CONICET | Buscador de Institutos y Recursos Humanos

Post-diarrhea hemolytic uremic syndrome (HUS) is the most common cause of acute renal failure in children younger than 5 years of age in Argentina. Clinical and histological renal damage has been strongly associated with Shiga toxin type 2 (Stx2) produced by O157 and non-O157 Escherichia coli (STEC). In addition, several STEC non-O157 produce Subtilase (SubAB) that may contribute to HUS pathogenesis. Stx2 binds to the globotriaosylceramide (Gb3) located on the plasma membrane of target cells. SubAB binds to the specific sialated glycans (Neu5Gc), a monosaccharide identified in several food products. In this work we have developed primary cultures of human glomerular endothelial cells (HGEC) to evaluate the action of Stx2 and SubAB toxins. HGEC were isolated from kidneys of pediatric patients undergoing nephrectomies. Glomeruli were isolated, treated with collagenase and cultured on 0.2 % gelatin coated flask. HGEC were confirmed as endothelial cells by positive staining for PECAM-1 and VWF. Gb3 expression was evaluated by immunofluorescence staining and TLC. HGEC were incubated with Stx2 or SubAB for 72 h and the viability was assessed by neutral red uptake. Stx2 (1 pg/μl) and SubAB (15 pg/μl) were able to cause the cytotoxic effects in the 50% of the cells (CD50). Necrosis and apoptosis of HGEC were analyzed by acridine orange-ethidium bromide staining. Stx2 (10 pg/μl) caused more necrosis than apoptosis with a significant increase at 4 h: 41.0 ± 15.3 % vs. 5.2 ± 0.1 % (necrosis vs. apoptosis, p <0.05, n = 3). On the contrary, SubAB (3 ng/μl) produced more apoptosis than necrosis at all times considered, being significantly higher at 6 h: 37.4 ± 2.5 % vs. 19.7 ± 4.6 %, and 24 h: 66.0 ± 11.6 % vs. 17.0 ± 5.0 % (apoptosis vs. necrosis, p <0.05, n=3). Finally, for studies examining the protective effect of Gb3 inhibition, HGEC were pretreated with C-9 (Genzyme Corp.), a new specific inhibitor for Gb3 biosynthesis. C-9 significantly neutralized, in a dose-dependent way, the inhibition in HGEC viability produced by 10 pg/μl Stx2 (p<0.05, n=4) when cells were preincubated with C-9 for 24 h (Table below). On the contrary, C-9 did not neutralize the cytotoxic effect caused by 15 pg/μl SubAB on HGEC under the same experimental conditions.

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