Introduction

Hemolytic uremic syndrome (HUS) is characterized by the triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury (AKI) [1]. Approximately 90 % of pediatric patients develop this syndrome after infection with Shigella dysenteriae, which produces true Shiga toxins, or Escherichia coli, some strains of which produce Shiga-like toxins. Shiga toxin was originally called verotoxin because Vero cells derived from the kidney epithelial cells of the African green monkey are hypersensitive to this toxin [2]. Subsequently, other toxins were called Shiga-like toxin because of their similarities to Shiga toxin in terms of their antigenicity and structure. Shiga-like toxin-1 differs from Shiga toxin by only 1 amino acid, whereas Shiga-like toxin-2 shares 56 % sequence homology with Shiga-like toxin-1. Although Shiga-like toxin-producing E. coli-HUS (STEC-HUS) strains most often trigger HUS, certain Shiga toxin-secreting strains of S. dysenteriae can also cause HUS. They are currently known as the Shiga toxin family, and the terms are often used interchangeably. HUS occurring from infection with STEC-HUS was formerly called diarrhea + HUS (D + HUS) or typical HUS.

In contrast, HUS that is not related to Shiga toxins and accounts for ~10 % of all HUS cases, is called atypical HUS (aHUS). Although STEC-HUS is relatively common in children, aHUS occurs in individuals of all ages and is often familial. The prognosis is very poor, with the first aHUS attack being associated with a mortality rate of ~25 %, and with ~50 % of cases resulting in end-stage renal disease requiring dialysis [3].

In recent years, abnormalities in the mechanisms underlying complement regulation have been focused on as causes of aHUS. Various genetic abnormalities in complement regulatory factors, including complement factor H, have been noted in 50–60 % of patients. The analysis of the pathology underlying this condition is currently progressing rapidly [4].

The differential diagnosis of aHUS from STEC-HUS or thrombotic thrombocytopenic purpura (TTP), another form of thrombotic microangiopathy (TMA) caused by a deficiency of ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), is not necessarily easy at the early stages of disease onset. However, if treatment is delayed, there is a high risk of this syndrome progressing to renal failure. Therefore, the Joint Committee of the Japanese Society of Nephrology and the Japan Pediatric Society (JSN/JPS) has developed diagnostic criteria for aHUS to enable its early diagnosis and to facilitate the timely initiation of appropriate treatment [5, 6]. We hope that the diagnostic criteria presented in this report will become familiar to as many clinicians as possible and that they will be used widely.

Definition of aHUS

aHUS is a type of TMA that differs from STEC-HUS and TTP, with the latter being caused by markedly reduced ADAMTS13 activity. aHUS is a syndrome characterized by the triad of microangiopathic hemolytic anemia, thrombocytopenia, and AKI, which is similar to STEC-HUS.

Guidelines for the diagnosis of aHUS

Definitive diagnosis

A definitive diagnosis of aHUS is made when the triad of microangiopathic hemolytic anemia, thrombocytopenia, and AKI is present. The disease should not be associated with Shiga toxins, and TTP should also be excluded.

The Joint Committee of the JSN/JPS defined microangiopathic hemolytic anemia based on a hemoglobin (Hb) level of <10 g/dL. The presence of microangiopathic hemolytic anemia should be confirmed based on increased serum lactate dehydrogenase levels, a marked decrease in serum haptoglobin levels, and the presence of red blood cell fragments in a peripheral blood smear.

Thrombocytopenia is defined as a platelet (PLT) count of <150,000/μL.

The definition of AKI has been updated, with the most recent definition given by the international guidelines group, the Kidney Disease: Improving Global Outcomes that integrates both the Risk, Injury, Failure, Loss, End-stage kidney disease and the Acute Kidney Injury Network classifications to facilitate identification. Thus, we recommend diagnosis based on the most recent guidelines, along with the following definitions. For pediatric cases, the serum creatinine should be increased to a level that is 1.5fold higher than the serum creatinine reference values based on age and gender issued by the Japanese Society for Pediatric Nephrology [7]. For adult cases, the diagnostic criteria for AKI should be used.

Guidelines for the diagnosis of aHUS

Definitive diagnosis

A definitive diagnosis of aHUS is made when the triad of microangiopathic hemolytic anemia, thrombocytopenia, and AKI is present. The disease should have no association with Shiga toxins, and TTP should also be excluded. Table 1 presents the definitions of microangiopathic hemolytic anemia, thrombocytopenia, and AKI that are established by the Joint Committee of the JSN/JPS.

Table 1 Definitions of microangiopathic hemolytic anemia, thrombocytopenia, and AKI that have been established by the joint committee of the JSN/JPS
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Probable diagnosis

A probable diagnosis of aHUS is made when 2 of the following 3 conditions are found: microangiopathic hemolytic anemia, thrombocytopenia, and AKI. The disease should have no association with Shiga toxins and TTP should be excluded.

Applicability of these diagnostic criteria

When we applied these diagnostic criteria to the Nara Medical University (NMU) TMA cohort, 15 out of 37 individuals who had all the data required for the assessment were diagnosed as having definitive aHUS. Since the data were recorded at one time point only, we speculate that the sensitivity of the diagnostic criteria would increase if we could assess data from multiple time points. The cut-off value for anemia, defined as an Hb level of <10 g/dL, and the cut-off value for thrombocytopenia, defined as a PLT count of <150,000/μL, are equivalent to those employed by the International Registry of Recurrent and Familial HUS/TTP [8]. We had considered using a cut-off value of a PLT count <100,000/μL for thrombocytopenia to reflect that used in the diagnostic criteria for STEC-HUS by the Japanese Society for Pediatric Nephrology (2000), but we only found 1 patient with a PLT count between 100,000 and 150,000/μL in the NMU cohort. Therefore, it is likely that this difference will not have a large impact on the sensitivity or specificity of our diagnostic criteria. Our diagnostic criteria include the category of “Probable” aHUS because we believe that this tentative diagnosis will help in the early diagnosis of aHUS and avoid delays in developing appropriate therapeutic approaches for patients with aHUS.

Evaluation of inappropriate complement activation

Abnormalities in complement regulation are among the main causes of aHUS. The diagnosis of aHUS that is caused by inappropriate complement activation has become more critical because eculizumab, a humanized anti-C5 monoclonal antibody, has been shown to be an effective therapeutic modality [9] that has been approved for the treatment of aHUS patients in Europe and the United States. Recently, Fan and colleagues evaluated genotype–phenotype relationships in 10 Japanese patients with aHUS and identified potentially causative mutations in complement factor H, C3, membrane cofactor protein, and thrombomodulin in 8 of the patients [10]. However, the definitive diagnosis of inappropriate complement activation in aHUS patients is difficult because some patients show normal serum levels of complement components [11] and there are a number of complement regulatory proteins, making it difficult to decide which complement regulatory protein is responsible for a particular patient developing aHUS.

Excluding Shiga toxin-producing E. coli infection

STEC-HUS is characterized by diarrhea accompanied by bloody stools. However, diarrhea may also be present in some aHUS cases. Diarrhea in aHUS can be a manifestation of ischemic colitis. In addition, enteritis that is not caused by STEC can trigger aHUS. Therefore, a diagnosis of STEC-HUS cannot be made based on symptoms alone, and the earlier nomenclature that used “D + HUS” to correspond with STEC-HUS and “D-HUS” to correspond with aHUS is not used at present [11]. The involvement of Shiga toxins should be confirmed by stool culture, the direct detection of Shiga toxins, or the detection of anti-lipopolysaccharide-IgM antibodies.

Excluding TTP

Conventionally, TTP has been diagnosed based on the classic pentad (microangiopathic hemolytic anemia, thrombocytopenia, labile psychoneurotic disorder, fever, and renal failure). However, the discovery of ADAMTS13 led to the finding that 60–90 % of patients with TTP have a marked reduction in the activity of ADAMTS13, to a level of <5 %, regardless of race. Therefore, when diagnosing aHUS, patients who have markedly reduced levels of ADAMTS13 activity (<5 %) should be diagnosed as having TTP, thereby ruling out a diagnosis of aHUS. However, some patients may show the classic TTP pentad and have normal or slightly reduced levels of ADAMTS activity. Therefore, if a patient has levels of ADAMTS13 activity ≥5 %, a differential diagnosis of aHUS or TTP may be necessary to account for other clinical symptoms.

Excluding TMA caused by other distinct factors

Diseases that evidently cause a clinical state of TMA, including disseminated intravascular coagulation, sclerodermatous kidney, and malignant hypertension, should be excluded when diagnosing aHUS.

When a probable case of aHUS is suspected

When a probable case of aHUS is suspected, samples that are necessary to determine the appropriate diagnosis should be collected, and the therapeutic strategy should be established after consultation with an institution that has extensive experience of managing aHUS cases.

Cases where aHUS should be strongly suspected

If there are features that are characteristic of HUS, aHUS should be strongly suspected if the following criteria are fulfilled, regardless of the presence of diarrhea: the patient is younger than 6 months of age; time of onset is unclear (latent onset); the patient has a history of HUS (recurrent case); the patient has a history of anemia of unknown cause; recurrent HUS after kidney transplantation; the patient has a family history of HUS (excluding cases of food poisoning); and, the patient has no diarrhea or bloody stools.

Classification of aHUS causes, excluding TTP caused by the ADAMTS13 defect

Table 2 classifies the causes of aHUS and presents methods to determine the causes.

Table 2 Classification and determination of the causes of aHUS, excluding TTP caused by the ADAMTS13 defect
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Discussion

Nineteen years after Gasser et al. [1] reported HUS, an interesting report was published in the Lancet [10]. This report indicated that although C3-predominant activity is initiated in the blood vessels in TMA patients, this is not observed in typical cases of HUS, suggesting that complement activation is involved in aHUS onset [12]. Subsequently, numerous researchers have elucidated further information on the pathology of aHUS. At present, the reported causes of aHUS include, complement regulation abnormalities, cobalamin metabolism disorder, infection with Streptococcus pneumoniae and other microorganisms, drugs, pregnancy, and autoimmune diseases.

The complement system plays an important role as part of the immune systems of living organisms. It is activated via 3 pathways, the classical, alternative, and lectin pathways. As a result of the activation of the host’s alternative and classical pathways, C5b-9, a membrane attack complex, is generated and destroys cells by forming transmembrane pores. The alternative pathway is involved in the onset of aHUS. Unlike the classical and lectin pathways, activation of the alternative pathway does not require initiators; it is continuously activated by the spontaneous hydrolysis of C3.

When complement proteins are inappropriately activated, there is a risk of inducing cell dysfunction within the host itself. Thus, humoral factors in the circulating plasma and several plasma membrane-bound factors are involved in the regulation of complement activation and act at various stages, such as the inactivation of C3b or C4b, and the inhibition of the generation of membrane attack complexes. The regulators involved in the alternative pathway include complement factors H and I, which are humoral factors, and membrane cofactor protein and thrombomodulin, which are membrane-bound factors. If these factors are abnormal, the subsequent failure of regulation will hyperactivate the complement proteins, leading to the onset of aHUS. Some cases of aHUS develop after trigger events, for example, infections of the respiratory tract and the gastrointestinal tract, and it is likely that activation of the complement cascade by these trigger events and the subsequent amplification of complement activation by the alternative pathway cannot be regulated in patients with deficiencies in complement regulation. Gain-of-function mutations in C3 and complement factor B, which are complement-activating factors, also cause hyperactivation of complement proteins and, ultimately, aHUS.

It has been reported that ~50 % of aHUS patients have genetic abnormalities in complement regulatory factors, including complement factor H. The frequency of the presence of certain mutations among aHUS cases, responsiveness to plasma therapy, prognosis of kidney function, and the recurrence rate after kidney transplantation, vary depending on the type of genetic abnormalities present [13]. Although plasmapheresis within 24 h of confirmation of the diagnosis has been recommended as the initial treatment for aHUS [14], its effects are not always satisfactory. The mortality or incidence of end-stage renal disease is considered to be between 70 and 80 %, and the recurrence rate after kidney transplantation may be as high as 80–90 %, particularly in patients with abnormal complement factor H, which is the most frequent abnormality [15].

In 2011, eculizumab (Soliris®, Alexion Pharmaceuticals), a terminal complement inhibitor, was approved as a new drug for the treatment of aHUS in Europe and the US. Eculizumab is a humanized recombinant immunoglobulin G2/4 monoclonal antibody directed against the complement component C5, which was developed as a treatment for paroxysmal nocturnal hemoglobinuria. By binding to complement component C5, the drug inhibits the generation of C5a and C5b-9, and thus subsequently inhibits the complement system.

There are a number of reports stating that only HUS that is associated with complement regulation abnormalities is defined as aHUS. On the basis of the current diagnostic criteria, we have defined aHUS to include all types of HUS that are not related to Shiga toxins or other distinct causes. In cases where aHUS is associated with complement dysregulation, the introduction of eculizumab may markedly change therapeutic strategies. It should be noted, however, that recommendations of specific therapeutic modalities are beyond the scope of the current diagnostic criteria. However, in cases where complement dysregulation is confirmed as the cause, treatment with eculizumab is established. Thus, it may be desirable to assign HUS associated with complement dysregulation a separate disease name rather than it being classified as “aHUS”, as in the case of definitive “complement-mediated TMA”.

As described in previous reports, aHUS is a disease that may frequently cause renal failure and be fatal if it is not appropriately diagnosed and treated at the early stages of disease onset. In Japan, aHUS may be misdiagnosed as HUS caused by Shiga toxins because clinicians are not sufficiently aware of aHUS, and consequently, treatment may be delayed. Thus, our diagnostic criteria include the category of “Probable” aHUS to ensure that the clinicians consider aHUS during diagnosis. Many issues should be addressed in the future, including the development of diagnostic strategies to diagnose cases of suspected aHUS, the establishment of insurance coverage for ADAMTS13 activity measurement testing that is necessary to differentiate aHUS from TTP, and the development of treatment guidelines. We hope that our diagnostic criteria will be used widely and will contribute to the diagnosis and treatment of aHUS patients.