Model Of Hemolytic Uremic Syndrome Research Articles

Detection and characterization of circulating microvesicles containing Shiga toxin type 2 in a rat model of Hemolytic Uremic Syndrome

Shiga toxin producing Escherichia coli (STEC) are foodborne pathogens that release Shiga toxin (Stx), virulence factor responsible for the development of Hemolytic Uremic Syndrome (HUS). Stx causes endothelial cell damage, which leads to platelets deposition and thrombi formation within the microvasculature. It has been described that Stx activates blood cells and induces the shedding of proinflammatory and prothrombotic microvesicles (MVs) containing the toxin. In this sense, it has been postulated that MVs containing Stx2 (MVs-Stx2+) can contribute to the physiopathology of HUS, allowing Stx2 to reach the target organs while evading the immune system. In this work, we propose that circulating MVs-Stx2+ can be a potential biomarker for the diagnosis and prognosis of STEC infections and HUS progression. We developed a rat HUS model by the intraperitoneal injection of a sublethal dose of Stx2 and observed: decrease in body weight, increase of creatinine and urea levels, decrease of creatinine clearance and histological renal damages. After characterization of renal damages, we investigated circulating total MVs and MVs-Stx2+ by flow cytometry at different times after Stx2 injection. Additionally, we evaluated the correlation of biochemical parameters such as creatinine and urea in plasma with MVs-Stx2+. As a result, we found a significant circulation of MVs-Stx2+ at 72 and 96 h after Stx2 injection, nevertheless no correlation with creatinine and urea plasma levels were detected. Our results suggest that MVs-Stx2+ may be an additional biomarker for the characterization and diagnosis of HUS progression. A further analysis is required in order to validate MVs-Stx2+ as biomarker of the disease.

Neutrophil Extracellular Traps Induced by Shiga Toxin and Lipopolysaccharide-Treated Platelets Exacerbate Endothelial Cell Damage

Hemolytic uremic syndrome (HUS) is the most common cause of acute renal failure in the pediatric population. The etiology of HUS is linked to Gram-negative, Shiga toxin (Stx)-producing enterohemorrhagic bacterial infections. While the effect of Stx is focused on endothelial damage of renal glomerulus, cytokines induced by Stx or bacterial lipopolysaccharide (LPS) and polymorphonuclear cells (PMNs) are involved in the development of the disease. PMN release neutrophil extracellular traps (NETs) to eliminate pathogens, although NETs favor platelets (Plts) adhesion/thrombus formation and can cause tissue damage within blood vessels. Since thrombus formation and occlusion of vessels are characteristic of HUS, PMN–Plts interaction in the context of Stx may promote netosis and contribute to the endothelial damage observed in HUS. The aim of this study was to determine the relevance of netosis induced by Stx in the context of LPS-sensitized Plts on endothelial damage. We observed that Stx2 induced a marked enhancement of netosis promoted by Plts after LPS stimulation. Several factors seemed to promote this phenomenon. Stx2 itself increased the expression of its receptor on Plts, increasing toxin binding. Stx2 also increased LPS binding to Plts. Moreover, Stx2 amplified LPS induced P-selectin expression on Plts and mixed PMN–Plts aggregates formation, which led to activation of PMN enhancing dramatically NETs formation. Finally, experiments revealed that endothelial cell damage mediated by PMN in the context of Plts treated with LPS and Stx2 was decreased when NETs were disrupted or when mixed aggregate formation was impeded using an anti-P-selectin antibody. Using a murine model of HUS, systemic endothelial damage/dysfunction was decreased when NETs were disrupted, or when Plts were depleted, indicating that the promotion of netosis by Plts in the context of LPS and Stx2 plays a fundamental role in endothelial toxicity. These results provide insights for the first time into the pivotal role of Plts as enhancers of endothelial damage through NETs promotion in the context of Stx and LPS. Consequently, therapies designed to reduce either the formation of PMN–Plts aggregates or NETs formation could lessen the consequences of endothelial damage in HUS.

Shiga Toxin 2 Triggers C3a-Dependent Glomerular and Tubular Injury through Mitochondrial Dysfunction in Hemolytic Uremic Syndrome.

Shiga toxin (Stx)-producing Escherichia coli is the predominant offending agent of post-diarrheal hemolytic uremic syndrome (HUS), a rare disorder of microvascular thrombosis and acute kidney injury possibly leading to long-term renal sequelae. We previously showed that C3a has a critical role in the development of glomerular damage in experimental HUS. Based on the evidence that activation of C3a/C3a receptor (C3aR) signaling induces mitochondrial dysregulation and cell injury, here we investigated whether C3a caused podocyte and tubular injury through induction of mitochondrial dysfunction in a mouse model of HUS. Mice coinjected with Stx2/LPS exhibited glomerular podocyte and tubular C3 deposits and C3aR overexpression associated with cell damage, which were limited by C3aR antagonist treatment. C3a promoted renal injury by affecting mitochondrial wellness as demonstrated by data showing that C3aR blockade reduced mitochondrial ultrastructural abnormalities and preserved mitochondrial mass and energy production. In cultured podocytes and tubular cells, C3a caused altered mitochondrial fragmentation and distribution, and reduced anti-oxidant SOD2 activity. Stx2 potentiated the responsiveness of renal cells to the detrimental effects of C3a through increased C3aR protein expression. These results indicate that C3aR may represent a novel target in Stx-associated HUS for the preservation of renal cell integrity through the maintenance of mitochondrial function.

Gene expression profile and injury sites in mice treated with Shiga toxin 2 and lipopolysaccharide as a Shiga toxin-associated hemolytic uremic syndrome model.

Shiga toxin 2 (Stx2) and lipopolysaccharide (LPS) contribute to the development of hemolytic uremic syndrome (HUS). Mouse models of HUS induced by LPS/Stx2 have been used for elucidating HUS pathophysiology and for therapeutic development. However, the underlying molecular mechanisms and detailed injury sites in this model remain unknown. We analyzed mouse kidneys after LPS/Stx2 administration using microarrays. Decreased urinary osmolality and urinary potassium were observed after LPS/Stx2 administration, suggestive of distal nephron disorders. A total of 1,212 and 1,016 differentially expressed genes were identified in microarrays at 6 h and 72 h after LPS/Stx2 administration, respectively, compared with those in controls. Ingenuity pathway analysis revealed activation of TNFR1/2, iNOS, and IL-6 signaling at both time points, and inhibition of pathways associated with lipid metabolism at 72 h only. The strongly downregulated genes in the 72-h group were expressed in the distal nephrons. In particular, genes associated with distal convoluted tubule (DCT) 2/connecting tubule (CNT) and principal cells of the cortical collecting duct (CCD) were downregulated to a greater extent than those associated with DCT1 and intercalated cells. Stx receptor globotriaosylceramide 3 (Gb3) revealed no colocalization with DCT1-specific PVALB and intercalated cell-specific SLC26A4 but did present colocalization with SLC12A3 (present in both DCT1 and DCT2), and AQP2 in principal cells. Gb3 localization tended to coincide with the segment in which the downregulated genes were present. Thus, the LPS/Stx2-induced kidney injury model represents damage to DCT2/CNT and principal cells in the CCD, based on molecular, biological, and physiological findings.

Carboxypeptidase B2 and N play different roles in regulation of activated complements C3a and C5a in mice

Background There are two basic carboxypeptidases in plasma. Carboxypeptidase B2 (CPB2) is activated from a circulating zymogen, proCPB2, and carboxypeptidase N (CPN) is constitutively active with both inactivating complement C3a and C5a. Aims To test the roles of CPB2 and CPN in complement-driven mouse models of cobra venom factor (CVF) challenge and hemolytic-uremic syndrome (HUS). Methods Cpb2-/- , Cpn-/- and wild-type (WT) mice were compared in an HUS model induced by Shiga toxin and lipopolysaccharide administration and following CVF administration. Results HUS was exacerbated in Cpb2-/- mice more than in Cpn-/- mice, compared with WT mice. Cpb2-/- mice developed the HUS clinical triad of microangiopathic hemolytic anemia, uremia and thrombocytopenia. Treatment with anti-C5 antibody improved survival of both Cpb2-/- and Cpn-/- mice. In contrast, when challenged acutely with CVF, the reverse phenotype was observed. Cpn-/- mice had markedly worse disease than Cpb2-/- mice, whereas the WT mice were resistant. Conclusions CPN and CPB2 play overlapping but non-redundant roles in regulating complement activation in vivo. The constitutively active CPN is key for inactivation of systemic C5a, whereas CPB2 functions as an on-demand supplementary anaphylatoxin inhibitor in inactivating excessive C5a formed locally.

Deficiency of Either Carboxypeptidase B2 or Carboxypeptidase N Exacerbated Disease Activity in a Mouse Model of Hemolytic Uremic Syndrome

Two different basic carboxypeptidases circulate in blood - carboxypeptidase N (CPN) and proCPB2. CPN is constitutively active, while proCPB2 is a zymogen, (also termed thrombin activatable fibrinolysis inhibitor, TAFI), and is activated by the endothelial thrombin/thrombomodulin complex to CPB2. Their kinetics of substrate cleavage are distinct but both can efficiently inactivate the complement anaphylatoxins, C3a and C5a. Hemolytic uremic syndrome (HUS) is caused by Shiga toxin (stx) producing strains of E. coli and is characterized by the triad of hemolysis, thrombocytopenia and uremia. We hypothesized that in a mouse model of HUS, Cpb2-/- and Cpn-/- mice would have prolonged C5a anaphylatoxin activity thus causing disease exacerbation.HUS was induced by stx2 and LPS administration in WT, Cpn-/- and Cpb2-/- mice. In Cpb2-/- mice, median time to death was earlier (60 hours, n=15) than in WT mice (96 hours: n=42, p<0.0001), and had greater kidney and liver damage shown by increases in ALT, AST, BUN and creatinine levels at 48 hours (creatinine Cpb2-/-: 1.01 mg/dL, WT: 0.25 mg/dL; Cpb2-/- control: 0.20 mg/dL and WT control: 0.19 mg/dL; Cpb2-/- p<0.0001 vs. all other groups, n>9). An increase in hemolysis was demonstrated by reduced RBC count and hemoglobin level plus an increase in total bilirubin and LDH. Profound thrombocytopenia (Cpb2-/-: 121,000/μL vs. WT: 217,000/μL; p=ns) developed in both genotypes (control Cpb2-/-: 1,001,000/μL vs. control WT: 1,141,000/μL; p=ns but vs. either Cpb2-/- or WT with HUS, p<0.0001) and thus the HUS clinical triad was present. Histology showed tubular epithelial necrosis in the kidney ante-mortem. Administration of either toxin separately caused milder disease without the characteristics of HUS and with no observed mortality. Induction of the disease depended on co-administration of both toxins. Treatment with anti-murine C5 antibody (0.75 mg every 24 hours from 3 hours before disease initiation) improved survival of both WT and Cpb2-/- mice with a median survival time of 168 hours for both genotypes (n=11, p=0.003 and <0.0001 respectively) and normalized the outcomes between the genotypes.Cpn-/- mice also died sooner (median time to survival 81.5 hours, n=28) than WT mice (96 hours, n=42, p=0.0002). The median survival time between Cpb2-/- and Cpn-/- mice was also significantly different (60 vs. 81.5 hours, p=0.0083). This is a first direct comparison of the role of CPN vs. CPB2 in regulating C5a activity in a disease relevant mouse model. Our study suggests that both CPB2 and CPN protect against HUS by inactivation of C5a with CPB2 having a greater effect than CPN. When Cpb2+/-/Cpn+/- mice are crossed, all expected genotypes are recovered in the expected Mendelian ratios including double deficient Cpb2-/-/Cpn-/- mice. Thus absence of both plasma basic carboxypeptidases is not essential for murine life. We are currently evaluating the Cpb2-/-/Cpn-/- mice in our HUS model. DisclosuresNo relevant conflicts of interest to declare.

Therapeutic use of a receptor mimic probiotic reduces intestinal Shiga toxin levels in a piglet model of hemolytic uremic syndrome

BackgroundHemolytic uremic syndrome (HUS) is a systemic and potentially fatal complication of gastroenteritis secondary to Shiga toxin-producing enterohemorrhagic Escherichia coli (EHEC) infection characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal damage. Shiga toxin (Stx), the toxin principle in HUS, is produced locally within the gut following EHEC colonization and is disseminated via the vasculature. Clinical development of HUS currently has no effective treatment and is a leading cause of renal failure in children. Novel post-exposure therapies are currently needed for HUS; therefore, the purpose of this study was to investigate the efficacy of a Stx receptor mimic probiotic in a porcine model of HUS. Edema disease, an infection of swine caused by host adapted Shiga toxin-producing Escherichia coli (STEC) and mediated by Shiga toxin 2e (Stx2e), shares many pathogenic similarities to HUS. In this study, three-week old piglets were inoculated with STEC and 24 hours later treated twice daily with a probiotic expressing an oligosaccharide receptor mimic for Stx2e to determine if the probiotic could reduce intestinal toxin levels.MethodsPiglets were orally inoculated with 1010 CFU of STEC strain S1191 eight days after weaning. Beginning day 1 post-inoculation, piglets were treated orally twice daily with 5 × 1011 CFU of either the receptor mimic probiotic or a sham probiotic for 10 days. Intestinal Stx2e levels were assessed daily via Vero cell assay. The efficacy of the probiotic at reducing intestinal Stx2e, vascular lesions, and clinical disease was evaluated with repeated measures ANOVA and Fisher’s exact test as appropriate.ResultsThe probiotic significantly reduced intestinal Stx2e, as reflected by decreased fecal toxin titers on days 3–8 post-inoculation (p < 0.01). Despite this reduction in intestinal toxin levels, however, the probiotic failed to reduce the incidence of vascular necrosis in target organs and had no effect on clinical disease.ConclusionsThe data suggest that post-exposure treatment with a Stx-binding probiotic is effective in reducing intestinal toxin burden. Future studies could target this approach for possible development of post-exposure interventions.

Oral administration of Shiga toxin-producing Escherichia coli induces intestinal and systemic specific immune response in mice

Hemolytic uremic syndrome (HUS) is the major complication of gastrointestinal infections with enterohemorrhagic Escherichia coli (EHEC) and is mediated by the production of Shiga toxins (Stx). Although it has been previously reported that not only HUS patients but healthy children have anti-Stx antibodies, very little is known about how these infections impact on mucosal immune system to generate a specific immune response. This work aimed to evaluate the immune responses elicited after a single oral dose of EHEC in a mouse model of HUS at weaning. We found sequential activation of T and B lymphocytes together with an increased percentage of IgA-bearing B cells in Peyer's patches and mesenteric lymph nodes. We also found fecal anti-EHEC IgA and serum anti-Stx2 IgG in EHEC-inoculated mice. Besides, these mice were partially protected against an intravenous challenge with Stx2. These data demonstrate that one episode of EHEC infection is enough to induce activation in the gut-associated lymphoid tissue, especially the B cell compartment, and lead to the production of specific IgA in mucosal tissue and the generation of systemic protection against Stx2 in a percentage of intragastrically inoculated mice. These data also support the epidemiologic observation that a second episode of HUS is very rare.

Intraperitoneal administration of Shiga toxin 2 induced neuronal alterations and reduced the expression levels of aquaporin 1 and aquaporin 4 in rat brain

Shiga toxin-producing Escherichia coli produces watery and hemorrhagic diarrhea, and hemolytic uremic syndrome (HUS) characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure. Central nervous system (CNS) complications are observed in around 30% of infant population with HUS. Common signs of severe CNS involvement leading to death include seizures, alteration of consciousness, hemiparesis, visual disturbances, and brain stem symptoms. The purpose of the present work was to study the effects of Shiga toxin 2 (Stx2) in the brain of rats intraperitoneally (i.p.) injected with a supernatant from recombinant E. coli expressing Stx2 (sStx2). Neurological alterations such as postural and motor abnormalities including lethargy, abnormal walking, and paralysis of hind legs, were observed in this experimental model of HUS in rats. Neuronal damage, as well as significant decrease in aquaporin 1 (AQP1) and aquaporin 4 (AQP4) expression levels were observed in the brain of rats, 2 days after sStx2 injection, compared to controls. Downregulation of aquaporin protein levels, and neuronal alterations, observed in brain of rats injected with sStx2, may be involved in edema formation and in neurological manifestations characteristic of HUS.

Chemokine Receptor CCR1 Disruption Limits Renal Damage in a Murine Model of Hemolytic Uremic Syndrome

Shiga toxin (Stx)-producing Escherichia coli is the main etiological agent that causes hemolytic uremic syndrome (HUS), a microangiopathic disease characterized by hemolytic anemia, thrombocytopenia, and acute renal failure. Although direct cytotoxic effects on endothelial cells by Stx are the primary pathogenic event, there is evidence that indicates the inflammatory response mediated by polymorphonuclear neutrophils and monocytes as the key event during HUS development. Because the chemokine receptor CCR1 participates in the pathogenesis of several renal diseases by orchestrating myeloid cell kidney infiltration, we specifically addressed the contribution of CCR1 in a murine model of HUS. We showed that Stx type 2-treated CCR1(-/-) mice have an increased survival rate associated with less functional and histological renal damage compared with control mice. Stx type 2-triggered neutrophilia and monocytosis and polymorphonuclear neutrophil and monocyte renal infiltration were significantly reduced and delayed in CCR1(-/-) mice compared with control mice. In addition, the increase of the inflammatory cytokines (tumor necrosis factor-α and IL-6) in plasma was delayed in CCR1(-/-) mice compared with control mice. These data demonstrate that CCR1 participates in cell recruitment to the kidney and amplification of the inflammatory response that contributes to HUS development. Blockade of CCR1 could be important to the design of future therapies to restrain the inflammatory response involved in the development of HUS.

In vitro modeling of the microvascular occlusion and thrombosis that occur in hematologic diseases using microfluidic technology

In hematologic diseases, such as sickle cell disease (SCD) and hemolytic uremic syndrome (HUS), pathological biophysical interactions among blood cells, endothelial cells, and soluble factors lead to microvascular occlusion and thrombosis. Here, we report an in vitro "endothelialized" microfluidic microvasculature model that recapitulates and integrates this ensemble of pathophysiological processes. Under controlled flow conditions, the model enabled quantitative investigation of how biophysical alterations in hematologic disease collectively lead to microvascular occlusion and thrombosis. Using blood samples from patients with SCD, we investigated how the drug hydroxyurea quantitatively affects microvascular obstruction in SCD, an unresolved issue pivotal to understanding its clinical efficacy in such patients. In addition, we demonstrated that our microsystem can function as an in vitro model of HUS and showed that shear stress influences microvascular thrombosis/obstruction and the efficacy of the drug eptifibatide, which decreases platelet aggregation, in the context of HUS. These experiments establish the versatility and clinical relevance of our microvasculature-on-a-chip model as a biophysical assay of hematologic pathophysiology as well as a drug discovery platform.

Mouse model for hemolytic uremic syndrome induced by outer membrane vesicles ofEscherichia coliO157:H7

Hemolytic uremic syndrome (HUS) is characterized by acute renal failure in children and is typically complicated with thrombocytopenia and hemolytic anemia. Although mouse models of HUS have been evaluated using Shiga toxin (STx) combined with or without lipopolysaccharide (LPS), no HUS model has been tested using purified outer membrane vesicles (OMVs) from STx-producing Escherichia coli (STEC) O157:H7. Accordingly, we investigated whether OMVs of STEC O157:H7 conveying STx2 and LPS can cause HUS-like symptoms in mice inoculated intraperitoneally. Three types of OMVs differing in LPS acylation status and STx2 amount were used to compare their ability to induce HUS-like symptoms. Native OMVs (nOMV) with fully hexa-acylated LPS caused HUS-like symptoms at 72-96h after dually divided injections of 1μg nOMV per animal. However, elevated doses of modified OMVs (mOMV) carrying mainly penta-acylated LPS were required to induce similar HUS signs. Moreover, mitomycin-C-induced OMVs (mcOMV) that were enriched with STx2 induced HUS-like symptoms similar to those of nOMV at the same dose. These results suggest that the OMVs of STEC O157:H7 potentiated with STx2 and fully hexa-acylated LPS can be utilized for inducing HUS-like symptoms in mice and could be the causative material involved in the development of HUS.

Alternative Pathway Activation of Complement by Shiga Toxin Promotes Exuberant C3a Formation That Triggers Microvascular Thrombosis

Shiga toxin (Stx)-producing E.coli O157:H7 has become a global threat to public health; it is a primary cause of diarrhea-associated hemolytic uremic syndrome (HUS), a disorder of thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure with thrombi occluding renal microcirculation. In this study, we explored whether Stx triggers complement-dependent microvascular thrombosis in in vitro and in vivo experimental settings of HUS. Stx induced on human microvascular endothelial cell surface the expression of P-selectin, which bound and activated C3 via the alternative pathway, leading to thrombus formation under flow. In the search for mechanisms linking complement activation and thrombosis, we found that exuberant complement activation in response to Stx generated an increased amount of C3a that caused further endothelial P-selectin expression, thrombomodulin (TM) loss, and thrombus formation. In a murine model of HUS obtained by coinjection of Stx2 and LPS and characterized by thrombocytopenia and renal dysfunction, upregulation of glomerular endothelial P-selectin was associated with C3 and fibrin(ogen) deposits, platelet clumps, and reduced TM expression. Treatment with anti-P-selectin Ab limited glomerular C3 accumulation. Factor B-deficient mice after Stx2/LPS exhibited less thrombocytopenia and were protected against glomerular abnormalities and renal function impairment, indicating the involvement of complement activation via the alternative pathway in the glomerular thrombotic process in HUS mice. The functional role of C3a was documented by data showing that glomerular fibrin(ogen), platelet clumps, and TM loss were markedly decreased in HUS mice receiving C3aR antagonist. These results identify Stx-induced complement activation, via P-selectin, as a key mechanism of C3a-dependent microvascular thrombosis in diarrhea-associated HUS.

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.

Characterization of Stx2 Tubular Response in a Rat Experimental Model of Hemolytic Uremic Syndrome

Background/Aims: In Argentina, hemolytic uremic syndrome (HUS) constitutes the most frequent cause of acute renal failure in children. The aim of our study was to analyze the early tubular response under the effect of Shiga toxin type 2 (Stx2) in a rat experimental model of HUS. Methods: Adult male Sprague-Dawley rats were injected intraperitoneally with culture supernatant from recombinant Escherichia coli expressing Stx2. Functional, histological, immunohistochemical and Western blot studies were performed at 48 h postinoculation. Results: Renal tubules showed the loss of the epithelial markers E-cadherin and β-catenin, and an increase in transforming growth factor-β1 expression. We detected the expression of α-smooth muscle actin in the interstitium and fibrosis in the periglomerular areas. Conclusion: Our results indicate that the early tubular response to the effects of Stx2 is related to an immunophenotype change of tubular cells and the presence of mild fibrosis in the interstitium.

Development of an experimental hemolytic uremic syndrome in rats

Escherichia coli strains producing Shiga toxins (Stxs) colonize the lower gastrointestinal tract and cause watery diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome (HUS). HUS is characterized by hemolytic anemia, thrombocytopenia, and acute renal failure. Oliguria associated with acute tubular necrosis and microangiopathic thrombosis has been reported as the most common cause of renal failure in Argentinean children. Our study was undertaken to obtain a model of HUS in rats that was similar to the clinical and renal histopathology findings described in humans. Rats were intraperitoneally inoculated with culture supernatant from recombinant E. coli expressing Stx2. Glomerular filtrate volume evaluated from clearance of creatinine resulted in a progressive reduction (from 53% at 24 h to 90% at 48 h). Urine volume increased significantly at 24 h but returned to normal levels at 48 h. Evidence of thrombocytopenia, anemia and leukocytosis was documented. Macroscopic analysis revealed a hyperemic peritoneal face with intestinal water accumulation. The kidneys were friable and congestive. Histopathological analysis showed glomerular and tubular necrosis as well as microangiopathic thrombosis. Our findings indicated vascular damage and kidney lesions similar to those described in humans with HUS.

Complement factor H and the hemolytic uremic syndrome

Immune recognition is coupled to powerful proinflammatory effector pathways that must be tightly regulated. The ancient alternative pathway of complement activation is one such proinflammatory pathway. Genetic susceptibility factors have been identified in both regulators and activating components of the alternative pathway that are associated with thrombotic microangiopathies, glomerulonephritides, and chronic conditions featuring debris deposition. These observations indicate that excessive alternative pathway activation promotes thrombosis in the microvasculature and tissue damage during debris accumulation. Intriguingly, distinct genetic changes in factor H (FH), a key regulator of the alternative pathway, are associated with hemolytic uremic syndrome (HUS), membranoproliferative glomerulonephritis (dense deposit disease), or age-related macular degeneration (AMD). A mouse model of HUS designed to mirror human mutations in FH has now been developed, providing new understanding of the molecular pathogenesis of complement-related endothelial disorders.

A Murine Model of HUS

Hemolytic uremic syndrome (HUS), which is caused by Shiga toxin-producing Escherichia coli infection, is the leading cause of acute renal failure in children. At present, there is no complete small animal model of this disease. This study investigated a mouse model using intraperitoneal co-injection of purified Shiga toxin 2 (Stx2) plus LPS. Through microarray, biochemical, and histologic analysis, it was found to be a valid model of the human disease. Biochemical and microarray analysis of mouse kidneys revealed the Stx2 plus LPS challenge to be distinct from the effects of either agent alone. Microarrays identified differentially expressed genes that were demonstrated previously to play a role in this disease. Blood and serum analysis of these mice showed neutrophilia, thrombocytopenia, red cell hemolysis, and increased serum creatinine and blood urea nitrogen. In addition, histologic analysis and electron microscopy of mouse kidneys demonstrated glomerular fibrin deposition, red cell congestion, microthrombi formation, and glomerular ultrastructural changes. It was established that this C57BL/6 mouse is a complete model of HUS that includes the thrombocytopenia, hemolytic anemia, and renal failure that define the human disease. In addition, a time course of HUS disease progression that will be useful for identification of therapeutic targets and development of new treatments for HUS is described.

Lipopolysaccharide Upregulates Renal Shiga Toxin Receptors in a Primate Model of Hemolytic Uremic Syndrome

Background: Although Shiga toxin (Stx) mediates classical hemolytic uremic syndrome (HUS), it is not fully understood why only some subjects exposed to Stx-expressing Escherichia coli develop HUS. We have previously shown in a baboon model of Stx-mediated HUS that coadministration of lipopolysaccharide (LPS) results in an augmented host response to otherwise subtoxic Stx1 doses. We used this model to test the hypothesis that LPS upregulates renal Stx receptor (Gb₃) expression. Methods: Juvenile baboons were treated with either Stx1 (100 ng/kg), LPS (1 mg/kg as two divided doses 24 h apart), or a sham injection of saline, and sacrificed and immediately autopsied at 72 h. Renal cortical tissue Gb₃ content was quantitated by lipid extraction and thin-layer chromatography, and Stx1 and Gb₃/CD77 immunostaining was assessed by quantitative immunofluorescent microscopy. Results: Compared to saline-in jected controls, LPS administration resulted in a 2.2-fold increase in renal cortical Gb₃ by chromatography (p < 0.01), a 2.5-fold increase in Stx1 staining (p = 0.003) and a 1.7-fold increase in CD77 immunostaining (p = 0.004). Stx treatment did not significantly alter either Stx or CD77 immunostaining. Conclusion: These observations suggest that LPS primes the host for Stx-mediated renal injury by upregulating renal Gb₃ expression.

Administration of Ricin Induces a Severe Inflammatory Response via Nonredundant Stimulation of ERK, JNK, and P38 MAPK and Provides a Mouse Model of Hemolytic Uremic Syndrome

Recent interest in the health consequences of ricin as a weapon of terrorism has led us to investigate the effects of ricin on cells in vitro and in mice. Our previous studies showed that depurination of the 28S rRNA by ricin results in the inhibition of translation and the coordinate activation of the stress-activated protein kinases JNK and p38 MAPK. In RAW 264.7 macrophages, ricin induced the activation of ERK, JNK, and p38 MAPK, the accumulation of mRNA encoding tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, the transcription factors c-Fos, c-Jun, and EGR1, and the appearance of TNF-alpha protein in the culture medium. Using specific inhibitors of MAPKs, we demonstrated the nonredundant roles of the individual MAPKs in mediating proinflammatory gene activation in response to ricin. Similarly, the intravenous administration of ricin to mice led to the activation of ERK, JNK, and p38 MAPK in the kidneys, and increases in plasma-borne TNF-alpha, IL-1beta, and IL-6. Ricin-injected mice developed the hallmarks of hemolytic uremic syndrome, including thrombotic microangiopathy, hemolytic anemia, thrombocytopenia, and acute renal failure. Microarray analyses demonstrated a massive proinflammatory transcriptional response in the kidneys, coincidental with the symptoms of hemolytic uremic syndrome. Therapeutic management of the inflammatory response may affect the outcome of intoxication by ricin.