Role of the endothelial glycocalyx in kidney disease and vascular dysfunction

Abstract

BackgroundAlbuminuria, an early marker of kidney disease, is strongly and independently associated with adverse cardiovascular outcomes, including heart attacks and strokes. Nonetheless, the mechanisms linking albuminuria and adverse cardiovascular events remain unclear. The endothelial glycocalyx, a friable but protective lining of all blood vessels, is damaged in both atherosclerosis and kidney disease. We examined whether factors in the plasma of patients with kidney disease could damage the endothelial glycocalyx, and sought underlying mechanisms. MethodsMetabolic labelling of glycosaminoglycans (GAGs) was achieved by culturing human glomerular endothelial cells in media containing 20 μCi/mL D-[3H3] glucosamine for 24 h. Monolayers were washed and subsequently exposed either to conditioned serum free media containing 10% plasma from patients with either heavy albuminuria (from an intrinsic renal disease) or from healthy controls for 24h, or to 100 μM hydrogen peroxide or vehicle for 4 h. The supernatant was then collected and applied to a di-ethyl-amino-ethyl fast flow column connected to a fast protein liquid chromatography system. Samples were applied to the column at 1 mL/min and subsequently merged with column matrix for 15 min. Bound proteoglycans were eluted with 7M urea (pH 6) and collected in 1 mL fractions. All fractions were quantified by liquid scintillation counting. FindingsExposure of glomerular endothelial cells to plasma from three patients with albuminuria resulted in reduced levels of GAGs into the media (control plasma 112 514 disintegrations per min (DPM) [SE 6153], albuminuric plasma 74 743 DPM [9607]; p<0·05 unpaired t test), indicating altered endothelial cell GAG metabolism that might represent decreased turnover. Because oxidative stress is regarded as a key mediator of endothelial dysfunction in kidney disease, we generated reactive oxygen species by exposing cells to hydrogen peroxide. This exposure resulted in a different pattern of GAG turnover: shedding of radiolabelled GAGs into the media was increased (vehicle 1302 [684] DPM D-[3H3] glucosamine-labelled heparan sulphate GAG, hydrogen peroxide 25 053 [6635] DPM D-[3H3] glucosamine-labelled heparan sulphate GAG; p<0·005 unpaired t test). InterpretationDamage to the endothelial glycocalyx in response to factors in albuminuric plasma provides a novel concept in understanding endothelial dysfunction in disease states. This work provides evidence that the albuminuric state itself might lead to systemic alterations in endothelial cell function, although our preliminary studies indicate that reactive oxygen species are unlikely to be involved in these changes. We are now seeking alternative pathways underlying the effects of albuminuric plasma on the endothelial glycocalyx, assessing whether the endothelial glycocalyx is damaged in blood vessels in situ in patients with kidney disease, and examining whether the endothelial glycocalyx can be restored (and endothelial cell function improved) by manipulating these pathways. Our long-term goal is to manipulate endothelial glycocalyx regulation to develop an entirely new class of treatments for vasculoprotection in kidney disease and other conditions. FundingNational Institute for Health Research.