Eliglustat, an inhibitor of Gb3 receptor synthesis, protects human microvascular endotelial cells from Shiga toxin type 2 cytotoxicity.

GOMEZ FERNANDO D; VELEZ DANIEL; BALESTRACCI ALEJANDRO; IBARRA CRISTINA; AMARAL MARÍA MARTA

Mar del Plata

Congreso; LXIV Reunión Anual de la Sociedad Argentina de Investigación Clínica.; 2019

Sociedad Argentina de Investigación Clínica

Hemolytic Uremic Syndrome associated to Shiga toxin (Stx)-producing E. coli infection is the most commoncause of acute renal failure (ARF) in children in Argentina. Stx2 binds the globotriaosylceramide(Gb3) receptor and causes direct damages on human renal microvascularendothelial cells (HGEC). In this work, we assayed the action of a Gb3synthesis inhibitor, Eliglustat (EG), to prevent the Stx2 cytotoxicity on humanrenal cells. Cell viability was analyzed by neutral red uptake and data areexpressed as mean ± SEM. Cell morphology analysis was evaluated by lightmicroscopy after staining with H&E. Cell counts were performed on fivefields and cell area values were obtained using Image J software. Necrosis andapoptosis were detected by flow cytometry after Annexin V-FITC/PI doublestaining assay. Non-cytotoxic concentrations of EG were established on HGECtreated with EG (0.05µM-50µM) for 120 h. While EG (50µM) caused a significantdecreased of cell viability (EG (50µM): 8.3 ± 0.9% vsCtrl: 100 ±2.7%, n = 3, p <0.05), EG (0.05-25µM) did not exhibit any cytotoxic effect. Next,HGEC were pre-treated with EG (0.05-25µM) at different times (2, 4, 6, 24 and48 h) and then incubated with Stx2 (0.5 ng/ml) for 72 h and in the presence ofEG. EG (0.5-10 µM) at all the times evaluated prevented the HGEC viability decreasecaused by Stx2 (n=5, p <0.05) and pre-incubation with EG (5 µM) for only 2 hwas enough to protect HGEC (73%). The maximum protection (100%) was obtained after24 h and 48 h of pre-treatment with 5 µM EG (EG 24 h and 48 h: 100.0 ± 2.6% vs Stx2:49.0 ± 7.9%,n = 5, p<0.05). EG (5 µM, 24 h) also prevented the cell detachment (80%) andswelling (81%) caused by Stx2. Finally, a significant prevention (86%) of necrosisinduced by Stx2 was obtained with EG (1µM, 24 h). We propose the EG as a drugto prevent the ARF and the consequent HUS development.&amp;lt;!-- /* Font Definitions */ @font-face{font-family:"Cambria Math";panose-1:2 4 5 3 5 4 6 3 2 4;mso-font-charset:0;mso-generic-font-family:roman;mso-font-pitch:variable;mso-font-signature:-536869121 1107305727 33554432 0 415 0;}@font-face{font-family:Calibri;panose-1:2 15 5 2 2 2 4 3 2 4;mso-font-charset:0;mso-generic-font-family:swiss;mso-font-pitch:variable;mso-font-signature:-469750017 -1073732485 9 0 511 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal{mso-style-unhide:no;mso-style-qformat:yes;mso-style-parent:"";margin:0cm;margin-bottom:.0001pt;mso-pagination:widow-orphan;font-size:12.0pt;font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:ES;mso-fareast-language:ES;}.MsoChpDefault{mso-style-type:export-only;mso-default-props:yes;font-size:10.0pt;mso-ansi-font-size:10.0pt;mso-bidi-font-size:10.0pt;font-family:"Calibri",sans-serif;mso-ascii-font-family:Calibri;mso-fareast-font-family:Calibri;mso-hansi-font-family:Calibri;mso-ansi-language:EN-US;mso-fareast-language:EN-US;}size:612.0pt 792.0pt;margin:70.85pt 3.0cm 70.85pt 3.0cm;mso-header-margin:36.0pt;mso-footer-margin:36.0pt;mso-paper-source:0;}div.WordSection1{page:WordSection1;}