Background: The podocytes and the glomerular endothelial cells (GEnC) are the two cellular components of the glomerular filtration ‘barrier’. We have recently shown that GEnC are covered by a thick layer of glycoproteins called glycocalyx which also acts a selective sieve to the passage of protein suggesting its contribution to the ‘barrier’ to protein in vivo. Diabetes, recognized by high glucose levels, is the commonest condition leading to dysfunction of this barrier leading to leakage of protein in the urine. Aims: To test the effects of high glucose on the structural components of GEnC glycocalyx and examine its functional relevance by analysing its barrier properties to the passage of water and albumin. Methods: We used conditionally immortalised human GEnC developed in our laboratory. For all experiments comparisons were made between cells cultured in control medium (with 5.5mM glucose and 20mM mannitol) and high glucose medium (25mM glucose). Expression of proteoglycan core proteins syndecan 1and 4, glypican-1, perlecan and versican was analysed by Western blotting. The expression of glycosaminoglycan chains was analysed by immunofluorescence and further quantified and characterized by radiolabelling studies (tritiated glucosamine and S³⁵). Barrier properties of GEnC monolayers were studied by real time trans-endothelial electrical resistance (TEER) and passage of labelled albumin. Results: High glucose reduced expression of total and sulphated glycosaminoglycan chains of glycocalyx by 55% and 62% respectively compared to controls (n=4, p<0.0001). Immunofluorescence further confirmed reduction in the heparan sulphate chains with preservation of endothelial junctions. There was however no significant change in the expression of proteoglycan core proteins. Functional studies showed no changes in passage of water and solutes after high glucose but a significant increase in the passage of labelled albumin by 30% relative to controls (n=10, p<0.001). Conclusions: High glucose milieu reduces the expression of sugar chains of glycocalyx without any alterations in their core proteins. This leads to a selective increase flux of albumin across GEnC monolayers without affecting the passage of water and solutes. These results support the role of GEnC glycocalyx in the pathophysiology of proteinuria in diabetes.