The interaction between renal endothelial and epithelial cells prevents the cytotoxicity caused by Shiga toxin type 2 and Subtilase cytotoxin

ALVAREZ, ROMINA S.; GIRARD, MAGALÍ CELESTE; JANCIC, CAROLINA; REPETTO, HORACIO A.; IBARRA, CRISTINA; AMARAL, MARÍA MARTA

Boston

Congreso; VTEC; 2015

Introduction: Post diarrheahemolytic uremic syndrome (HUS), a complication of Shiga toxin (Stx)-producing E.coli (STEC) infection, is the most common cause of acute renal failure(ARF) in children in Argentina. Stx2 damages renal microvascular endothelialand proximal tubule and collecting duct epithelial cells. Subtilase cytotoxin(SubAB), identified in STEC producing Stx, may also contribute to renalpathogenesis. In this work, we study the effects of Stx2 and SubAB on primarycultures of human glomerular endothelial cells (HGEC), on cultures of humantubular epithelial cell line (HK-2) and on co-cultures of HGEC/HK-2. Methods:Cells were grown on one side (monolayers) or both sides (bilayers) of apermeable support. The epithelial and/or endothelial barrier integrity wasestablished by measuring the transcellular electrical resistance. HGEC, HK-2 orHGEC/HK-2 were incubated with Stx2 (1 ng/ml), SubAB (150 ng/ml) or both(co-incubation) for 72 h. Cell viability was studied by neutral red uptake.IL-6, IL-8 and MCP-1 were quantified by ELISA. The TNF-α activity was evaluated by cytotoxicity on L929 cellline. Results and Discussion: The cytotoxicity of Stx2 or SubAB wassignificantly higher on monolayers than bilayers and the co-incubation, withboth toxins, had not additive or synergistic effects (n=6, NS). After treatmentof HGEC monolayers with SubAB, Stx2 or Stx2+SubAB, a differential release ofpro-inflammatory cytokines was detected. Stx2 increased the presence of activeTNF-α and SubAB induced a higherrelease of IL-6. The soluble mediators released by HGEC establish apro-inflammatory state that could be neutralized by the presence of HK-2. Thisfact could explain why the damages caused by the toxins in HGEC and HK-2monolayers were significantly higher than those observed in HGEC/HK-2 bilayers.Studies are in progress to elucidate if these pro-inflammatory cytokines aremodulated by the glomerular and tubular cells cross-talk. Implications :These results suggest that this model of co-culture of human renalendothelium and epithelium would be more representative to study the mechanismsthat lead to ARF in patients with HUS.