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

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.

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

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.