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

Am J Physiol Renal Physiol. 2005 Apr;288(4):F748-56. doi: 10.1152/ajprenal.00202.2004. Epub 2004 Dec 7.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Blotting, Western
  • Cells, Cultured
  • Endothelial Cells / cytology
  • Endothelial Cells / drug effects
  • Endothelial Cells / physiology
  • Glucosamine / pharmacokinetics
  • Humans
  • Kidney Glomerulus / cytology
  • Kidney Glomerulus / physiology*
  • Protein Processing, Post-Translational / drug effects*
  • Protein Synthesis Inhibitors / pharmacology*
  • Proteoglycans / genetics*
  • Proteoglycans / metabolism
  • Puromycin / pharmacology*
  • RNA, Messenger / analysis
  • Sulfates
  • Transferases / genetics
  • Transferases / metabolism
  • Tritium

Substances

  • Protein Synthesis Inhibitors
  • Proteoglycans
  • RNA, Messenger
  • Sulfates
  • Tritium
  • Puromycin
  • Transferases
  • Glucosamine