An investigation into the genetic and functional basis of proteinuric kidney disease

Chronic kidney disease affects 13% of the UK population, with 59,000 UK patients either on dialysis or dependent on a kidney transplant for the treatment of kidney failure. Worldwide 1.9 million patients receive these kidney replacement therapies, with many more unable to access these expensive and limited resources.

Kidney disease is often associated with abnormal leak of protein into the urine, for example in rare inherited diseases and in diabetes, the leading cause of kidney failure worldwide. This protein leak is due to failure of the filtration barrier, by which healthy kidneys act as selective sieves, filtering waste products and excess water, but keeping important proteins in the blood. Unique cells called podocytes are key component of this barrier, but because they do not replicate and are difficult to alter genetically, podocytes present specific challenges for research.

In order to develop more effective, safer treatments, we need to understand more about how damage to podocytes and the filtration barrier leads to protein leak and disease.

Genome sequencing projects in patients have begun to reveal many genes associated with a severe protein leak. However, because humans carry many genetic differences from each other it can be difficult to pinpoint which gene from a shortlist of candidates actually causes the disease. The aim of the project is to address this problem, by applying recent advances in techniques to manipulate genes precisely and efficiently. These methods will be used to develop and study models of kidney disease carrying patient mutations. By measuring the effects of gene disruption on cells and organisms, we will identify new disease genes, leading directly to a genetic diagnosis for more patients with rare kidney diseases. This genetic diagnosis can help patients and families, even if it does not immediately change treatment, by quantifying risk in different individuals and helping with planning.

The models of human gene variation produced in this project will lead to more detailed study of kidney biology at the level of cells and tissues. One further aim of the project is to explore ways to connect genes to disease, using sequencing techniques to measure the landscape of 'switched on' genes. These techniques will gather and compare information at the level of individual cells and kidney tissues. In the future this approach may have clinical applications in human tissue such as kidney biopsy samples.

In the long term, better tools for investigating genetic differences and greater understanding of the effects of mutations on pathways and cellular systems will provide new targets for treating kidney disease, improve outcomes for individuals and reduce the health and economic burdens of chronic kidney disease.

The aim of the project is to discover novel genetic causes of renal disease, in the process developing and characterising models to link genes to function at the cellular and whole animal level.

These models will provide an opportunity to examine the molecular basis of kidney disease and therefore a secondary aim is to develop tools to explore the transcriptomic profile of affected cells and tissues, linking genes to function.

Proteinuria is a hallmark of many kidney diseases and indicates dysfunction of the glomerular filtration barrier, and in particular podocyte loss and de-differentiation. Genetic manipulation of podocytes has been historically challenging due to their terminal differentiation and resistance to transfection.

The project will optimise methods for precise CRISPR gene editing in immortalised podocytes, and apply these to generate both CRISPR podocyte lines and murine models mimicking human variants. Functional validation tools are increasingly needed to establish causation for many rare variants identified in large scale whole genome sequencing projects. The project will begin with variants identified using whole genome sequencing in a national cohort of patients with severe nephrotic disease (NephroS, University of Bristol).

Access to cells and tissues from the in vitro and in vivo models provides an opportunity to study the pathways and mechanisms by which the human genetic variants lead to clinical phenotypes. The outcomes of this research will be novel genetic insights into kidney disease, the development of clinically relevant tools to model and test for the effects human genetic variation on renal pathology, and discoveries and methods that will improve the diagnosis and treatment of human disease.

Several groups beyond the academic community may benefit from this project.

1. Patients with the genetic variants modelled in the study.
This project uses genetic data from a large national registry of patients with severe renal protein leak, the nephrotic syndrome, to identify new genes causing this disease. Therefore it can have a direct impact by providing a genetic diagnosis for patients. This may apply initially only to 1 or 2 families or individuals, but we will go on to look for alterations in these genes within patients enrolled in two large national genetic studies, and this is likely to lead to more diagnoses. The impact of genetic diagnosis can be far reaching: screening of other family members can be offered, and negative testing can identify potential kidney donors in a family. With more accurate clinicopathological diagnoses, it will be possible to give more accurate predictions about outcomes and conduct more productive trials of new therapy in these individuals.

2. Local people with genetic kidney disease.
Regionally, renal patients with hereditary disease will benefit from the clinical genetic service in Oxford linked to my research. Access to specialist services will increase patient participation in trials and involvement in patient groups. With earlier diagnosis, monitoring, and new treatments emerging for genetic renal disease, the clinical goal of the service will be better patient experiences and health.

3. The wider renal patient community and public
A better understanding the underlying mechanisms of kidney disease is a prerequisite for future development of personalised and targeted treatments.
Genes and pathways identified in this project may lead to the development of new therapies, and the tools for modelling genetic variation could be scaled up to test drugs.

Renal biopsy is a routine investigation in the evaluation of the patient with renal disease, but interpretation relies on microscopic appearances and specific stains. Tools developed in his project could be applied to human biopsy tissue to give a more detailed view of cellular activity, providing a rich dataset to better classify renal disease.

My public engagement programme of 'pop up' activities, and my communications plan, will raise the public profile of genetic testing, genetic research and kidney disease, amongst patients, hospital visitors and staff, and the wider public at science events and online.

This is an opportunity for education and dialogue about potentially controversial or misunderstood research techniques, including gene editing and management of patient genetic data. Engaging the public can inform research priorities, and enable people to make informed choices about their own data and health.

4. Economics and Policy making
Better diagnostics will reduce the cost of futile testing, and accelerate treatment or at least reduce inappropriate interventions. The aim of better treatments and earlier diagnosis is to reduce the number of patients reaching end stage kidney disease. Haemodialysis costs £25,000 per patient per year, in addition to the costs of managing co-morbidity and lost earnings due to chronic ill health, so even a small change can have major socioeconomic benefits.
Efficient validation tools for human genetic variants, as developed in this project, will improve the scope and success rates of clinical genetic programmes, providing evidence to justify investment and policy supporting clinical integration of next generation sequencing.

5. Researchers working on the project
The academic clinical trainees and research assistant working on this project will gain new skills and expertise, supporting their professional development. I hope to inspire those working on the project to pursue or progress their research careers, armed with a toolkit of skills and enthusiasm for kidney biology and genetics.