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

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

Boston

Congreso; VTEC 2015; 2015

IntroductionPost diarrhea hemolytic 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 endothelial and proximal tubule and collecting duct epithelial cells. Subtilase cytotoxin (SubAB), identified in STEC producing Stx, may also contribute to renal pathogenesis. In this work, we study the effects of Stx2 and SubAB on primary cultures of human glomerular endothelial cells (HGEC), on cultures of human tubular epithelial cell line (HK-2) and on co-cultures of HGEC/HK-2.MethodsCells were grown on one side (monolayers) or both sides (bilayers) of a permeable support. The epithelial and/or endothelial barrier integrity was established by measuring the transcellular electrical resistance. HGEC, HK-2 or HGEC/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 cell line. Results and DiscussionThe cytotoxicity of Stx2 or SubAB was significantly higher on monolayers than bilayers and the co-incubation, with both toxins, had not additive or synergistic effects (n=6, NS). After treatment of HGEC monolayers with SubAB, Stx2 or Stx2+SubAB, a differential release of pro-inflammatory cytokines was detected. Stx2 increased the presence of active TNF-α and SubAB induced a higher release of IL-6.The soluble mediators released by HGEC establish a pro-inflammatory state that could be neutralized by the presence of HK-2. This fact could explain why the damages caused by the toxins in HGEC and HK-2 monolayers were significantly higher than those observed in HGEC/HK-2 bilayers. Studies are in progress to elucidate if these pro-inflammatory cytokines are modulated by the glomerular and tubular cells cross-talk.ImplicationsThese results suggest that this model of co-culture of human renal endothelium and epithelium would be more representative to study the mechanisms that lead to ARF in patients with HUS.