney transplant to treat their SRNS, experience recurrence of the condition suggests that the cause is not just a result of intrinsic kidney disease. The recurrence of the disease in transplanted patients (often within minutes or hours of the graft being perfused) and the fact that immunosuppressive drug therapy and plasma exchange have proven to be useful in treating the recurrence of SRNS led to the 'circulating toxic factor hypothesis' in the pathogenesis of the disease.
The kidney filtration barrier is made up of two cell types: glomerular endothelial cells and podocytes. We have and others have shown that the podocyte is specifically damaged in SRNS and also provided robust evidence that the toxic SRNS factor belongs to a class of proteins known as proteases. Proteases bind to specific receptors (PARs) on the surface of cells leading to changes in cell biology and the purpose of this application is to identify the cellular signalling pathways that mediate these effects and determine if we can block proteins specifically involved in that pathway, as new drug targets with minimal toxicity, to treat this devastating disease.
Identifying the cellular mechanisms underlying the development of SRNS is essential given its clinical importance and is a critical step in designing and developing targeted therapeutic approaches to deal with this problem.

Technical Summary

The leading cause of acquired established renal failure (ERF) in children is Steroid Resistant Nephrotic Syndrome (SRNS). This devastating disease usually leads to ERF despite the use of prolonged and toxic immunosuppression. The rapid recurrence of disease (within hours) in transplanted patients has contributed to the 'circulating factor hypothesis' acting via unknown receptor(s) to dysregulate the kidney podocyte. Our observations shed light on novel disease mechanisms linking the activation of protease receptors, especially PAR-1, which signal to the podocyte cytoskeleton resulting in changes in podocyte motility and functional breakdown. We have shown with existing MRC funding that transgenic expression of constitutively active PAR-1 on podocytes (pod-PAR-1active) results in dramatic proteinuria and a phenocopy of severe SRNS. Excitingly, biopsy samples from human SRNS corroborate increased expression of activated PAR-1. In vitro studies reveal a signalling 'platform' downstream of PAR-1, comprising the podocyte specific protein podocin, VASP, TRPC6 and b3 integrin. The latter two are highly attractive targets for clinical therapy with novel compounds.
We now wish to demonstrate a direct role for TRPC6 and b3 integrin in mediating PAR-1 mediated proteinuria, so that specific compounds can be tested in vivo. We will achieve this by:
(1) Continuing current work to cross the pod-PAR-1active mouse with a pod-TRPC6 knockout mouse, to attenuate proteinuria.
(2) Cross the pod-PAR-1active mouse with a podocyte specific b3 integrin deleted mouse, to attenuate proteinuria.
(3) Correlate these findings utilising a national patient cohort with SRNS, to investigate active PAR-1 on podocytes in biopsy specimens, thus for the first time stratify patients with 'circulating factor disease'.
(4) Test specific inhibitors of b3 integrin and TRPC6 using our model, in a collaboration with industry partners, to deliver rationalisation for clinical trials in this population

Planned Impact

Realising the objective of this programme of research will benefit the following groups:
1. Biomedical scientists wishing to understand the normal biochemistry and physiology of the kidney especially those interested in the mechanisms through which the glomerular filtration barrier is maintained and regulated.
2. Biochemists seeking to understand the regulation of actin dynamics and the intra and inter molecular interactions of proteins.
3. Patients with glomerular disease and clinicians - The main non-academic beneficiaries will be patients with kidney disease, which is an ever-growing scourge in the developed world with major health economic implications. Improved understanding of the normal biochemistry and physiology of the kidney will in the future lead to further identification of the causes of proteinuria and kidney disease. Understanding the mechanisms underlying the normal functioning of the glomerular filtration barrier does have the potential to lead to new targets/diagnostics/possible therapeutic interventions that impact on this community by combating both kidney failure and also associated cardiovascular disease. The likely timescale for this is 5-10 years. We have numerous industrial links especially with Evotec and these companies are actively working towards anti-proteinuric therapies. We will be ideally placed to share in the development and testing of new agents. We have clinical trial experience alongside our laboratory track record: the PIs in the Academic Renal Unit include 4 academic clinicians who can lead translational application of this work.
4. The general public. The other non-academic beneficiaries will be the public: we are committed to public engagement and the PIs have many years of experience between them of addressing lay groups including patients, carers and the general public. The SRNS Rare Disease Group is a national group with multiprofessional and lay membership, led by Professor Saleem, and organises annual or biannual patient away days.
5. Patient organizations and Charities- specific charities and patient organisations such as Kidney Research UK and the nephrotic syndrome trust (NeST) will be better able to inform patients about research that will benefit their own disease in the next 5-10 years.
6. Industry - by forming additional partnerships with industrial/pharmaceutical companies we will develop new assays and drug/compound pipelines based on new regulatory mechanisms in the podocyte that we identify in this programme, thus creating commercial opportunities for a worldwide market.
7. The UK economy. The UK currently has over 40,000 patients on renal replacement therapies, around 700 per million population, total cost over £700 million per year, over 2% of NHS budget being spent on 0.1% of the population. Recent UK government figures show that chronic kidney disease affects at least 3% of the population. New understanding of the normal functioning of the glomerualr filtration barrier will lead to new research avenues in to the causes of glomerular diseases.

Publications

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Preston R (2019) A role for OCRL in glomerular function and disease in Pediatric Nephrology

 
Description Dissociation of CD2AP from the slit diaphragm leads to transcriptional changes in WT1, causing podocyte phenotypic change.
Amount £58,607 (GBP)
Funding ID ST2/2010 
Organisation Kidney Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2010 
End 10/2013
 
Description Global Challenges Research Fund
Amount £586,833 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 03/2017 
End 03/2019
 
Description Innovate UK KTP scheme
Amount £150,000 (GBP)
Funding ID KTP 11232 
Organisation Innovate UK 
Sector Public
Country United Kingdom
Start 09/2020 
End 12/2021
 
Description MICA: NURTuRE - changing the landscape of renal medicine to foster a unified approach to stratified medicine
Amount £2,561,603 (GBP)
Funding ID MR/R013942/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 06/2018 
End 07/2022
 
Description MICA: Signalling pathways to proteinuria - part II. Establishment of b3 integrin and TRPC6 as tractable renal disease targets
Amount £507,855 (GBP)
Funding ID MR/R003017/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 12/2017 
End 11/2020
 
Description Signalling pathways to Proteinuria
Amount £517,412 (GBP)
Funding ID MR/L002418/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 11/2013 
End 08/2017
 
Description Stratified Medicine
Amount £3,140,000 (GBP)
Funding ID MR/R013942/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 04/2018 
End 05/2022
 
Description Trans-national cohorts of nephrotic syndrome - a unified approach to a global chronic disease
Amount £532,743 (GBP)
Funding ID MR/P024297/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 03/2017 
End 03/2020
 
Title Glomsphere development 
Description The glomerulus is the filtration unit of the kidney. Injury to any component of this specialised structure leads to impaired filtration and eventually fibrosis and chronic kidney disease. Current two and three dimensional (2D and 3D) models that attempt to recreate structure and interplay between glomerular cells are imperfect. Most 2D models are simplistic and unrepresentative, and 3D organoid approaches are currently difficult to reproduce at scale and do not fit well with current industrial drug-screening approaches. Here we report a rapidly generated and highly reproducible 3D co-culture spheroid model (GlomSpheres), better demonstrating the specialised physical and molecular structure of a glomerulus. Co-cultured using a magnetic spheroid formation approach, conditionally immortalised (CI) human podocytes and glomerular endothelial cells (GEnCs) deposited mature, organized isoforms of collagen IV and Laminin. We demonstrate a dramatic upregulation of key podocyte (podocin, nephrin and podocalyxin) and GEnC (pecam-1) markers. Electron microscopy revealed podocyte foot process interdigitation and endothelial vessel formation. Incubation with pro-fibrotic agents (TGF-ß1, Adriamycin) induced extracellular matrix (ECM) dysregulation and podocyte loss, which were attenuated by the anti-fibrotic agent Nintedanib. Incubation with plasma from patients with kidney disease induced acute podocyte loss and ECM dysregulation relative to patient matched remission plasma, and Nintedanib reduced podocyte loss. Finally, we developed a rapid imaging approach to demonstrate the model's usefulness in higher throughput pharmaceutical screening. GlomSpheres therefore represent a robust, scalable, replacement for 2D in vitro glomerular disease models. 
Type Of Material Model of mechanisms or symptoms - human 
Year Produced 2021 
Provided To Others? Yes  
Impact Collaborative tool developed with UCB pharma Further development with funding and project plans Also licensed to Purespring Therapeutics 
 
Description Travere industry funding for Endothelin project 
Organisation Retrophin
Country United States 
Sector Private 
PI Contribution Set up spheroid models in Bristol Renal laboratory, for testing of angiotensin and endothelin inhibitors in our cellular models
Collaborator Contribution Funding a post-doc Supply of compounds for detailed testing
Impact Started 2020
Start Year 2020
 
Title THERAPY 
Description The application provides gene therapies for treating monogenic forms of nephrotic syndrome. 
IP Reference US2021402008 
Protection Patent application published
Year Protection Granted 2021
Licensed Yes
Impact Licensed as part of University spinout of Purespring Therapeutics
 
Company Name PURESPRING THERAPEUTICS LIMITED 
Description World's first renal gene therapy ocmpany Series A funding of £45M from Syncona Professor Saleem is Founder and Chief Scientific Officer 
Year Established 2020 
Impact 4 patents filed