A global approach to prevent secondary microvessel complications in diabetes

Lead Research Organisation: University of Bristol
Department Name: Bristol Medical School

Abstract

People with diabetes have high blood sugar. This can act like a toxin, causing damage to the small blood vessels all around the body and making them leakier. Leaky small blood vessels in the kidney and eye, can lead to diabetic kidney disease and diabetic eye disease (secondary complications).

All blood vessels are lined with a protective seaweed-like layer called the glycocalyx. People with diabetes have a thinner glycocalyx. People with diabetes also have more proteins in the blood (like heparanase) that can act like scissors and chop up the glycocalyx. This happens early in diabetes before secondary blood vessel complications develop, such as diabetic kidney or eye disease. We believe that damage to the glycocalyx in diabetes can lead to leakiness in all blood vessels. We have shown that repairing the glycocalyx in diabetes stops blood vessels from leaking in the kidney. We have some data to suggest this may also happen in the eye. If damage to the glycocalyx is the same in all blood vessels in diabetes, then we may be able to protect them in the same way.

Little is known about the glycocalyx in the eye and whether it is involved in the development of diabetic eye disease. We aim to understand how the glycocalyx normally prevents leakiness of small blood vessels in the eye. We then aim to find out whether diabetes affects the glycocalyx in the eye. Our main and final aim is to use a drug to stop proteins in the blood, like heparinase, from chopping up the glycocalyx. We will then find out if the small blood vessels in the eye and kidney are protected in diabetes and work better as a result. This work will be done in rats with diabetes.

If we can make the small blood vessels in the eye and kidney work better in diabetes, it is likely that all other small blood vessels will also work better. This will mean that we can use one drug to prevent all small blood vessel complications in diabetes.

Technical Summary

Objective 1: To establish the structural changes in e-Glx at retinal endothelial cell clefts, caused by HS loss, that are associated with increased solute flux.
i. Human retinal endothelial cells: HS will be disrupted by enzymatic removal (heprainase III) or knock down (shRNA to the HS biosynthesis enzyme, Ext1). Solute permeability will be assessed across monolayers using electrical cell impedence sensing (ECIS) and labelled albumin passage. E-Glx structure and junctional cleft parameters will be assessed by electron microscopy (EM).
ii. Rat retina: E-Glx HS will be removed using an i.v. bolus of heparinase III. Retinal solute flux will be assessed by sodium fluorescein angiography (NaF). HS removal and albumin leak will be confirmed by immunofluorescence on retina. E-Glx structure and junctional cleft parameters will be quantified by EM.

Objective 2: To establish structural endothelial glycocalyx HS changes in retinal endothelial cells in diabetes
Adult male Wistar rats will receive a single dose of streptozotocin (STZ, 50mg/Kg) or vehicle. Retinal NaF will be measured at 2wk and 4wk post STZ. Urine albumin creatinine ratios (uACR) will be determined at baseline and endpoint. HS removal, retinal albumin leak, e-Glx structure and junctional cleft parameters will be confirmed as in aim 1ii. E-Glx HS immunofluorescence and heparanase activity will be carried out on healthy versus diabetic retina from human donor eyes.

Objective 3: To protect e-Glx HS and reduce microvascular permeability in experimental diabetes.
Diabetes will be induced in rats as in aim 2. A heparanase inhibitor will be administered i.p. daily for the last 2wk in cohort 1: 4wk post-STZ and cohort 2: 12wk post-STZ. E-Glx HS changes will confirmed by immunofluorescence on fixed retina and glomeruli. E-Glx structural and junctional cleft parameters in the retina and glomerulus quantified as above. Functional microvascular changes will be confirmed by NaF angiography and uACR.

Planned Impact

Realising the objective of this programme of research will benefit the following groups:

1. Diabetic patients with and without secondary complications- The main non-academic beneficiaries will be patients with diabetes, which is an ever-growing scourge in the world (8.5% of the adult population world-wide), with major health economic implications. The likely timescale to treatment following this project is 7 years because this treatment is already being investigated clinically. We have numerous industrial links and these companies are currently actively working towards anti-proteinuric therapies. We will be ideally placed to share in the development and testing of new agents. We have clinical trial experience alongside our laboratory track record: the PIs include 2 academic clinicians who can lead translational application of our work.

2. The general public. The other non-academic beneficiaries will be the public: we are committed to public engagement and the PIs have many years of experience between them of addressing lay groups including patients, carers and the general public

3. Charities - This research programme will make conceptual changes in diabetic kidney disease and other vascular diseases in diabetes and so influence the direction of future research into avenues following the targets identified. This will allow more efficient use of scarce financial resources spent by charities on research.

4. Patient organizations - specific charities will be better able to inform patients about research that will benefit their own disease in the next 5-10 years.
5. Academic Researchers - as described under academic beneficiaries

6. Clinicians - understanding and developing treatments for patients with secondary diabetic complications, both by identifying targets and subsequent design of clinical trials, will allow clinicians to participate in the scientific advances being made as well as informing patients of new treatment options on the horizon.

7. Industry - by forming additional partnerships with industrial/pharmaceutical companies we will develop new drug/compound pipelines based on targets that we identify in this programme, thus creating commercial opportunities for a worldwide market.

8. The UK economy. Diabetes currently costs 10% of the total NHS budget with 5 million diabetic individuals in the UK. The majority of these patients will die from secondary complications. This project will target the complications associated with diabetes, in order to cut back the majority of NHS spending on this condition.
 
Description A patient PPI group has been set up
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Participation in a guidance/advisory committee
 
Description A global approach to prevent secondary microvessel complications in diabetes
Amount £404,309 (GBP)
Funding ID MR/T031921/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 07/2020 
End 07/2023
 
Description Adiponectin receptor agonists prevent the development of kidney disease through protection of the glomerular endothelial glycocalyx
Amount £211,026 (GBP)
Funding ID RP_005_20221128 
Organisation Kidney Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2023 
End 10/2026
 
Description Aldosterone-induced endothelial glycocalyx dysfunction, a potential therapeutic target in proteinuria?
Amount £250,592 (GBP)
Funding ID MR/M018237/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 07/2015 
End 07/2018
 
Description Endothelial glycocalyx restoration to prevent proteinuria-associated vascular damage
Amount £200,168 (GBP)
Funding ID BHF PG/20/10187 
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2021 
End 03/2024
 
Description Investigating the role of glomerular endothelial glycocalyx dysfunction in the renal injury of pre-eclampsia
Amount £301,435 (GBP)
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2022 
End 09/2025
 
Description Targeting endothelial-erythrocyte glycocalyx exchange for the novel diagnosis and treatment of renal disease
Amount £1,086,513 (GBP)
Funding ID MR/W024187/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 09/2022 
End 10/2027
 
Description Vascular endothelial glycocalyx dysfunction as a therapeutic target in sepsis-associated acute kidney injury (sAKI)
Amount £293,771 (GBP)
Funding ID PG/22/11121 
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2023 
End 01/2026
 
Title Glomerular albumin permeability assay 
Description This assay uses isolated glomeruli from kidneys of rodents/humans. Kidneys are perfused with a red membrane label (R18) and and greeen labelled albumin (AF488-BSA). The decline in fluorescence intensity of labelled albumin over time, within a single capillary loop, will be imaged and analysed using confocal microscopy. This allows the relative permeability to albumin to be calculated. This is a very powerful and novel assay whihc allows comparisons of glomerular albumin permeability bewteen groups (i.e. with/without diabetes) 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? Yes  
Impact We can determine the relative glomerular albumin permeabilty of isolated human glomeruli for the first time. We can also determine the effects of stimuli on protein passage in a very controllesd system. For example, we can ezymatically remove the glycocalyx layer from the microvessels in the kidney and determine how that affects protein passage within short time frames. 
 
Description Heparanase inhibitor 
Organisation Victoria University of Wellington
Department Ferrier Research Institute
Country New Zealand 
Sector Academic/University 
PI Contribution The Ferrier Institute provides a novel heparanase inhibitor that we use in various disease models to show protection of endothelial glycocalyx and rescue of vascular function
Collaborator Contribution They provide the inhibitor
Impact Publication in preparation Monica Gamez's PhD thesis MRC grant
Start Year 2017
 
Description Horizon ERC Synergy application 
Organisation Keele University
Country United Kingdom 
Sector Academic/University 
PI Contribution We are writing a Horizon application together and have secured in-person funds for travel for all PIs to meet in-person to write the bid.
Collaborator Contribution Lead PIs
Impact Cardiovasc Diabetol. 2024 Feb 1;23(1):50. doi: 10.1186/s12933-024-02133-1.
Start Year 2017
 
Description Horizon ERC Synergy application 
Organisation Victoria University of Wellington
Country New Zealand 
Sector Academic/University 
PI Contribution We are writing a Horizon application together and have secured in-person funds for travel for all PIs to meet in-person to write the bid.
Collaborator Contribution Lead PIs
Impact Cardiovasc Diabetol. 2024 Feb 1;23(1):50. doi: 10.1186/s12933-024-02133-1.
Start Year 2017
 
Description Invited workshop hosted by KRUK, Diabetes UK and JDRF 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Clinicians, academics, those with lived experience and charity workers met to discuss the research gaps in diabetes and diabetic kidney disease. A publication is planned.
Year(s) Of Engagement Activity 2024
URL https://www.kidneyresearchuk.org/2024/02/14/diabetes-and-kidney-research-charities-team-up-to-tackle...
 
Description Patient participation group 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Patients, carers and/or patient groups
Results and Impact 8 patients met online to discuss a format for our new patient participation group. The meeting was 2 hr long and we plan to reflect on this meeting in our next meetings moving forward.
Year(s) Of Engagement Activity 2022,2023
 
Description Press release- heparanase paper 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact A press release was co-ordinated with the publication of our Cardiovascular Diabetology manuscript.
Year(s) Of Engagement Activity 2024
URL https://www.ukri.org/news/new-drug-could-prevent-diabetic-eye-and-kidney-disease/