The impact of primary and secondary epithelial senescence on renal function and fibrosis

Chronic Kidney Disease (CKD) affects over 850 million people worldwide. It is a disease process which causes the kidneys to become scarred; a term we call 'fibrosis'. Injuries can range from simple things; like short periods of infection and low blood pressure to more complicated and rarer diseases which affect the kidney tissue itself. Regardless of the initial injury and regardless of this initial injury going away completely, we believe that the kidney is often permanently damaged. Often we don't measure this in blood or urine tests; these become normal again. But we know from observational data that having one such episode, even if brief and completely resolved, puts a person at significantly higher risk of developing progressive CKD. At present, we don't have any treatments to stop or slow this progression and this can ultimately lead to complete kidney failure and the requirement for regular dialysis or kidney transplantation. We also know that in individuals with CKD, although the scarring is in the kidneys, their chance of heart disease, and death from heart disease is significantly increased.

There are certain processes and cellular changes which occur in response to these injuries (not just in a kidney, but in other organs too) which we believe contribute to this progressive scarring. If they can be targeted and blocked it could prevent this inevitable scar formation.

This PhD will focus on one such pathway, and a group of cells; called 'senescent cells'. These are cells which are damaged but do not die in a normal programmed way. We know that these cells can persist and communicate with other cells in the body to promote scarring. We find senescent cells in human kidneys in lots of different kidney diseases, we find more in older kidneys and more in the aftermath of kidney injury. So far; we know that we can treat and remove these cells safely in mice and that the mice live longer and healthier lives. If we can study these cells and their signalling pathways in greater detail we believe this would be a potentially very successful treatment and would impact significantly on human kidney disease.

My supervisor's group's recent work has shown that more than one kind of senescent cell exists in the kidney. Some of these cells ('primary senescent cells') occur in direct response to kidney injury. Others ('secondary senescent cells') are generated later in response to signals coming from the primary senescent cells. Important experimental work in the liver has shown that by blocking the signals from the primary senescent cells - it is possible to prevent secondary senescence from happening, and this helps the liver to regenerate after injuries without scarring. It seems likely that similar pathways will contribute to kidney scarring - but this has yet to be tested.

In my fellowship, I will use a novel type of mouse where I can generate senescent cells in one particular cell type ('epithelial cells') in the kidney using a drug treatment with no associated kidney injury. This will allow me to understand how primary senescent cells lead to both secondary senescence and scarring by studying mice and kidney cells growing in the laboratory. By understanding these processes, I hope to discover how they can be blocked in the laboratory, in mice, and eventually in studies in patients - leading to novel treatments to prevent progressive fibrosis in the kidney and other organs.

In this fellowship, I will address the hypothesis that secondary senescence is critical for kidney fibrosis, dysfunction and leukocyte recruitment after injury. I will use our system of transgenic conditional primary (1ry) renal proximal tubular epithelial cell (PTEC) senescence induction to answer 2 questions:

i) Is 1ry PTEC senescence in the absence of renal injury sufficient to induce 2ry PTEC senescence and fibrosis in vitro and in the murine kidney in vivo?

I will use our validated in-house transgenic models of conditional genetic senescent cell (SC) induction and fluorescent labelling in PTECs with either normal TGFb1 expression or with SC TGFb1 knockout to test the effect of 1ry senescence on 2ry PTEC senescence transmission / fibroblast activation in vitro and in vivo. I will:
a) Determine the transcriptome and secretome of 1ry / 2ry PTEC SCs in vitro
b) Assess 2ry SC induction via cell contact, media transfer and TGFb1 signalling, and the ability of TGFb signalling blockade to inhibit these pathways in vitro
c) Test the impact of 1ry PTEC senescence on fibroblast activation in vitro/in vivo
d) Characterise 2ry PTEC senescence, leukocyte kinetics and fibrosis in response to targeted SC induction in vivo and the necessity for TGFb1 release by 1ry SCs

ii) Can early/late depletion / inhibition of SCs prevent fibrosis in vivo?

Using our transgenic model of 1ry SC generation and renal fibrosis, I will test the ability of senolytic therapy with ABT-263 or TGFb1 inhibition with Galunisertib to:
a) Establish if SC killing is sufficient to prevent fibrosis
b) Quantify the therapeutic window where senolytic treatment is effective
c) Test the ability of early TGFbetaRI inhibition to inhibit 2ry senescence and fibrosis (representing a translational target in kidney disease)
d) Sort, single cell sequence and identify 1ry and 2ry SCs in vivo in the kidney
e) Determine the transcriptional trajectories of 1ry and 2ry SCs after injury/therapy

Scientists
The field of senescence research is young but rapidly expanding, with emerging evidence implicating senescent cells as causative in multiple diseases of aging including organ dysfunction and fibrosis. It is also recognised that senescent cells contribute to the prevention of neoplasia, and to successful wound healing. Our existing, unpublished data demonstrates that subsets of senescent cells exist in the fibrotic kidney- and over the course of my fellowship I will use my transgenic mouse studies and pharmacological interventions to unpick the precise identity and behaviour of primary and secondary senescent cells - defining their functions and impact on organ fibrosis. This data will be complementary to studies in press by our collaborators looking at liver fibrosis and our single cell analysis will extend our understanding of the processes underlying the beneficial and deleterious effects of senescence. This data will be of value to scientists seeking to understand the roles of senescence throughout the body, and researchers interested in how signalling pathways and cell fate decisions impact on overall organ function and fibrosis.

Clinicians
Fibrotic CKD forms a major component of the clinical workload of nephrologists with its impact reaching deep into other specialities. There is a major unmet need for therapies which halt progressive kidney damage. One of the key goals of this research is to define the contribution of primary and secondary senescence to renal fibrosis. I hope to both establish this, and determine signalling pathways such as TGFb release which could be amenable to therapeutic intervention; and with that, substantial clinical gain. Within this, I anticipate there being information discovered and published of downstream benefit in many other clinical realms. At present, there are several clinical trials and studies within humans, looking at the impact of senolytics within respiratory, orthopaedic and endocrinology contexts and I expect the work of this fellowship to be able to contribute to this rapidly expanding and exciting field.

Patients with organ fibrosis
Fibrosis is a common finding in progressive dysfunction of multiple organ systems including but not limited to the kidney, heart, lungs and liver. I believe that this fellowship will impact in time on the lives of patients for whom at present there are no effective treatments and provide valuable information on fundamental scientific pathways as yet undefined. We believe that it has clear translational potential and could lead to novel therapeutic interventions in future years.

We will test the efficacy of both senolytic and anti-fibrotic treatments. At present, there are already four clinical trials underway evaluating the efficacy of senolytics in human disease, and agents blocking TGFbeta signalling are also in Phase II clinical studies in other organ diseases. Thus, should this fellowship confirm potential benefit; this would impact significantly on patients with kidney disease, including kidney transplantation. There would also be the potential to extend our work to other organ systems, with the goal of reducing the excess morbidity and mortality attributable to organ fibrosis.

Economic Impact on Healthcare expenditure
More than 1.8 million people in the UK have CKD, with a further million thought to be undiagnosed. Data from 2010 estimate the annual cost at £1.45 billion, rising as our population ages. In addition the significant excess cardiovascular morbidity associated with CKD adds an extra £175million per annum treating MIs and strokes within this group. People with CKD have longer hospital stays, increased propensity for infections and higher levels of frailty. Thus costs covering solely health provisions pertaining to CKD likely grossly underestimate expenditure. There are therefore major long-term economic benefits to developing therapies to reduce the incidence and morbidity associated with CKD