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.

Postdiarrheal hemolytic uremic syndrome (HUS) affects children under 5 years old and is responsible for the development of acute and chronic renal failure, particularly in Argentina. This pathology is a complication of Shiga toxin (Stx)-producing Escherichia coli infection and renal damage is attributed to Stx types 1 and 2 (Stx1, Stx2) produced by Escherichia coli O157:H7 and many other STEC serotypes. It has been reported the production of Subtilase cytotoxin (SubAB) by non-O157 STEC isolated from cases of childhood diarrhea. Therefore, it is proposed that SubAB may contribute to HUS pathogenesis. The human kidney is the most affected organ because very Stx-sensitive cells express high amounts of biologically active receptor. In this study, we investigated the effects of Stx2 and SubAB on primary cultures of human glomerular endothelial cells (HGEC) and on a human tubular epithelial cell line (HK-2) in monoculture and coculture conditions. We have established the coculture as a human renal proximal tubule model to study water absorption and cytotoxicity in the presence of Stx2 and SubAB. We obtained and characterized cocultures of HGEC and HK-2. Under basal conditions, HGEC monolayers exhibited the lowest electrical resistance (TEER) and the highest water permeability, while the HGEC/HK-2 bilayers showed the highest TEER and the lowest water permeability. In addition, at times as short as 20–30 minutes, Stx2 and SubAB caused the inhibition of water absorption across HK-2 and HGEC monolayers and this effect was not related to a decrease in cell viability. However, toxins did not have inhibitory effects on water movement across HGEC/HK-2 bilayers. After 72 h, Stx2 inhibited the cell viability of HGEC and HK-2 monolayers, but these effects were attenuated in HGEC/HK-2 bilayers. On the other hand, SubAB cytotoxicity shows a tendency to be attenuated by the bilayers. Our data provide evidence about the different effects of these toxins on the bilayers respect to the monolayers. This in vitro model of communication between human renal microvascular endothelial cells and human proximal tubular epithelial cells is a representative model of the human proximal tubule to study the effects of Stx2 and SubAB related to the development of HUS.

BACKGROUND AND PURPOSEInfections with a strain of Escherichia coli producing Shiga toxins could be one of the causes of fetal morbidity and mortality in pregnant women. We have previously reported that Shiga toxin type 2 (Stx2) induces preterm delivery in pregnant rats. In this study, we evaluate the role of TNF-a, PGs and NO in the Stx2-induced preterm delivery. EXPERIMENTAL APPROACHPregnant rats were treated with Stx2 (0.7 ng g -1) and killed at different times after treatment. Placenta and decidua were used to analyse NOS activity by the conversion of L-[ 14 C]arginine into L-[ 14 C]citrulline, levels of PGE2 and PGF2a assessed by radioimmunoassay, and cyclooxygenase (COX) proteins by Western blot. TNF-a level was analysed in serum by ELISA and by cytotoxicity in L929 cells. The inhibitor of inducible NOS, aminoguanidine, the COX-2 inhibitor, meloxicam, and the competitive inhibitor of TNF-a, etanercept, were used alone or combined to inhibit NO, PGs and TNF-a production respectively, to prevent Stx2-induced preterm delivery. KEY RESULTSStx2 increased placental PGE2 and decidual PGF2a levels as well as COX-2 expression in both tissues. Aminoguanidine and meloxicam delayed the preterm delivery time but did not prevent it. Etanercept blocked the TNF-a increase after Stx2 treatment and reduced the preterm delivery by approximately 30%. The combined action of aminoguanidine and etanercept prevented Stx2-induced preterm delivery by roughly 70%. CONCLUSION AND IMPLICATIONSOur results demonstrate that the increased TNF-a and NO induced by Stx2 were the predominant factors responsible for preterm delivery in rats. Abbreviations

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Cyclooxygenase (COX)-2, the inducible isoform of cyclooxygenase, plays a role in the process of uterine decidualization and blastocyst attachment. On the other hand, overexpression of COX-2 is involved in the proliferation of the endometrial tissue during endometriosis. Deregulation of the renin-angiotensin-system plays a role in the pathophysiology of endometriosis and pre-eclampsia. Angiotensin II increases intracellular Ca2+ concentration by targeting phospholypase C-gamma in endometrial stromal cells (ESC). A key element of the cellular response to Ca2+ signals is the activity of the Ca2+- and calmodulin-dependent phosphatase calcineurin. Our first aim was to study whether angiotensin II stimulated Cox-2 gene expression in rat ESC and to analyze whether calcineurin activity was involved. In cells isolated from non-pregnant uteri, COX-2 expression -both mRNA and protein- was induced by co-stimulation with phorbol ester and calcium ionophore (PIo), as well as by angiotensin II. Pretreatment with the calcineurin inhibitor cyclosporin A inhibited this induction. We further analyzed the role of the calcineurin/NFAT signaling pathway in the induction of Cox-2 gene expression in non-pregnant rat ESC. Cyclosporin A abolished NFATc1 dephosphorylation and translocation to the nucleus. Cyclosporin A also inhibited the transcriptional activity driven by the Cox-2 promoter. Exogenous expression of the peptide VIVIT -specific inhibitor of calcineurin/NFAT binding- blocked the activation of Cox-2 promoter and the up-regulation of COX-2 protein in these cells. Finally we analyzed Cox-2 gene expression in ESC of early-pregnant rats. COX-2 expression -both mRNA and protein- was induced by stimulation with PIo as well as by angiotensin II. This induction appears to be calcineurin independent, since it was not abrogated by cyclosporin A. In conclusion, angiotensin II induced Cox-2 gene expression by activating the calcineurin/NFAT signaling pathway in endometrial stromal cells of non-pregnant but not of early-pregnant rats. These results might be related to differential roles that COX-2 plays in the endometrium.