Aldosterone-induced endothelial glycocalyx dysfunction, a potential therapeutic target in proteinuria?

In health the kidney act as a filter, keeping cells and proteins within our blood whilst allowing toxins to pass into the urine. The presence of a significant quantity of protein in the urine (proteinuria) indicates that the kidney filtering units, called glomeruli, have been damaged. Proteinuria is common affecting 4% of healthy adults and over 20 % of those with chronic kidney disease. It is important because the presence of proteinuria places individuals at a much higher risk of progressive kidney failure, heart attacks and strokes.

In order for protein to leave the blood and enter the urine it has to pass through a number of cell layers collectively called the glomerular filtration barrier. This forms a key part of our kidneys and normally prevents protein from entering the urine. The barrier consists of two types of cell, podocytes and endothelial cells. The endothelial cells form the innermost layer of cells within our blood vessels. Endothelial cells are covered by an additional protective layer called the glycocalyx. This layer is produced by the endothelial cells and is a mix of proteins and sugars. It forms a jelly like covering on the cells surface. This layer has been found to have a number of important jobs. It regulates the passage of cells and proteins from the blood into the tissues. It detects how fast the blood is moving helping to regulate tissues blood flow and it prevents blood from clotting unnecessarily

So far a number of conditions have been shown to affect the glycocalyx, I believe that a hormone called Aldosterone is likely to damage the glycocalyx and that this damage results in protein leaking into the urine. Aldosterone is a hormone that helps animals regulate the amount of salt and water in their bodies. Its main action is on kidney cells to make them keep sodium within the body and loose potassium into the urine. When we are healthy the level of aldosterone in our blood is tightly regulated however the levels are increased in a number of conditions including; high blood pressure, obesity, renal failure, sleep disorders and by some medications (including ACE inhibitors and angiotensin receptor blockers which are commonly used in the UK to treat high blood pressure).

Clinical studies in patients with proteinuria have shown that blocking aldosterone reduces the amount of protein that leaks from the blood into the urine preserving kidney function. Blocking aldosterone also helps patients with poor heart function. Kidney doctors are reluctant to prescribe aldosterone-blocking drugs, however, because of their side effects. In kidney disease potassium levels in the blood can become high. Blocking aldosterone can worsen this problem. If the potassium level becomes too high it can send the heart into a dangerous rhythm. This means most kidney patients miss out on an important drug that could help preserve their kidney function. My provisional work on human cells in the lab has shown that aldosterone damages the glycocalyx. The next step in my research is to confirm that aldosterone damages the glycocalyx in living animals. I think it is likely that aldosterone causes cells to make an enzyme called heparanase. When this is released into the blood this enzyme removes heparan sulphate from the glycocalyx damaging its structure. I believe if I can prevent this process I can protect the glycocalyx and potentially reduce proteinuria without causing high potassium levels.

I will be working in the University of Bristol laboratories within a group focused on the glycocalyx within the kidney. The group has an excellent international reputation for work involving the glycocalyx and is led by renal physicians. In my role as a clinician I know that there is a clinical need for further therapies to reduce proteinuria. Studying this novel pathway may yield new therapeutic targets suitable for clinical practice free from the side effect of high potassium.

Hypothesis- Aldosterone acts on glomerular endothelial cells (GEnC), to initiate a chain of biochemical events that result in damage to the endothelial glycoclayx (Glx) and proteinuria.

Research objectives
1. Demonstrate aldosterone disrupts the GEnC Glx with functional effects including increased protein permeability
2. Confirm this effect is dependent on the direct action of aldosterone on GEnC
3. Review if GEnC Glx disruption is caused by heparanase release from GEnC

Methodology- Human conditionally immortalised glomerular endothelial cells (CiGEnC) will be used in vitro. Glx response to Ald, and protection by heparanase inhibition will be assessed by immunofluorescence (IF) of key Glx components, shear stress sensitivity and Glx permeability to FITC-labeled BSA. In vivo aldosterone exposure will be assessed using osmotic minipumps to administer 0.6micg/g/day aldosterone to FBV/N mice to render them proteinuric by 4/52. Glomerular endothelial MR knock out mice (Tie2-rtTA, Tet-O-Cre, MRflox/flox mice) will be used to confirm these effects are due to GEnC MR receptors. Outcome measures will include proteinuria and Glx damage. Glx will be directly visualised and permeability assessed using multiphoton microscopy.2 Immunofluorescence will be used to quantify key glomerular Glx components and electron microscopy used to assess alterations in Glx thickness. Spironolactone and SST0001 (specific heparanase inhibitor) will be used to confirm the roles of MR/heparanase in the process.

Opportunities- Proteinuria places patients at high risk of progressive kidney disease and systemic vascular disease. Proteinuria results from a failure of the glomerular filtration barrier of which the Glx is a vital part. Ald has been shown to cause proteinuria and damage the Glx in vitro. Clarifying the effects of Ald in vivo and mapping a pathway potentially amenable to pharmacological intervention could provide a novel series of therapeutic targets for future research.

The impact of this study can be divided into immediate gains in knowledge and potential long-term gains. The long term gains can to divided into development of research tools, individuals training, commercial applications for heparanase inhibition. The immediate implications of establishing a causal link between aldosterone excess and glycocalyx damage have been outlined in the "academic beneficiaries" section. Longer term the implications of endothelial protection are far reaching.
1. Patients with proteinuria- As discussed previously a causal link has yet to be established between proteinuria and chronic kidney disease (CKD) progression however we know that medications capable of reducing proteinuria delay CKD progression. Providing clinicians with additional medications to reduce proteinuria in the future could significantly reduce the risk of CKD progression.

2. The NHS/ tax paying public- Renal replacement therapy includes dialysis and renal transplantation. There is currently a shortfall in donor organ supply and so the majority of patient with end stage renal disease require dialysis. In 2009-10 the cost of CKD to the English NHS was £1.44 billion, approximately 1.3% of the total NHS spending. More than half this sum went towards RRT, which provided for just 2% of the CKD population. Economic modeling suggest 7000 excess strokes and 12000 excess MIs occurred in the CKD population in 2009-10 relative to matched controls1. This excess represents an additional £174 million spend. In addition there is a human impact, dialysis, strokes and MIs (if survived) are associated with significant reductions in individuals quality of life and social dependence. Discovering a method of endothelial protection that could simultaneously reduce proteinuria - slowing CKD progression, and protect individuals with CKD from vascular complications could therefore save the NHS millions and reduce the burden of disease.

3. The general public. Another non-academic beneficiaries will be the public: we are committed to public engagement and the applicants have many years of experience between them of addressing lay groups including patients, carers and the general public. Improved education about the importance of urine testing for protein will have tangible health benefits in a relatively short timescale.

4. Charities - This research program will make conceptual changes in an important area of glomerular biology and disease and so influence the direction of future research. Targeting the endothelial glycocalyx therapeutically will become much more viable once tools become available to study the glycolayx. The methods pioneered in this fellowship will be made freely available. This will allow more efficient use of scarce financial resources spent by charities on research.

5. Patient organizations - specific charities such as the nephrotic syndrome trust and diabetes UK patient groups will be better able to inform patients about research that will benefit their own disease.

6. Commercial partners- If a causal link is established between aldosterone excess, endothelial mineralocorticoid receptors, heparanase production and glycocalyx damage then our commercial collaborators would be an ideal team to help us investigate this area further. This project is being run/ supervised by 3 clinically active renal physicians who will be ideally placed to drive forward future research. The compound SST0001 specifically inhibits heparanase and Sigma Tau have already entered phase 1 trials for the treatment of myeloma. By the time of completion of this fellowship they will have significant experience of human dosing using this product. This will allow us to consider human trials of heparanase inhibition in proteinuria in future studies.

1. Kerr, M., Bray, B., Medcalf, J., O'Donoghue, D.J. & Matthews, B. Estimating the financial cost of chronic kidney disease to the NHS in England. Nephrol Dial Transplant 27 Suppl 3, iii73-80,2012