The discovery and validation of biomarkers of renal epithelial senescence in the human kidney.

Chronic kidney disease (CKD) is common; it affects 8% of the UK population. It is important because patients with kidney disease are at higher risk of death, particularly from heart problems. It is often progressive and, in some patients, leads to kidney failure meaning they need to start dialysis or get a kidney transplant to survive. A major clinical need exists for non-invasive tests to identify 'high-risk' patients to target with emerging therapies.

Senescence is the term used to describe cells that have stopped functioning and regenerating normally. Senescent cells accumulate in aged and injured kidneys, and drive scarring and loss of function. In recent studies, giving medications to mice that target and kill senescent cells (known as senolytic medications) allowed them to live longer in good health and improved the function of multiple organs including the kidneys. At present, the only methods available for measuring senescence in the kidney require a kidney biopsy but this if often not performed due to the risk of bleeding and is a key limitation of trials of senolytic medications that are now underway in humans.

This fellowship will test whether levels of proteins in the urine can act as non-invasive 'biomarkers' of senescence levels in the kidney. In this study, I will have available a large collection of patient urine and biopsy samples. My laboratory group has already identified 11 proteins that have been shown to link with senescence in experimental models of CKD and are potential biomarkers. In paired samples from 100 patients, the levels of the 11 candidate biomarkers will be measured in the urine whilst the amount of senescence in their kidneys will be measured using biopsy samples. I will determine which biomarkers most closely match levels of the senescence on the biopsy. The urine samples from 20 patients (the 10 with the highest and lowest levels of senescence respectively) will undergo additional testing to see if there are other proteins, that relate more closely with the amount of senescence on the biopsy that we are not yet aware of.

The top performing 5 biomarkers from the above studies will then be taken forward to the next phase. Urinary levels of these biomarkers will be measured in samples from 600 patients with kidney disease with over 450 of these patients having 5-year follow-up data available. This will allow me to determine if our urinary biomarkers of senescence can be used to predict which patients develop a more rapid decline in the kidney function.

Finally, I will measure urinary biomarker levels in a study already ongoing which is testing a senolytic medication, known as ABT-263, on human kidneys being perfused outside the body. Using samples from before and after treatment, I will be able to determine if urinary levels of the senescence biomarkers change following a treatment to deplete senescent cells. Identifying markers that are measured non-invasively and fall with successful senescent cell depletion, will be an important tool in future studies using senolytic medications in humans.

Senescent (dysfunctional, permanently growth arrested) cells are widely recognised to drive fibrosis and organ dysfunction. Their depletion improves the health of multiple organs including the kidney and prolongs lifespan in aged mice. This fellowship will test the hypothesis that biomarkers present in human urine will correlate to senescent cell number in the kidney and predict adverse renal outcomes in humans.

Building on existing studies in our lab, I will address three aims:
1) Determine which candidate biomarkers of senescence in patient urine samples best correlate with senescent cell numbers in the kidney.
2) Determine if levels of the most promising urinary biomarkers predict 5-year patient outcomes.
3) Test the ability of urine biomarker measurement to identify successful pharmacological depletion of senescent cells.

Aim 1 will measure 11 existing candidate biomarkers in urine from 100 patients from the seNSOR renal biorepository using ELISA, and correlate these with senescence quantified histologically using p16INK4a and p21CIP1 in matched renal biopsy samples. Additionally, urine LC-MS/MS analysis will be used to identify any novel factors unique to human CKD in a subset of patients with the highest and lowest levels of histological senescence.

Aim 2 will correlate up to 5 of the best-performing biomarkers alone and in combination with existing markers of CKD progression risk in 600 patients (all of whom have 4+ years, and >450 having 5+ years follow-up) testing the ability of senescence quantification to predict patients at high risk of rapid progression.

Aim 3 will analyse urine collected in an ongoing study (funded by Kidney Research UK and CSO) treating human kidneys undergoing normothermic perfusion ex vivo with vehicle control or the senolytic drug ABT-263. Pre- and post-treatment samples will be available and provide data supporting a non-invasive urine test that can be used to assess the efficacy of senolytic therapy in humans.