Targeting endothelial-erythrocyte glycocalyx exchange for the novel diagnosis and treatment of renal disease

Endothelial cells form a continual lining within our blood vessels. They are covered by a protective layer called the glycocalyx. This layer is made from a mix of proteins and sugars. It forms a jelly like covering on the cells surface. This glycocalyx performs several 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. When the glycocalyx fails to perform these roles blood vessels cease to function optimally, which subsequently results in organs becoming damaged. Damage to the delicate glycocalyx layer occurs in several human diseases. In diabetes damage to the endothelial glycocalyx happens very early on, before predisposed patients develop detectable kidney disease. In animal models of diabetes, I have shown that intervening at this early stage when the glycocalyx is damaged (but before protein starts to be detectable in the urine) can prevent the development of kidney disease. In humans not everyone with diabetes develops glycocalyx damage or kidney disease. We believe that patients that develop a damaged glycocalyx early in the disease course will be the most likely to subsequently develop diabetic kidney damage. Identifying these high-risk patients would allow clinicians to prioritise which patients to see more regularly and which patients to treat more aggressively with existing medications.

Currently, human glycocalyx damage is not easily detectable with a clinical test. However, I have developed a novel blood test to study glycocalyx changes that could be developed for clinical practice. In this project I will confirm that my test is giving us a true picture of how the glycocalyx changes throughout the body by simultaneously studying the endothelial glycocalyx at multiple sites. This study needs to be performed in animal models of disease to allow us access to all the tissue we need. To perform this study in the UK would need large numbers of diabetic animals. However, by collaborating with a research group in Los Angeles I can re-use a technique that I developed to measure glycocalyx depth changes during my PhD. Using their cutting edge microscope, I can image blood vessels deep inside anesthetised mice and measure how the glycocalyx is changing inside the organs and skin as diabetes develops. I can measure the glycocalyx depth at these sites every week and compare the changes I detect with my blood test to prove that my test is reflecting the changes we see.

To check that my test can predict which diabetic patients will go on to develop damage to their blood vessels I will include my test in the established European study 'BEAt-DKD'. This study is currently enrolling participants and will allow me to compare my test to the current gold standard tests of endothelial damage and collect follow-up data to test my hypothesis that we can predict who will develop detectable diabetic nephropathy. I will simultaneously work on the development of my test by working with computer scientists at the University of Bristol to refine and automate the analysis whilst integrating machine learning into the programming. Machine learning should let the computer get better and better at performing the analysis for us as it learns from its mistakes to become a faster and more reliable with time.

Finally, I will be use the information I learn by studying changes in the endothelial glycocalyx to develop a novel method of reapplying key parts of the glycocalyx to the blood vessel wall. My method will target the areas of the circulation where the glycocalyx is most damaged by focusing repairs on areas where the blood does not flow smoothly. This novel repair system could be used when patients are at the highest risk of acute glycocalyx damage e.g. after trauma and sepsis, or in patients with chronic glycocalyx damage e.g. patients on haemodialysis.

The endothelial glycocalyx (EnGlx) is composed of interwoven proteins and sugars. It lines blood vessels, limiting permeability to macromolecules, regulating local blood flow and clotting, and restricting the interaction with circulating cells. In diabetes damage to glomerular EnGlx results in increased albumin leakage. During the earliest phase of diabetic nephropathy (DN) the renal tubules re-absorb and metabolise filtered albumin, preventing detectable albuminuria. By the time albuminuria is detectable extensive damage has occurred and the potential impact of reno-protective strategies (e.g. angiotensin converting enzyme inhibition) are limited. However, DN does not develop in all diabetic patients. I believe that if we could detect Glx damage we could identify the subset of patients at the highest risk of subsequently developing DN. I have developed a lectin-based method to assess the EnGlx on fixed human tissue and shown that patients with DN have a damaged EnGlx. I have since developed a blood assay to detect Glx damage and shown that it accurately predicts glomerular Glx changes and directly measured glomerular albumin permeability in rats. In addition, I have demonstrated a novel mechanism of Glx damage and repair using an in vitro human cell model. A patent covering this novel work is being submitted. I now plan to test the predictive power of my test in human diabetic patients by collaborating with the BEAt-DKD research team in Exeter whilst investigating its effectiveness in patients with minimal change nephrotic syndrome by establishing my own study in Bristol. I will also continue work on the novel mechanism of Glx repair I have discovered, testing if it can be harnessed to deliver 'pre-formed Glx' selectively to areas of endothelial damage. In addition, I will work with the computer sciences department to develop software to further automate my assay, ensuring that it can be used in large scale trials and clinical practice in the future.