Reactive Oxygen Species Modulate the Barrier Function of the Human Glomerular Endothelial Glycocalyx

Abstract

Reactive oxygen species (ROS) play a key role in the pathogenesis of proteinuria in glomerular diseases like diabetic nephropathy. Glomerular endothelial cell (GEnC) glycocalyx covers the luminal aspect of the glomerular capillary wall and makes an important contribution to the glomerular barrier. ROS are known to depolymerise glycosaminoglycan (GAG) chains of proteoglycans, which are crucial for the barrier function of GEnC glycocalyx. The aim of this study is to investigate the direct effects of ROS on the structure and function of GEnC glycocalyx using conditionally immortalised human GEnC. ROS were generated by exogenous hydrogen peroxide. Biosynthesis and cleavage of GAG chains was analyzed by radiolabelling (S35 and 3H-glucosamine). GAG chains were quantified on GEnC surface and in the cell supernatant using liquid chromatography and immunofluorescence techniques. Barrier properties were estimated by measuring trans-endothelial passage of albumin. ROS caused a significant loss of WGA lectin and heparan sulphate staining from the surface of GEnC. This lead to an increase in trans-endothelial albumin passage. The latter could be inhibited by catalase and superoxide dismutase. The effect of ROS on GEnC was not mediated via the GAG biosynthetic pathway. Quantification of radiolabelled GAG fractions in the supernatant confirmed that ROS directly caused shedding of HS GAG. This finding is clinically relevant and suggests a mechanism by which ROS may cause proteinuria in clinical conditions associated with high oxidative stress.

Modeling Loss of Microvascular Wall Homeostasis during Glycocalyx Deterioration and Hypertension that Impacts Plasma Filtration and Solute Exchange

“…These results are in line with experimental observations, where a significant increase in macro-molecular flux across microvessel wall was observed both after exposure to H2O2 [25] and after treatment with heparan-sulfate-degrading enzymes [24]. Depending on the enzymes used, it was noticed a 1.4 to 3-fold increase in the trans-vascular albumin flux after enzyme-treatment compared to the initial macro-molecular flux, supporting our predictions [24].…”

“…Glycocalyx degeneration, induced by reactive oxygen species exposure, has been shown to lead to endothelial dysfunction, with possible onset of microalbuminuria or proteinuria (i.e. the presence of an excess of albumin or other serum proteins in the urine) in clinical diseases [24,25], and subsequently to vascular inflammation and cardiovascular disease progression [26].…”

Protein-Bound Uremic Toxins Stimulate Crosstalk between Leukocytes and Vessel Wall

“…A chronic inflammatory status and increased oxidative stress, both associated with vascular dysfunction in CKD, contribute to severe atherosclerosis and cardiovascular morbidity and mortality in these patients, 3,54-56 but they have also been linked to glycocalyx disruption. 57,58 Of note, IS has been shown repeatedly to induce generation of reactive oxygen species in the endothelium. 14,32,59 Also, hypervolemia, a common problem in CKD and dialysis patients, can alter the endothelial surface layer, probably through atrial natriuretic peptide, which causes shedding of glycocalyx constituents into the blood.…”

The Endothelial Glycocalyx and One-Lung Ventilation Induced Lung Injury—New Focus

“…The main factors leading to vascular endothelial glycocalyx damage include inflammatory cytokines (11) and oxidative stress (12). Destruction of glycocalyx structure due to inflammatory injury is one of the early features of endothelial activation.…”

“…Meanwhile, after Ischemia-reperfusion, a large number of oxygen free radicals generated in local sugar can lead to rapid destruction of EG, while superoxide dismutase (SOD) can significantly improve glycocalyx damage (28). NO and its reactive metabolites such as peroxynitrite can directly nitrosylate tyrosine residues on endothelial proteins, resulting in barrier dysfunction (12). Thus, both inflammatory cy- (Figure 3).…”