Semin Immunopathol DOI 10.1007/s00281-014-0416-x

REVIEW

Hemolytic uremic syndrome Caterina Mele & Giuseppe Remuzzi & Marina Noris

Received: 6 December 2013 / Accepted: 19 January 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Hemolytic uremic syndrome (HUS) is a thrombotic microangiopathy defined by thrombocytopenia, nonimmune microangiopathic hemolytic anemia, and acute renal failure. The most frequent form is associated with infections by Shigalike toxin-producing bacteria (STEC-HUS). Rarer cases are triggered by neuraminidase-producing Streptococcus pneumoniae (pneumococcal-HUS). The designation of aHUS is used to refer to those cases in which an infection by Shigalike toxin-producing bacteria or S. pneumoniae can be excluded. Studies performed in the last two decades have documented that hyperactivation of the complement system is the pathogenetic effector mechanism leading to the endothelial damage and the microvascular thrombosis in aHUS. Recent data suggested the involvement of the complement system in the pathogenesis of STEC-HUS and pneumococcal-HUS as well. Clinical signs and symptoms may overlap among the different forms of HUS; however, pneumococcal-HUS and aHUS have a worse prognosis compared with STEC-HUS. Early diagnosis and identification of underlying pathogenetic mechanism allows instating specific support measures and therapies. In clinical trials in patients with aHUS, complement inhibition by eculizumab administration leads to a rapid and sustained normalization of hematological parameters with improvement in long-term renal function. This review summarizes current concepts about the epidemiological findings, This article is a contribution to the special issue on Immunopathology of Glomerular Diseases - Guest Editors: P. Ronco and J. Floege C. Mele : G. Remuzzi : M. Noris (*) IRCCS Istituto di Ricerche Farmacologiche “Mario Negri”, Clinical Research Center for Rare Diseases “Aldo e Cele Daccò”, Via Camozzi, 3, Ranica, Bergamo 24020, Italy e-mail: [email protected] G. Remuzzi Unit of Nephrology and Dialysis, Azienda Ospedaliera Papa Giovanni XXIII, Bergamo, Italy

the pathological and clinical aspects of STEC-HUS, pneumococcal-HUS, and aHUS, and their diagnosis and management. Keywords STEC-HUS . Pneumococcal-HUS . aHUS . Complement system activation . Eculizumab

Definition and classification In the early 1950s, two students in medicine working with Carlos Gianantonio at the Italian Hospital of Buenos Aires saw three subsequent children presenting with bloody diarrhea, edema, and convulsion. In the first two patients, who died within few days, a diagnosis of encephalitis was made. However, urea values evaluated in the third child were very high, and this led the students to suspect that the children had actually a renal disease [1]. Those were indeed childhood cases of hemolytic uremic syndrome (HUS), a new syndrome described shortly after by Gasser and colleagues in 1955 in a clinical report of five children with acute renal failure (ARF) who died with renal cortical necrosis [2]. Clinically defined by thrombocytopenia, nonimmune microangiopathic hemolytic anemia, and acute kidney failure, HUS is a microvascular occlusive disorder that belongs to the category of diseases known as thrombotic microangiopathies (TMAs) [3, 4]. The term TMA defines a histologic lesion found in the arterioles and capillaries and characterized by thickening of the vascular walls, prominent endothelial swelling and detachment, and subendothelial accumulation of proteins and cell debris. In patients with TMA, formation of fibrin and plateletrich thrombi occurs in the microcirculation obstructing vessel lumina, leading to end-organ ischemia and infarction. Thrombocytopenia is partly due to consumption in microthrombi; however, labeling studies have shown that platelets are also removed in the reticuloendothelial system. Microangiopathic

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hemolytic anemia is due to the fragmentation of erythrocytes as a consequence of the abnormal high levels of shear stress in obstructed vessels. In patients with HUS, organ dysfunction includes major involvement of the kidneys, although heart, lungs, gastrointestinal tract, pancreas, and especially the brain can be affected [4]. In common medical language, HUS is broadly classified as typical (or post-diarrheal or diarrhea-positive D+) and atypical [5]. The former is associated with infections by Shiga-like toxin- (Stx-) producing bacteria, such as enterohemorrhagic Escherichia coli (STEC) or Shigella dysenteriae, and is more appropriately referred to as STEC-HUS. Several strains of E. coli (mostly the serotype 0157:H7, but also other serotypes such as O111:H8, O103:H2, O123, O26, O145, and O104:H4) isolated from human cases with diarrhea were found to produce Stxs [6]. STEC-HUS is acquired as a foodborne illness or from a contaminated water supply and begins with a history of bloody diarrhea in the majority of cases. It affects predominantly children, except in epidemics when it may occur in individuals with a wider range of ages, and represents the most frequent form of HUS [7]. Approximately 5 % of HUS cases in children are not associated with Stx-producing bacteria and result from infection by neuraminidase-producing Streptococcus pneumoniae. These forms are classified as pneumococcal-HUS (or neuraminidase-associated HUS) [8, 9]. The designation of atypical HUS (aHUS) has been historically used to describe rare cases of the disease (less than 10 %) in which infections by Stx-producing bacteria or S. pneumoniae can be excluded. Atypical HUS can be hereditary, affecting members of the same family several years apart [10], or sporadic. During the last two decades, genetic or acquired defects leading to the dysregulation of the complement system, an important effector mechanism of the innate immune system, have been discovered in about 60 % of patients with aHUS [3]. There are many other forms of aHUS that are associated with a variety of conditions, including viral (human immunodeficiency virus and H1N1 influenza A) and other bacterial infections, parasites (Plasmodium falciparum), oral contraceptives, calcineurin inhibitors, illicit drugs, cancer chemotherapy and ionizing radiation, malignant hypertension, bone marrow or solid organ transplantation, autoimmune disorders (e.g., systemic lupus erythematosus, scleroderma, antiphospholipid antibody disease), malignancy, pregnancy, hemolysis, elevated liver enzymes, and low platelet syndrome [3], and, in children, methyl malonic aciduria with homocystinuria, cblC type, a rare hereditary defect of cobalamin metabolism [11]. These forms are frequently called secondary aHUS. However, the term does not account for the evidence that many of the above conditions often act as trigger of the disease in individuals with a genetic background leading to complement dysregulation. For instance, a substantial proportion of aHUS cases occurring during pregnancy or

post-partum have been found to be associated with complement gene mutations [12]. Along with this, triggering/ underlying clinical conditions have been reported in up to 70 % of patients with complement gene mutations, showing that both genetic predisposition and a precipitating event are required for the development of the disease [13]. The classification of aHUS proposed by the European Pediatric Research Group in 2006 [5] should therefore be refreshed taking into account both the genetic background and etiologic trigger; however, this goes beyond the purpose of this article. This review summarizes the epidemiological findings and the pathological and clinical aspects of STEC-HUS, pneumococcal-HUS, and aHUS forms for which the underlying genetic or acquired abnormalities have been identified. In addition, the piece focuses on the role of activation of the complement system as a common pathogenetic mechanism leading to endothelial injury and microvascular thrombosis in the various forms of HUS [14] and the implications for therapy. Secondary aHUS forms with still undetermined mechanisms and cobalamin-associated-HUS will be no further discussed, since they should be deepened in relation to the specific associated disorders.

Epidemiology STEC-HUS The annual incidence of STEC-HUS is about 2/100,000 in adults and 6.1/100,000 in children under the age of 5 years [3]. It may be sporadic or present as an outbreak. It occurs worldwide but is most widely reported in countries with less developed medical services. Some studies indicate that rural populations are more at risk than urban populations [15, 16], and the incidence is higher in warmer months, peaking from June to September [17]. High rates of STEC-HUS have been reported in the regions of South America, especially Argentina, where HUS is endemic with an incidence five to ten times higher than in North America. In Argentina, STEC-HUS is the main cause of ARF in children and the second commonest cause of chronic kidney failure, accounting for 20 % of renal transplants in children and adolescents [18]. Conversely, to the best of our knowledge, in Mexico, there have been no reports of HUS or hemorrhagic colitis in association with E. coli O157:H7 infection. The reason for this lack of E. coli O157:H7-associated diseases has been explained by finding antibodies against the O157 lipopolysaccharide (LPS) in up to 69 % of serum and maternal milk samples from Mexicans [19]. In 2011, several European countries, particularly Northern Germany, experienced one of the largest STEC-HUS outbreaks ever reported. About 4,000 individuals suffered from

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E. coli O104:H4 infection, with more than 800 of them who developed HUS. Almost 90 % of affected patients were adults and, compared to previous STEC epidemics, there was a higher prevalence of affected young and middle-aged women [6]. STEC is transmitted by contaminated food and water, directly from one person to another and occasionally through occupational exposure. Most foodborne outbreaks have been traced to food derived from cattle, especially ground beef and raw milk [20]. In Argentina, the high incidence of STEC-HUS has been associated with the extensive cattle farming and the high consumption of meat. About 20 % of Argentine children start to eat meat at 5 months of age, and 80 % of them have meat in their diets at least three times a week. Eighty percent of the meat consumed is undercooked [21]. In addition to cattle, bacteria have been isolated from deer, sheep, goats, horses, dogs, birds, and flies. Shedding by ruminants is particularly common, suggesting that these animals provide a specific niche for the bacterium. In the past few years, fruits and vegetables have accounted for a growing number of recognized STEC infections. Radish sprouts have been implicated in several O157:H7 outbreaks in Japan [20]. In the USA, fresh produces such as lettuce, apple cider, unpasteurized apple juice, and alfalfa sprouts have been implicated. A widespread outbreak associated with spinach in North America had dramatically higher than typical rates of both hospitalization (52 %) and HUS (16 %), due to the emergence of a new variant of O157:H7 serotype that has acquired several gene mutations that likely contributed to more severe disease [22]. While some produce-associated outbreaks may be due to cross-contamination from meat products, others are more likely to reflect direct contamination in the field with feces of wild or domestic animals. STEC-HUS was considered a disease of white people. However, on October 1992, thousands of patients were affected by bloody diarrhea in Swaziland, an African country [23]. The patients were mainly men who drank untreated water while working in the fields. E. coli infected the carcasses of cattle that died after a prolonged drought then heavy rains washed them into rivers and contaminated the surface water. E. coli 0157 was isolated from the stools of affected patients, cattle dung, and randomly collected water samples. Sprouts may pose a special hazard, since pathogens present in trace amounts in seed may multiply during sprouting. The consumption of sprouts was identified as the most likely vehicle of infection in the recent O104:H4 outbreak in Germany [6, 24]. The chain of transmission appeared to have started in Egypt with fecal contamination of fenugreek seeds by either humans or farm animals. During sprout germination, bacteria multiplied and produced large amounts of toxins and were then diffused with food provided to restaurants and consumers. It has been suggested that the higher prevalence

of women in this outbreak reflects a gender-specific dietary preference [6]. Waterborne outbreaks of STEC O157 infection have occurred as a result of drinking and swimming in unchlorinated water [25]. Person-to-person transmission occurs in day care and chronic care facilities, settings that combine a high potential for transmission with a population at increased risk for severe outcomes [20]. Pneumococcal-HUS Pneumococcal-associated HUS is a rare but potentially fatal disease that may complicate pneumonia or, less frequently, meningitis caused by S. pneumoniae [26]. It occurs mainly in children under 2 years of age, while in adults, it is extremely rare [27], but it is possible that pneumococcal-HUS is underdiagnosed. It comprises ∼5 % of cases of HUS in children and 40 % of cases are not associated with STEC [28, 29]. Atypical HUS Atypical HUS is an extremely rare disease. Very few sources of data are available regarding the incidence of this condition, severely limiting our understanding of its true epidemiology. The annual incidence of aHUS is thought to be about 2 per million for adults [29] and 3.3 per million in children younger than 18 years [30]. Atypical HUS occurs at any ages, from the neonatal period to the adult age [13, 31]. Onset during childhood (≤18 years) appears slightly more frequent than during adulthood (approximately 60 and 40 % of cases, respectively) [13, 32]. Seventy percent of children have the first episode of the disease before the age of 2 years and approximately 25 % before the age of 6 months [31]. Therefore, onset before the age of 6 months is strongly suggestive of aHUS, as less than 5 % of STEC-HUS occurs in this age group [33]. Both sexes are equally affected in childhood [31], while there is a female preponderance in adults [34]. A familial occurrence is observed in approximately 20 % of patients. In these cases, the disease has either autosomal recessive or dominant patterns of inheritance [3].

Laboratory exams Microangiopathic hemolytic anemia and thrombocytopenia are the laboratory hallmarks of HUS [3, 35]. Laboratory results indicate the presence of a usually severe microangiopathic hemolytic anemia, highlighted by low hemoglobin levels (460 U/l), reflecting not only hemolysis but also diffuse tissue ischemia. The latter mechanism is highlighted by reports of patients with high LDH levels persisting after normalization of platelet counts

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and hemoglobin levels [36]. Other indicators of intravascular hemolysis include hyperbilirubinemia, reticulocyte counts uniformly elevated, and low or undetectable haptoglobin concentrations. The peripheral smear reveals increased schistocyte numbers, with polychromasia and often nucleated red blood cells. Detection of fragmented erythrocytes together with a negative Coombs’ test is crucial to confirm the microangiopathic nature of the hemolytic anemia, although patients with pneumococcal-HUS may have a positive direct Coombs’ test result. Moderate leukocytosis may accompany hemolytic anemia. Consumption of platelets in thrombi causes a remarkable reduction of platelet count (platelets ≤150×109/l). The presence of giant platelets in the peripheral smear and/or reduced platelet survival time is consistent with peripheral consumption. In children with STEC-HUS, the duration of thrombocytopenia is variable and does not correlate with the course of renal disease [37]. Bone marrow biopsy specimens usually show erythroid hyperplasia and an increased number of megakaryocytes. Prothrombin time, partial thromboplastin time, fibrinogen level, and coagulation factors are generally normal. Mild fibrinolysis with minimal elevation in fibrin degradation products, however, may be observed. Evidence of kidney involvement is present in all patients with HUS (by definition). Elevated serum creatinine levels, low glomerular filtration rates (GFR), microscopic hematuria, and subnephrotic proteinuria are the most consistent findings.

The third lesion, “cortical necrosis,” patchy or rarely diffuse to the whole superficial cortex, is due to the acute cortical ischemia after obstruction of the local microcirculation. It is mainly observed in the most severe forms of STEC-HUS and generally associated with prolonged anuria at the acute phase of the disease. It is irreversible and, if extensive, correlates with a failure to recover kidney function. However, it should be underlined that biopsies are rarely taken in HUS patients, especially during the period of thrombocytopenia because of the risk of bleeding. Therefore, much of the correlations between the various forms of HUS and pathological findings have been obtained with tissues collected at post-mortem, which represent a later stage in the process. Other pathological changes such as mesangiolysis occur, but the cause is unclear. Deposition of fibrin or fibrinogen in the glomeruli and in the mesangium as well as within the vessel walls is seen by immunofluorescence. Deposits of complement activation products have also been reported in kidney biopsies (Fig. 1a b) [40, 41]. Unless there is a rapid resolution of glomerular thrombosis, the lesion progresses to global glomerular sclerosis, downstream ischemia, interstitial fibrosis, and tubular atrophy. Following an episode of HUS with moderate or severe nephron loss, surviving glomeruli and nephrons undergo compensatory hypertrophy, as with any other nephron destructive process [42].

Clinical features and pathogenesis Pathology

STEC-HUS: clinical features and diagnosis

Broadly, three types of TMA lesion are seen in patients with HUS [38]. In the most common “glomerular TMA,” glomerular capillary walls are thickened, with widening of the subendothelial space. Endothelial cells may appear swollen, obstructing the capillary, or detached from the basement membrane. These lesions are mainly observed in glomerular capillaries and preglomerular arterioles, whereas larger arteries are only rarely involved. The mesangial matrix often has a fibrillar appearance. Glomeruli may be enlarged, with capillaries distended by red cells and platelet–fibrin thrombi that may also be observed in the afferent arterioles. Glomerular TMA is a characteristic of HUS induced by STEC infections and is, to some extent, reversible [39]. The second type, “arterial TMA,” involves arterioles and interlobular arteries. Intimal edema and proliferation, necrosis of the arterial wall, luminal narrowing, and thrombosis are observed. Glomeruli appear ischemic and shrunken, with wrinkling of the basement membranes of the collapsed capillaries. Arterial TMA is more often seen in aHUS forms, in relapsing disease, and associated with both severe hypertension and poor prognosis.

STEC infections cause a spectrum of clinical signs ranging from asymptomatic carriage to non-bloody diarrhea, hemorrhagic colitis, HUS, and death. The average interval between ingestion of STEC and illness manifestation is approximately 3 days, although this can vary between 2 and 12 days. Illness typically begins with severe abdominal cramping and nonbloody diarrhea which becomes hemorrhagic in 70 % of cases usually within 1 or 2 days [20]. Vomiting occurs in 30 to 60 % of cases and fever is reported in up to 30 % of patients during this initial phase of the disease. The percentage of cases that progressed to HUS ranged from 3 to 9 % in a series of sporadic cases to about 20 % or more in some outbreaks [20]. The diagnosis of HUS is usually made 6 to 10 days after the onset of diarrhea, once kidney failure supervenes. Renal involvement presents with oligoanuria and is often associated with hypertension. Microscopic hematuria and mild proteinuria are common and rarely gross hematuria occurs [4]. STEC-HUS is not a benign disease. Seventy percent of patients require red blood cell transfusions, and 40–50 % need dialysis for an average duration of approximately 10 days, while the remainder have milder renal involvement without

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a

b

Fig. 1 Immunohistochemical staining of C3 and C9 in kidney biopsies from aHUS patients. a Strong endothelial C3 deposition in a glomerulus with acute ischemic alterations from a patient with aHUS and a C3

mutation. b Diffuse subendothelial and mesangial localization of C9 staining in a glomerulus from a patient with aHUS and a CFH mutation

the need for dialysis [20, 43]. Mortality for infants and young children in industrialized countries decreased with the introduction of dialysis and intensive care facilities, though still 1– 2 % of patients die during the acute phase of the disease. More than 90 % of childhood cases of STEC-HUS fully recover from the acute disease. However, a meta-analysis of 49 published studies (3,476 patients, including children and adults, mean follow-up of 4.4 years) describing the long-term prognosis of patients who survived an episode of STEC-HUS reported death or permanent end-stage renal disease (ESRD) in 12 % of patients and a GFR below 80 ml/min/1.73 m2 in 25 % [43]. Extrarenal manifestations are an important cause of added morbidity and are the main cause of death. Central nervous system disturbances are common and usually present early in the course of the illness; indeed, about 25 % of patients have neurological involvement, including stroke, seizure, and coma [20]. A rare but well-recognized complication is pancreatic involvement that can lead to overt diabetes mellitus [44]. It usually appears a week or more after the onset of HUS. Cardiomyopathy and myocarditis are described and may occur several weeks after onset. They may or may not be secondary to hypertension or vascular volume overload [45]. Disease presentation and outcome were particularly severe during the STEC O104:H4 German outbreak. Out of the 845 HUS cases, 50 died in Germany and 15 in other countries [46, 47]. Compared to previous STEC epidemics, there was a higher incidence of dialysis-dependent ARF (20 versus 6 %) and death (6 versus 1 %) [46]. Nearly half of the patients presented with neurological symptoms and 20 % of the patients suffered seizures. The severe clinical phenotype was explained by the lack of previous immunity to this novel STEC strain and its exceptional virulence [46]. Genomic analysis showed that the DNA sequence of E. coli O104:H4 outbreak strain is 93 % identical to the genomic sequence of enteroaggregative E. coli strains that form fimbriae which facilitate adhesion to the intestinal wall [48]. In addition, E. coli O104:H4 has acquired the ability to produce Stx [49], presumably through infection by a Stx-encoding phage

from a Stx-producing E. coli enterohemorrhagic strain. The combination of these two virulence factors would lead to increased gut colonization and thus the release of increased quantities of toxin into the circulation [46]. Moreover, while enterohemorrhagic E. coli are found in the gastrointestinal tract of ruminants, enteroaggregative E. coli appear to have their reservoir in humans [49]. This might explain why E. coli O104:H4 strain has acquired new resistances to antibiotics most commonly used in human disease [6]. Diagnosis of STEC-HUS depends on the detection of E. coli O157:H7 and other Stx-producing bacteria and their products in stool cultures. When infection by STEC is suspected, physicians should ensure that stool specimens are collected promptly and specifically cultured for the organism. In one study, detection of E. coli O157 in stool cultures declined from over 90 % for stools collected during the first 6 days of illness to 33 % for stools collected later [50]. Unlike most E. coli, serotype O157:H7 does not ferment sorbitol rapidly and thus forms colorless colonies on sorbitol containing MacConkey agar (SMAC). The use of SMAC provides a simple, inexpensive, and generally reliable method of screening stools for E. coli O157. Suspect colonies can be assayed for the O157 antigen with commercially available antiserum or latex agglutination kits. Enzyme-linked immunosorbent assays to detect O157 LPS or Stxs can further enhance detection [20]. Unfortunately, none of the major non-O157 serotypes have a known biochemical marker such as the lack of sorbitol fermentation to facilitate screening in the clinical laboratory. The polymerase chain reaction is being increasingly used in laboratory practice to detect Stxencoding genes. The sensitivity and specificity of this technique have been proven using DNA directly isolated from stool specimens, with or without prior broth enrichment. In the latter case, this test significantly shortened the turnaround time providing same-day results [51]. Convalescent-phase serum samples can be assayed for antibodies to O157 or to other specific strain-derived LPS, although this test is not commercially available and does not provide a diagnosis acutely [20].

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STEC-HUS: pathogenetic mechanisms After ingestion of contaminated food or water, STEC colonizes the intestinal mucosa, producing an “attaching/effacing” effect, whereby bacteria closely adhere to the epithelial cells, induce destruction of brush border villi, and likely cause watery or, most often, bloody diarrhea [52]. Once adhered to the intestinal epithelial cells, STEC produces and releases Stxs into the gut lumen within a few days after bacterial colonization. It is assumed that Stxs reach the kidney and other target organs via bloodstream after translocation across the intestinal epithelium. Free Stxs have not been detected in the sera of HUS patients so far, but Stx binding to neutrophils has been demonstrated [53]. The latter study indeed demonstrates that the Toll-like receptor 4 (TLR4) specifically recognizes and binds Stxs in human neutrophils as documented by the finding that Stx was displaced upon neutrophil treatment with TLR4 agonists and antagonists [53]. Other circulating human blood cells, such as erythrocytes, platelets [54], and monocytes [55], have been suggested to serve as Stx carriers. The process of Stx translocation across the epithelium is still unclear, but multiple routes have been proposed, including transcytosis by the polarized gastrointestinal cells and paracellular transport during polymorphonuclear leukocyte transmigration [56]. Two types of Stxs exist, Stx-1 and Stx-2, which are almost identical to the toxin produced by S. dysenteriae type 1 [57]. Both are 70-kDa AB5 holotoxins comprised of a single 32kDa A subunit and five 7.7-kDa B subunits. Despite their similar sequences, Stx-1 and Stx-2 cause different degrees and types of tissue damage as documented by the higher pathogenicity of strains of E. coli that produce only Stx-2 than strains that produce Stx-1 alone [58, 59]. In a study of children who became infected by STEC, E. coli strains producing Stx-2 were most commonly associated with HUS, whereas most strains isolated from children with diarrhea alone or remaining asymptomatic only produced Stx-1 [60]. Binding of Stxs to target cells is dependent on B subunits and occurs via the glycolipid cell surface receptor globotriaosylceramide (Gb3), predominantly expressed on endothelium [3]. The density of the Gb3 receptor mediates the susceptibility of individual vascular beds to the action of the Stxs and is particularly high in the kidney, as well as in the colon and brain [61]. Stx-mediated injury to the gastrointestinal microvasculature is the proposed mechanism leading to bloody diarrhea in STEC infections. In the case of HUS, kidney injury is mainly due to the binding of Stxs to renal endothelial cells, although evidence that Stx can also bind to podocytes, mesangial cells, and proximal tubules exists [62]. Interestingly, Gb3 represents the metabolite that accumulates in biological fluids, different organs, and vascular endothelium of patients with Fabry disease, an X-linked lysosomal storage disorder caused by deficiency of the lysosomal

hydrolase, α-galactosidase A. In these patients, Gb3 concentrations in urine and blood have been studied as potential biomarkers of disease severity [63]. So far, a possible relationship (albeit theoretical) between STEC-HUS and Fabry disease has never been addressed. It seems indeed an intriguing subject for future investigations. For many years, it has been assumed that the only relevant biologic activity of Stxs was to induce cell death by modifying the ribosome and blocking protein synthesis. However, it has been shown that treatment of endothelial cells with sublethal doses of Stxs exerts minimal influence on protein synthesis, altering instead endothelial cell gene expression and phenotype. Stxs upregulate mRNA expression and protein levels of chemokines [64, 65], chemokine receptors [65], and cell adhesion molecules [66, 67], which favor leukocyte recruitment. Stxs also increase endothelial tissue factor activity and directly activate platelets [68] and inflammatory cells [55]. Overall, Stxs favor leukocyte-dependent inflammation and induce loss of thromboresistance in endothelial cells leading to microvascular thrombosis, as documented by the formation of organized thrombi upon whole blood flowing on human microvascular endothelial cells pre-exposed to Stxs [67, 69]. In addition, it has been recently shown that Stx can directly interact with the von Willebrand factor (VWF). This interaction causes a delay in the cleavage of VWF by a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), providing a new molecular explanation for thrombus formation in STEC-HUS [70]. In vitro, tubular epithelial and mesangial cells are as susceptible to the cytotoxic effects of Stxs as endothelial cells [71]. Stxs inhibit water absorption across human renal tubular epithelial cell monolayers, which may contribute to the early events in the pathogenesis of renal dysfunction in STECHUS. Evidence is also emerging that complement system activation at renal endothelial level may contribute to microangiopathic lesions in STEC-HUS [14].

STEC-HUS: complement system activation The complement system plays an important role in the innate immune defense and in the maintenance of tissue homeostasis, leading to removal of foreign and host apoptotic cells by induction of cell lysis, phagocytosis, and inflammation [72]. It consists of a complex network of plasma proteins, organized in three distinct activation pathways (the alternative, the classical, and the lectin pathways), and a common terminal pathway (Fig. 2). The activation of the three pathways leads to the formation of proteases (complement C3 convertases) that cleave the central complement component C3. The cleavage of C3 generates large amounts of C3b and C3a, a peptide mediator of inflammation.

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Classical pathway

Lectin pathway

C1q, C1r, C1s

Alternative pathway

MBL, MASP

C2, C4

C1-inh

Tick-over hydrolysis

C3(H2O)

C1-inh

CFB CFI C4BP DAF MCP CR1 CFH CFI MCP CR1

C4BP DAF MCP CR1

C2aC4b

C3(H2O)Bb

CP/LP C3 convertase

AP fluid-phase C3 convertase

THDB

C3

CFH CFI

C3a

C3

C2aC4b

Amplification loop

C3a

CFB CFD

C4bC2aC3b

C3bBb

CP/LP C5 convertase

CFH DAF CR1

THDB

AP surface C3 convertase

C3

C3bBb

C5a

C3b

(C3b)2Bb

C5b C6-C9

CFH CFI MCP CR1

AP surface C5 convertase

C5a

C5b-9 (MAC)

THDB

C3a

C5

Clusterin, Vitronectin, CD59

THDB

C3b

C3b

C5

CFD

CFH DAF

Fig. 2 Schematic representation of the three pathways of complement activation. The classical (CP), the lectin (LC), and the alternative pathways (AP) of complement activation. The CP is activated by the binding of Fc region of IgG or IgM antibodies to the complement component C1 that circulates as a serum molecular complex comprising one molecule of C1q, two molecules of C1r, and two molecules of C1s (C1 complex or C1qr2s2). The LP is triggered by mannose binding lectin (MBL) associated serine proteases (MASP-1, MASP-2, and MASP-3). The CP and LP converge into the cleavage of complement components C2 and C4 leading to the formation of the CP/LP C3 convertase (C4bC2a). The AP is continuously activated in plasma by low-grade hydrolysis of C3 forming C3(H2O). The latter binds to factor B (CFB), which in turn is cleaved by factor D to form the AP fluid-phase C3 convertase. Both CP/

LP and AP C3 convertases cleave C3 into C3a, an anaphylotoxin, and C3b, the main effector molecule of the complement system. Once formed, C3b contributes to the formation of the AP surface C3 convertase that cleaves additional C3 molecules, resulting in an amplification loop. In addition, C3b contributes to the formation of the C5 convertases that cleave the complement component C5 producing the anaphylatoxin C5a and C5b. C5b initiates the late events of complement activation leading to the formation of the membrane-attack complex (MAC or C5b-9 complex). Self surfaces are protected from complement damage by protein regulators: C1-inh C1 inhibitor; CFI complement factor I; C4BP C4 binding protein; DAF decay accelerating factor; MCP membrane cofactor protein; CR1 complement receptor 1; CFH complement factor H; THBD trombomodulin

The alternative pathway (AP) undergoes constant lowgrade activation in fluid phase, so-called tick-over mechanism, by low rate spontaneous formation of C3(H2O) due to the hydrolysis of the reactive internal thioesther bond within the C3. Plasma complement factor B (CFB) binds to C3(H2O) and the plasma protease complement factor D cleaves bound CFB to Ba and Bb, the latter remaining associated with C3(H 2 O) to form the fluid-phase AP C3 convertase (C3(H2O)Bb), which in turn cleaves many molecules of C3 to C3a and C3b. Much of this C3b is quickly neutralized by hydrolysis in blood. However, some C3b molecules can attach covalently to the cell surfaces, where they bind CFB, and form the surface AP C3 convertase (C3bBb). The surface AP C3 convertase cleaves additional C3 molecules, resulting in a positive feedback loop.

Instead, triggering of the classical (CP) and lectin (LP) pathways relies on the recognition of pathogens or altered self cell surfaces by antibodies and pattern recognition molecules. CP and LP converge on C4 cleavage, leading to the formation of the CP/LP C3 convertase (C4bC2a) on target surfaces, with subsequent proteolysis of C3. Once C3b is formed, it can feed into the AP. Thus, once C3b is formed, activation of all pathways of complement can be enhanced through the amplification loop of the AP. The binding of C3b to the C3 convertases forms the C5 convertases that cleave the complement component C5 producing C5a and C5b. C5a, as well as C3a, is a potent anaphylatoxin that acts as chemoattractant for phagocytes and induces endothelial activation. C5b initiates the “late” events of complement activation, in which the terminal

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complement components (C5b, C6 to C9) interact to each other to form the membrane-attack complex (MAC, C5b-9 complex), which creates a pore in the cell membranes leading to their lysis. C3b molecules can bind to the surfaces of both pathogens and host cells. Self surfaces are protected from complement damage by a set of regulators that are either membraneanchored, such as membrane cofactor protein (MCP or CD46), decay accelerating factor, CD59, and the anticoagulant and complement inhibitory molecule thrombomodulin (THBD), or plasmatic such as complement factor H (CFH). These regulatory proteins favor the cleavage of C3b to inactive C3b (iC3b) by complement factor I (CFI) (cofactor activity), dissociate C3-convertase and C5-convertase (decay acceleration activity), or prevent C9 assembly into the C5b-9 complex. As early as 1970s, it was noticed that some STEC-HUS patients had low C3 plasma levels [73], and more recent studies have confirmed C3 reduction in severe cases of STEC-HUS [74, 75]. Nonetheless, serum levels of C3 fall within normal limits in the majority of affected patients. When more sensitive assays were used, signs of complement activation were consistently found in most cases. In 1980, Monnens et al. [76] showed increased levels of breakdown products of the two components of the AP C3 convertase, C3 (C3b, C3c, C3d) and CFB (Ba), in the plasma of children who were likely to have had STEC-HUS. More recently, increased plasma levels of another CFB activation fragment, Bb, and of sC5b9, the plasma form of MAC, were found in 17 children with STEC-HUS studied during the acute phase [77]. All these parameters rapidly normalized after resolution of the acute episode [77]. Similar results were obtained in a Swedish cohort of ten children, all of whom displayed elevated plasma levels of C3a and sC5b-9 at disease onset, with normalization during recovery [78]. The above findings point to the activation of the AP of complement in STEC-HUS through to C5b9 assembly. A preliminary report confirmed in 18 Argentine children with STEC-HUS an increase in Bb, sC5b-9, and C3c levels during the acute phase of the disease (Ferraris Jorge, Complement activation in typical hemolytic uremic syndrome. Poster at the Sixteenth Congress of the International Pediatric Nephrology Association, August 30–September 3, 2013, Shanghai, China). Bb and sC5b-9 values normalized within a week from admission. Bb values significantly correlated with blood urea nitrogen, LDH, and platelet count. Of relevance, oliguric patients significantly show higher plasma Bb level versus non-oliguric patients. In addition, the above Swedish report described higher levels of platelet-derived and leukocyte-derived microparticles bearing C3 and C5b-9 in blood from patients with STEC-HUS compared with healthy controls [78]. Evidence shows that Stxs might directly contribute to complement activation as documented by C3 deposition on

microvascular endothelial cell lines exposed to Stx-1 and then perfused with human serum [69]. Upon perfusion with whole blood, microvascular endothelial cells pretreated with Stxs had a larger cell surface covered by thrombi than did cells pre-exposed to medium alone, a phenomenon fully prevented by adding the soluble form of the complement inhibitor, complement receptor 1 (sCR1). Endothelial complement deposition and loss of thromboresistance depended on Stx-induced upregulation of the membrane adhesion molecule P-selectin, which has been shown to bind C3b with high affinity [69, 79], thereby triggering the AP. The functional relevance of this mechanism has been demonstrated through the effect of P-selectin blockade, which (1) substantially limited Stx-induced endothelial C3 deposition and thrombus formation in vitro and (2) reduced glomerular C3 staining in vivo in a murine model of STECHUS obtained by coinjection of Stx-2 and LPS [69]. CFBdeficient mice treated with Stx-2 and LPS exhibited less thrombocytopenia and were protected against glomerular abnormalities and renal function impairment, indicating the involvement of complement activation via the AP in the glomerular thrombotic process in mice with HUS [69]. Evidence showing that Stx-2 activates complement in the fluid phase is also available. Orth et al. [80] documented the formation of sC5b-9 upon incubation of human serum with Stx-2. This process was concentration-dependent, occurred predominantly via the AP, and did not involve degradation of the complement regulators CFH or CFI. Thus, the mechanisms underlying complement activation by Stx-2 in the fluid phase remain unknown. Pneumococcal-HUS: clinical features and pathogenesis In 1971, Fischer and colleagues first proposed an association between HUS and infection with neuraminidase-producing organisms such as S. pneumoniae [81]. The preceding infection is usually severe and invasive, and patients may present with septicemia, meningitis, and/or pneumonia with empyema. Patients with pneumococcal-HUS usually have a severe clinical picture with microangiopathic hemolytic anemia, respiratory distress, neurological involvement, and coma. Mortality in the acute phase is 25 % [26]. Neuraminidase produced by S. pneumoniae cleaves Nacetylneuraminic acid from the glycoproteins on the cell membrane of erythrocytes, platelets, and glomerular cells [82]; this exposes the normally hidden ThomsenFriedenreich antigen (T-antigen), which can then react with anti-T IgM antibodies naturally present in human plasma [83]. This antigen–antibody reaction occurs more frequently in infants and children and causes poly-agglutination of red blood cells in vitro. This is the reason why, unlike in other forms of HUS, in pneumococcal-HUS, there is a positive Coombs’ test. T-anti-T interaction on red cells, platelets, and

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endothelium was thought to explain the pathogenesis, whereas the pathogenic role of the anti-T cold antibody in vivo is uncertain [84]. T-antigen exposure on red cells is detected using the lectin hypogea. Recently, Szilagyi and colleagues reported a detailed investigation of the complement system profile including the analysis of different variants of the complement genes in five patients with pneumococcal-HUS [27]. In the acute phase, patients had decreased levels of C3 and C4 and decreased residual activity of the CP and AP, indicating severe complement consumption. Three out of five patients carried mutations and/or risk haplotypes in complement genes, suggesting that complement gene variants may contribute to the development of pneumococcal-HUS. Atypical HUS: clinical features At first flares of the disease, symptoms are characterized by the presence of the complete triad of HUS in most patients [35]. Arterial hypertension is frequent and often severe. Approximately 20 % of patients have a progressive onset with subclinical anemia and fluctuating thrombocytopenia for weeks or months and preserved normal renal function at diagnosis. In addition, some patients have no anemia or thrombocytopenia, and the only manifestation of an active renal TMA is hypertension and proteinuria and a progressive increase of serum creatinine. Although TMA predominantly affects renal vessels in aHUS by definition, the lesion can involve the microvasculature of other organs (heart, intestines, pancreas, lungs, and especially the brain) in about 20 % of patients [13, 31]. The most frequent (~10 %) extrarenal symptoms are neurological, including irritability, somnolence, confusion, convulsions, encephalopathy, cerebrovascular accidents, hemiparesia, hemiplexia, and coma [3, 13, 32]. Myocardial infarction, which can cause sudden death [13, 85], has been observed in as many as 3 % of patients, in addition to cardiomyopathy, heart failure, and peripheral ischemic heart disease [86, 87]. Evidence is also emerging that either thrombosis or stenosis of medium and large arteries may complicate disease course, and such disorders occur even after renal function is lost [88, 89]. Failure of different organs, as well as death, can occur unpredictably at any time, either very quickly or following prolonged symptomatic or asymptomatic disease progression [3, 13, 31, 87]. In contrast to STEC-HUS, which tends to occur as a single event, aHUS is a chronic condition and involves a poorer prognosis [13, 31, 32]. Half of the children and the majority of adults need dialysis at admission, and until very recent years when the anti-C5 monoclonal antibody eculizumab was introduced [90], about 50 % of patient never recovered from renal function. After the first episode, mortality has been reported to be higher in children than in adults, but progression to ESRD was more frequent in adults [91]. At 3 to 5 years after onset, 36

to 48 % [13, 92] of children and 64 to 67 % [13, 92] of adults died or had reached ESRD. Atypical HUS: pathogenesis To date, one or more genetic or acquired abnormalities in the complement system have been documented in nearly 60 % of patients with aHUS (Table 1) [13]. CFH mutations, CFH-CFHR hybrid genes, and anti-CFH autoantibodies CFH is the most important plasma regulator of the AP of complement. It is composed of 20 domains called short consensus repeats (SCRs), encoded by 23 exons. The four N-terminal SCRs 1–4 mediate the complement regulatory functions of the protein that acts as cofactor for CFI-mediated inactivation of C3b; competes with CFB for C3b binding; and accelerates the decay of the C3 convertase [93]. In addition to regulating complement in the fluid phase, CFH protects host surfaces by binding to polyanions such as the glycosaminoglycans [94]. CFH has two glycosaminoglycan binding domains in SCRs 6–8 and SCRs 19–20, which have different sulfate specificities resulting in the C-terminal domains (SCRs 19–20) being predominantly responsible for binding to renal endothelium [95]. The association between aHUS and low CFH levels had been described since the 1980s [96]. However, it is only in 1998 that Warwicker and colleagues described for the first time mutations in CFH gene (CFH) in aHUS [97]. Since then, more than 100 CFH mutations (http://www.FH-HUS.org) have been identified in adults and children with sporadic or familial aHUS. The mutations are of all types: nonsense, missense, small deletion/insertion. Mutations in CFH are the most frequent genetic abnormality in aHUS patients as they account for 20 to 30 % of cases [13, 87, 98]. Some patients had homozygous mutations, but most of them were heterozygous. More than 50 % of mutations cluster in SCR19–20. Some mutations are associated with quantitative deficit of Table 1 Summary of genetic and acquired abnormalities associated with atypical hemolytic uremic syndrome

Genetic/acquired abnormalities

Frequency (%)

CFH mutations CFH/CFHR hybrid genes CFH autoantibodies MCP mutations CFI mutations C3 mutations CFB mutations THBD mutations Combined complement abnormalities DGKE mutations

20–30 3–5 5–10 10–15 4–10 2–10 1–4 3–4 3.4 n.d.

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CFH, while the majority (including most SCR19–20 mutations) are associated with normal levels of CFH and result in a mutant protein that is unable to bind to and regulate complement on endothelial cells and platelets. Plasma C3 level is decreased in 30–50 % of patients with heterozygous mutations [13, 87]. CFH gene resides in the regulators of complement activation cluster and is in close proximity to the genes CFHR1-5 encoding five CFH-related proteins. CFH and CFHRs share a high degree of sequence identity. This homology predisposes to gene conversions and genomic rearrangements through nonallelic homologous recombination (NAHR) and microhomology-mediated end joining. A hybrid gene comprising the first 21 CFH exons and the last 2 CFHR1 exons [99] was shown to have arisen through NAHR. Another hybrid gene consisting of the first 22 CFH exons and the last 5 CFHR3 exons was reported in aHUS [100]. More recently, the finding of a novel NAHR event that results in an additional copy of CFHR3 and the formation of a novel hybrid gene consisting of the first four exons of CFHR1 and the last two exons of CFH has been reported in a 14-year-old aHUS patient [101]. Overall hybrid genes account for 3–5 % of aHUS patients and result in gene products with decreased complement regulatory activity on endothelial surfaces. In 2005, an acquired CFH defect due to anti-CFH IgG autoantibodies was described in aHUS patients [102]. These cases account for 5–10 % of patients [13, 86, 103] and around 25–50 % of pediatric cases [104, 105]. Ninety percent of patients with anti CFH-antibodies have a complete deficiency of CFHR1 and CFHR3 associated with a homozygous deletion of CFHR1 and CFHR3 [86, 106, 107], a polymorphism also observed at a frequency of 4 % in healthy Caucasians that do not develop anti-CFH antibodies. In more recent studies, some aHUS patients with CFHR1 deficiency resulting from CFHR1 mutations or from a deletion incorporating CFHR1 and CFHR4 developed anti-CFH antibodies [103, 108]. This may suggest that deficiency of CFHR1 alone is the predominant predisposing factor for autoantibodies. Mapping of the epitopes initially suggested that the antiCFH antibodies bound predominantly to SCR19 and 20 [102, 109]; however, it has been recently reported that the antibody response was polyclonal to multiple epitopes throughout CFH [110]. Cross-reactivity with CFHR1 [103, 110] and CFHR2 [110] has been found also. Several studies have shown various functional consequences of anti-CFH antibodies. They reduce CFH binding to C3b [109, 110] and other C3 fragments [110] perturb CFH-mediated cell surface protection [109, 110], and in some individuals, they also impair cofactor activity [110] or decay accelerating activity [102]. Plasma C3 levels are decreased in 40–60 % of patients with anti-CFH antibodies. CFH autoantibodies form immune complexes in the serum [110], which may explain the low CFH levels seen in 28 % of the cases.

MCP mutations MCP is a surface-bound complement regulatory protein that acts as a cofactor for the CFI-mediated cleavage of C3b and C4b molecules that are deposited on host cells [111]. The association between mutations in the corresponding gene (MCP) and aHUS was first described by Noris et al. and Richards et al. in 2003 [112, 113]. More than 40 different MCP mutations have been identified so far, accounting for 10–15 % of cases, and most are heterozygous (http://www. FH-HUS.org). The majority cluster in the extracellular domains SCRs 1–4, critical for regulation. MCP mutant proteins bind C3b weakly and have low cofactor activity [32]. Most patients (~75 %) have decreased MCP expression on peripheral leucocytes. Less frequently, MCP expression is normal, but the protein is dysfunctional. C3 levels in MCPmutated patients are most often normal, an expected finding as MCP alterations are not expected to activate complement in the fluid phase [13]. CFI mutations CFI is a serum serine protease that functions as a critical mediator of complement regulation by cleaving C3b and C4b in the presence of its cofactors (CFH for C3b, C4BP for C4b, MCP and CR1 for both). CFI mutations were first described in 2004 [114]. About 40 CFI mutations have been reported in patients with aHUS, all heterozygous [13, 32, 115, 116]. According to published series, CFI mutations account for 4 to 10 % of patients. Eighty percent of them cluster in the serine protease domain. Approximately 50 % of mutations induce a default of protein secretion, and some are secreted but have proteolytic activity disrupted with altered degradation of C3b/C4b in the fluid phase and on surfaces [115, 116]. C3 level can be decreased (observed in 20–30 % of patients) while CFI level is normal or vice versa [13, 87, 114, 115]. Mutations in the AP C3 convertase components Heterozygous gain-of-function mutations affecting genes encoding the AP C3 convertase components, CFB (CFB) and C3 (C3), were first described in 2007 and 2008, respectively [117, 118]. Most C3 mutations induce a defect on the ability of complement regulators to bind to C3b and lead to severe impairment of degradation of mutant C3b [118, 119]. More recently, two mutations in C3 have been reported in patients with aHUS leading to mutant proteins that bind to CFB with higher affinity, resulting in increased C3 convertase formation [120, 121]. These mutations caused increased complement activation on glomerular endothelium and platelets. Plasma C3 levels are low in 70–80 % of patients [13, 118, 119]. C3 mutations account for 2 to 10 % of aHUS patients [13, 98, 118]. CFB mutations result in a “super-B” which induces an increased stability and activity of the C3 convertase, resistant to decay by CFH, with enhanced formation of C5b-9

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complexes and deposition of C3 fragments at endothelial cell surfaces [120]. CFB-mutated patients exhibit a permanent activation of the AP with very low C3. Plasma CFB levels may be normal or low. Mutations in CFB are rare, accounting for only 1–4 % of aHUS patients [13, 87, 98, 120, 122]. THBD mutations THBD facilitates the activation of protein C by thrombin and enhances thrombin-mediated activation of plasma pro-carboxypeptidase B (TAFI), an inhibitor of fibrinolysis that also inactivates complement-derived anaphylatoxins C3a and C5a [123]. THBD has also been shown to downregulate the AP by accelerating CFImediated inactivation of C3b in the presence of cofactors [124]. Heterozygous mutations in the gene encoding THBD (THBD) have been found in 3–4 % of patients with aHUS [13, 98, 124]. THBD mutations have been reported in association with aHUS in two additional independent cohorts [98, 125]. Cells expressing these mutants inactivate C3b less efficiently than cells expressing wild-type THBD. These data document a functional link between complement and coagulation. C3 levels are decreased in half of THBD-mutated patients. Combined complement abnormalities Some patients have been reported with mutations in more than one complement genes or mutations in one complement gene in addition to anti-CFH autoantibodies [13, 87, 98, 115, 126]. In a report from four European cohorts, 27 out of 795 (3.4 %) aHUS patients screened for complement genes had combined mutations [126]. Of note, only 8–10 % of patients with CFH, C3, or CFB mutations carried abnormalities in other genes, suggesting that mutations in CFH, C3, or CFB alone may be sufficient to cause aHUS. In contrast, ∼25 % of patients with a mutation in MCP or CFI had a second or third mutation in other complement genes. DGKE mutations Very recently, homozygous or compound heterozygous mutations in the gene encoding for the diacylglycerol kinase ε (DGKE) co-segregated with aHUS in nine unrelated kindreds [127]. Mutation carriers presented with aHUS before 1 year of age had persistent hypertension, hematuria, and proteinuria and developed chronic kidney disease with age. DGKE is expressed in endothelium, platelets, and podocytes. It is apparently unrelated to the complement cascade and the mechanism by which DGKE mutations cause aHUS remains to be elucidated. Incomplete penetrance and triggering factors Mutations in complement genes can be found in healthy family members. Incomplete penetrance has been reported in approximately 50 % for individuals carrying mutations in CFH, CFI, MCP,

CFB, and C3 [13, 32]. Thus, it can be inferred that the genetic alterations are important but are not sufficient for the development of aHUS. As discussed above, patients may carry mutations in more than one gene or mutations combined with autoantibodies; otherwise, they may carry a mutation in combination with common at-risk genetic variants (SNPs and haplotype blocks) in CFH [13, 128–131] or MCP [128, 130] or CFHR1 [108]. It has been observed that the penetrance increased as the number of alterations in a patient increased. Even in the situation in which a patient has multiple genetic/ acquired risk factors, aHUS may not occur until middle age, suggesting that a triggering stimulus is required for the disease to manifest (multiple hits theory) [129]. Precipitating events, most commonly upper respiratory tract infections or gastroenteritis, have been reported in more than half of patients [13, 31]. Although the association with diarrhea has been well established with STEC-HUS, diarrhea also preceded aHUS in up to 24 % of patients [13]. However, it remains unclear whether the diarrheal episode acted as a trigger or was a consequence of the diffuse character of the TMA. Other infectious triggers such as varicella [132], H1N1 influenza [133], and, interestingly, STEC-diarrhea [13, 31, 86, 134] have been reported in patients who were investigated for aHUS because of a fulminant course, a familial incidence of the disease, or the subsequent occurrence of relapses. Pregnancy is a frequent triggering event in women [3, 12, 13]: as many as 20 % of women with aHUS experience the disease at pregnancy, 80 % of them during the post-partum period [12]. Thus, it is likely that aHUS results from an otherwise innocuous stimulus that triggers the AP and sets off a selfamplifying cycle that cannot be controlled appropriately in genetically susceptible individuals. Genotype–phenotype correlations The age of onset varies according to complement abnormality [13, 31]. The disease manifests mostly in childhood (≤18 years), with the exception of patients carrying CFI and C3 mutations. The earliest onset (0 to 1 years) was in patients with DGKE, CFH, or THBD mutations [13, 127]. Most patients with anti-CFH antibodies developed the disease from 9 to 13 years of age [86]. However, in 12 to 50 % of individuals, the disease occurred after the age of 25 years (up to 83 years). Outcomes vary according to the underlying complement alteration [13, 31, 92]. Individuals with mutations in CFH, CFI, or C3 had poor prognosis. About 60–70 % of patients with CFH, CFI, and C3 mutations lost renal function or died during the presenting episode or developed ESRD following relapses [3, 13, 32]. Patients with THBD mutations also had poor outcome, with evolution to ESRD in 46 % of patients at 1 year and 54 % at 3 years follow-up [13]. The prognosis of aHUS with CFB mutations is also poor [117]. In patients with anti-CFH-associated HUS, 35 % [86] to 60 % [13] of individuals died or reached ESRD within 3 years follow-up.

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Although in patients with MCP mutations recurrences are frequent, long-term outcome is good with 80 % of dialysisfree patients [3, 13, 32]. However, rare patients with MCP mutations had severe disease, immediate ESRD, intractable hypertension, and coma [126]. Concerning patients with combined complement gene mutations, in those with either CFH or CFI, the presence of mutations in other genes did not modify outcome. Conversely, in individuals with MCP mutations combined with a mutation in another complement gene, the prognosis was worse than in patients with MCP mutation alone [126].

Differential diagnosis Due to the rapid and severe progression of TMA, the diagnostic process must involve an immediate (first 24 h) identification of the responsible syndrome in order to instate initial support measures. Figure 3 displays the algorithm for the differential diagnosis and the corresponding therapy. Detection of Stx or positive STEC cultures in patients with TMA is diagnostic of STEC-HUS [135]. In cases of suspected pneumococcal-HUS, infection by S. pneumoniae is diagnosed by identification of positive bacteriological cultures of body

fluids (sputum/cerebral spinal fluid/blood/pus). The direct Coombs’ test is positive in over 90 % of patients with pneumococcal-HUS [136]; however, its specificity has not been tested. The demonstration of red cell agglutination by the lectin hypogea is the easiest way to demonstrate that circulating neuraminidase is present. The presence of extrarenal manifestations in aHUS can make ruling out other forms of TMA, especially thrombotic thrombocytopenic purpura (TTP), difficult. Atypical HUS and TTP generally have different clinical presentation, with predominant neurologic involvement in TTP and renal involvement in HUS, but symptoms may overlap [137]. In the case of TTP, intravascular thrombosis is the consequence of a severely deficient activity of ADAMTS13 [138], due genetic or acquired abnormalities. Thus, the diagnosis of TTP requires a direct demonstration that plasma activity of ADAMTS13 is ≤5 %. After excluding infections by STEC and neuraminidase-producing S. pneumoniae, and severe ADAMTS13 deficiency, the diagnosis should be orientated towards aHUS [35]. Complement analysis in cases of aHUS should include serum levels of C3, C4, CFH, and CFI. Low C3 levels may be observed in patients with mutations in CFH, CFI, CD46, C3, and CFB and may point to a complement-mediated

Diagnosis of TMA:

Critically ill patients - CNS signs - Dialysis dependent ARF

- Low Platelet Count - Increased serum LDH - Fragmented erythrocytes in the peripheral smear

Urgent plasma exchange with fresh frozen plasma

≤5% ADAMTS13 activity

TTP Continue plasma exchange with fresh frozen plasma

Anti-ADAMTS13 autoantibodies Consider also immunosuppressive therapy including Rituximab

No evidence of STEC or S. pneumoniae infection and >5% ADAMTS13 activity

aHUS

Evidence of STEC infection

Evidence of S. pneumoniae infection

aHUS with anti-CFH Ab

STEC-HUS

pneumococcal-HUS

Plasma exchange and immunosuppressive therapy

Conservative therapy Plasma exchange?

Antibiotics Conservative therapy Plasma exchange?

No or incomplete response

Consider Eculizumab therapy

Fig. 3 Proposed algorithm for differential diagnosis and therapy of STEC-HUS, pneumococcal-HUS, TTP, and complement-related aHUS. ADAMTS13 a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13; LDH lactate dehydrogenase; TMA thrombotic

microangiopathy; TTP thrombotic thrombocytopenic purpura; HUS hemolytic uremic syndrome; aHUS atypical hemolytic uremic syndrome; STEC-HUS Shiga-toxin producing E. coli; CNS central nervous system; ARF acute renal failure; anti-CFH Ab anti-factor H antibodies

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process; however, normal C3 levels do not exclude the presence of a complement gene mutation or anti-CFH autoantibodies [139]. Fluorescence-activated cell sorter analysis of peripheral blood mononuclear cells provides a quick screening option for MCP mutations, although genetic analysis still is required to detect all changes. Full analysis of diseaseassociated genes and screening for anti-CFH antibodies is recommended, in particular in the perspective of kidney transplantation for patients who progressed to ESRD. Reference laboratories in several countries are equipped for genetic and antibody screening. However, although new sequencing techniques are cutting cost and timing, a complete screening still requires several days of work. Treatment of acute episodes (plasma therapy or eculizumab when available, see below) should be started very rapidly after clinical diagnosis, without waiting for results of genetic and anti-CFH antibody tests. A complete clinical history is imperative, including age at onset, family history, triggering factors, and an exhaustive physical examination. In children, age, clinical context, and symptoms at presentation most often allow to differentiate patients as having STEC-HUS, pneumococcal-HUS, or aHUS. On the opposite, clinical presentation is more confusing in adults and aHUS has to be suspected whatever the clinical context. In newborns and children less than 6 months of age, hereditary aHUS is the first-line diagnosis, but pneumococcal-HUS also needs urgent recognition and treatment [136]. Post-diarrheal STEC-HUS largely predominates in children from 6 months to 5 years of age, but aHUS comes next. In this age group, again, the diagnosis of pneumococcalHUS must not be delayed. Preadolescents and adolescents mostly have aHUS, predominantly associated with anti-CFH antibodies.

Treatment STEC-HUS Supportive care Typical management of STEC-HUS patients relies on supportive care of electrolyte and water imbalance, anemia, hypertension, and renal failure. Administration of intravenous fluid and sodium as soon as a STEC infection is suspected (that is within the first 4 days of illness, even before culture results are available) appears to limit the severity of ARF and the need for renal replacement therapy [140]. The importance of early hydration was later confirmed by the same group in a multicentric cohort of 50 HUS children, only half of whom received intravenous fluids in the first 4 days of diarrhea: oligoanuria developed in 84 % of the children who received no intravenous fluids, compared to 52 % of the children given any intravenous fluid in that same interval [141]. HUS patients are mostly children presenting with diarrhea and, often, vomit, pain, and distress,

which are considerably worsened by oral intake [142]. A certain degree of dehydration is highly likely in these children and prompt initiation of intravenous fluid administration is a simple measure to avoid renal hypoperfusion, which could possibly synergize with Stx in inducing kidney injury. What is the ideal composition and rate of intravenous fluid administration deserves further investigation. The Washington group recommend early administration, as soon as bloody diarrhea is diagnosed, of a 20-ml/kg fluid bolus of isotonic crystalloid (normal saline, normal saline with 5 % dextrose, or lactated Ringer’s solution), followed by maintenance volumes, keeping a close eye on signs of fluid overload, urine output, renal function, and electrolytes [142]. Avoidance of hypotonic fluids, due to the increased risk of hyponatremia in hospitalized children, is becoming standard in pediatric care [143]. Some studies in adult patients are shedding light on the putative nephrotoxicity of chloride-rich fluids, like normal saline [144, 145], an aspect that deserves further investigation also in the pediatric setting. Up to 80 % of patients receive packed red blood cells for symptomatic anemia [146]. Careful blood pressure control and renin-angiotensin system blockade may be particularly beneficial in the long-term for those patients who have chronic kidney disease after an episode of STEC-HUS. Thus, 8–15year treatment with ACE inhibitors after severe STEC-HUS normalized blood pressure, reduced proteinuria, and improved long-term GFR [147]. Bowel rest is important for the enterohemorrhagic colitis associated with STEC-HUS. Antimotility agents should not be administered, since they may prolong the persistence of E. coli in the intestinal lumen and therefore increase patient exposure to its toxin. Nonsteroidal anti-inflammatory drugs should also be avoided because of a theoretical risk of worsening of gastrointestinal bleeding and/or ARF. Heparin and antithrombotic agents should be avoided since they may increase the risk of bleeding. Perhaps, the most controversial issue in the therapy for STEC infections is the use of antibiotics. A study has reported a strong association between receipt of antibiotics and the eventual development of HUS [148]. A possible explanation for the increased risk of HUS after antibiotic administrations is that antibiotic-induced injury to the bacterial membrane might favor the acute release of large amounts of preformed toxin. Alternatively, antibiotic therapy might give STEC a selective advantage if bacteria are not as readily eliminated from the bowel as are the normal intestinal flora. Moreover, several antimicrobial drugs, particularly the quinolones, trimethoprim, and furazolidone, are potent inducers of the expression of the Stx gene and may increase the level of toxin in the intestine. Although the possibility of a cause-and-effect relationship between antibiotics and HUS was challenged by a meta-analysis of 26 reports [149], at present, there is no indication to prescribe antibiotics. An interesting exception

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may be azithromycin as its use appeared to have some benefit on the duration of bacterial shedding in adult patients from the German O104:H4 epidemic [150]. In contrast to STEC-HUS, hemorrhagic colitis and HUS caused by S. dysenteriae type 1 should be treated with antibiotics as treatment shortens the duration of diarrhea, decreases the incidence of complications, and reduces the risk of transmission by shortening the duration of bacterial shedding. Plasma therapy The efficacy of specific treatments in adult patients is difficult to evaluate, since most information is derived from uncontrolled series that may include also aHUS cases. In particular, no prospective, randomized trials are available to definitely establish whether plasma infusion (PI) or plasma exchange (PE) offers specific benefit as compared to supportive treatment alone. However, comparative analyses of two large series of patients treated [151] or not treated [152] with plasma suggest that plasma therapy may dramatically decrease overall mortality of STEC-HUS. PI or PE should therefore be considered, particularly in adult patients with severe ARF and central nervous system involvement. Eculizumab Eculizumab is a recombinant, humanized, monoclonal Ig that targets C5 and blocks the C5 cleavage to C5a and C5b, preventing the generation of the proinflammatory peptide C5a and MAC. Evidence that uncontrolled complement activation may contribute to microangiopathic lesions of STEC-HUS [69, 77] provided the background for eculizumab therapy in three children with severe STEC-HUS who fully recovered with this treatment [75]. These encouraging results prompted nephrologists to use eculizumab in the STEC O104:H4 HUS outbreak in Germany. However, no significant difference was reported in treatment efficacy between patients who received eculizumab together with PE and those who received PE alone [153]. The study findings, however, might also be interpreted in a totally different way. Patients with more severe disease received more intensive treatment with PE and eculizumab. Thus, the similar outcomes observed in the different treatment groups might reflect the superior benefit of PE, with or without eculizumab, given to sicker patients compared with use of conservative therapy alone restricted to patients with less severe disease [154]. Of relevance, all nine cases of STEC-HUS from the 0104:H4 outbreak in France who were treated with eculizumab had a favorable outcome with rapid normalization of hematological parameters and renal function and neurological improvement [155]. Whether eculizumab is a useful adjunct for the treatment of the most severe forms of STEC-HUS should be clarified by prospective randomized, controlled trials. Kidney transplantation For those patients with STEC-HUS who progress to ESRD, kidney transplant is effective and safe and graft survival at 10 years is even better than in control

children with other diseases [156]. However there are rare cases in which disease recurred after transplantation, a complication more commonly observed in aHUS [157, 158]. Two patients with a clinical history of STEC-HUS lost the graft for HUS recurrence [40]. Before planning a second renal transplantation, the two patients underwent complement gene screening that revealed the presence of a heterozygous CFI mutation in the first patient and a heterozygous MCP mutation in the second patient. The same mutation was found in the mother of the latter patient who donated the kidney. This finding argued that the two cases originally diagnosed as STEC-HUS had indeed aHUS triggered by STEC infection on a genetic background of impaired complement regulation. Genetic screening should be performed before kidney transplantation in all patients who developed ESRD following STEC-HUS since they may be undiagnosed cases of aHUS, at risk of posttransplant recurrence. Pneumococcal-HUS The prognosis of patients with pneumococcal-HUS is strongly dependent on the effectiveness of antibiotic therapy. In theory, plasma either infused or exchanged is contraindicated, since adult plasma contains antibodies against the T-antigen that may accelerate polyagglutination and hemolysis. Thus, patients should be treated only with antibiotics and washed red cells. In some cases, however, plasma therapy, occasionally in combination with steroids, has been associated with recovery [83].

Atypical HUS Plasma therapy Plasma therapy in the form of PE or PI has been the gold standard of aHUS therapy since 1980s and was essentially the only therapy available until recently. The efficacy of plasma therapy is presumed to be related to its ability to deliver normal levels of complement proteins and, when plasma is exchanged by apheresis, to remove mutant regulators, anti-CFH antibodies, and hyperfunctional complement components. Trials of plasma therapy are scanty, so reported successes or failures of therapy should be interpreted cautiously. Plasma therapy efficacy differs according to the underlying complement defect. In a report from the International Registry of HUS/TTP, plasma treatment induced complete or partial (hematological remission with renal sequelae) remission in 63, 25, 57, 88, and 75 % of patients with CFH, CFI, C3, and THBD mutations, or anti-CFH autoantibodies, respectively [13]. Thirty to 40 % of patients with CFB mutations are responsive to plasma therapy [3, 117, 118]. The prognosis of aHUS due to MCP mutations is not influenced by plasma therapy because MCP is not a circulating protein [13].

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In clinical practice, because genetic information about complement abnormalities is not available in short time frames, the consensus-based guidelines recommended that an empiric plasma therapy should be started within 24 h of diagnosis of aHUS [159, 160]. The first-line plasma therapy should be PE, with exchange of one to two plasma volumes (60–75 ml/kg) per session. When PE cannot be performed, PI of 10–20 ml/kg should be given if the patient is not volume overloaded and/or hypertensive and does not show symptoms of cardiac failure. In the case of aHUS due to anti-CFH antibodies, PE is strongly recommended mainly in association with immunosuppressants (corticosteroids and azathioprine or mycophenolate-mofetil) [86]. Data on the effect of rituximab, an anti-CD20 antibody, and intravenous immunoglobulin administration on such circumstances are scanty and inconsistent [86, 105]. Studies are necessary to clarify the indications for use of these therapies. Initially, plasma therapy should be performed daily and the dose titrated until hemoglobin levels, platelet count, and LDH are substantially improved or even normalized. Once laboratory parameters have been controlled, plasma therapy can be withdrawn slowly, although individuals with genetic defects in the complement system are frequently plasma-dependent and require long-term plasma therapy to maintain remission [161]. In these cases, the modality and the interval between sessions must be determined individually. Persistence of hemolysis or lack of improvement in thrombocytopenia after a few days of therapy should be considered an indication to shift to eculizumab (Fig. 3). Eculizumab The activation of the terminal complement pathway is essential for the development of the endothelial lesion that characterizes aHUS [162, 163]. Eculizumab, previously used for paroxysmal nocturnal hemoglobinuria, was approved both by the European Medicines Agency and by the US Food and Drug Administration in 2011 for the treatment of aHUS after successful trials in adults and adolescents [90]. It is not clear, however, how long eculizumab therapy should be extended and which is the ideal treatment regimen to be administered. This issue is also relevant because of the unaffordable high cost of the drug. Conceivably, chronic life time treatment with eculizumab at doses able to persistently block the complement cascade might be indicated to prevent disease recurrence in genetic forms. However, whether and to which extent this applies to all patients with aHUS associated with complement genetic abnormalities is unknown. Reasonably, different underlying genetic defects, different clinical courses before eculizumab therapy, and different residual complement activity while on eculizumab therapy should be taken into account when strategies of chronic eculizumab therapy are planned. On the other hand, the risk of sensitization associated with chronic eculizumab exposure or with its deposition in tissues suggests that careful treatment tapering up to withdrawal whenever possible should be attempted in most cases under

tight control of disease and complement activity. Levels of serum C3 and plasma sC5b-9 are not suitable markers of complement activation in aHUS, since they have been found normal in a substantial proportion of patients, even in the acute phase [35] (Noris M. et al., personal communication). An ex vivo assay recently set up by our group detected complement activation at endothelial level in all tested patients with aHUS. Ex vivo complement deposits normalized after eculizumab treatment, paralleled clinical remission, and guided adjusting the interval between doses to the minimum necessary to block complement at endothelial level, thus avoiding drug overexposure and waste of money (Galbusera M. et al., personal communication). A concern with eculizumab treatment is the risk for infection with encapsulated bacterial organisms, particularly Neisseria meningitis, as a result of terminal complement blockade [164]. Therefore, patients must receive meningococcal vaccination before being treated with eculizumab at least 1 week before treatment. Antibiotic prophylaxis is highly recommended because not all serotypes are covered by vaccination. In pediatric patients, vaccination against Haemophilus influenzae and pneumococci is also necessary [165]. Organ transplantation The outcome of kidney transplantation in aHUS is poor and is largely predicted by the underlying genetic alteration [166]. Disease recurred in 64–78 % of transplanted patients with CFH, CFI, and C3 mutations and graft failure occurred in 75–95 %. All the grafts from patients with CFH-CFHR1 hybrid genes were lost for recurrence. The incidence of recurrence in patients with anti-CFH antibodies is 33 %, lower than that in patients with CFH mutations. A reduction in autoantibody levels with PE, steroids, and/or rituximab enabled successful renal transplantation in few patients [86]. The three reported patients with CFB mutations [117, 120] and one with THBD mutation [124] who were transplanted lost the graft for recurrence. Conversely, the lowest incidence of recurrence was observed in patients with MCP mutations (20 %); this can be explained by the fact that MCP is highly expressed in the kidney and a graft that brings normal MCP protein corrects the defect. The subgroup of patients with MCP mutations who experienced recurrence in the graft likely carried additional genetic abnormalities. In a large European survey [126], high incidence of recurrence (about 65 %) was observed in patients with MCP mutations combined with mutations in other complement genes, contrasting with the good graft outcome among patients carrying an isolated MCP mutation. The concomitant presence of genetic dysfunction in circulating proteins that could not be corrected by a kidney transplant likely contributed to the higher risk of recurrences in the former group. Renal transplantation may be successful in patients with DGKE mutations: among three transplanted patients, none experienced recurrence [127].

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Although most studies have shown that PE fails to prevent graft loss in patients with recurrent posttransplant HUS, a preemptive PI/PE strategy has been successful associated with a trend to decreased incidence of recurrences. However, in some of the patients, delayed recurrence occurred when plasma therapy was tapered [167]. Because CFH is synthesized predominantly in the liver, simultaneous kidney and liver transplant was performed in two children with aHUS and CFH mutations with the rationale of correcting the genetic complement abnormality defect, thus preventing disease recurrence in the transplanted kidney [168, 169]. However, both cases were complicated by premature liver failure with microvascular thrombosis and complement deposition. The first child recovered after a second liver transplantation and had no symptoms of HUS for 3 years even in concomitance of infections [168]. This case offered the proof of concept that liver transplantation cures aHUS associated with CFH mutations. The second case died few days after transplant because of primary liver nonfunction [169]. It was reasoned that the surgical stress with ischemia/ reperfusion-induced complement activation in a liver could not be regulated because of functional CFH deficiency [170]. A modified approach [171] included extensive PE before surgery to provide enough normal CFH until the liver graft recovered synthetic functions. This procedure was successful in seven patients, including one with preserved renal function who received an isolated liver transplant [171–175]. However, a child with a CFH mutation developed severe hepatic thrombosis and fatal encephalopathy [171]. The risks of kidney and liver transplantation have limited widespread diffusion of this option and ask for a careful assessment of benefits in candidate patients. Perioperative use of eculizumab during simultaneous kidney and liver transplant could be of help to inhibit complement activity, thereby reducing the risk of graft thrombosis. This possibility is supported by a recent report of an 11month-old child with aHUS and a CFH mutation, who received a combined kidney and liver transplant together with perioperative PE and eculizumab. At 19 months posttransplant, she has excellent kidney and liver graft functions without signs of disease recurrence [176]. Eculizumab was also efficiently used as prophylaxis to prevent posttransplant aHUS recurrence in ten patients receiving an isolated kidney transplant [165, 177–180] in whom a high recurrence risk was predicted from identified genetic abnormalities. The more common strategy was based on eculizumab alone, which started immediately before transplantation. Only one patient lost the graft for immediate arterial thrombosis at day 1 posttransplant. The other nine had a successful recurrence-free posttransplant course. No patient with preserved graft had discontinued the drug so far [178]. Whether aHUS transplanted patients should continue lifelong eculizumab prophylaxis or whether it can be stopped at any time and used as rescue therapy if clinical signs of

relapse occur remain to be established in controlled trials. When compared to kidney–liver transplant, eculizumab has the lower short-term risk and more effectiveness in preventing recurrences; however, the disadvantages are the need of chronic treatment, the potential long-term effects of C5inhibition on the already heavily immunosuppressed transplant recipients, and the extremely high costs. In low-income and poor countries, the high costs prevent eculizumab use, and such limitation applies to the large majority of patients worldwide. Kidney–liver transplant is associated with higher shortterm complications but provides surgical correction of the genetic abnormality and could be widely available due to lower monetary cost. Eighteen patients have been reported in literature who received eculizumab to treat posttransplant aHUS recurrence [125, 178, 181–185]. In all patients, hematological features rapidly normalized following the start of eculizumab and renal function also improved in the subsequent months. Both single-dose eculizumab and eculizumab discontinuation after several months were associated with subsequent relapses in four out of four patients and resulted in graft loss in three of them despite re-initiation of eculizumab [183]. Similarly, few days of delay in eculizumab infusion in two patients were associated with relapses [178, 185]. Thus, another lesson that could be drawn from previous experience is that eculizumab should not be discontinued or if so it should be done with extreme caution. This issue has particular relevance taking into consideration that the cost of this medication is so high that even in high-income countries, public health systems and private insurances tend to limit eculizumab use [43, 186, 187]. Acknowledgments This work has been partially supported by grants from Fondazione ART per la Ricerca sui Trapianti ONLUS (Milano Italy) and the European Community (FP7 Grant 2012-305608 EURenOmics). The authors wish to thank Dr. Claudio Tripodo (University of Palermo, Italy) for kindly providing the images of C3 and C9 staining in kidney biopsies from HUS patients.

References 1. Gianantonio CA, Vitacco M, Mendilaharzu F, Gallo GE, Sojo ET (1973) The hemolytic-uremic syndrome. Nephron 11:174–192 2. Gasser C, Gautier E, Steck A, Siebenmann RE, Oechslin R (1955) Hemolytic-uremic syndrome: bilateral necrosis of the renal cortex in acute acquired hemolytic anemia. Schweiz Med Wochenschr 85: 905–909 3. Noris M, Remuzzi G (2009) Atypical hemolytic-uremic syndrome. N Engl J Med 361:1676–1687 4. Ruggenenti P, Noris M, Remuzzi G (2001) Thrombotic microangiopathy, hemolytic uremic syndrome, and thrombotic thrombocytopenic purpura. Kidney Int 60:831–846 5. Besbas N, Karpman D, Landau D, Loirat C, Proesmans W, Remuzzi G, Rizzoni G, Taylor CM, Van de Kar N, Zimmerhackl LB (2006) A classification of hemolytic uremic syndrome and thrombotic

Semin Immunopathol

6.

7.

8.

9.

10. 11.

12.

13.

14.

15.

16.

17.

18.

19.

20. 21.

thrombocytopenic purpura and related disorders. Kidney Int 70: 423–431 Frank C, Werber D, Cramer JP, Askar M, Faber M, an der Heiden M, Bernard H, Fruth A, Prager R, Spode A, Wadl M, Zoufaly A, Jordan S, Kemper MJ, Follin P, Muller L, King LA, Rosner B, Buchholz U, Stark K, Krause G (2011) Epidemic profile of Shigatoxin-producing Escherichia coli O104:H4 outbreak in Germany. N Engl J Med 365:1771–1780 Tarr PI, Gordon CA, Chandler WL (2005) Shiga-toxin-producing Escherichia coli and haemolytic uraemic syndrome. Lancet 365: 1073–1086 Copelovitch L, Kaplan BS (2010) Streptococcus pneumoniae-associated hemolytic uremic syndrome: classification and the emergence of serotype 19A. Pediatrics 125:e174–e182 Shimizu M, Yokoyama T, Sakashita N, Sato A, Ueno K, Akita C, Ohta K, Kitano E, Hatanaka M, Kitamura H, Saikawa Y, Yachie A (2012) Thomsen-Friedenreich antigen exposure as a cause of Streptococcus pyogenes-associated hemolytic-uremic syndrome. Clin Nephrol 78:328–331 Kaplan BS, Chesney RW, Drummond KN (1975) Hemolytic uremic syndrome in families. N Engl J Med 292:1090–1093 Sharma AP, Greenberg CR, Prasad AN, Prasad C (2007) Hemolytic uremic syndrome (HUS) secondary to cobalamin C (cblC) disorder. Pediatr Nephrol 22:2097–2103 Fakhouri F, Roumenina L, Provot F, Sallee M, Caillard S, Couzi L, Essig M, Ribes D, Dragon-Durey MA, Bridoux F, Rondeau E, Fremeaux-Bacchi V (2010) Pregnancy-associated hemolytic uremic syndrome revisited in the era of complement gene mutations. J Am Soc Nephrol 21:859–867 Noris M, Caprioli J, Bresin E, Mossali C, Pianetti G, Gamba S, Daina E, Fenili C, Castelletti F, Sorosina A, Piras R, Donadelli R, Maranta R, van der Meer I, Conway EM, Zipfel PF, Goodship TH, Remuzzi G (2010) Relative role of genetic complement abnormalities in sporadic and familial aHUS and their impact on clinical phenotype. Clin J Am Soc Nephrol 5:1844–1859 Noris M, Mescia F, Remuzzi G (2012) STEC-HUS, atypical HUS and TTP are all diseases of complement activation. Nat Rev Nephrol 8:622–633 Crump JA, Sulka AC, Langer AJ, Schaben C, Crielly AS, Gage R, Baysinger M, Moll M, Withers G, Toney DM, Hunter SB, Hoekstra RM, Wong SK, Griffin PM, Van Gilder TJ (2002) An outbreak of Escherichia coli O157:H7 infections among visitors to a dairy farm. N Engl J Med 347:555–560 Haack JP, Jelacic S, Besser TE, Weinberger E, Kirk DJ, McKee GL, Harrison SM, Musgrave KJ, Miller G, Price TH, Tarr PI (2003) Escherichia coli O157 exposure in Wyoming and Seattle: serologic evidence of rural risk. Emerg Infect Dis 9:1226–1231 Boyce TG, Swerdlow DL, Griffin PM (1995) Escherichia coli O157:H7 and the hemolytic-uremic syndrome. N Engl J Med 333: 364–368 Palermo MS, Exeni RA, Fernandez GC (2009) Hemolytic uremic syndrome: pathogenesis and update of interventions. Expert Rev Anti Infect Ther 7:697–707 Navarro A, Eslava C, Hernandez U, Navarro-Henze JL, Aviles M, Garcia-de la Torre G, Cravioto A (2003) Antibody responses to Escherichia coli O157 and other lipopolysaccharides in healthy children and adults. Clin Diagn Lab Immunol 10:797–801 Mead PS, Griffin PM (1998) Escherichia coli O157:H7. Lancet 352:1207–1212 Rivas M, Miliwebsky E, Chinen I, Roldan CD, Balbi L, Garcia B, Fiorilli G, Sosa-Estani S, Kincaid J, Rangel J, Griffin PM (2006) Characterization and epidemiologic subtyping of Shiga toxinproducing Escherichia coli strains isolated from hemolytic uremic syndrome and diarrhea cases in Argentina. Foodborne Pathog Dis 3: 88–96

22. Manning SD, Motiwala AS, Springman AC, Qi W, Lacher DW, Ouellette LM, Mladonicky JM, Somsel P, Rudrik JT, Dietrich SE, Zhang W, Swaminathan B, Alland D, Whittam TS (2008) Variation in virulence among clades of Escherichia coli O157:H7 associated with disease outbreaks. Proc Natl Acad Sci U S A 105:4868–4873 23. Isaacson M, Canter PH, Effler P, Arntzen L, Bomans P, Heenan R (1993) Haemorrhagic colitis epidemic in Africa. Lancet 341:961 24. Buchholz U, Bernard H, Werber D, Bohmer MM, Remschmidt C, Wilking H, Delere Y, an der Heiden M, Adlhoch C, Dreesman J, Ehlers J, Ethelberg S, Faber M, Frank C, Fricke G, Greiner M, Hohle M, Ivarsson S, Jark U, Kirchner M, Koch J, Krause G, Luber P, Rosner B, Stark K, Kuhne M (2011) German outbreak of Escherichia coli O104:H4 associated with sprouts. N Engl J Med 365:1763–1770 25. Keene WE, McAnulty JM, Hoesly FC, Williams LP Jr, Hedberg K, Oxman GL, Barrett TJ, Pfaller MA, Fleming DW (1994) A swimming-associated outbreak of hemorrhagic colitis caused by Escherichia coli O157:H7 and Shigella sonnei. N Engl J Med 331:579–584 26. Brandt J, Wong C, Mihm S, Roberts J, Smith J, Brewer E, Thiagarajan R, Warady B (2002) Invasive pneumococcal disease and hemolytic uremic syndrome. Pediatrics 110:371–376 27. Szilagyi A, Kiss N, Bereczki C, Talosi G, Racz K, Turi S, Gyorke Z, Simon E, Horvath E, Kelen K, Reusz GS, Szabo AJ, Tulassay T, Prohaszka Z (2013) The role of complement in Streptococcus pneumoniae-associated haemolytic uraemic syndrome. Nephrol Dial Transplant 28:2237–2245 28. Mizusawa Y, Pitcher LA, Burke JR, Falk MC, Mizushima W (1996) Survey of haemolytic-uraemic syndrome in Queensland 1979– 1995. Med J Aust 165:188–191 29. Constantinescu AR, Bitzan M, Weiss LS, Christen E, Kaplan BS, Cnaan A, Trachtman H (2004) Non-enteropathic hemolytic uremic syndrome: causes and short-term course. Am J Kidney Dis 43:976– 982 30. Ohanian M, Cable C, Halka K (2011) Eculizumab safely reverses neurologic impairment and eliminates need for dialysis in severe atypical hemolytic uremic syndrome. Clin Pharmacol 3:5–12 31. Sellier-Leclerc AL, Fremeaux-Bacchi V, Dragon-Durey MA, Macher MA, Niaudet P, Guest G, Boudailliez B, Bouissou F, Deschenes G, Gie S, Tsimaratos M, Fischbach M, Morin D, Nivet H, Alberti C, Loirat C (2007) Differential impact of complement mutations on clinical characteristics in atypical hemolytic uremic syndrome. J Am Soc Nephrol 18:2392–2400 32. Caprioli J, Noris M, Brioschi S, Pianetti G, Castelletti F, Bettinaglio P, Mele C, Bresin E, Cassis L, Gamba S, Porrati F, Bucchioni S, Monteferrante G, Fang CJ, Liszewski MK, Kavanagh D, Atkinson JP, Remuzzi G (2006) Genetics of HUS: the impact of MCP, CFH, and IF mutations on clinical presentation, response to treatment, and outcome. Blood 108:1267–1279 33. Milford DV, Taylor CM, Guttridge B, Hall SM, Rowe B, Kleanthous H (1990) Haemolytic uraemic syndromes in the British Isles 1985–8: association with verocytotoxin producing Escherichia coli. Part 1: Clinical and epidemiological aspects. Arch Dis Child 65:716–721 34. Sullivan M, Erlic Z, Hoffmann MM, Arbeiter K, Patzer L, Budde K, Hoppe B, Zeier M, Lhotta K, Rybicki LA, Bock A, Berisha G, Neumann HP (2010) Epidemiological approach to identifying genetic predispositions for atypical hemolytic uremic syndrome. Ann Hum Genet 74:17–26 35. Loirat C, Fremeaux-Bacchi V (2011) Atypical hemolytic uremic syndrome. Orphanet J Rare Dis 6:60 36. Gilbert RD, Fowler DJ, Angus E, Hardy SA, Stanley L, Goodship TH (2013) Eculizumab therapy for atypical haemolytic uraemic syndrome due to a gain-of-function mutation of complement factor B. Pediatr Nephrol 28:1315–1318

Semin Immunopathol 37. Kaplan BS, Proesmans W (1987) The hemolytic uremic syndrome of childhood and its variants. Semin Hematol 24:148–160 38. Habib R (1992) Pathology of the hemolytic uremic syndrome. In: TR Kaplan B, Moake J (eds) Hemolytic uremic syndrome and thrombotic thrombocytopenic purpura. Marcel Dekker, New York, p 315 39. Inward CD, Howie AJ, Fitzpatrick MM, Rafaat F, Milford DV, Taylor CM (1997) Renal histopathology in fatal cases of diarrhoea-associated haemolytic uraemic syndrome. British Association for Paediatric Nephrology. Pediatr Nephrol 11:556–559 40. Alberti M, Valoti E, Piras R, Bresin E, Galbusera M, Tripodo C, Thaiss F, Remuzzi G, Noris M (2013) Two patients with history of STEC-HUS, posttransplant recurrence and complement gene mutations. Am J Transplant 13:2201–2206 41. Landau D, Shalev H, Levy-Finer G, Polonsky A, Segev Y, Katchko L (2001) Familial hemolytic uremic syndrome associated with complement factor H deficiency. J Pediatr 138:412–417 42. Moghal NE, Ferreira MA, Howie AJ, Milford DV, Raafat E, Taylor CM (1998) The late histologic findings in diarrhea-associated hemolytic uremic syndrome. J Pediatr 133:220–223 43. Garg AX, Suri RS, Barrowman N, Rehman F, Matsell D, RosasArellano MP, Salvadori M, Haynes RB, Clark WF (2003) Longterm renal prognosis of diarrhea-associated hemolytic uremic syndrome: a systematic review, meta-analysis, and meta-regression. JAMA 290:1360–1370 44. Grodinsky S, Telmesani A, Robson WL, Fick G, Scott RB (1990) Gastrointestinal manifestations of hemolytic uremic syndrome: recognition of pancreatitis. J Pediatr Gastroenterol Nutr 11:518–524 45. Abu-Arafeh I, Gray E, Youngson G, Auchterlonie I, Russell G (1995) Myocarditis and haemolytic uraemic syndrome. Arch Dis Child 72:46–47 46. Ruggenenti P, Remuzzi G (2011) A German outbreak of haemolytic uraemic syndrome. Lancet 378:1057–1058 47. Blaser MJ (2011) Deconstructing a lethal foodborne epidemic. N Engl J Med 365:1835–1836 48. Mellmann A, Harmsen D, Cummings CA, Zentz EB, Leopold SR, Rico A, Prior K, Szczepanowski R, Ji Y, Zhang W, McLaughlin SF, Henkhaus JK, Leopold B, Bielaszewska M, Prager R, Brzoska PM, Moore RL, Guenther S, Rothberg JM, Karch H (2011) Prospective genomic characterization of the German enterohemorrhagic Escherichia coli O104:H4 outbreak by rapid next generation sequencing technology. PLoS One 6:e22751 49. Qin J, Cui Y, Zhao X, Rohde H, Liang T, Wolters M, Li D, Belmar Campos C, Christner M, Song Y, Yang R (2011) Identification of the Shiga toxin-producing Escherichia coli O104:H4 strain responsible for a food poisoning outbreak in Germany by PCR. J Clin Microbiol 49:3439–3440 50. Tarr PI, Neill MA, Clausen CR, Watkins SL, Christie DL, Hickman RO (1990) Escherichia coli O157:H7 and the hemolytic uremic syndrome: importance of early cultures in establishing the etiology. J Infect Dis 162:553–556 51. Gerritzen A, Wittke JW, von Ahsen N, Wolff D (2012) Direct faecal PCR for diagnosis of Shiga-toxin-producing Escherichia coli. Lancet Infect Dis 12:102 52. Donnenberg MS, Tacket CO, James SP, Losonsky G, Nataro JP, Wasserman SS, Kaper JB, Levine MM (1993) Role of the eaeA gene in experimental enteropathogenic Escherichia coli infection. J Clin Invest 92:1412–1417 53. Brigotti M, Carnicelli D, Arfilli V, Tamassia N, Borsetti F, Fabbri E, Tazzari PL, Ricci F, Pagliaro P, Spisni E, Cassatella MA (2013) Identification of TLR4 as the receptor that recognizes Shiga toxins in human neutrophils. J Immunol 191:4748–4758 54. Cooling LL, Walker KE, Gille T, Koerner TA (1998) Shiga toxin binds human platelets via globotriaosylceramide (Pk antigen) and a novel platelet glycosphingolipid. Infect Immun 66:4355–4366

55. van Setten PA, Monnens LA, Verstraten RG, van den Heuvel LP, van Hinsbergh VW (1996) Effects of verocytotoxin-1 on nonadherent human monocytes: binding characteristics, protein synthesis, and induction of cytokine release. Blood 88:174–183 56. Hurley BP, Thorpe CM, Acheson DW (2001) Shiga toxin translocation across intestinal epithelial cells is enhanced by neutrophil transmigration. Infect Immun 69:6148–6155 57. O'Brien AD, Lively TA, Chang TW, Gorbach SL (1983) Purification of Shigella dysenteriae 1 (Shiga)-like toxin from Escherichia coli O157:H7 strain associated with haemorrhagic colitis. Lancet 2:573 58. Cimolai N, Carter JE, Morrison BJ, Anderson JD (1990) Risk factors for the progression of Escherichia coli O157:H7 enteritis to hemolytic-uremic syndrome. J Pediatr 116:589–592 59. Scotland SM, Willshaw GA, Smith HR, Rowe B (1987) Properties of strains of Escherichia coli belonging to serogroup O157 with special reference to production of Vero cytotoxins VT1 and VT2. Epidemiol Infect 99:613–624 60. Jenkins C, Willshaw GA, Evans J, Cheasty T, Chart H, Shaw DJ, Dougan G, Frankel G, Smith HR (2003) Subtyping of virulence genes in verocytotoxin-producing Escherichia coli (VTEC) other than serogroup O157 associated with disease in the United Kingdom. J Med Microbiol 52:941–947 61. O'Loughlin EV, Robins-Browne RM (2001) Effect of Shiga toxin and Shiga-like toxins on eukaryotic cells. Microbes Infect 3:493– 507 62. Chaisri U, Nagata M, Kurazono H, Horie H, Tongtawe P, Hayashi H, Watanabe T, Tapchaisri P, Chongsa-nguan M, Chaicumpa W (2001) Localization of Shiga toxins of enterohaemorrhagic Escherichia coli in kidneys of paediatric and geriatric patients with fatal haemolytic uraemic syndrome. Microb Pathog 31:59–67 63. Auray-Blais C, Cyr D, Ntwari A, West ML, Cox-Brinkman J, Bichet DG, Germain DP, Laframboise R, Melancon SB, Stockley T, Clarke JT, Drouin R (2008) Urinary globotriaosylceramide excretion correlates with the genotype in children and adults with Fabry disease. Mol Genet Metab 93:331–340 64. Zoja C, Angioletti S, Donadelli R, Zanchi C, Tomasoni S, Binda E, Imberti B, te Loo M, Monnens L, Remuzzi G, Morigi M (2002) Shiga toxin-2 triggers endothelial leukocyte adhesion and transmigration via NF-kappaB dependent up-regulation of IL-8 and MCP1. Kidney Int 62:846–856 65. Petruzziello-Pellegrini TN, Yuen DA, Page AV, Patel S, Soltyk AM, Matouk CC, Wong DK, Turgeon PJ, Fish JE, Ho JJ, Steer BM, Khajoee V, Tigdi J, Lee WL, Motto DG, Advani A, Gilbert RE, Karumanchi SA, Robinson LA, Tarr PI, Liles WC, Brunton JL, Marsden PA (2012) The CXCR4/CXCR7/SDF-1 pathway contributes to the pathogenesis of Shiga toxin-associated hemolytic uremic syndrome in humans and mice. J Clin Invest 122:759–776 66. Morigi M, Micheletti G, Figliuzzi M, Imberti B, Karmali MA, Remuzzi A, Remuzzi G, Zoja C (1995) Verotoxin-1 promotes leukocyte adhesion to cultured endothelial cells under physiologic flow conditions. Blood 86:4553–4558 67. Morigi M, Galbusera M, Binda E, Imberti B, Gastoldi S, Remuzzi A, Zoja C, Remuzzi G (2001) Verotoxin-1-induced up-regulation of adhesive molecules renders microvascular endothelial cells thrombogenic at high shear stress. Blood 98:1828–1835 68. Karpman D, Papadopoulou D, Nilsson K, Sjogren AC, Mikaelsson C, Lethagen S (2001) Platelet activation by Shiga toxin and circulatory factors as a pathogenetic mechanism in the hemolytic uremic syndrome. Blood 97:3100–3108 69. Morigi M, Galbusera M, Gastoldi S, Locatelli M, Buelli S, Pezzotta A, Pagani C, Noris M, Gobbi M, Stravalaci M, Rottoli D, Tedesco F, Remuzzi G, Zoja C (2011) Alternative pathway activation of complement by Shiga toxin promotes exuberant C3a formation that triggers microvascular thrombosis. J Immunol 187:172–180

Semin Immunopathol 70. Lo NC, Turner NA, Cruz MA, Moake J (2013) Interaction of Shiga toxin with the A-domains and multimers of von Willebrand factor. J Biol Chem 288:33118–33123 71. Hughes AK, Stricklett PK, Kohan DE (1998) Cytotoxic effect of Shiga toxin-1 on human proximal tubule cells. Kidney Int 54:426– 437 72. Ricklin D, Hajishengallis G, Yang K, Lambris JD (2010) Complement: a key system for immune surveillance and homeostasis. Nat Immunol 11:785–797 73. Cameron JS, Vick R (1973) Letter: Plasma-C3 in haemolyticuraemic syndrome and thrombotic thrombocytopenic purpura. Lancet 2:975 74. Robson WL, Leung AK, Fick GH, McKenna AI (1992) Hypocomplementemia and leukocytosis in diarrhea-associated hemolytic uremic syndrome. Nephron 62:296–299 75. Lapeyraque AL, Malina M, Fremeaux-Bacchi V, Boppel T, Kirschfink M, Oualha M, Proulx F, Clermont MJ, Le Deist F, Niaudet P, Schaefer F (2011) Eculizumab in severe Shiga-toxinassociated HUS. N Engl J Med 364:2561–2563 76. Monnens L, Molenaar J, Lambert PH, Proesmans W, van Munster P (1980) The complement system in hemolytic-uremic syndrome in childhood. Clin Nephrol 13:168–171 77. Thurman JM, Marians R, Emlen W, Wood S, Smith C, Akana H, Holers VM, Lesser M, Kline M, Hoffman C, Christen E, Trachtman H (2009) Alternative pathway of complement in children with diarrhea-associated hemolytic uremic syndrome. Clin J Am Soc Nephrol 4:1920–1924 78. Stahl AL, Sartz L, Karpman D (2011) Complement activation on p l a t e l e t - l e u k o c y t e c o m p l e x e s a n d m i c ro p a r t i c l e s i n enterohemorrhagic Escherichia coli-induced hemolytic uremic syndrome. Blood 117:5503–5513 79. Del Conde I, Cruz MA, Zhang H, Lopez JA, Afshar-Kharghan V (2005) Platelet activation leads to activation and propagation of the complement system. J Exp Med 201:871–879 80. Orth D, Khan AB, Naim A, Grif K, Brockmeyer J, Karch H, Joannidis M, Clark SJ, Day AJ, Fidanzi S, Stoiber H, Dierich MP, Zimmerhackl LB, Wurzner R (2009) Shiga toxin activates complement and binds factor H: evidence for an active role of complement in hemolytic uremic syndrome. J Immunol 182:6394–6400 81. Fischer K, Poschmann A, Oster H (1971) Severe pneumonia with hemolysis caused by neuraminidase. Detection of cryptantigens by indirect immunofluorescent technic. Monatsschr Kinderheilkd 119: 2–8 82. Cochran JB, Panzarino VM, Maes LY, Tecklenburg FW (2004) Pneumococcus-induced T-antigen activation in hemolytic uremic syndrome and anemia. Pediatr Nephrol 19:317–321 83. McGraw ME, Lendon M, Stevens RF, Postlethwaite RJ, Taylor CM (1989) Haemolytic uraemic syndrome and the Thomsen Friedenreich antigen. Pediatr Nephrol 3:135–139 84. Eder AF, Manno CS (2001) Does red-cell T activation matter? Br J Haematol 114:25–30 85. Sallee M, Daniel L, Piercecchi MD, Jaubert D, Fremeaux-Bacchi V, Berland Y, Burtey S (2010) Myocardial infarction is a complication of factor H-associated atypical HUS. Nephrol Dial Transplant 25: 2028–2032 86. Dragon-Durey MA, Sethi SK, Bagga A, Blanc C, Blouin J, Ranchin B, Andre JL, Takagi N, Cheong HI, Hari P, Le Quintrec M, Niaudet P, Loirat C, Fridman WH, Fremeaux-Bacchi V (2010) Clinical features of anti-factor H autoantibody-associated hemolytic uremic syndrome. J Am Soc Nephrol 21:2180–2187 87. Loirat C, Noris M, Fremeaux-Bacchi V (2008) Complement and the atypical hemolytic uremic syndrome in children. Pediatr Nephrol 23:1957–1972 88. Loirat C, Macher MA, Elmaleh-Berges M, Kwon T, Deschenes G, Goodship TH, Majoie C, Davin JC, Blanc R, Savatovsky J, Moret J, Fremeaux-Bacchi V (2010) Non-atheromatous arterial stenoses in

atypical haemolytic uraemic syndrome associated with complement dysregulation. Nephrol Dial Transplant 25:3421–3425 89. Bekassy ZD, Kristoffersson AC, Cronqvist M, Roumenina LT, Rybkine T, Vergoz L, Hue C, Fremeaux-Bacchi V, Karpman D (2013) Eculizumab in an anephric patient with atypical haemolytic uraemic syndrome and advanced vascular lesions. Nephrol Dial Transplant 28:2899–2907 90. Legendre CM, Licht C, Muus P, Greenbaum LA, Babu S, Bedrosian C, Bingham C, Cohen DJ, Delmas Y, Douglas K, Eitner F, Feldkamp T, Fouque D, Furman RR, Gaber O, Herthelius M, Hourmant M, Karpman D, Lebranchu Y, Mariat C, Menne J, Moulin B, Nurnberger J, Ogawa M, Remuzzi G, Richard T, Sberro-Soussan R, Severino B, Sheerin NS, Trivelli A, Zimmerhackl LB, Goodship T, Loirat C (2013) Terminal complement inhibitor eculizumab in atypical hemolytic-uremic syndrome. N Engl J Med 368:2169–2181 91. Rathbone J, Kaltenthaler E, Richards A, Tappenden P, Bessey A, Cantrell A (2013) A systematic review of eculizumab for atypical haemolytic uraemic syndrome (aHUS). BMJ Open 3:e003573 92. Fremeaux-Bacchi V, Fakhouri F, Garnier A, Bienaime F, DragonDurey MA, Ngo S, Moulin B, Servais A, Provot F, Rostaing L, Burtey S, Niaudet P, Deschenes G, Lebranchu Y, Zuber J, Loirat C (2013) Genetics and outcome of atypical hemolytic uremic syndrome: a nationwide French series comparing children and adults. Clin J Am Soc Nephrol 8:554–562 93. Schmidt CQ, Herbert AP, Kavanagh D, Gandy C, Fenton CJ, Blaum BS, Lyon M, Uhrin D, Barlow PN (2008) A new map of glycosaminoglycan and C3b binding sites on factor H. J Immunol 181: 2610–2619 94. Ferreira VP, Pangburn MK, Cortes C (2010) Complement control protein factor H: the good, the bad, and the inadequate. Mol Immunol 47:2187–2197 95. Clark SJ, Ridge LA, Herbert AP, Hakobyan S, Mulloy B, Lennon R, Wurzner R, Morgan BP, Uhrin D, Bishop PN, Day AJ (2013) Tissue-specific host recognition by complement factor H is mediated by differential activities of its glycosaminoglycan-binding regions. J Immunol 190:2049–2057 96. Thompson RA, Winterborn MH (1981) Hypocomplementaemia due to a genetic deficiency of beta 1H globulin. Clin Exp Immunol 46:110–119 97. Warwicker P, Goodship TH, Donne RL, Pirson Y, Nicholls A, Ward RM, Turnpenny P, Goodship JA (1998) Genetic studies into inherited and sporadic hemolytic uremic syndrome. Kidney Int 53: 836–844 98. Maga TK, Nishimura CJ, Weaver AE, Frees KL, Smith RJ (2010) Mutations in alternative pathway complement proteins in American patients with atypical hemolytic uremic syndrome. Hum Mutat 31: E1445–E1460 99. Venables JP, Strain L, Routledge D, Bourn D, Powell HM, Warwicker P, Diaz-Torres ML, Sampson A, Mead P, Webb M, Pirson Y, Jackson MS, Hughes A, Wood KM, Goodship JA, Goodship TH (2006) Atypical haemolytic uraemic syndrome associated with a hybrid complement gene. PLoS Med 3:e431 100. Francis NJ, McNicholas B, Awan A, Waldron M, Reddan D, Sadlier D, Kavanagh D, Strain L, Marchbank KJ, Harris CL, Goodship TH (2012) A novel hybrid CFH/CFHR3 gene generated by a microhomology-mediated deletion in familial atypical hemolytic uremic syndrome. Blood 119:591–601 101. Eyler SJ, Meyer NC, Zhang Y, Xiao X, Nester CM, Smith RJ (2013) A novel hybrid CFHR1/CFH gene causes atypical hemolytic uremic syndrome. Pediatr Nephrol 28:2221–2225 102. Dragon-Durey MA, Loirat C, Cloarec S, Macher MA, Blouin J, Nivet H, Weiss L, Fridman WH, Fremeaux-Bacchi V (2005) Antifactor H autoantibodies associated with atypical hemolytic uremic syndrome. J Am Soc Nephrol 16:555–563

Semin Immunopathol 103. Moore I, Strain L, Pappworth I, Kavanagh D, Barlow PN, Herbert AP, Schmidt CQ, Staniforth SJ, Holmes LV, Ward R, Morgan L, Goodship TH, Marchbank KJ (2010) Association of factor H autoantibodies with deletions of CFHR1, CFHR3, CFHR4, and with mutations in CFH, CFI, CD46, and C3 in patients with atypical hemolytic uremic syndrome. Blood 115:379–387 104. Hofer J, Janecke AR, Zimmerhackl LB, Riedl M, Rosales A, Giner T, Cortina G, Haindl CJ, Petzelberger B, Pawlik M, Jeller V, Vester U, Gadner B, van Husen M, Moritz ML, Wurzner R, Jungraithmayr T (2013) Complement factor H-related protein 1 deficiency and factor H antibodies in pediatric patients with atypical hemolytic uremic syndrome. Clin J Am Soc Nephrol 8:407–415 105. Sinha A, Gulati A, Saini S, Blanc C, Gupta A, Gurjar BS, Saini H, Kotresh ST, Ali U, Bhatia D, Ohri A, Kumar M, Agarwal I, Gulati S, Anand K, Vijayakumar M, Sinha R, Sethi S, Salmona M, George A, Bal V, Singh G, Dinda AK, Hari P, Rath S, Dragon-Durey MA, Bagga A (2013) Prompt plasma exchanges and immunosuppressive treatment improves the outcomes of anti-factor H autoantibodyassociated hemolytic uremic syndrome in children. Kidney Int. doi:10.1038/ki.2013.373 106. Jozsi M, Licht C, Strobel S, Zipfel SL, Richter H, Heinen S, Zipfel PF, Skerka C (2008) Factor H autoantibodies in atypical hemolytic uremic syndrome correlate with CFHR1/CFHR3 deficiency. Blood 111:1512–1514 107. Zipfel PF, Edey M, Heinen S, Jozsi M, Richter H, Misselwitz J, Hoppe B, Routledge D, Strain L, Hughes AE, Goodship JA, Licht C, Goodship TH, Skerka C (2007) Deletion of complement factor H-related genes CFHR1 and CFHR3 is associated with atypical hemolytic uremic syndrome. PLoS Genet 3:e41 108. Abarrategui-Garrido C, Martinez-Barricarte R, Lopez-Trascasa M, de Cordoba SR, Sanchez-Corral P (2009) Characterization of complement factor H-related (CFHR) proteins in plasma reveals novel genetic variations of CFHR1 associated with atypical hemolytic uremic syndrome. Blood 114:4261–4271 109. Jozsi M, Strobel S, Dahse HM, Liu WS, Hoyer PF, Oppermann M, Skerka C, Zipfel PF (2007) Anti factor H autoantibodies block Cterminal recognition function of factor H in hemolytic uremic syndrome. Blood 110:1516–1518 110. Blanc C, Roumenina LT, Ashraf Y, Hyvarinen S, Sethi SK, Ranchin B, Niaudet P, Loirat C, Gulati A, Bagga A, Fridman WH, SautesFridman C, Jokiranta TS, Fremeaux-Bacchi V, Dragon-Durey MA (2012) Overall neutralization of complement factor H by autoantibodies in the acute phase of the autoimmune form of atypical hemolytic uremic syndrome. J Immunol 189:3528–3537 111. Richards A, Kathryn Liszewski M, Kavanagh D, Fang CJ, Moulton E, Fremeaux-Bacchi V, Remuzzi G, Noris M, Goodship TH, Atkinson JP (2007) Implications of the initial mutations in membrane cofactor protein (MCP; CD46) leading to atypical hemolytic uremic syndrome. Mol Immunol 44:111–122 112. Noris M, Brioschi S, Caprioli J, Todeschini M, Bresin E, Porrati F, Gamba S, Remuzzi G (2003) Familial haemolytic uraemic syndrome and an MCP mutation. Lancet 362:1542–1547 113. Richards A, Kemp EJ, Liszewski MK, Goodship JA, Lampe AK, Decorte R, Muslumanoglu MH, Kavukcu S, Filler G, Pirson Y, Wen LS, Atkinson JP, Goodship TH (2003) Mutations in human complement regulator, membrane cofactor protein (CD46), predispose to development of familial hemolytic uremic syndrome. Proc Natl Acad Sci U S A 100:12966–12971 114. Fremeaux-Bacchi V, Dragon-Durey MA, Blouin J, Vigneau C, Kuypers D, Boudailliez B, Loirat C, Rondeau E, Fridman WH (2004) Complement factor I: a susceptibility gene for atypical haemolytic uraemic syndrome. J Med Genet 41:e84 115. Bienaime F, Dragon-Durey MA, Regnier CH, Nilsson SC, Kwan WH, Blouin J, Jablonski M, Renault N, Rameix-Welti MA, Loirat C, Sautes-Fridman C, Villoutreix BO, Blom AM, Fremeaux-Bacchi V (2010) Mutations in components of complement influence the

outcome of factor I-associated atypical hemolytic uremic syndrome. Kidney Int 77:339–349 116. Kavanagh D, Richards A, Noris M, Hauhart R, Liszewski MK, Karpman D, Goodship JA, Fremeaux-Bacchi V, Remuzzi G, Goodship TH, Atkinson JP (2008) Characterization of mutations in complement factor I (CFI) associated with hemolytic uremic syndrome. Mol Immunol 45:95–105 117. Goicoechea de Jorge E, Harris CL, Esparza-Gordillo J, Carreras L, Arranz EA, Garrido CA, Lopez-Trascasa M, Sanchez-Corral P, Morgan BP, Rodriguez de Cordoba S (2007) Gain-of-function mutations in complement factor B are associated with atypical hemolytic uremic syndrome. Proc Natl Acad Sci U S A 104:240–245 118. Fremeaux-Bacchi V, Miller EC, Liszewski MK, Strain L, Blouin J, Brown AL, Moghal N, Kaplan BS, Weiss RA, Lhotta K, Kapur G, Mattoo T, Nivet H, Wong W, Gie S, Hurault de Ligny B, Fischbach M, Gupta R, Hauhart R, Meunier V, Loirat C, Dragon-Durey MA, Fridman WH, Janssen BJ, Goodship TH, Atkinson JP (2008) Mutations in complement C3 predispose to development of atypical hemolytic uremic syndrome. Blood 112:4948–4952 119. Lhotta K, Janecke AR, Scheiring J, Petzlberger B, Giner T, Fally V, Wurzner R, Zimmerhackl LB, Mayer G, Fremeaux-Bacchi V (2009) A large family with a gain-of-function mutation of complement C3 predisposing to atypical hemolytic uremic syndrome, microhematuria, hypertension and chronic renal failure. Clin J Am Soc Nephrol 4:1356–1362 120. Roumenina LT, Jablonski M, Hue C, Blouin J, Dimitrov JD, Dragon-Durey MA, Cayla M, Fridman WH, Macher MA, Ribes D, Moulonguet L, Rostaing L, Satchell SC, Mathieson PW, SautesFridman C, Loirat C, Regnier CH, Halbwachs-Mecarelli L, Fremeaux-Bacchi V (2009) Hyperfunctional C3 convertase leads to complement deposition on endothelial cells and contributes to atypical hemolytic uremic syndrome. Blood 114:2837–2845 121. Roumenina LT, Frimat M, Miller EC, Provot F, Dragon-Durey MA, Bordereau P, Bigot S, Hue C, Satchell SC, Mathieson PW, Mousson C, Noel C, Sautes-Fridman C, Halbwachs-Mecarelli L, Atkinson JP, Lionet A, Fremeaux-Bacchi V (2012) A prevalent C3 mutation in aHUS patients causes a direct C3 convertase gain of function. Blood 119:4182–4191 122. Tawadrous H, Maga T, Sharma J, Kupferman J, Smith RJ, Schoeneman M (2010) A novel mutation in the complement factor B gene (CFB) and atypical hemolytic uremic syndrome. Pediatr Nephrol 25:947–951 123. Weiler H, Isermann BH (2003) Thrombomodulin. J Thromb Haemost 1:1515–1524 124. Delvaeye M, Noris M, De Vriese A, Esmon CT, Esmon NL, Ferrell G, Del-Favero J, Plaisance S, Claes B, Lambrechts D, Zoja C, Remuzzi G, Conway EM (2009) Thrombomodulin mutations in atypical hemolytic-uremic syndrome. N Engl J Med 361:345–357 125. Fan X, Yoshida Y, Honda S, Matsumoto M, Sawada Y, Hattori M, Hisanaga S, Hiwa R, Nakamura F, Tomomori M, Miyagawa S, Fujimaru R, Yamada H, Sawai T, Ikeda Y, Iwata N, Uemura O, Matsukuma E, Aizawa Y, Harada H, Wada H, Ishikawa E, Ashida A, Nangaku M, Miyata T, Fujimura Y (2013) Analysis of genetic and predisposing factors in Japanese patients with atypical hemolytic uremic syndrome. Mol Immunol 54:238–246 126. Bresin E, Rurali E, Caprioli J, Sanchez-Corral P, Fremeaux-Bacchi V, Rodriguez de Cordoba S, Pinto S, Goodship TH, Alberti M, Ribes D, Valoti E, Remuzzi G, Noris M (2013) Combined complement gene mutations in atypical hemolytic uremic syndrome influence clinical phenotype. J Am Soc Nephrol 24:475–486 127. Lemaire M, Fremeaux-Bacchi V, Schaefer F, Choi M, Tang WH, Le Quintrec M, Fakhouri F, Taque S, Nobili F, Martinez F, Ji W, Overton JD, Mane SM, Nurnberg G, Altmuller J, Thiele H, Morin D, Deschenes G, Baudouin V, Llanas B, Collard L, Majid MA, Simkova E, Nurnberg P, Rioux-Leclerc N, Moeckel GW, Gubler

Semin Immunopathol

128.

129.

130.

131.

132.

133.

134.

135.

136. 137. 138. 139.

140.

141.

142. 143. 144.

MC, Hwa J, Loirat C, Lifton RP (2013) Recessive mutations in DGKE cause atypical hemolytic-uremic syndrome. Nat Genet 45: 531–536 Esparza-Gordillo J, Goicoechea de Jorge E, Buil A, Carreras Berges L, Lopez-Trascasa M, Sanchez-Corral P, Rodriguez de Cordoba S (2005) Predisposition to atypical hemolytic uremic syndrome involves the concurrence of different susceptibility alleles in the regulators of complement activation gene cluster in 1q32. Hum Mol Genet 14:703–712 Esparza-Gordillo J, Jorge EG, Garrido CA, Carreras L, LopezTrascasa M, Sanchez-Corral P, de Cordoba SR (2006) Insights into hemolytic uremic syndrome: segregation of three independent predisposition factors in a large, multiple affected pedigree. Mol Immunol 43:1769–1775 Fremeaux-Bacchi V, Kemp EJ, Goodship JA, Dragon-Durey MA, Strain L, Loirat C, Deng HW, Goodship TH (2005) The development of atypical haemolytic-uraemic syndrome is influenced by susceptibility factors in factor H and membrane cofactor protein: evidence from two independent cohorts. J Med Genet 42:852–856 Caprioli J, Castelletti F, Bucchioni S, Bettinaglio P, Bresin E, Pianetti G, Gamba S, Brioschi S, Daina E, Remuzzi G, Noris M (2003) Complement factor H mutations and gene polymorphisms in haemolytic uraemic syndrome: the C-257T, the A2089G and the G2881T polymorphisms are strongly associated with the disease. Hum Mol Genet 12:3385–3395 Kwon T, Belot A, Ranchin B, Baudouin V, Fremeaux-Bacchi V, Dragon-Durey MA, Cochat P, Loirat C (2009) Varicella as a trigger of atypical haemolytic uraemic syndrome associated with complement dysfunction: two cases. Nephrol Dial Transplant 24:2752–2754 Allen U, Licht C (2011) Pandemic H1N1 influenza A infection and (atypical) HUS—more than just another trigger? Pediatr Nephrol 26:3–5 Fang CJ, Fremeaux-Bacchi V, Liszewski MK, Pianetti G, Noris M, Goodship TH, Atkinson JP (2008) Membrane cofactor protein mutations in atypical hemolytic uremic syndrome (aHUS), fatal Stx-HUS, C3 glomerulonephritis, and the HELLP syndrome. Blood 111:624–632 Bitzan M, Schaefer F, Reymond D (2010) Treatment of typical (enteropathic) hemolytic uremic syndrome. Semin Thromb Hemost 36:594–610 Copelovitch L, Kaplan BS (2008) Streptococcus pneumoniae-associated hemolytic uremic syndrome. Pediatr Nephrol 23:1951–1956 Remuzzi G (1987) HUS and TTP: variable expression of a single entity. Kidney Int 32:292–308 Sadler JE (2008) Von Willebrand factor, ADAMTS13, and thrombotic thrombocytopenic purpura. Blood 112:11–18 Kavanagh D, Richards A, Fremeaux-Bacchi V, Noris M, Goodship T, Remuzzi G, Atkinson JP (2007) Screening for complement system abnormalities in patients with atypical hemolytic uremic syndrome. Clin J Am Soc Nephrol 2:591–596 Ake JA, Jelacic S, Ciol MA, Watkins SL, Murray KF, Christie DL, Klein EJ, Tarr PI (2005) Relative nephroprotection during Escherichia coli O157:H7 infections: association with intravenous volume expansion. Pediatrics 115:e673–e680 Hickey CA, Beattie TJ, Cowieson J, Miyashita Y, Strife CF, Frem JC, Peterson JM, Butani L, Jones DP, Havens PL, Patel HP, Wong CS, Andreoli SP, Rothbaum RJ, Beck AM, Tarr PI (2011) Early volume expansion during diarrhea and relative nephroprotection during subsequent hemolytic uremic syndrome. Arch Pediatr Adolesc Med 165:884–889 Tarr PI, Neill MA (2001) Escherichia coli O157:H7. Gastroenterol Clin North Am 30:735–751 Cavari Y, Pitfield AF, Kissoon N (2013) Intravenous maintenance fluids revisited. Pediatr Emerg Care 29:1225–1228 Chowdhury AH, Cox EF, Francis ST, Lobo DN (2012) A randomized, controlled, double-blind crossover study on the effects of 2-L

infusions of 0.9% saline and plasma-lyte(R) 148 on renal blood flow velocity and renal cortical tissue perfusion in healthy volunteers. Ann Surg 256:18–24 145. Yunos NM, Bellomo R, Hegarty C, Story D, Ho L, Bailey M (2012) Association between a chloride-liberal vs chloride-restrictive intravenous fluid administration strategy and kidney injury in critically ill adults. JAMA 308:1566–1572 146. Bitzan M (2009) Treatment options for HUS secondary to Escherichia coli O157:H7. Kidney Int Suppl: S62-6 147. Van Dyck M, Proesmans W (2004) Renoprotection by ACE inhibitors after severe hemolytic uremic syndrome. Pediatr Nephrol 19: 688–690 148. Wong CS, Jelacic S, Habeeb RL, Watkins SL, Tarr PI (2000) The risk of the hemolytic-uremic syndrome after antibiotic treatment of Escherichia coli O157:H7 infections. N Engl J Med 342:1930– 1936 149. Safdar N, Said A, Gangnon RE, Maki DG (2002) Risk of hemolytic uremic syndrome after antibiotic treatment of Escherichia coli O157:H7 enteritis: a meta-analysis. JAMA 288: 996–1001 150. Nitschke M, Sayk F, Hartel C, Roseland RT, Hauswaldt S, Steinhoff J, Fellermann K, Derad I, Wellhoner P, Buning J, Tiemer B, Katalinic A, Rupp J, Lehnert H, Solbach W, Knobloch JK (2012) Association between azithromycin therapy and duration of bacterial shedding among patients with Shiga toxin-producing enteroaggregative Escherichia coli O104:H4. JAMA 307:1046– 1052 151. Dundas S, Murphy J, Soutar RL, Jones GA, Hutchinson SJ, Todd WT (1999) Effectiveness of therapeutic plasma exchange in the 1996 Lanarkshire Escherichia coli O157:H7 outbreak. Lancet 354:1327–1330 152. Carter AO, Borczyk AA, Carlson JA, Harvey B, Hockin JC, Karmali MA, Krishnan C, Korn DA, Lior H (1987) A severe outbreak of Escherichia coli O157:H7–associated hemorrhagic colitis in a nursing home. N Engl J Med 317:1496–1500 153. Kielstein JT, Beutel G, Fleig S, Steinhoff J, Meyer TN, Hafer C, Kuhlmann U, Bramstedt J, Panzer U, Vischedyk M, Busch V, Ries W, Mitzner S, Mees S, Stracke S, Nurnberger J, Gerke P, Wiesner M, Sucke B, Abu-Tair M, Kribben A, Klause N, Schindler R, Merkel F, Schnatter S, Dorresteijn EM, Samuelsson O, Brunkhorst R (2012) Best supportive care and therapeutic plasma exchange with or without eculizumab in Shiga-toxin-producing E. coli O104: H4 induced haemolytic-uraemic syndrome: an analysis of the German STEC-HUS registry. Nephrol Dial Transplant 27:3807– 3815 154. Ruggenenti P, Remuzzi G (2012) Thrombotic microangiopathy: E. coli O104:H4 German outbreak: a missed opportunity. Nat Rev Nephrol 8:558–560 155. Delmas Y, Vendrely B, Clouzeau B, Bachir H, Bui HN, Lacraz A, Helou S, Bordes C, Reffet A, Llanas B, Skopinski S, Rolland P, Gruson D, Combe C. 2013. Outbreak of Escherichia coli O104:H4 haemolytic uraemic syndrome in France: outcome with eculizumab. Nephrol Dial Transplant 0: 1–9 doi: 10.1093/ndt/gft470 156. Ferraris JR, Ramirez JA, Ruiz S, Caletti MG, Vallejo G, Piantanida JJ, Araujo JL, Sojo ET (2002) Shiga toxin-associated hemolytic uremic syndrome: absence of recurrence after renal transplantation. Pediatr Nephrol 17:809–814 157. Artz MA, Steenbergen EJ, Hoitsma AJ, Monnens LA, Wetzels JF (2003) Renal transplantation in patients with hemolytic uremic syndrome: high rate of recurrence and increased incidence of acute rejections. Transplantation 76:821–826 158. Loirat C, Niaudet P (2003) The risk of recurrence of hemolytic uremic syndrome after renal transplantation in children. Pediatr Nephrol 18:1095–1101 159. Ariceta G, Besbas N, Johnson S, Karpman D, Landau D, Licht C, Loirat C, Pecoraro C, Taylor CM, Van de Kar N, Vandewalle J,

Semin Immunopathol Zimmerhackl LB (2009) Guideline for the investigation and initial therapy of diarrhea-negative hemolytic uremic syndrome. Pediatr Nephrol 24:687–696 160. Taylor CM, Machin S, Wigmore SJ, Goodship TH (2010) Clinical practice guidelines for the management of atypical haemolytic uraemic syndrome in the United Kingdom. Br J Haematol 148: 37–47 161. Nathanson S, Fremeaux-Bacchi V, Deschenes G (2001) Successful plasma therapy in hemolytic uremic syndrome with factor H deficiency. Pediatr Nephrol 16:554–556 162. de Jorge EG, Macor P, Paixao-Cavalcante D, Rose KL, Tedesco F, Cook HT, Botto M, Pickering MC (2011) The development of atypical hemolytic uremic syndrome depends on complement C5. J Am Soc Nephrol 22:137–145 163. Salant DJ (2011) Targeting complement C5 in atypical hemolytic uremic syndrome. J Am Soc Nephrol 22:7–9 164. Wong EK, Goodship TH, Kavanagh D (2013) Complement therapy in atypical haemolytic uraemic syndrome (aHUS). Mol Immunol 56:199–212 165. Campistol JM, Arias M, Ariceta G, Blasco M, Espinosa M, Grinyo JM, Praga M, Torra R, Vilalta R, Rodriguez de Cordoba S (2013) An update for atypical haemolytic uraemic syndrome: diagnosis and treatment. A consensus document. Nefrologia 33:27–45 166. Noris M, Remuzzi G (2013) Managing and preventing atypical hemolytic uremic syndrome recurrence after kidney transplantation. Curr Opin Nephrol Hypertens 22:704–712 167. Hirt-Minkowski P, Schaub S, Mayr M, Schifferli JA, Dickenmann M, Fremeaux-Bacchi V, Steiger J (2009) Haemolytic uraemic syndrome caused by factor H mutation: is single kidney transplantation under intensive plasmatherapy an option? Nephrol Dial Transplant 24:3548–3551 168. Remuzzi G, Ruggenenti P, Codazzi D, Noris M, Caprioli J, Locatelli G, Gridelli B (2002) Combined kidney and liver transplantation for familial haemolytic uraemic syndrome. Lancet 359:1671–1672 169. Remuzzi G, Ruggenenti P, Colledan M, Gridelli B, Bertani A, Bettinaglio P, Bucchioni S, Sonzogni A, Bonanomi E, Sonzogni V, Platt JL, Perico N, Noris M (2005) Hemolytic uremic syndrome: a fatal outcome after kidney and liver transplantation performed to correct factor h gene mutation. Am J Transplant 5:1146–1150 170. Koskinen AR, Tukiainen E, Arola J, Nordin A, Hockerstedt HK, Nilsson B, Isoniemi H, Jokiranta TS (2011) Complement activation during liver transplantation—special emphasis on patients with atypical hemolytic uremic syndrome. Am J Transplant 11:1885– 1895 171. Saland JM, Ruggenenti P, Remuzzi G (2009) Liver-kidney transplantation to cure atypical hemolytic uremic syndrome. J Am Soc Nephrol 20:940–949 172. Haller W, Milford DV, Goodship TH, Sharif K, Mirza DF, McKiernan PJ (2010) Successful isolated liver transplantation in a child with atypical hemolytic uremic syndrome and a mutation in complement factor H. Am J Transplant 10:2142–2147 173. Jalanko H, Peltonen S, Koskinen A, Puntila J, Isoniemi H, Holmberg C, Pinomaki A, Armstrong E, Koivusalo A, Tukiainen E, Makisalo H, Saland J, Remuzzi G, de Cordoba S, Lassila R, Meri S, Jokiranta TS (2008) Successful liver-kidney transplantation in two children with aHUS caused by a mutation in complement factor H. Am J Transplant 8:216–221

174. Forbes TA, Bradbury MG, Goodship TH, McKiernan PJ, Milford DV (2013) Changing strategies for organ transplantation in atypical haemolytic uraemic syndrome: a tertiary case series. Pediatr Transplant 17:E93–E99 175. Saland JM, Shneider BL, Bromberg JS, Shi PA, Ward SC, Magid MS, Benchimol C, Seikaly MG, Emre SH, Bresin E, Remuzzi G (2009) Successful split liver-kidney transplant for factor H associated hemolytic uremic syndrome. Clin J Am Soc Nephrol 4:201– 206 176. Tran H, Chaudhuri A, Concepcion W, Grimm PC (2013) Use of eculizumab and plasma exchange in successful combined liverkidney transplantation in a case of atypical HUS associated with complement factor H mutation. Pediatr Nephrol. doi:10.1007/ s00467-013-2630-5 177. Zimmerhackl LB, Hofer J, Cortina G, Mark W, Wurzner R, Jungraithmayr TC, Khursigara G, Kliche KO, Radauer W (2010) Prophylactic eculizumab after renal transplantation in atypical hemolytic-uremic syndrome. N Engl J Med 362:1746–1748 178. Zuber J, Le Quintrec M, Krid S, Bertoye C, Gueutin V, Lahoche A, Heyne N, Ardissino G, Chatelet V, Noel LH, Hourmant M, Niaudet P, Fremeaux-Bacchi V, Rondeau E, Legendre C, Loirat C (2012) Eculizumab for atypical hemolytic uremic syndrome recurrence in renal transplantation. Am J Transplant 12:3337–3354 179. Nester C, Stewart Z, Myers D, Jetton J, Nair R, Reed A, Thomas C, Smith R, Brophy P (2011) Pre-emptive eculizumab and plasmapheresis for renal transplant in atypical hemolytic uremic syndrome. Clin J Am Soc Nephrol 6:1488–1494 180. Krid S, Roumenina LT, Beury D, Charbit M, Boyer O, FremeauxBacchi V, Niaudet P (2012) Renal transplantation under prophylactic eculizumab in atypical hemolytic uremic syndrome with CFH/ CFHR1 hybrid protein. Am J Transplant 12:1938–1944 181. Le Quintrec M, Zuber J, Moulin B, Kamar N, Jablonski M, Lionet A, Chatelet V, Mousson C, Mourad G, Bridoux F, Cassuto E, Loirat C, Rondeau E, Delahousse M, Fremeaux-Bacchi V (2013) Complement genes strongly predict recurrence and graft outcome in adult renal transplant recipients with atypical hemolytic and uremic syndrome. Am J Transplant 13:663–675 182. Davin JC, Gracchi V, Bouts A, Groothoff J, Strain L, Goodship T (2010) Maintenance of kidney function following treatment with eculizumab and discontinuation of plasma exchange after a third kidney transplant for atypical hemolytic uremic syndrome associated with a CFH mutation. Am J Kidney Dis 55:708–711 183. Nurnberger J, Philipp T, Witzke O, Opazo Saez A, Vester U, Baba HA, Kribben A, Zimmerhackl LB, Janecke AR, Nagel M, Kirschfink M (2009) Eculizumab for atypical hemolytic-uremic syndrome. N Engl J Med 360:542–544 184. Al-Akash SI, Almond PS, Savell VH Jr, Gharaybeh SI, Hogue C (2011) Eculizumab induces long-term remission in recurrent posttransplant HUS associated with C3 gene mutation. Pediatr Nephrol 26:613–619 185. Alachkar N, Bagnasco SM, Montgomery RA (2012) Eculizumab for the treatment of two recurrences of atypical hemolytic uremic syndrome in a kidney allograft. Transpl Int 25:e93–e95 186. Barrow M (2013) Health chiefs refuse to pay for lifesaving kidney treatment. In The Times 16 January 187. Laurance J (2013) At what cost? Live saving drug withheld. In The Independent 26 May 2013