4-year PhD Studentship: Can glomerular podocyte damage cause widespread vascular permeability in cardiovascular disease?

Cardiovascular disease is the leading cause of premature death in chronic kidney disease [1] and proteinuria is an independent risk factor of cardiovascular mortality [2]. Reducing proteinuria can prevent cardiovascular complications [1], suggesting a causative link. Blood vessels are coated with a protective layer, the endothelial glycocalyx (eGlx). Damage to eGlx results in increased vascular permeability, including in glomeruli (proteinuria). Proteinuric kidney diseases are associated with widespread vascular permeability and eGlx damage. We have shown that targeting eGlx can restore vascular integrity in the glomerular [3] and systemic [4] circulation. Our preliminary data shows directly that proteinuria, caused by targeted glomerular podocyte damage, results in increased systemic permeability/leak. This is correlated with decreased eGlx depth. Heparanase (HPSE), which cleaves heparan sulphate (HS), an essential component of eGlx, is upregulated by injured podocytes in proteinuric disease [5]. We hypothesise that podocyte-induced HPSE upregulation causes vascular eGlx damage in proteinuric disease.

Aims and objectives

Hypothesis: Podocyte HPSE upregulation causes widespread vascular permeability

Aim 1: Create adeno-associated virus to express heparanase in mouse podocytes

An adeno-associated virus (AAV) approach will be used to drive targeted expression in podocytes, since a conditional, inducible HPSE mouse has not been made and we have specific and unique expertise in driving gene expression in podocytes

I. Validate in vitro

Aim 2: Induce podocyte expression of heparanase in adult mice

Ubiquitous HPSE expression induces proteinuria. We will drive podocyte-HPSE expression in mice in vivo and demonstrate the impact on eGlx HS and on vascular permeability/leak in microvessels at a distance from the kidney.

I. Confirm that this is a model of proteinuria

II. Quantify vascular permeability/leak in mesenteric, coronary and retinal microvessels

III. Quantify eGlx depth in mesenteric, coronary and retinal microvessels

Methodology

Aim 1: Create adeno-associated virus to express heparanase in mouse podocytes

I. Validate in vitro

Human HPSE with a myc tag will be ligated into AAV2/9, containing a human minimal nephrin promotor (AAV-HPSE). Mouse conditionally immortalised podocytes will be infected with multiplicity of infection (MOI) of 1x105. Infection will be quantified by RNA extraction and qPCR for viral particles. Transduction will be confirmed by HPSE activity and protein expression by Western blotting.

Aim 2: Induce podocyte expression of heparanase in adult mice

I. Confirm that this is a model of proteinuria

Six mice will be tail vein injected with AAV-HPSE. Urine will be collected weekly to assess proteinuria. Local and systemic HPSE activation/expression will be quantified.

II. Quantify vascular permeability/leak in mesenteric, coronary and retinal microvessels

Vascular permeability/leak will be quantified in these vessel beds using labelled albumin/NaCl and in vivo confocal microscopy or using transmission electron microscopy to estimate edema.

III. Quantify eGlx depth in mesenteric, coronary and retinal microvessels

Mice will be sacrificed at 4wk post AAV injection. EGlx depth will be quantified using confocal imaging and analysis or transmission electron microscopy

(typically three years but can be up to four years).

How to apply for this project

This project will be based in Bristol Medical School - Translational Health Sciences in the Faculty of Health Sciences at the University of Bristol.

Please visit the Faculty of Health Sciences website for details of how to apply