CONICET | Buscador de Institutos y Recursos Humanos

IntroductionPost diarrhea hemolyticuremic syndrome (HUS) is the most common cause of acute renal failure (ARF) inchildren in Argentina. It is well established that Shiga toxin type 2 (Stx2)causes direct damage to glomerular endothelial cells and tubular epithelialcells. Recently, it has been reported that Subtilase cytotoxin (SubAB) may bealso contribute to the pathogenesis of HUS. Previously, we have reported thatStx2 decreased cell viability and inhibited the water absorption across (Jw) monolayersof human glomerular endothelial cells (HGEC) and human renal epithelial cells (cellline HK-2) in culture. These effects were significantly attenuated on HGEC/HK-2bilayers. In this work, we studied the SubAB contribution to ARF developmentand the possible modulation of SubAB effects by endothelium-epithelium communication. MethodsCells were grown onone side (monolayer) or both sides (bilayer) of a permeable support. Theepithelial and/or endothelial barrier integrity was established by measuring thetranscellular electrical resistance. Cell viability was studied by neutral red uptake.The Jw was determined across monolayers or bilayers placed in a modified Ussingchamber connected to an electro-optical device. IL-6, IL-8 and MCP-1 were quantifiedby ELISA. Results and DiscussionSubAB (1.5 mg/ml) inhibited the Jw across HGEC (26 %) and HK-2 (22%) after 30 min ofincubation, but not across the HGEC/HK-2 bilayers. At later times (72 h), a lowdose of SubAB (0.15 mg/ml) caused areduction of HGEC, HK-2 and HGEC/HK-2 viability. This effect was independent ofendothelium?epithelium interaction (HGEC: 67.3 ± 2.6 %; HK-2: 67.4 ± 5.0 %;HGEC/HK-2: 74.2 ± 2.7 % vs Ctrl: 100%, p <0.05, n = 6). However, even at the higher dose (1.5 µg/ml), SubAB doesnot produce significant changes in cell viability of HGEC and HK-2, after 30min, 60 min and 90 min of treatment. After 24 h of incubation with SubAB (1.5 mg/ml), a differential release of pro-inflammatory cytokines andchemokines from HGEC and HK-2 was observed. ImplicationsThese results showthat endothelium-epithelium interactionmodulates the cytotoxic action of SubAB in renal cells and may contribute tothe early events of ARF.

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