Primary human glomerular endothelial cells produce proteoglycans, and puromycin affects their posttranslational modification

Abstract

This article describes the possible role of the endothelial cell-surface coat, containing proteoglycans (PGs) with connected glycosaminoglycans (GAGs), in maintaining glomerular permselectivity. Primary human glomerular endothelial cells (HGEC) in culture were treated with the nephrosis-inducing agent puromycin aminonucleoside (PAN). Analysis was made by TaqMan real-time PCR, Western blot analysis, and by metabolic labeling with [(35)S]sulfate. The HGECs express several PGs: syndecan, versican, glypican, perlecan, decorin, and biglycan, which may contribute to the glomerular charge barrier. PAN treatment downregulated both the protein expression (by 25%) and the mRNA expression (by 37 +/- 6%, P < 0.001, n = 8) of versican compared with control. Transferases important for chondroitin and heparan sulfate biosynthesis were also significantly downregulated by PAN, resulting in less sulfate groups, shorter GAG chains, and reduced PG net-negative charge. Moreover, analysis of the cell media after PAN treatment revealed a reduced content of [(35)S]sulfate-labeled PGs (40% of control). We conclude that PAN may cause proteinuria by affecting the endothelial cell-surface layer and not only by disrupting the foot process arrangement of the podocytes. Thus the endothelium may be a more important component of the glomerular barrier than hitherto acknowledged.