title: Verocytotoxin inhibits mitogenesis and protein synthesis in purified human glomerular mesangial cells without affecting cell viability: evidence for two distinct mechanisms.
abstract: Acute renal failure is one of the hallmarks of the hemolytic uremic syndrome (HUS). Infection with a verocytotoxin (VT)- or Shiga-like toxin (SLT)-producing Escherichia coli has been strongly implicated in the etiology of the epidemic form of HUS. The functional receptor for these closely related toxins appears to be a glycosphingolipid, globotriaosylceramide (Gb3). Endothelial damage in the glomeruli and arterioles of the kidney induced by VT is believed to play a crucial role in the pathogenesis of HUS. However, little information is available regarding the effects of VT on mesangial cells, which also play an important role in glomerular function. In this study, the effects of VT on human mesangial cells in vitro were investigated. Mesangial cells were enriched by collecting hillock-shaped outgrowths derived from adult human glomeruli and subsequently purified by elimination of contaminating epithelial cells by immunoseparation with ulex europaeus lectin-I (UEA-I)-coated dynabeads. The obtained and subcultured mesangial cell populations were >98% pure. Their mesangial nature was established by the presence of a-smooth muscle cell actin in highly confluent cultures and the absence of cytokeratin or platelet/endothelial cell adhesion molecule-1. Mesangial cells bound VT to bands of Gb3 and a closely related glycolipid, which is similar to a glycolipid involved in the VT-dependent cytokine production in monocytes. VT did not induce the release of cytokines or chemokines in mesangial cells. In VT-susceptible cells, binding of VT to Gb3 causes cell death by the inhibition of protein synthesis. Although protein synthesis was inhibited in mesangial cells, all cells remained viable, both under basal and tumor necrosis factor-alpha-stimulated conditions. However, the marked reduction in protein synthesis may impair a proper response of the cells in conditions of increased demand of newly synthesized proteins. Furthermore, VT markedly inhibited DNA synthesis and proliferation of mesangial cells. The inhibition of mitogenesis was also found with the B-subunit of VT-1 alone, albeit to a lesser extent, without a significant effect on protein synthesis. Because the inhibition of protein synthesis involves the A-subunit, this suggests that two distinct mechanisms contribute to the effects of VT on protein synthesis and mitogenesis. Intracellular routing of VT (A- and B-subunits) may vary between cell types and result in differential effects on human mesangial cells when compared with other cell types.
authors.affiliation: Department of Pediatrics, University Hospital, Nijmegen, The Netherlands.
authors.forename: P A
authors.initials: PA
authors.lastname: Van Setten
authors.forename: V W
authors.initials: VW
authors.lastname: van Hinsbergh
authors.forename: L P
authors.initials: LP
authors.lastname: Van den Heuvel
authors.forename: T J
authors.initials: TJ
authors.lastname: van der Velden
authors.forename: N C
authors.initials: NC
authors.lastname: van de Kar
authors.forename: R J
authors.initials: RJ
authors.lastname: Krebbers
authors.forename: M A
authors.initials: MA
authors.lastname: Karmali
authors.forename: L A
authors.initials: LA
authors.lastname: Monnens
mesh_terms: D058186:Acute Kidney Injury / Q000209:etiology / Q000378:metabolism; D000328:Adult; D001427:Bacterial Toxins / Q000737:chemistry / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004261:DNA Replication / Q000187:drug effects; D004927:Escherichia coli Infections / Q000139:chemically induced; D005920:Glomerular Mesangium / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006017:Glycolipids / Q000378:metabolism; D006131:Growth Inhibitors / Q000494:pharmacology*; D006463:Hemolytic-Uremic Syndrome / Q000209:etiology / Q000378:metabolism; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects*; D014176:Protein Biosynthesis; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D022622:Shiga Toxin 1; D014281:Trihexosylceramides / Q000235:genetics / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D001427:Bacterial Toxins; D006017:Glycolipids; D006131:Growth Inhibitors; D011500:Protein Synthesis Inhibitors; D022622:Shiga Toxin 1; D014281:Trihexosylceramides; D014409:Tumor Necrosis Factor-alpha; C018549:globotriaosylceramide