title: 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 (S(35) and (3)H-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.
authors: Academic Renal Unit, Clinical Sciences North Bristol, Southmead Hospital, Bristol, United Kingdom. anuragsing@hotmail.com
authors: Anurag
authors: A
authors: Singh
authors: Raina D
authors: RD
authors: Ramnath
authors: Rebecca R
authors: RR
authors: Foster
authors: Emma C
authors: EC
authors: Wylie
authors: Vincent
authors: V
authors: Fridén
authors: Ishita
authors: I
authors: Dasgupta
authors: Borje
authors: B
authors: Haraldsson
authors: Gavin I
authors: GI
authors: Welsh
authors: Peter W
authors: PW
authors: Mathieson
authors: Simon C
authors: SC
authors: Satchell
mesh_terms: D000418:Albumins / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival; D042783:Endothelial Cells / Q000378:metabolism*; D019276:Glycocalyx / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000378:metabolism*; D006801:Humans; D007678:Kidney Glomerulus / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014909:Wheat Germ Agglutinins / Q000378:metabolism
chemical_list: D000418:Albumins; D006025:Glycosaminoglycans; D017382:Reactive Oxygen Species; D014909:Wheat Germ Agglutinins