Influence of communication between human renal glomerular endothelial cells and tubular epithelial cells on the action of Subtilase cytotoxin

ALVAREZ, ROMINA S.; GIRARD, MAGALÍ CELESTE; JANCIC, CAROLINA; PATON, ADRIENNE W.; PATON, JAMES C.; IBARRA, CRISTINA; AMARAL, MARÍA MARTA

Boston

Congreso; VTEC; 2015

Introduction: Post diarrheahemolytic uremic syndrome (HUS) is the most common cause of acute renal failure(ARF) in children in Argentina. It is well established that Shiga toxin type 2(Stx2) causes direct damage to glomerular endothelial cells and tubularepithelial cells. Recently, it has been reported that Subtilase cytotoxin(SubAB) may be also contribute to the pathogenesis of HUS. Previously, we havereported that Stx2 decreased cell viability and inhibited the water absorptionacross (Jw) monolayers of human glomerular endothelial cells (HGEC) and humanrenal epithelial cells (cell line HK-2) in culture. These effects weresignificantly attenuated on HGEC/HK-2 bilayers. In this work, we studied theSubAB contribution to ARF development and the possible modulation of SubABeffects by endothelium-epithelium communication. Methods: Cells were grown onone side (monolayer) or both sides (bilayer) of a permeable support. Theepithelial and/or endothelial barrier integrity was established by measuringthe transcellular electrical resistance. Cell viability was studied by neutralred uptake. The Jw was determined across monolayers or bilayers placed in amodified Ussing chamber connected to an electro-optical device. IL-6, IL-8 andMCP-1 were quantified by ELISA. Results and Discussion: SubAB (1.5 μg/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 μg/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 does not produce significant changes incell viability of HGEC and HK-2, after 30 min, 60 min and 90 min of treatment.After 24 h of incubation with SubAB (1.5 μg/ml), a differential release of pro-inflammatory cytokines andchemokines from HGEC and HK-2 was observed. Implications: These results showthat endothelium-epithelium interaction modulates the cytotoxic action of SubABin renal cells and may contribute to the early events of ARF.