Horacio A. Repetto

Action of shiga toxin type-2 and subtilase cytotoxin on human microvascular endothelial cells.

The hemolytic uremic syndrome (HUS) associated with diarrhea is a complication of Shiga toxin (Stx)-producing Escherichia coli (STEC) infection. In Argentina, HUS is endemic and responsible for acute and chronic renal failure in children younger than 5 years old. The human kidney is the most affected organ due to the presence of very Stx-sensitive cells, such as microvascular endothelial cells. Recently, Subtilase cytotoxin (SubAB) was proposed as a new toxin that may contribute to HUS pathogenesis, although its action on human glomerular endothelial cells (HGEC) has not been described yet. In this study, we compared the effects of SubAB with those caused by Stx2 on primary cultures of HGEC isolated from fragments of human pediatric renal cortex. HGEC were characterized as endothelial since they expressed von Willebrand factor (VWF) and platelet/endothelial cell adhesion molecule 1 (PECAM-1). HGEC also expressed the globotriaosylceramide (Gb3) receptor for Stx2. Both, Stx2 and SubAB induced swelling and detachment of HGEC and the consequent decrease in cell viability in a time-dependent manner. Preincubation of HGEC with C-9 −a competitive inhibitor of Gb3 synthesis-protected HGEC from Stx2 but not from SubAB cytotoxic effects. Stx2 increased apoptosis in a time-dependent manner while SubAB increased apoptosis at 4 and 6 h but decreased at 24 h. The apoptosis induced by SubAB relative to Stx2 was higher at 4 and 6 h, but lower at 24 h. Furthermore, necrosis caused by Stx2 was significantly higher than that induced by SubAB at all the time points evaluated. Our data provide evidence for the first time how SubAB could cooperate with the development of endothelial damage characteristic of HUS pathogenesis.

A Glucosylceramide Synthase Inhibitor Protects Rats Against the Cytotoxic Effects Of Shiga Toxin-2

Postdiarrhea hemolytic uremic syndrome is the most common cause of acute renal failure in children in Argentina. Renal damage has been strongly associated with Shiga toxin (Stx), which binds to the globotriaosylceramide (Gb3) receptor on the plasma membrane of target cells. The purpose of the study was to evaluate the in vivo effects of C-9, a potent inhibitor of glucosylceramide synthase and Gb3 synthesis, on kidney and colon in an experimental model of hemolytic uremic syndrome in rats. Rats were i.p. injected with supernatant from recombinant Escherichia coli expressing Stx2 (sStx2). A group of these rats were orally treated with C-9 during 6 d, from 2 d prior until 4 d after sStx2 injection. The injection of sStx2 caused renal damage as well as a loss of goblet cells in colonic mucosa. Oral treatment with C-9 significantly decreased rat mortality to 50% and reduced the extension of renal and intestinal injuries in the surviving rats. The C-9 also decreased Gb3 and glucosylceramide expression levels in rat kidneys. It is particularly interesting that an improvement was seen when C-9 was administered 2 d before challenge, which makes it potentially useful for prophylaxis.

Focal glomerulosclerosis in children: an argentinian experience

Twenty-six children presenting with idiopathic nephrotic syndrome and a histological diagnosis of focal glomerulosclerosis were studied retrospectively to evaluate their response to treatment, outcome and clinicopathological correlations. Twenty-two patients (84.6%) were steroid resistant; of these, 8 of the 19 with focal segmental glomerulosclerosis and 2 of the 3 with focal global within 16 weeks of starting therapy. Seven patients relapsed after a CY-induced remission, but 5 of them became steroid responsive. After an average follow-up of 83 months, 17 patients are in remission with normal renal function, 3 patients have persistent nephrotic range proteinuria and 6 patients are in chronic renal failure. Persistence of proteinuria, a high percentage of segmentally selerotic glomeruli and diffuse mesangial proliferation were indicators of poor prognosis. We believe longer courses of CY therapy than those traditionally utilized are responsible for the relatively good results in our patients.

Renal functional reserve in children with a previous episode of haemolytic-uraemic syndrome

Renal function [creatinine clearance (CCr)] and renal functional reserve (RFR) was measured in 16 children who had had haemolytic-uraemic syndrome (HUS) an average of 6.6±0.72 years previously. All patients had normal plasma creatinine and blood pressure and only 3 had proteinuria, which was mild in every instance. Patients were studied whilst ingesting three diets which provided an average of 1.5, 2.1 and 3.1 g protein/kg body weight per day, respectively. Diets were administered over three consecutive periods of 7 days each andCCr was measured on the 7th day of each diet. Values tended to correlate with protein intake. They were in the normal range when patients were taking 1.5 and 2.1 g protein diets and increased markedly in 13 of the 16 patients (P<0.001) when they ingested the high-protein diet (3.1 g). The effect on glomerular filtration rate (GFR)-measured byCCr and inulin clearance (Cin)-of an acute oral protein load was studied in 12 of the HUS patients and four control subjects. In the control periods, prior to the protein load, values forCCr were similar in the HUS and control subjects (104.0±11.0 vs 121.6±10.1 ml/min per 1.73 m2, NS). HoweverCin values were significantly reduced in HUS patients (59.5±9.2 vs 102.7±12.4 ml/min per 1.73 m2, (P<0.025). TheCCr/Cin ratio in the patients averaged 2.10 compared with 1.13 in controls. Acute protein loading was accompanied by an increase inCin in all controls but in only 8 of the 12 patients. Baseline values forCin did not correlate with the presence or absence of protein-stimulated enhancement ofCin. TheCCr/Cin ratios after protein loading remained twice as high in HUS patients as in controls. The data indicate thatCCr is not an accurate indicator of GFR in children who have had acute renal injury. Tubular secretion of creatinine represents a greater proportion of excreted creatinine in these children, may maintain serum creatinine in the normal range and mask the decrease in GFR. The study also emphasizes the problems of measuring RFR in these children.

Effects of Escherichia Coli Subtilase Cytotoxin and Shiga Toxin 2 on Primary Cultures of Human Renal Tubular Epithelial Cells

Shiga toxin (Stx)-producing Escherichia coli (STEC) cause post-diarrhea Hemolytic Uremic Syndrome (HUS), which is the most common cause of acute renal failure in children in many parts of the world. Several non-O157 STEC strains also produce Subtilase cytotoxin (SubAB) that may contribute to HUS pathogenesis. The aim of the present work was to examine the cytotoxic effects of SubAB on primary cultures of human cortical renal tubular epithelial cells (HRTEC) and compare its effects with those produced by Shiga toxin type 2 (Stx2), in order to evaluate their contribution to renal injury in HUS. For this purpose, cell viability, proliferation rate, and apoptosis were assayed on HRTEC incubated with SubAB and/or Stx2 toxins. SubAB significantly reduced cell viability and cell proliferation rate, as well as stimulating cell apoptosis in HRTEC cultures in a time dependent manner. However, HRTEC cultures were significantly more sensitive to the cytotoxic effects of Stx2 than those produced by SubAB. No synergism was observed when HRTEC were co-incubated with both SubAB and Stx2. When HRTEC were incubated with the inactive SubAA272B toxin, results were similar to those in untreated control cells. Similar stimulation of apoptosis was observed in Vero cells incubated with SubAB or/and Stx2, compared to HRTEC. In conclusion, primary cultures of HRTEC are significantly sensitive to the cytotoxic effects of SubAB, although, in a lesser extent compared to Stx2.

A Glucosylceramide Synthase Inhibitor Prevents the Cytotoxic Effects of Shiga Toxin-2 on Human Renal Tubular Epithelial Cells

Shiga toxin-2 binds to the globotriaosyl-ceramide 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 hemolytic uremic syndrome. The cytotoxic effect of Shiga toxin-2 was assayed on primary cultures of hu- man renal tubular epithelial cells treated with a new specific inhibitor of glucosylceramide synthase (C-9), the rate- limiting first step in the glycosphingolipid biosynthetic pathway. The treatment of the cells with 1-5 �M C-9 for at least 24 h significantly neutralized the action of 1 ng/ml Shiga toxin-2 on cell viability. The expression levels of globotriaosyl- ceramide significantly decreased when cells were incubated with 1 �M C-9 for 48 h. We propose here that prevention of globotriaosyl-ceramide synthesis by the C-9 could be a novel substrate inhibition therapy to neutralize Shiga toxin-2 ac- tion in renal epithelial cells.

Síndrome urémico hemolítico inducido por Escherichia coli enterohemorrágica

Hemolytic uremic syndrome (HUS) is characterized by microangiopathic hemolytic anemia, plaquetopenia and kidney damage. It is the leading cause of acute renal failure in pediatric age and the second for chronic renal failure. Shiga toxin-producing Escherichia coli (STEC) is the first etiologic agent of HUS being its main reservoir cattle and transmitted via contaminated food. At present, there is no specific treatment to reduce the progression of HUS. The study of the mechanisms by which STEC infects and Shiga toxin induces HUS can help to find new strategies to prevent this disease.

Prevention of renal damage caused by Shiga toxin type 2: Action of Miglustat on human endothelial and epithelial cells

Typical hemolytic uremic syndrome (HUS) is responsible for acute and chronic renal failure in children younger than 5 years old in Argentina. Renal damages have been associated with Shiga toxin type 1 and/or 2 (Stx1, Stx2) produced by Escherichia coli O157:H7, although strains expressing Stx2 are highly prevalent in Argentina. Human glomerular endothelial cells (HGEC) and proximal tubule epithelial cells are very Stx-sensitive since they express high levels of Stx receptor (Gb3). Nowadays, there is no available therapy to protect patients from acute toxin-mediated cellular injury. New strategies have been developed based on the Gb3 biosynthesis inhibition through blocking the enzyme glucosylceramide (GL1) synthase. We assayed the action of a GL1 inhibitor (Miglustat: MG), on the prevention of the renal damage induced by Stx2. HGEC primary cultures and HK-2 cell line were pre-treated with MG and then incubated with Stx2. HK- 2 and HGEC express Gb3 and MG was able to decrease the levels of this receptor. As a consequence, both types of cells were protected from Stx2 cytotoxicity and morphology damage. MG was able to avoid Stx2 effects in human renal cells and could be a feasible strategy to protect kidney tissues from the cytotoxic effects of Stx2 in vivo.

Ouabain Protects Human Renal Cells against the Cytotoxic Effects of Shiga Toxin Type 2 and Subtilase Cytotoxin

Hemolytic uremic syndrome (HUS) is one of the most common causes of acute renal failure in children. The majority of cases are associated with Shiga toxin (Stx)-producing Escherichia coli (STEC). In Argentina, HUS is endemic and presents the highest incidence rate in the world. STEC strains expressing Stx type 2 (Stx2) are responsible for the most severe cases of this pathology. Subtilase cytotoxin (SubAB) is another STEC virulence factor that may contribute to HUS pathogenesis. To date, neither a licensed vaccine nor effective therapy for HUS is available for humans. Considering that Ouabain (OUA) may prevent the apoptosis process, in this study we evaluated if OUA is able to avoid the damage caused by Stx2 and SubAB on human glomerular endothelial cells (HGEC) and the human proximal tubule epithelial cell (HK-2) line. HGEC and HK-2 were pretreated with OUA and then incubated with the toxins. OUA protected the HGEC viability from Stx2 and SubAB cytotoxic effects, and also prevented the HK-2 viability from Stx2 effects. The protective action of OUA on HGEC and HK-2 was associated with a decrease in apoptosis and an increase in cell proliferation. Our data provide evidence that OUA could be considered as a therapeutic strategy to avoid the renal damage that precedes HUS.

[The defense of our language: a barren effort?].

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