Effects of a glucosylceramide synthase inhibitor on an experimental model of Hemolytic Uremic Syndrome in rats

SILBERSTEIN CLAUDIA; COPELAND DIANE P; CHIANG WEI-LIEN; REPETTO HORACIO A; IBARRA CRISTINA

Buenos Aires

Simposio; VTEC2009: 7th International Symposium on Shiga Toxin (Verocytotoxin)-producing Escherichia coli infections.; 2009

Asociación Argentina de Microbiología

Post-diarrhea hemolytic uremic syndrome (HUS) is the most common cause of acute renal failure in children in Argentina. Renal damages have been strongly associated with Shiga toxins (Stxs) produced by Escherichia coli O157:H7 and other related strains. These strains express Stx1 and/or Stx2, although those that express only Stx2 are highly prevalent in Argentina. Stx2 binds to the globotriaosylceramide (Gb3) receptor on the plasma membrane of target cells. The high level expression of this receptor in renal epithelial cells may account, at least in part, for acute renal failure observed in children with HUS. We have recently observed that C-9 (Genzyme Corp.), a specific inhibitor of glucosylceramide (GL1) synthase, decreases Gb3 expression levels and prevents the cytotoxic effects of Stx2 on human renal tubular epithelial cells. The purpose of the study: To evaluate the in vivo effects of C-9 on the kidney, small intestine and colon in an experimental model of HUS in rats. Methods: To develop a model of HUS in rats, male Sprague Dawley rats (4 weeks old) were injected intraperitoneally with 1ml of filtered culture supernatant from recombinant E. coli expressing Stx2 (1 × 104 DC50 in Vero cells). Some of these rats were orally treated with C-9 (5-10 mg/100g body weight/day) for 48h previous to the Stx2 injection and during Stx2 treatment. Some animals were orally treated with C-9 and injected with control supernatants that did not contain Stx2. Control rats were injected with control supernatants without the C-9 treatment. Twenty-four hours after the Stx2 injection, rats were anesthetized and different organs were dissected and fixed. Histopathological and immunohistochemical analysis for Stx2 binding were performed on kidney, small intestine and colon sections. Results: Immunohistochemical analysis using a specific antibody against Stx2 showed positive stain in renal tubular epithelial cells and glomerular podocytes in rats injected with supernatant containing Stx2. The binding of Stx2 coincided with the extension of tubular necrosis, congestion and glomerular mesangial proliferation. Stx2 also bound to intestinal endothelial cells, and to the apical membranes and cytoplasm of enterocytes located in colon and small intestine crypts. Interstitial cells from the small intestine were positive for Stx2. In colonic mucosa, Stx2 produced a loss of goblet cells and a distortion of the crypts. Villous mononuclear infiltrate was detected in the small intestine. The treatment with C-9 produced a large decrease in Stx2 binding, which coincided with a decrease in the extent of the renal and intestinal injuries. Renal and intestinal sections from rats injected with control supernatant, treated or not with C-9, were negative for Stx2 staining and did not show any lesions. Conclusion: We propose that prevention of Gb3 synthesis by GL1 synthase inhibitors such as C-9 could be a novel substrate inhibition therapy to prevent the binding of Stx2 to the receptor and consequently neutralize the cytotoxic activity of Stx2 in target cells. Key words: Globotriaosylceramide, Glucosylceramide synthase inhibitor, kidney, small intestine, colon, Shiga toxin-2.