Aliskiren inhibits renin-mediated complement activation.

Certain kidney diseases are associated with complement activation although a renal triggering factor has not been identified. Here we demonstrated that renin, a kidney-specific enzyme, cleaves C3 into C3b and C3a, in a manner identical to the C3 convertase. Cleavage was specifically blocked by the renin inhibitor aliskiren. Renin-mediated C3 cleavage and its inhibition by aliskiren also occurred in serum. Generation of C3 cleavage products was demonstrated by immunoblotting, detecting the cleavage product C3b, by N-terminal sequencing of the cleavage product, and by ELISA for C3a release. Functional assays showed mast cell chemotaxis towards the cleavage product C3a and release of factor Ba when the cleavage product C3b was combined with factor B and factor D. The renin-mediated C3 cleavage product bound to factor B. In the presence of aliskiren this did not occur, and less C3 deposited on renin-producing cells. The effect of aliskiren was studied in three patients with dense deposit disease and this demonstrated decreased systemic and renal complement activation (increased C3, decreased C3a and C5a, decreased renal C3 and C5b-9 deposition and/or decreased glomerular basement membrane thickness) over a follow-up period of four to seven years. Thus, renin can trigger complement activation, an effect inhibited by aliskiren. Since renin concentrations are higher in renal tissue than systemically, this may explain the renal propensity of complement-mediated disease in the presence of complement mutations or auto-antibodies.

A novel mechanism of bacterial toxin transfer within host blood cell-derived microvesicles.

Shiga toxin (Stx) is the main virulence factor of enterohemorrhagic Escherichia coli, which are non-invasive strains that can lead to hemolytic uremic syndrome (HUS), associated with renal failure and death. Although bacteremia does not occur, bacterial virulence factors gain access to the circulation and are thereafter presumed to cause target organ damage. Stx was previously shown to circulate bound to blood cells but the mechanism by which it would potentially transfer to target organ cells has not been elucidated. Here we show that blood cell-derived microvesicles, shed during HUS, contain Stx and are found within patient renal cortical cells. The finding was reproduced in mice infected with Stx-producing Escherichia coli exhibiting Stx-containing blood cell-derived microvesicles in the circulation that reached the kidney where they were transferred into glomerular and peritubular capillary endothelial cells and further through their basement membranes followed by podocytes and tubular epithelial cells, respectively. In vitro studies demonstrated that blood cell-derived microvesicles containing Stx undergo endocytosis in glomerular endothelial cells leading to cell death secondary to inhibited protein synthesis. This study demonstrates a novel virulence mechanism whereby bacterial toxin is transferred within host blood cell-derived microvesicles in which it may evade the host immune system.

Eculizumab in an anephric patient with atypical haemolytic uraemic syndrome and advanced vascular lesions.

BACKGROUND: Atypical haemolytic uraemic syndrome (aHUS) is associated with dysfunction of the alternative pathway of complement. Disease activity subsides as renal failure progresses but recurs upon renal transplantation, indicating that viable renal tissue contributes to disease activity. We present evidence of cerebrovascular occlusive disease indicating that vascular injury may occur in the absence of kidneys. METHODS: A currently 12-year-old girl developed renal failure at the age of 20 months. She underwent bilateral nephrectomy and renal transplantation but lost the transplant due to recurrences. She was on haemodialysis for 7 years. At 10 years of age she developed a transient ischaemic attack. Imaging, genetic investigation and mutation characterization were performed. RESULTS: Imaging demonstrated occlusion and stenosis of the carotid arteries. Two complement mutations, a novel mutation in factor B and a previously described mutation in factor I, and the H3-factor H haplotype, were identified. The factor B mutation, L433S, did not induce excessive complement activation in vitro. Measurement of C3 degradation products indicated ongoing complement activation. In spite of the patient being anephric, treatment was initiated with eculizumab, a humanized anti-C5 antibody that blocks terminal complement activation. She underwent a successful kidney transplant 9 months later and has not developed a recurrence or progression of vascular stenosis 1 year later. CONCLUSIONS: The course of disease in this patient with aHUS suggests that complement-mediated vascular injury may occur in the total absence of renal tissue and overt recurrences. To our knowledge, this is the first description of eculizumab treatment in an anephric aHUS patient.

Intestinal damage in enterohemorrhagic Escherichia coli infection.

Enterohemorrhagic Escherichia coli (EHEC) infection leads to marked intestinal injury. Sigmoid colon obtained from two children during EHEC infection exhibited abundant TUNEL-positive cells. To define which bacterial virulence factors contribute to intestinal injury the presence of Shiga toxin-2 (Stx2), intimin and the type III secretion system were correlated with symptoms and intestinal damage. C3H/HeN mice were inoculated with Stx2-producing (86-24) and non-producing (87-23) E. coli O157:H7 strains and 86-24 mutants lacking eae, encoding intimin (strain UMD619) or escN regulating the expression of type III secretion effectors (strain CVD451). Severe symptoms developed in mice inoculated with 86-24 and 87-23. Few mice inoculated with the mutant strains developed severe symptoms. Strain 86-24 exhibited higher fecal bacterial counts, followed by 87-23, whereas strains UMD619 and CVD451 showed minimal fecal counts. More TUNEL-positive cells were found in proximal and distal colons of mice inoculated with strain 86-24 compared with strains 87-23 and CVD451 (p ≤ 0.01) or UMD619 (p < 0.05, proximal colon, p < 0.01, distal colon). The results show that strains 86-24 and 87-23 exhibited better colonic persistence and more symptoms, presumably due to the presence of intimin and type III secretion effectors. Extensive intestinal mucosal cell death was related to the presence of Stx2.

Biologically active ADAMTS13 is expressed in renal tubular epithelial cells.

ADAMTS13 mRNA, which encodes the von Willebrand factor-cleaving protease, has been detected in a variety of tissues, including the kidney. The aim of our study was to characterize tubular expression and bioactivity of ADAMTS13. ADAMTS13 mRNA was detected in cultured primary human renal tubular epithelial cells (HRTEC) and in A498 cells, a human renal carcinoma cell line, by real-time PCR. Protein was detected using immunofluorescence and immunoblotting. Immunoblots demonstrated that the protein was secreted. The protease was proteolytically active in both cell lysates and cleaved the FRETS­VWF73 substrate. ADAMTS13 was demonstrated in situ in the renal cortex by immunohistochemistry. Protease was detected in both the proximal and distal renal tubules in normal renal tissue (n=3) as well as in patients with tubular disorders (n=3). Immunoblotting revealed that ADAMTS13 was present in the urine of patients with tubulopathy (n=5) but not in normal urine. ADAMTS13 in urine had a molecular size similar to that in plasma, which indicates that the protease originates in the tubuli because such large proteins do not normally pass the glomerular filter. In conclusion, human renal tubular epithelial cells synthesize biologically active ADAMTS13 which may, after release from tubuli, regulate hemostasis in the local microenvironment.

Shiga toxin and lipopolysaccharide induce platelet-leukocyte aggregates and tissue factor release, a thrombotic mechanism in hemolytic uremic syndrome.

BACKGROUND: Aggregates formed between leukocytes and platelets in the circulation lead to release of tissue factor (TF)-bearing microparticles contributing to a prothrombotic state. As enterohemorrhagic Escherichia coli (EHEC) may cause hemolytic uremic syndrome (HUS), in which microthrombi cause tissue damage, this study investigated whether the interaction between blood cells and EHEC virulence factors Shiga toxin (Stx) and lipopolysaccharide (LPS) led to release of TF. METHODOLOGY/PRINCIPAL FINDINGS: The interaction between Stx or LPS and blood cells induced platelet-leukocyte aggregate formation and tissue factor (TF) release, as detected by flow cytometry in whole blood. O157LPS was more potent than other LPS serotypes. Aggregates formed mainly between monocytes and platelets and less so between neutrophils and platelets. Stimulated blood cells in complex expressed activation markers, and microparticles were released. Microparticles originated mainly from platelets and monocytes and expressed TF. TF-expressing microparticles, and functional TF in plasma, increased when blood cells were simultaneously exposed to the EHEC virulence factors and high shear stress. Stx and LPS in combination had a more pronounced effect on platelet-monocyte aggregate formation, and TF expression on these aggregates, than each virulence factor alone. Whole blood and plasma from HUS patients (n = 4) were analyzed. All patients had an increase in leukocyte-platelet aggregates, mainly between monocytes and platelets, on which TF was expressed during the acute phase of disease. Patients also exhibited an increase in microparticles, mainly originating from platelets and monocytes, bearing surface-bound TF, and functional TF was detected in their plasma. Blood cell aggregates, microparticles, and TF decreased upon recovery. CONCLUSIONS/SIGNIFICANCE: By triggering TF release in the circulation, Stx and LPS can induce a prothrombotic state contributing to the pathogenesis of HUS.

Antibodies to intimin and Escherichia coli secreted proteins A and B in patients with enterohemorrhagic Escherichia coli infections.

Enterohemorrhagic Escherichia coli produce an attaching and effacing lesion upon adhering to the intestinal epithelium. Bacterial factors involved in this histopathology include the intimin adhesin and E. coli secreted proteins (Esps) A and B. In this study we investigated the serum antibody responses to recombinant E. coli O157:H7 intimin, EspA, and EspB by immunoblotting. Canadian patients with O157:H7 infection (n=10), Swedish patients with O157:H7 (n=21), non-O157 (n=18), or infection from which the serotype was not available (n=3), and asymptomatic household members (n=25) were studied and compared with Canadian (n=20) and Swedish controls (n=52). In Canadian patients, IgG antibodies to intimin, EspA, and EspB were analyzed, in Swedish patients and their household members IgA, IgG, and IgM antibodies to EspA and EspB were studied. Patients and household members mounted an antibody response to the antigens. Significantly more patients developed an acute response to EspB compared with controls (P<0.01 Canadian patients, P<0.0001 Swedish patients). EspB IgA, IgG, and IgM had a specificity of 100%, 86%, and 86%, positive predictive value of 100%, 83%, and 81%, and sensitivity of 57%, 69%, and 63%, respectively, and appear to be an appropriate assay for the detection of EHEC infection. In cases of hemolytic uremic syndrome or hemorrhagic colitis this assay may be useful when a fecal strain has not been isolated, or in epidemics of non-O157 infection.