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Hemolytic uremic syndrome (HUS), the leading cause of acute renal failure in children (< 3 years), is mainly related to Shiga toxins (Stx)-producing Escherichia coli (STEC) infections. STEC are confined to the gut resulting in hemorrhagic colitis, whereas Stx are delivered in blood to target kidney and brain, with unclear mechanisms, triggering HUS in 5 to 15% of infected children. Stx were found on circulating cells, free in sera (soluble Stx) or in blood cell-derived microvesicles (particulate Stx), whereby the relationship between these forms of circulating toxins is unclear. Here, we have examined 2,846 children with bloody diarrhea and found evidence of STEC infection in 5%. Twenty patients were enrolled to study the natural course of STEC infections before the onset of HUS. In patients, Stx were found to be associated to circulating cells and/or free and functionally active in sera. In most children, Stx were bound to neutrophils when high amounts of toxins were found in feces. Time-course analysis showed that Stx increased transiently in patients' sera while the decrease of toxin amount on leukocytes was observed. Notably, patients who recovered (85%) displayed different settings than those who developed HUS (15%). The distinctive feature of the latter group was the presence in blood of particulate Stx2 (Stx2 sedimented at g -forces corresponding to 1 μm microvesicles) the day before diagnosis of HUS, during the release phase of toxins from circulating cells. This observation strongly suggests the involvement of blood cell-derived particulate Stx2 in the transition from hemorrhagic colitis to HUS., Competing Interests: G.A. reports grants from Progetto Organizzazione non Lucrativa di Utilità Sociale–Alice Associazione per la Lotta alla Sindrome Emolitico Uremica, during the conduct of the study. M.B. reports grants from Progetto Organizzazione non Lucrativa di Utilità Sociale–Alice Associazione per la Lotta alla Sindrome Emolitico Uremica, during the conduct of the study. X.H. reports grants from USDA-ARS National Program NP108, CRIS project 2030–42000–049–00D, during the conduct of the study., (Georg Thieme Verlag KG Stuttgart · New York.)

Complement factor H (CFH) regulates complement activation in host tissues through its recognition of polyanions, which mediate CFH binding to host cell surfaces and extracellular matrix, promoting the deactivation of deposited C3b. These polyanions include heparan sulfate (HS), a glycosaminoglycan with a highly diverse range of structures, for which two regions of CFH (CCP6-8 and CCP19-20) have been implicated in HS binding. Mutations/polymorphisms within these glycosaminoglycan-binding sites have been associated with age-related macular degeneration (AMD) and atypical hemolytic uremic syndrome. In this study, we demonstrate that CFH has tissue-specific binding properties mediated through its two HS-binding regions. Our data show that the CCP6-8 region of CFH binds more strongly to heparin (a highly sulfated form of HS) than CCP19-20, and that their sulfate specificities are different. Furthermore, the HS binding site in CCP6-8, which is affected by the AMD-associated Y402H polymorphism, plays the principal role in host tissue recognition in the human eye, whereas the CCP19-20 region makes the major contribution to the binding of CFH in the human kidney. This helps provide a biochemical explanation for the genetic basis of tissue-specific diseases such as AMD and atypical hemolytic uremic syndrome, and leads to a better understanding of the pathogenic mechanisms for these diseases of complement dysregulation.

The macrophage has previously been implicated in contributing to the renal inflammation associated with hemolytic-uremic syndrome (HUS). However, there is currently no in vivo model detailing the contribution of the renal macrophage to the kidney disease associated with HUS. Therefore, renal macrophage recruitment and inhibition of infiltrating renal macrophages were evaluated in an established HUS mouse model. Macrophage recruitment to the kidney was evident by immunohistochemistry 2 h after administration of purified Stx2 and peaked at 48 h postinjection. Mice administered a combination of Stx2 and lipopolysaccharide (LPS) showed increased macrophage recruitment to the kidney compared to mice treated with Stx2 or LPS alone. Monocyte chemoattractants were induced in the kidney, including monocyte chemoattractant protein 1 (MCP-1/CCL2), macrophage inflammatory protein 1alpha (MIP-1alpha/CCL3), and RANTES (CCL5), in a pattern that was coincident with macrophage infiltration as indicated by immunohistochemistry, protein, and RNA analyses. MCP-1 was the most abundant chemokine, MIP-1alpha was the least abundant, and RANTES levels were intermediate. Mice treated with MCP-1, MIP-1alpha, and RANTES neutralizing antibodies had a significant decrease in Stx2 plus LPS-induced macrophage accumulation in the kidney, indicating that these chemokines are required for macrophage recruitment. Furthermore, mice exposed to these three neutralizing antibodies had decreased fibrin deposition in their kidneys, implying that macrophages contribute to the renal damage associated with HUS.

Background/aims: Shiga toxin (Stx) and lipopolysaccharide (LPS) both participate in the pathogenesis of post-diarrheal (D+) hemolytic uremic syndrome (HUS), but little is known about factors that modulate the host response to these toxins. Prostacyclin (PGI(2)) is a potent renal vasodilator and inhibitor of platelet aggregation and adhesion. An inability to produce PGI(2) in response to endothelial cell injury could drive the pathogenic cascade. We therefore used a baboon model of HUS to measure PGI(2 )production following the administration of Stx and LPS., Methods: Shiga toxin-1 (Stx-1), with and without LPS, was administered intravenously to baboons in various doses and schedules. 6-keto-PGF(1)alpha, the stable metabolite of PGI(2), was measured by ELISA in the plasma and urine., Results: Plasma concentrations did not change significantly. Urine values increased significantly in some groups, but not in others, and HUS developed both in animals that did and did not exhibit a significant increase in urinary PGI(2) production., Conclusions: Renal PGI(2) biosynthesis appears to be affected by the dose and rate of Stx administration, and the timing of LPS infusion. PGI(2) does not protect our primate model from developing HUS., (Copyright 2002 S. Karger AG, Basel)

Haemolytic uraemic syndrome (HUS) is characterized by haemolytic anaemia, thrombocytopenia and renal failure. Infection with enterohaemorrhagic Escherichia coli (EHEC), mainly O157:H7, has been strongly implicated as the major cause of HUS in children. The pathogenesis of HUS caused by the infection is not well understood and the defined sites of Stx in kidney of EHEC-infected humans has not been clearly demonstrated. The aim of this study was to investigate and compare the locations of Stx deposition in kidneys of paediatric and geriatric patients who died from enterohaemorrhagic E. coli O157 (EHEC) associated HUS, using an immunoperoxidase staining of the tissues. The study revealed that binding of Stx was relatively less and limited only to the renal tubules of an adult case (81 years old), while more binding was found at both renal tubules and glomeruli of an infant case (21 months old). The Stx binding in the infant's glomeruli was at podocytes, mesangial and endothelial cells. It has been known that young children are more susceptible than adults to HUS. One possibility for this is that the more extensive binding of the Stx to the kidney tissue of the paediatric patient might be due to the higher synthesis and expression of Stx receptors, i.e. Gb(3), in infants and less so in the aged individuals. However, other alternatives are possible, for example, the difference in stage of HUS in individual patients. Thus it is too early to draw any conclusion on this enigma and further investigation is required., (Copyright 2001 Academic Press.)

Infection of Shiga toxin (Stx)-producing Escherichia coli induces hemolytic uremic syndrome (HUS) in 10 to 15% of cases in infants and young children. Although the endothelial cell damage induced by Stx is widely believed to be a primary event of renal dysfunction in HUS, the precise mechanism remains to be elucidated. We were able to examine the kidney obtained at autopsy of a child who died after HUS associated with Stx-producing Escherichia coli O157:H7 infection, and immunohistochemistry indicated the deposition of Stxl and Stx2 in a portion of the distal tubular epithelia. To our knowledge, this is the first report to show the presence of Stx in human tissue of a patient with HUS, and the results obtained in this study provide evidence that Stx indeed migrates into the kidney and binds to renal tubules during Stx-producing Escherichia coli infection.

Renal thrombotic microangiopathy is characterized by glomerular and vascular thrombosis. The persistancy of fibrin deposits may result from imbalance between plasminogen activation and inhibition. In the present study, we used immunohistochemistry and in situ hybridization techniques to determine the localization of urokinase-type (u-PA) and tissue-type (t-PA) plasminogen activators, type 1 plasminogen activator inhibitor (PAI-1) and membrane receptor for u-PA (uPA-R) antigen and their sites of synthesis in renal thrombotic microangiopathy (N = 10) as compared to acute tubular necrosis (N = 5) and normal human kidneys (N = 7). We found an induction of PAI-1 and uPA-R expression in glomeruli and in arterial walls in renal thrombotic microangiopathy. In addition, the induction of uPA-R expression was also found in some tubular epithelial cells. In most case, local synthesis of PAI-1 and uPA-R was confirmed by in situ hybridization with the corresponding cDNA probes. In contrast, using similar techniques PAI-1 and uPA-R antigens and messenger RNAs could not be detected in normal kidneys. In both renal thrombotic microangiopathy and normal kidneys, t-PA mRNA was detected in large amounts in all glomeruli and in vascular endothelial cells, but t-PA antigen was only detected in a limited number of glomerular and arterial endothelial cells, whereas it was strongly expressed by all venous endothelial cells. Although u-PA antigen was found in almost all tubular sections, u-PA mRNA was only found in tubular epithelial cells in the deep cortex and the outer medulla. Our results indicate that there is an up-regulation of PAI-1 and u-PA-R expression in the glomeruli and in the arterial walls of thrombotic microangiopathy. The local release of PAI-1 could play a role in the persistancy of fibrin deposition and the further development of fibrotic lesions. Whether uPA-R plays a pathogenic role in the development of glomerular and vascular lesions, or is involved in the repair process of these lesion, remains to be elucidated.

Gastrointestinal infection with verotoxin-producing Escherichia coli (VTEC) has been strongly implicated in the etiology of the hemolytic uremic syndrome (HUS), the leading cause of pediatric acute renal failure. The binding of fluorescein-conjugated VT1 overlaid on to frozen human renal sections has been examined. Sections from biopsies of infants aged < 2 years were compared with those from adult autopsies. VT primarily stained distal convoluted tubules, particularly those adjacent to glomeruli, and collecting ducts. VT-binding was detected within the infant glomerulus but not the adult. Binding of the toxin was removed when the section was pretreated with alpha-galactosidase, confirming the receptor-binding specificity for globotriaosyl ceramide (gal alpha 1-4gal beta 1-4 glucosylceramide), the glycolipid receptor for VT. These studies may suggest that differential localization of this glycolipid in the pediatric renal glomerulus is a risk factor for the development of HUS following infection with VTEC.

This study addresses the basis for regional microvascular susceptibility to bacterial toxins implicated in hemolytic uremic syndrome. The results indicate a relationship between the degree of Shiga toxin sensitivity of human endothelial cells from different sources and the amount of globotriaosylceramide (Gb3) glycosphingolipid receptor for Shiga toxin expressed by these cells. Cell viability and protein synthesis of renal endothelial cells were reduced to 50% by 1 pM Shiga toxin, while umbilical vein cells were not affected by > 1 nM toxin. Similarly, basal levels of Gb3 were approximately 50 times higher in renal endothelial cells than in the umbilical endothelial cells. Pre-exposure of umbilical endothelial cells to tumor necrosis factor-alpha or bacterial lipopolysaccharide increased Gb3 content 4-6-fold coincident with increases in sensitivity to cytotoxic and protein synthesis inhibitory effects of Shiga toxin. Lipopolysaccharide induction of both Gb3 and sensitivity to Shiga toxin cytotoxic action in umbilical endothelial cells was dependent on the structure of lipopolysaccharide. Neither tumor necrosis factor-alpha nor lipopolysaccharide altered the Shiga toxin sensitivity or the Gb3 content of renal endothelial cells. These data indicate that differential endothelial expression of glycolipid receptors for Shiga toxins may be responsible for localized involvement of the kidney in hemolytic uremic syndrome.

Previous studies have reported various abnormalities in prostacyclin (PGI2) synthesis and metabolism in hemolytic-uremic syndrome (HUS). However, the conclusions of most of these studies are based on in vitro or ex vivo experiments that only give an indirect estimate of the actual biosynthesis in vivo. We studied the urinary excretion of PGI2 metabolites, taken as a marker of the actual biosynthesis, in six children with HUS during the acute phase of the disease and again when remission was achieved. Eight age- and sex-matched healthy children were studied as controls. Since HUS is also associated with platelet activation and consumption, we also studied the urinary excretion of thromboxane A2 (TxA2) metabolites. Urinary PGI2 and TxA2 metabolites were assessed by radioimmunoassay after high-performance liquid chromatography (HPLC) purification. Urinary excretion of the PGI2 hydrolysis product, 6-keto-PGF1 alpha, was significantly reduced in children with acute HUS as compared with controls, indicating a defective renal synthesis of PGI2. A significant inverse correlation was found between urinary 6-keto-PGF1 alpha and blood urea nitrogen (BUN), as well as plasma creatinine. At remission, urinary 6-keto-PGF1 alpha levels increased to values higher than those of controls. By contrast, the urinary excretion of the major PGI2 beta-oxidation product, 2,3-dinor-6-keto-PGF1 alpha, was comparable to controls, indicating normal systemic PGI2 biosynthesis. The urinary excretion of both TxA2 hydrolysis product, TxB2, and the major beta-oxidation metabolite, 2,3-dinor-TxB2, were lower than normal in the acute phase of HUS if expressed as absolute values.(ABSTRACT TRUNCATED AT 250 WORDS)

Forty-six patients who developed a Hemolytic Uremic Syndrome (HUS) during the period 1970-1976, were examined ten years later. Thirty-two individuals had no signs of renal disease whereas fourteen showed at least one abnormality. In the latter group a urinary osmolality below 800 mosmole per kg water was the most frequent defect found (eight cases). Three adolescents had both hypertension and proteinuria, which are considered as important late sequelae.

The localization of intrarenal cross-linked fibrin was examined by the effect of monochloroacetic acid treatment on the kidney sections. In acute glomerulonephritis or in mild diffuse or focal proliferative type of nephritis, cross-linked fibrin was observed mainly within glomerular capillary walls. Extension of cross-linked fibrin deposit over the mesangium or sclerotic area was seen in moderate to severe proliferative type of nephritis or in membranoproliferative glomerulonephritis. In hemolytic uremic syndrome or disseminated intravascular coagulation syndrome, cross-linked fibrin was detected within glomeruli and vessels.